phytochemistry and pharmacology 2.1...

ETHNOPHARMACOLOGICAL VALIDATION OF MEDICINAL PLANTS TREATING SKIN DISEASES IN HYDERABAD KARNATAKA REGION 41

CHAPTER-II

Phytochemistry and pharmacology

2.1 Introduction

Study on natural products is always an interesting target for scientists over

decades, especially on plants. Since the beginning of human civilization, medicinal

plants have been used by mankind for its therapeutic value. Nature has been a source of

medicinal agents for thousands of years and an impressive number of modern drugs have

been isolated from natural sources. Many of these isolations were based on the uses of

the agents in traditional medicine. The plant -based, traditional medicine systems

continues to play an essential role in health care, with about 80% of the world‘s

inhabitants relying mainly on traditional medicines for their primary health care

(Owolabi et al ., 2007). Historically, plants (fruits, vegetables, medicinal herbs, etc.)

have provided a good source of a wide variety of compounds, such as phenolic

compounds, nitrogen compounds, vitamins, terpenoids and some other secondary

metabolites, which are rich in valuable bioactivities like antioxidant, anti - inflammatory,

antitumor, antimutagenic, anti carcinogenic, antibacterial, orantiviral activities (Maridass

and Britto, 2008). In many oriental countries (India, China etc), the traditional herbal

medicines have been widely used for thousands of years. Herbal plants have become the

main object of chemists, biochemist, and pharmaceutics. Their research plays an

important role for discovering and developing new drugs, which are having hopefully

more effectiveness and no side actions like most synthetic modern drugs. Besides

focusing on chemistry of compounds from any plant, the studies of herbal plant s are

based on folkloric reputation and traditional uses. In addition, the isolation and

identification on these plants are due to the activities of their extracts and fractions. This

shows clearly that studies on herbal plants are requirements and demands of natural

products scientist or researchers. From natural products, a number of herbal drugs have

been developed into form of food supplements, nutraceuticals, and complementary/

alternative medicine.

In India, medicinal plants have been used as natural medicine since the days of

Vedic glory (Gupta et al., 2008). Therefore, study an on herbal plant profoundly not only

to discover active compounds but also to find the effective mechanism of them to


develop into drugs for treatment of skin diseases including other. Furthermore, the

studies also supplied general constituents and effects that can encourage the use of herbal

plants as ―food‖ for intensifying health and prevent diseases.

Importance of Drugs Discovery from Plants – Right now rapid development is

continuously happening in the field of chemistry of medicinal research. Despite this

rapid development, many plant derived drugs are still cannot be synthetically produced.

Two reason stands behind the statements. Some compounds such as atropine and

resperine are still too expensive to be synthesized; and many useful drugs also still

cannot be synthesized such as morphine, cocaine, ergotamine and digitalis (Ernavitha

2008). Thus, the isolation of plant derived drugs still holds important rules in drug

discovery. Once plant derived drugs is isolated, then it can act as the lead compound

which is a good starting point in developing new drug. It can allow the design and

rational planning of the new drugs as well as bio mimetic synthesis development and

discovery of new biological activity not yet related to the known compounds

(Hamburger and Hostettmann, 1991). One example is salicylic acid that originally

synthesized to found replacement for phenol as antiseptic. Further finding reported its

antipyretic and ant rheumatic activities (Sneader, 2005).

Medicinal plants produce bioactive compounds used mainly for medicinal

purposes. These compounds either act on different systems of animals including man,

and/or act through interfering in the metabolism of microbes infecting them. The

microbes may be pathogenic or symbiotic. In either way the bioactive compounds from

medicinal plants play a determining role in regulating host-microbe interaction in favour

of the host. So the identification of bioactive compound in plants, their isolation,

purification and characterization of active ingredients in crude extracts by various

analytical methods is important. The medicinal properties of plants could be based on the

antioxidant, antimicrobial, antipyretic effects of the phytochemicals in them (Adesokan

et al., 2008).

Infectious diseases caused by bacteria, fungi, viruses and parasites are still a

major threat to public health, despite the tremendous progress in human medicine. Their

impact is particularly large in developing countries due to the relative unavailability of

medicines and the emergence of widespread drug resistance (Okeke et al., 2005).


Research on new antimicrobial substances must therefore be continued and all possible

strategies should be explored. Besides small molecules from medicinal chemistry,

natural products are still major sources of innovative therapeutic agents for vario us

conditions, including infectious diseases (Clardy and Walsh, 2004). Only a minute

portion of the available diversity among fungi, marine fauna and flora, bacteria and

plants has yet been explored and ample opportunities lie theoretically ahead. Current

research on natural molecules and products primarily focuses on plants since they can be

sourced more easily and selected on the basis of their ethno-medicinal use (Verpoorte et

al., 2005). However, the chemical complexity of many natural products and the lack of

assurance of a renewable supply have created a diminishing interest by the

pharmaceutical industry, which in turn endorses the pivotal role of academia and public

organisations in the protracted exploration and evaluation of natural products. Use o f

ethnopharmacological knowledge is one attractive way to reduce empiricism and

enhance the probability of success in new drug-finding efforts (Patwardhan, 2005). Any

effort to identify pharmacological action entails the access to both robust bioassays and

targeted collections of compounds and extracts for testing. Specific hurdles for

ethnopharmacology include either the isolation and characterisation of bioactive

molecules in the extract and the problem of ―reisolation‖ of known bioactive compounds

or the standardization of plant extracts. In addition, fractionation of extracts frequently

leads to a reduction or loss of biological activity by compound break-down or loss of

additive or synergistic effects between analogue constituents. Validation and select ion of

primary screening assays are pivotal to guarantee sound selection of extracts or

molecules with relevant pharmacological action and worthy following-up. Primary

bioassays are generally designed for rapid screening of large numbers of products or

extracts. They are simple, easy to implement and produce results quickly and preferably

at low cost. Compounds or extracts with a specific activity at a non-toxic dose, so-called

―hits‖, then need further evaluation in secondary or specialized in vitro bioassays and in

animal models to define ―lead‖ status. Advanced assessment of kinetic and toxicological

properties will ultimately define full ‗proof-of concept‘ and ‗development- candidate‘

status (Verkman, 2004). A recent review of the literature (Rios and Recio, 2005)

revealed that still too many articles on natural products claim so-called ―exciting‖

antimicrobial activities, despite major flaws in used methodologies. Most frequent are

the lack of sound criteria for activity, the omission of appropriate in-test controls, the


inclusion of unrealistically high assay dosages and the nature of the bioassay itself

(selection of target rganism, endpoints, etc.). In an effort to provide some guidance on

how to improve quality of screening against infectious organisms,

The present chapter focused on Phytochemistry and antidermatophytic activity of

ethno-medicinal plants in detail with two steps.

In step 1, screening of 61 ethno-medicinal plants results presented.

In step 2, selected 07 plants detailed Phytochemistry and antidermatophytic activity is

presented.

2.2 Review of literature

The maximum plants used for medicinal purpose have been identified, and their

uses are documented and described by different authors (Nadkarni, 1954; Dastur, 1985;

Saradamma, 1990), but the efficacy of many of these plants is yet to be verified.

Moreover, natural plant extracts have been tested in the laboratory against fungi and

bacteria. Natural plant products yield extracts with antineoplastic, antimicrobial,

antifungal and antiviral activities (Lau et al., 1993). In the past few decades, a worldwide

increase in the incidence of fungal infections has been observed as well as a rise in the

resistance of some species of fungus to different fungicides used in medicinal practice.

Fungi are one of the most neglected pathogens, as demonstrated by the fact that the

amphotericin B, a polyene antibiotic discovered as long ago as 1956, is still used as

―gold standard‖ for antifungal therapy. The two decades have witnessed a dramatic rise

in the incidence of life threatening system of fungal infections. The challenge has been to

develop effective strategies for the treatment of candidiasis and other fungal diseases,

considering the increase in opportunistic fungal infections in human immunodeficiency

virus-positive patients and in others who are immunocompromised due to cancer

chemotherapy and the indiscriminate use of antibiotics. Invasive fungal infections are

associated with high rates of morbidity and mortality. The majority of clinically used

antifungals have limitations that include one of more of incomplete spectra of activity,

toxicities, poor stability, lack of oral availability and high cost and their frequent use has

led to the emergence of resistant strains. It is generally accepted that improved drugs

that ideally, act on different antifungal targets are needed.


A review of the literature on the evaluation of medicinal plant extracts showed

that many studies on antifungal activities have been carried out in recent years.

Antimicrobial acitivity of Indigofera suffruticosa was studied by Sonia Periera Leite et

al., (2006), the MIC value to dermatophyte strains were 2500 μg/ml against

Trichophyton rubrum (LM-09, LM-13) and Microsporum canis. Antidermatophyte

activities of Eucalyptus camaldulensis in comparison with griseofulvin were studied by

Mehraban et al., (2005). E. camaldulensis showed antifungal activity against all the

dermatophytes tested with MIC values ranging from 0.4 to 1.6 mg/ml using inhibitory

zone estimation, 0.4–1.6 mg/ml using agar dilution method and 0.2–1.6 mg/ml using

broth dilution method and minimum fungicidal concentration (MFC) of the extracts

ranged from 0.8 to 6.4 mg/ml. The five hydroethanolic extracts of Terminalia

glaucescens and Anogeissus leiocarpus appeared to be the most active, their MICs

ranging from 0.25 mg/ml to 4 mg/ml (Batawila, 2005).

During screening of 20 essential oils against Epidermophyton flocosum and

Microsporum gypseum, oils of Ocimum gratissimum and Trachyspermum ammi

exhibited strong antidermatophytic properties (Tiwari et al., 2003). The in vitro activity

of Malaleuca alternifolia oil against dermaophytes were determined and MICs for all

fungi ranged from 0.004% to 2.5% and minimum fungicidal concentrations ranged from

<0.03% to 8.0%. Antidermatophytic activities of Azadirachta indica was evaluated by

Ranganathan et al., (1996), and found that MIC of neem seed extract was found to be

lower than that of neem leaf when tested against different species of Trichophyton and

Epidermophyton floccosum. Gadhi et al., (2001), in the screening of antidermatophytic

efficiency against human pathogenic fungi, found the hexane fraction most effect (MIC

range: 64-2048 μg/ml), whereas the butanol fraction was the least active (MIC range:

1024 μg/ml) and the most susceptible fungi were E. floccosum and T. violaceum in

contrast to T. mentagrophytes and T. rubrum which were less sensitive to the fractions

tested. Rai and Acharya (1999) in the screening studies of some Asteraceous plants for

antimycotic activity found that the maximum antimycotic activity against F. oxysporum

and T. mentagrophytes was exhibited by flower extract of Tagetes erecta followed by

whole plant of T. patula and leaf extract of T. erecta whereas, extracts of Emilia

sonchifolia, Tridax procumbens and Cichorum intybus exhibited the minimum inhibitory

effect on T. mentagrophytes. Ali Shtayeh and Abu Ghdeib (1999), reported on the

antifungal activity of plant extracts against dermatophytes, extracts of Capparis spinosa


and Juglans regia completely prevented growth of M. canis and T. violaceum which was

found the most susceptible being completely inhibited by 50% of the extracts followed

by M. canis and T. mentagrophytes which were completely inhibited by only 23 and

14% of the extracts, respectively. In an antifungal screening of Nelumbo nucifera

rhizome extract inhibited the growth of all the yeast and fungal organisms tested (Pulok

Mukherjee, 1995). The essential oils of Cinnamomum tamala and Citrus maxima

exhibited absolute inhibition of mycelial growth on dermatopytes viz., Trichophton

mentagrophytes and Microsporum audouini (Dubey etal., 1998).

Wrightia tinctoria leaf hexane, methanol and ethanol extracts were screened

against skin bacteria and dermatophytes by in vitro. Wrightia tinctoria leaves possessed

potent antimicrobial properties against dermatophytic microbes. In particular, methanol

and ethanol extracts were active against bacteria and hexane extract was active against

dermatophytic fungi, suggesting that the active principles may be useful in the topical

treatment of superficial skin infections (Kannan et al., 2006).

The extract of the different plant species reduced colony growth of the three

dermatophytes by 36 to 100% compared with the control treatment. Antimycotic activity

of the extract against the three dermatophytes varied significantly (P<0.05) between test

plants (Ali-Shtayeh and Suheil, 1999).

Pratibha Yadav and Dubey (1993), during screening of 12 essential oils of higher

plants against two ring worm fungi Trichophyton mentagrophytes and M. audounii,

found the oils of plants viz., Cinnamomum tamala, Citrus maxima, Cymbpogon citrates,

Eucalyptus citriodora, Eupatorium cannabinum, Nepeta hindostana, Ocimum canum

showed absolute toxicity against both the test fungi. Antidermatophytic ac tivity of

Neem was investigated against 88 clinical isolates of dermatophytes by Pankajalakshmi

and Taralakshmi (1994). The ethanolic extract was found to be more active inhibiting

90% (MIC 90) of the isolates at a concentration of 100 µg/ml. Turmeric oil and

curcumin, isolated from Curcuma longa L. were studied against 15 isolates of

dermatophytes, by Apisariyakul et al., (1995), who reported, all 15 isolates of

dermatophytes could be inhibited by turmeric oil at dilutions of 1:40-1:320 and none of

the isolates were inhibited by curcumin.

According to Maoz and Neeman (1998), of the 10 plant extracts tested against

M. canis and T. rubrum. Inula viscosa showed maximal inhibitory effect, especially

against T. rubrum (MIC of 0.625%). Garg and Rajashree Jain (1999), studied antifungal


activity of essential oils from the fruits of Luvunga scandens and found the oil exhibited

very good to moderate inhibitory effect against the fungi and insisted the susceptibility

of the oil towards dermatophytes is interesting and can be exploited against dermal

infections. In the study of antifungal activity of the different extracts of Symphytum

sylvaticum, maximum in root alkaloid extract and Echinidine-N-Oxide exhibited strong

inhibitory activity for nine fungal culture out of ten (except C. albicans) at 200 µg/ml

(Murat Kartal et al., 2001). Karunyal Samuel et al, (2001), reported that aqueous

extract of Allium sativum bulbs inhibited Trichophyton rubrum at a given concentration

(200 mg of bulbs/ml) by disc diffusion method. Crude ethanol extract of Curcuma longa

exhibited an inhibition zone range of 6.1 to 26.0 mm against 29 clinical strains of

dermaophytes (Mansuang Wuthi-adamlert, 2002).

Several plant extracts showed antifungal activity against 13 human pathogenic

fungi and compared to the activities of Amphotericin B and Ketoconazole, and the plant-

derived antifungal berberine by Flicker (2002), the most powerful ones were extracts of

ginger and butternut that displayed antifungal activity against a wide variety of fungi.

Lucia Kiokoo Hasimot Esouza et al., (2003), in the evaluatin of essential oil and the

aqueous, hexane and 98% methanolic fractions from Hyptis ovalifolia leaves reported

that the most biologically active was the essential oil from the leaves which inhibied 57

isiolates of dermatophytes (95%) at a concentration <500 µg/ml. Antifungal activity of

Piper guineense was studied by Ngono Ngane et al., (2003), using filamentous fungi and

yeasts. The results indicated significant antifungal effect.

The increasing resistance to antifungal compounds and the reduced number of

available drugs led us search for therapeutic alternative among aromatic plants and their

essential oils, used for empirically antifungal properties. In recent years, these reports

have involved mainly the Lamiaceae and Asteraceae families. The antifungal effect on

Candida albicans growth of the essential oils from several species of the Lamiaceae

family, Satureja Montana L., Lavandula angustifolia Mill, Lavandula hybrida

Reverchon, Origanum vulgare L., Rosmarinus officinalis L. and six chemotypes of

Thymus vulgaris L. were studied. The greatest efficiency was obtained with the essential

oil from the T. vulgaris thymol chemotype (IC50 of 0.016 µg/ml). From two of these

genera, Lavandula and Rosmarinus, extensive works on the antifungal activity of their

essential oils have been reported. The essential oils and the aqueous, hexane and 98%


methanolic fractions from Hyptis ovalifolia leaves were evaluated for their antifungal

activity in vitro against 60 strains of dermatophytes. The extracts inhibited growth of the

dermatophytes tested at different concentrations. The most biologically active was the

essential oil from the leaves which inhibited 57 isolates (95%) at a concentration of 500

μg/ml.

Farukh Ali and Iqbal Ahmad (2003) reported antifungal activity of 22

traditionally used Indian medicinal plants against five filamentous fungi and a yeast

Candida albicans of clinical origin. Broad spectrum antimicrobial activity (with

antibacterial and antifungal) was detected among crude extracts of various medicinal

plants.

Effect of leaf and seed extracts of Azadirachta indica were evaluated against

various dermatophytes by Natarajan et al., (2003), the MIC of neem seed extracts was

31 µg/ml for all the dermatophytes tested. The neem seed extract at 15 µg/ml

concentrations (below MIC) was observed to be sufficient for distorting the growth

pattern of the organisms tested. Verastegui et al., (1996) investigated the antifungal

activity of several widely distributed plants in the vegetation of Northern Mexico and the

Southern U.S.A. The plants were evaluated on the growth of yeast and moulds: Candida

albicans, Candida krusei, Candida rugos, Cryptococcus neoformans, Cryptococcus

laurentis, Crptococcus labidus, Microsporum canis, Microsporum gypseum,

Trichophyton tonsurans, Epidermophyton floccosum and Sporotrix schenckii. The

extracts analysed showed good antifungal activity against more than one organism.

Extracts of Mitracarpus villosus leaves and inflorescences were investigated

against T.rubrum, M. gypseum, C. albicans, A. niger and Fusarium solani. The aqueous

extracts and the glycerol vehicle control did not inhibit any of the fungi tested. The zones

of inhibition produced by the ethanol extracts ranged from 10 to 20.5 mm (Irobi and

Daramola, 1993).

Another screening for antifungal agents was done by Schmourlo et al., (2005) on

medicinal and fruit bearing plants used against skin diseases by the Brazilian population.

The results, evaluated by the diameter of the inhibition zone of fungal growth, indicate

that six plant species, among the sixteen investigated, showed significant activity against

three fungi: Candida albicans, Trichophyton rubrum and Cyptococcus neoformans.

Duarte et al., (2005) screened ethanol extracts of the leaves and/or roots of thirty five


medicinal plants commonly used in Brazil for anti-Candida albicans activity. Extracts

from thirteen plants showed activity.

In recent years, there have also been a large number o f antifungal screening

programmes of medicinal plants used in the traditional medicine of Eastern Europe and

Africa. Tadeg et al., (2005), investigated the antifungal activities of some selected

traditional Ethiopian medicinal plants used in the treatment of skin disorders.

Hydroalcohol extracts of Acokanthera schimperi (D.C) Benth et Hook. (Apocyanaceae),

Calpurna aurea L. (Leguminoseae), Kalanchoe petitiana (Engl.) Cufod. (Crassulaceae),

Lippia adonensis Hochst. (verbenaceae), Malva parviflora L. (Malvaceae), Olinia

rochetiana L. (Oliniaceae) Phytolacca dodecandra L Herit (Phytolaccaceae) and

Verbuscum sinaiticum Bentham (Scophulariaceae) were screened for antifungal activity

against different strains of fungi which are known to cause different types of skin

infections. Of all the plants tested, L. adoensis and O. rochetiana were found to be the

most active species against fungal strains.

Seventy seven crude extracts from leaves and stem barks of fifteen Gabonese

plants used in traditional medicine were evaluated from their antifungal activities by

Lamidi et al., (2005). The methanol extract of Polyalthia suaveolens Engler and Dicls

(Polygonaceae) displayed good antifungal activity on all the strains tested with IC50

values (inhibitory concentration required for 50% inhibition) of 1 mg/ml. Zaidi and

Crow (2005) reported the antifungal activity of the following four important medicinal

plants from Balochistan, Pakistan: Grewia erythraea Schwein F. (Tiliaceae),

Hymenocrater sessilifolius Fisch. (Lamiaceae), Vincetoxicum stocksii (Asclepidiaceae)

and Zygophyllum fabago L. (Zygophyllaceae). The extracts of Z. fabago and V. stocksii

showed good activity against Candida albicans. In an antifungal screening programme

by Phongpaichit et al., (2005), thirty six extracts derived from ten plant species used by

traditional Thai healers were assayed for their antifungal activity against clinical isolates

of Candida albicans, Cryptococcus neoformans and Micropsorum gypseum.

Besides antifungal screening programmes, a review of the literature on the

pharmacological evaluation of plant extracts shows that many studies into their

antifungal activity have been carried out in recent years. These reports concern mainly

the Asteraceae and Liliaceae families. Plants from the genus Pterocaulon (Asteraceae),

known as quitoco, are used to treat problems popularly diagnosed as mycoses, which

may have a fungic etiology. In order to validate this traditional practice, the crude


methanol extracts from the aerial parts of three species of Pterocaulon, Pterocaulon

aloppecuroides (Lam.) D.C., Pterocaulon balansue Chodat and Pterocaulon polystachyu

D.C., grown in Southern Brazil were analysed for the in vitro antifungal activity against

a panel of standardized and clinical opportunistic pathogenic yeasts and filamentous

fungi, including dermatophytes (Stein, 2005). The crude methanol extract of P.

polystachyun was the most active. Liozzo et al., (2004) investigated the antifungal

activity of methanol, ethyl acetate, dichloromethane, n- hexne, n-butanol and chloroform

of Senecio inaequidens D.C. and Senecio vulgaris L. The hexane extract of S. vulgaris

showed significant activity against T. tonsurans (IC50 of 0.031 mg/ml). Examples of

other antifungal crude extracts from the Asteraceae family also included aqueous and

petroleum ether extracts of Spilanthes calva D.C. which were active towards Fusarium

oxysporum and T. mentagrophytes (Rai et al., 2001).

In the Liliaceae family, reports on the antifungal activity concern mainly the

Allium genus. By using an agar dilution assay, the antifungal activity of aqueous extracts

prepared from Allium cepa L. and Allium sativum L. were evaluated against Malassezia

furfur, Candida albicans as well as several strains of various dermatophyte species by

Shams et al., (2006). The results indicate that onion and garlic might be promising

sources of drugs for the treatment of fungal associated diseases from the important

pathogenic genera Candida, Malassezia and the dermatophytes. Similar studies on the

antifungal activity of onion and garlic was also investigated on two important

dermatophytes, Trichophyton rubrum and Trichophyton mentagrophytes by Ghahfarokhi

et al., (2004); Iwalokun et al., (2004).

The antifungal activity of Nigella sativa L. (Ranunculaceae) seed was tested by

Aljabre et al., (2005) against eight species of dermatophytes: four species of

Trichophytum rubrum and one each to Trichophyton interdigitale, Trichophyton

mentagrophytes, Epidermophyton floccosum and Microsporum canis. These results

denote the potentiality of N. sativa as a source for antidermatophytic drugs, and support

its use in folk medicine for the treatment of fungal skin infections.

In in vitro activity of Melaleuca alternifolia oil against dermatophytes Hammer

et al., (2002) found that MICs of tea tree oil for all fungi ranged from 0.004% to 0.25%

and minimum fungicidal concentrations (MFCs) ranged from <0.03% to 8.0%.

The antifungal activity of a crude extract from Yucca gloriosa L. (Agavaceae)

flowers, named alexin, was investigated in vitro against a panel of human pathogenic


fungi and yests, as well as dermatophytes and filamentus species (Favel et al., 2005).

Alien has a broad spectrum of antifungal activity for all the tested yeast strains, except

for Candida lusitaniae and Candida kefyr. It was also active against several clinical

Candida isolates known to be resistant to the usual antifungal agents. One member of the

Nyctaginaceae family, Boerhavia diffusa L., was active against the dermatophytic

species of Microsporum gypseum, Microsporum fulvum and Microsporum canis

(Agrawal et al, .2003).

Crude methanol extracts and fractions from the aerial parts of seven species of

Hypericum growing in Southern Brazil were analysed for their in vitro antifungal

activity against a panel of standardized and clinical opportunistic pathogenic yeasts and

filamentus fungi, including dermatophytes (Fenner et al., 2005). Rojas et al., (2004)

investigated the antifungal activity of Gentianella itida Griseb. The most susceptible

microorganisms were Candida albicans, Trichophyton mentagrophytes and

Microsporum gypseum. The antifungal activity was concentrated in the 90% methanol

and non-soluble fractions. Reports on the antifungal activity of medicinal species

belonging to the Myrtaceae family include the herbal food clove Syzygium aromaticum L

(Taguchi et al., 2005) and extracts of Eucalyptus globules Labill. Eucalyptus muculata

Hook. and Eucalyptus vaminalis Labill. Which significantly inhibited the growth of the

fungus T. mentagrophytes (Takahashi et al., 2004). In the zingiberaceae family, the

ethanol extract of Curcuma longa L and A. galanga were also found to possess good

antifungal activities against T. longifusus (Khattak et al., 2005).

Based on an ethnobotanical approach, the dragon‘s b lood collected from Croton

urucurana Baill. bark was tested for antifungal activity against five dermatophytes by

the paper disk diffusion method (Gurgel et al., 2005).

In an attempt made by Pyun and Shin (2006) to develop stable and antifungal

agents from natural products (daily food stuffs in particular), the activity of essential oils

from Allium fistulosum L., A. sativum and A.cepa (liliaceae) were investigated against

three Trichophyton species responsible for severe mycoses in humans. Among the oils

tested A. sativum oil exhibited the strongest inhibition of growth of Trichophyton rubrum

with and IC50 value of 61 µg/ml, while the activities of A. cepa and A. fistulosum were

relatively mild. The antifungal activity of hexane, ethyl acetate and methano l extracts of

45 medicinal plants of minimum inhibitory concentration for each extract against human

pathogenic fungi (T. rubrum, T. mentagrophytes, T. simii, E. floccosum, C. albicans) was


determined by Duraipandiyan V, Ignacimuthu S (2011). One of the review paper

published by P. Saranraj and S. Sivasakthi (2014) from Tamil Nadu on antimicrobial

properties of various medicinal plants. The water extract, methanol, free flavonoids

and bound flavonoids of Allium sativum, Cymbopogon martinii and Catharanthus

roseus were screened for their antimycotic activities using disc diffusion method

against human pathogenic dermatophytes determined by Seema Bhadauria and Padma

Kumar (2011).

Review of Literature on selected plants

2.2.1 Annona reticulata L.

A. reticulata L. belongs to Annonaceae, this is rich in phenolic compounds. The

genus Annona consists of about 119 species (Thang et al., 2013). A. reticulata L. is a

semi-evergreen and small deciduous (Baskar et al., 2007). It is commonly called as

custard apple, bullock's-heart or ox-heart (Chang et al., 1993).

The common names of A. ret iculata L. are in English, it is known as Custard

apple, Jamaican apple, Sugar apple, Netted custard apple, Bullock's heart, Sweetsop, In

Malaysia it is known as Lonang, Nona kapri, while in Thailand as Noinong. In Spanish

as Anona colorada, Anona deseso Anona deredecilla, Anona roja, Corazón, Anona

rosada, Mamon in French known as Corossol sauvage, Bois Cachi man, Coeur de boeuf,

cachiman, while in Hindi it is called as Luvun, Ramphal, Nonai

(http://en.wikipedia.org/wi ki/A. reticulata L..).

Ethnomedicinal value: Decoction of the bark or dried or pulverized unripe fruit is used

in annoreticuin, bullatacin, squamosine, rolliniastatin(Chang et al., 1993), reticullaci

none, rolli niastati n-2, molvizari n (Maeda U et al., 1993), 1 4-hydroxy-25-deoxy-rol li

nicin (Hisham A et al., 1994). Bul lataci n and a novel bi oacti ve monotetrahydrofuran

acetogeni n, reticulatacin, and kaurane diterpenes have been isolated from the bark of the

A. reticulata L. (Annonaceae) by bi oacti vi ty-di rected fractionation the treatment of

dysentery and diarrhea (Duke et al., 1993). Crushed leaves or paste prepared from the

flesh are use as poultice for abscesses and are also use for ulcers. Fruits are having

anthelmintic properties. The root bark is use in toothache and is placed around the gums

to get relief from toothache and roots of the plant are used in the form of a prepared

decoction for fever (Duke et al., 1993). Decoction of the leaves is use mostly in relieving

http://en.wi/

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malaria and syphilis (http://www.globinmed.com/i). The roots used for epilepsy

http://www.worldagroforestrycentre.org/sea/Products/A). The plant has been used as an

antiinflammatory agent in wound healing, anti-anxiety, anti-stress, anti-mutagenic, and

spasmolytic agent. Leaf and stem extract shows inotropic, positive chronotropic and

spasmolytic activities (http://www.stuartxchange.org/Anonas.).

Phytochemistry

Terpenes namely spathenelol, muurolene, copaene and eudesmol were also

reported (Saad et al., 1991). A novel compound cytotoxic 7-1actone acetogenin was

isolated by Chang et al. squamocin from ethyl acetate extract of seeds of A. reticulata L.

(Anonymous, 1994). Annonaretin a, a new triterpenoid was chemically investigated from

the leaves of A. reticulata L. by Shung T, Wu et al.

N-fatty acyl tryptamines were also reported from A. reticulata L. (FR et al.,

1993). Ogunwande and Ekundayo has obtained hydrodistilled oil from the leaves of A.

reticulata L. from in Nigeria.

Two cyclopepti des, the cycl oheptapepti de cycloreticulin C, cyclo(Pro1-Gly 2-

Gln3-Pro4-Pro5-Tyr6-Val7) and the cyclohexapeptide glabrin A, cyclo( Pro1-G ly2-L eu3-V

al 4-Ile5-Tyr6) were isolated using methanol seeds extract of the of A. reticulata. Whereas

Anonymous, (1994) and Chang et al., (1993), Contributed the sequence and three-

dimensional structure of cycloreticulins A and B, new cyclooctapeptides was identified.

Thirty nine compounds were characterized by Jirovetz L et al., (1998). Among

these, 18 monoterpenes amounting to sesquiterpenes totaling 52.9% and one aromatic

esters making upto, the oil contained (E, E) farnesyl acetate, ar-turmerone, benzyl

benzoate and gamma-terpinene as the major constituents.

Pharmacology:

Nine antiinflammatory compounds were characterized from the leaves of A.

reticulata L, by Thang et al., 2013. Mondal et al., (2007) reported anthelmintic activity

using aqueous leaf extract.

Analgesic and CNS depressant results were reported by Bhalke and Chavan

(2011) from ethyl acetate, methanol and petroleum ether extracts of A. ret iculata L. All

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the extracts exhibited significant central analgesic activity in the hot plate method in

mice. All the extract showed statistically significant mild to moderate central nervous

system depressant activity assessed by locomotor activity assay and pentobarbitone

sleeping time test.

An antioxidant study was carried out on three well known species of Annona.

Among these studies A. reticulata L. leaves extracts was showed better activity in

quenching DPPH and superoxide radicals (Thang 2013).

The effective cytotoxic activity was recorded by some of these compounds

against Hep.G2, Hep.2, 3, 15, KB and CCM2, four cancer cell- lines. In the next step,

isolation and purifying annonacin was studied, for biological activity.

Aetogenins are a important molecules from plants belonging to Annonaceae,

having potentials of antineoplastic agents. That main five annonaceous acetogenins

which are solamin, annoreticulin-9-one, annomonicin, squamone, and rolliniastatin are

having cytotoxic activities. Acetogenins isolated from the seeds of A. ret iculata L.

The ethanoliextract exhibited a significant in v itro and in v ivo inhibitory activity

against melanoma tumor cells. Alkaloids are also known to possess cytotoxic properties.

Ethanol and aqueous extract of roots of Annona ret iculata, are evaluated for the in

v ivo, against melanoma cells in mice for anticancer activity and also in v itro for

inhibitory activity on MDA- MB-435 human melanoma cells.Simultaneously, ethanol

extracts in v itro inhibition towards the vero cell line proliferation was found to be lower

in comparison with cancer cell lines (Yuan et al., 2003). Suresh et al (2011) investigated

in v itro cytotoxic and human recombinant caspase inhibitory using leaves.

Squamocin was isolated from the seeds of A. ret iculata L. was also analysed for

its biological effects and proved that squamocin is a cytotoxic constituent for all the

cancer cell lines tested (Baskar et al., 2007, Chang et al 1993, Ogunwande IA and

Olusegun E 2006, Yuan et al., 2003).

Nature has provided us with a huge count of flora and fauna. Some of the natural

medicinal plants are so common that we use them in daily life without knowing their

medicinal importance. A. reticulata L. is the best example of it. The extensive survey


literature reviewed that A. reticulata L. is an important medicinal plant with diverse

pharmacological spectrum. Few novel chemical constituent isolated from the A.

reticulata L. showed anti-cancer, properties for bladder cancer and various cancer cell

lines too. It‘s found to be a chemopreventive agent in cancer therapy. Further

evaluation is needed to be carried out on A. reticulata L. in order to explore concealed

areas and their practical clinical application, which can be used for the welfare of the

mankind.

2.2.2. Annona squamosa L.

A. squamosa L. (Annonaceae), commonly known as seethaphal, it is a native of

West Indies. Folkloric record reported the use of A. squamosa as an insecticidal, an anti-

tumor agent, anti-diabetic, antioxidant, anti- lipidimic and antiinflammatory agent

which has been characterized due to the presence of the cyclic peptides.

The leaves were applied on the ulcers and wounds. A leaf decoction was taken in

case of dysentery in traditional reports,anti- fertility and antitumour activities were

observed in mice and rats. The young leaves of Annona squamosa L. were used

extensively due to its anti-diabetic activity (Annie Shirwaikar et.al., 2004).

The past phytochemical investigations made on the plant have proved that

they possess a wide variety of compounds like acetogenins which were responsible

for anti- feedant, anti-malarial, cytotoxic and the immunosuppressive activities.

Diterpenes which was isolated from the A. squamosa L. possess the anti-HIV principle

and the anti-platelet aggregation activity. The partially purified flavonoids were

reported from the same source as the responsible agent for the anti-microbial and other

pesticidal activities. Some lignans and other hydroxyl ketones were also found to be

present in this plant. The number of alkaloids that was reported from this plant

belongs to different categories such as aporphine and benzoquinazoline. The above

provided evidences suggested that the plant is known for its various medicinal

values (Dinesh et al., 2011).

This plant also playing an important role in ethno medicine, that include anti-

fertility and antitumour. The young leaves have been using against anti-diabetes. (Annie

Shirwaikar et.al., 2004). Its leaves were used as the insecticidal and antispasmodic


agents that were used in the treatment of rheumatism and painful spleen. The

plant was also reported treating analgesic, antiinflammatory, anti-pyretic, anti-ulcer and

antiseptic and abortifacient activities. While other various phytochemical,

pharmacological, anti-bacterial and anti-ovulatory studies was performed using seeds

(Chavan et al., 2010).

The roots were found to be effective as a drastic purgative and in the

acute dysentery (Mukhlesur Rahman et al., 2005). The hot aqueous extract of A.

squamosa L. leaves was investigated to possess a significant hypoglycemic and

anti-diabetic activity (Rajesh Kumar Gupta et al., (2008) Dos Santos and Sant'Ana

(2001).

Phytochemical importance

A numerous acetogenins were isolated from the seeds of A. squamosa L. For the

most part, they were found to be a mono- or adjacent bis-THF-ring bearing compounds.

Annonaceous acetogenins were a group of compounds that were isolated so far only

from the Annonaceae family, but were recently reported to be present in the family of

Vitaceae. These compounds were characterized by the presence of terminal g-

methyl-glactone and by the presence of a long aliphatic chain bearing

tetrahydrofuranic THF and tetrahydropyranic rings, and the epoxy rings and (or)

the double bonds. They were reported to inhibit the first complex of mitochondrial

respiratory chain (NADH-ubiquinone oxydo-reductase), and also exhibits

parasiticide, insecticide and other cytotoxic activities, and were also represented as

the anti-tumoral candidates (Idensi Bajin ba Ndob et al., 2009).

The discovery of a compound uvaricin in 1982 was the first report on the

Annonaceous acetogenins, found to act as an in vivo active anti- leukemia (P-388) agent

that hasinvigorated a wide interest in the family of Annonaceae (Dos Santos AF and

Sant'Ana 2001).

Annonaceous acetogenins, was recently proved to inhibit the ATP production

at a similar site of action and at the higher levels of potency as a rotenone, i.e., at the

NADH-ubiquinone oxido-reductase, complex I in the mitochondrial electron-

transport chain (Landolta et al., 1995).


Chavan et al., (2010). Isolated Caryophyllene oxide from petroleum ether bark

extract of A. squamosa L. Whereas few new annonaceous acetogenins discoverd by

Craig Hopp et al., (1998). namely (2,4-cis and trans)-squamolinone, (2,4-cis and

trans)-9-oxo-asimicinone, and bullacin B (Craig Hopp et al., 1998).

The Chromatographic purification of the seeds of n-BuOH soluble fraction was

resulted in the isolation of seven cyclic peptides which were named as

cyclosquamosins A -G. Cyclic peptides were the molecules possessing a wide range of

biological activities. Hence, the Conformational determination of such cyclic peptides

plays an important role, because of their biological activities that were known to be

closely related with their conformational states. Recently, there was a report on the

conformations of a list of cyclic heptapeptides, such as hymenamide, pseudostellarin D,

and yunnanin A, and segetalins D and E10. Two bis-tetrahydrofuran acetogenins,

squamocin-O1 and squamocin-O2, were the compounds isolated from a MeOH extract

of the seeds of A. squamosa L. (Hiroshi Arayaa et al., 2002).

The isolation of three new bioactive acetogenins by Craig hopp D et al.,

(1998). namely 4-deoxy annoreticuin, cis-4deoxyannoreticuin, and (2,4-cis and

trans)-squamoxinone Two more new Annonaceous acetogenins called as the

squamostanin-C and squamostanin-D were isolated from the 95% EtOH seed extract of

the A. squamosa L.

Another similar compound namely Rollicosin was isolated from Rollinia

mucosa and Squamostolide from the A.squamosa L. These compounds contain a

partial skeleton of an ordinary Annonaceous acetogenins with two c- lactone

moieties on both the sides of an aliphatic chain. Rollicosin can be generated fro m the

oxidative degradation of the ordinary acetogenins such as murisolin and/or from

the cis-murisolin and also squamostolide from solamin and/or from the cis-solamin.

Moreover, these compounds were found to be helpful to investigating the role of

the terminal hydroxylated lactone moiety instead of the hydroxylated THF moiety with

long aliphatic chain that could be seen in the ordinary acetogenins for its bioactivity

(Hidefumi Makabe et al., 2006).


Pharmacological importance

A. squamosa L. seeds were generally thrown away as the waste materials. But,

they too were found to possess certain insecticidal, anti-ovulatory, abortifacient and

antiimplantation properties. The extract from the seeds were evaluated to know their

ameliorative effect in the regulation of hyperthyroidism in the mouse model. Serum

triiodothyronine (T3), thyroxine (T4) concentrations, hepatic glucose-6-phospatase

(G-6-Pase) and 5‘-mono-deiodinase (5‘DI) activity were determined as the end

parameters to assess the alterations in the thyroid function. And also certain other

parameters like hepatic lipid peroxidation (LPO), superoxide dismutase (SOD) and

catalase (CAT) activitieswere also investigated to reveal its hepatotoxic effect. The

TLC, UV spectra and HPLC analyses revealed the presence of quercetin in the

given test sample. This proves that the anti-thyroidal role of A. squamosa L. seed

extract could have been mediated by the quercetin. Further, the seed extract was

found to decrease the hepatic lipid peroxidation which has suggested that it is safe

and possess anti-peroxidative nature. Quercetin was also found to decrease the hepatic

LPO (Panda, Kar, 2007).

The antimicrobial activities of the plant compounds such as Petroleum ether

extract (PE), CHCl3 extract (CE), EtOH extract (EE), annotemoyin-1,

annotemoyin-2, squamocin and cholesteryl glucopyranoside showed maximum

inhibition against the gram positive organisms such as B. subtilis B. cereus, B.

megaterium, Staphylococcus aureus S. b-haemolytica, Sarcina lutea and the gram

negative organisms such as E. coli, S. dysenteriae, S. shiga, S. flexneriae, S.sonnei,

Salmonella typhi, P. aeruginosa, Klebsiella spp. The cytotoxicity of the plant

extracts was studied by the brine shrimp lethality bioassay and the LC50 values of

the petroleum ether and chloroform extracts were calculated by Mukhlesur Rahman et

al., (2005).

Anti-oxidants are the compounds responsible for the protection of living

organism from the damage caused by the abnormal production of reactive oxygen

species concomitant lipid peroxidation, protein damages and others including DNA

strand breaking etc. The aqueous extract of the A. squamosa L. significantly reduced

the triglyceride and total cholesterol levels with a gradual increase in the HDL


cholesterol level in the treated diabetic rats when compared to that of the

untreated diabetic rats (control) Rajesh Kumar Gupta et al., (2008).

Anti- inflammatory activity was proved using Caryophyllene oxide which was

isolated from an unsaponified petroleum ether extract from the bark of A. squamosa

L. (Chavan et al., 2010). Mujeeb Mohd et al., (2009) was investigated antidiabetic and

hypoglycemic activity using ethanolic extract of A. squamosa leaves.

Dinesh, Yadav et al., (2011). Investigated anti-ulcer activity in chloroform and

hexane fractions. The attenuated the formation of ulcer in CRU, PL, HA model and

also displayed anti-secretory activity in vivo with the decrease in plasma gastrin level.

Cytoprotection of A. squamosa L. was apparent with protection in AL, ASP models

and enhanced mucin level in PL. (+)-Omethylarmepavine, N-methylcorydaldine,

lanuginosine, were found to be the active principles of the plant which may serve as

the initial point for the designing of novel semi-synthetic and synthetic compounds

as the antiulcer agents in the future.

The ethanolic extracts of A. squamosa L. was evaluated against the adult forms

and egg masses of Biomphalaria glabrata. A. squamosa L. was used from the

traditional period as the toxic agent against the snail and then the experimental studies

of the seed, root, stem, bark and leaf‘s ethanolic extract was also found to show

the molluscicidal activity against the adult snail at a maximum concentration of 100

ppm(Hiroshi Arayaa et al., 2002).

Seed extract of A. squamosa L. produced a compound isosquamocin whichcould

be used as a promising pesticide for the protection of the plants. The genotoxicity of the

compound was also evaluated by the comet assay and other related studies which

revealed the fact that the genotoxicity and biochemical effects of A. squamosa L.

may not cause any risk to humans in a large magnitude. However, the dosages

have to be further established by the development of other mutagenic tests to

make the moderate usage in order to reduce the health risk of humans (Paramjit

Grover et al., 2009).


A cyclic octapeptide, cyclosquamosin B which was isolated from the seeds

ofAnnona squamosa was found to show a potential vasore laxant effect on the rat

aorta. The vasorelaxant effect caused by the cyclosquamosin might be attributed

significantly to the inhibition of calcium influx from the extracellular space via

voltage-dependent calcium channels (Hiroshi MoritA et al., 2006).

Twelve different acetogenins with diverse stereo chemical structures and

configurations namely asimicin18, squamocin18, squamocin-D18,

desacetyluvaricin18, Isodesacetyluvaricin18, squamostatin-D18, squamostatin-E18,

squamostatin B18, squamostatin-A18, 12, 15-cis-squamostatin-A19, 4-

deoxyannoreticuin20, and cis-4-deoxyannoreticuin20 were evaluated for their ability to

inhibit the growth of cancer cell lines using MTT method. (Haijun Yang et al., 2009).

The hepatoprotective effect of the alcoholic and water extract of A.

squamosa L. was evaluated in the hepatotoxic induced animals in order to explore its

usage for the treatment of hepatotoxicity in the human (Mohamed Saleem et al.,

2008). The protective effect of the 98% methanolic extract of A. squamosa L. on

isoniazid-rifampicin- induced hepatotoxicity was also evaluated in the rats and was

found that they also showed a protective effect against the liver injury (Mohamed

saleem et al., 2011).

The larvicidal and the growth regulating activities of A. squamosa L. was

reported against An.stephensi and other mosquitoes. The high potency of A. squamosa L

as a larvicide against mosquito species was evaluated but the active compound that

possess a toxic substance against the larval species has to be identified by Marta,

Souza et al., (2008).

In the past insecticidal studies, the common housefly Musca domestica

(Diptera: Muscidae) which is an important mechanical vector of many bacterial

and pathogenic microbes of human and animals have become resistant to the chemical

insecticides. Annonaceous acetogenins which were extracted from the tree leaves,

bark and seeds possess the insect anti- feedant properties. The larvicidal activities of

the ethanolic extracts of A. squamosa L leaves against the Musca domestica was

evaluated by Audrey Leatemia and Isman 2004).


The anthelmintic activity of the extracts and the isolated compounds o f A.

squamosa L. seeds were evaluated on the egg hatching of H. contortus. Compound one

which was isolated from the ethyl acetate extract inhibited the egg hatching of H.

contortus at the concentration of about 25 mg /ml and the structure of compound

one was determined as a C37 trihydroxy adjacent bistetrahydrofuran acetogenin by

the spectroscopic analysis (Souza et al., 2008). The anthelmintic activity of the A.

squamosa L. seed extract against the adult earthworm, Pheritima posthuma was

also investigated and was found that the 98% methanolic extract showed

theeffective anthelmintic activity causing the death of earthworms (Srilakshmi et al.,

2011).

Corchorus olitorius L.

Corchorus olitorius L. is belongs to family Tiliaceae, native of tropical Africa,

Asia, and has since spread to Australia, South America and some parts of Europe. C.

olitorius L. is an annual, much-branched herb 90-120 cm tall with glabrous stems, leaves

6-10 cm long and 3.5-5 cm broad, with pale yellow flowers and black trigonous seeds

(Kirtikar and Basu 1975).

Ethnomedicinal Importance

C. olitorius L. has huge medicinal values. The dried material is known as

"nalita." Injections of olitoriside markedly improve cardiac insufficiencies and have no

cumulative attributes; hence, it can serve as a substitute for strophanthin. It is used as

deobstruent, diuretic, lactagogue, purgative tonic. Tussah jute is a folk remedy for

aches and pains, dysentery, enteritis, fever, dysentery, pectoral pains, and tumors

(Duke and Wain, 1981; List and Horhammer, 1969-1979). Ayurvedics use the leaves for

ascites, pain, piles, and tumors. Elsewhere the leaves are used for cystitis, dysuria,

fever, and gonorrhea. The cold infusion is said to restore the appetite and strength

(Duke, 1981).

Phytochemistry

The action of the seed extract can be attributed to phytochemical content of the

extract. Of these flavanoids (Taoying Zhou et al., 2009, Kaku Nakagawa et al., 2004),

alkaloids (Day Cartwright 1990), saponins (George Francis et al., 2002) have been


reported to have hypoglycaemic effect. Several researchers have reported plant extracts

(hypoglycaemic agents) with several combinations of phytochemicals to which the

reported phytochemicals belong (Ocho-Anin Atchibri et al., 2010, Atangwho et al.,

2009) of these Adeneye and Adeyemi (Adeneye et al., 2009) reported the

phytochemicals, alkaloids, flavonoids, tannins and glycosides possessed by the aqueous

seed extract of Hunteria umbellate has hypoglycaemic effects in normoglycaemic,

glucose and nicotine- induced hyperglycaemic rats. It therefore would mean that the

hypoglycaemic action of the the seed extract of C. olitorius L. could be due to the

phytotochemicals present singly or in combination.

Seventeen active nutrients compounds reported in leaves of C. olitorius L.

including protein, fat, carbohydrate, fiber, ash, Calcium, Potassium, iron, sodium,

phosphorous, beta-carotene, thiamine, riboflavin, niacin, ascorbic acid etc (Calleja,

2010). Leaves contain oxydase and chlorogenic acid. The folic acid content is

substantially higher than that of other folacinrich vegetables, ca 800 micrograins per

100g (ca 75% moisture) or ca 3200 micrograms on a zero moisture basis (Chen and

Saad, 1981). This green, leafy vegetable is rich in beta-carotene for good eyesight, iron

for healthy red blood cells, calcium for strong bones and teeth, and vitamin C for

smooth, clear skin, strong immune cells, and fast wound -healing. Vitamins A, C and E

present in Saluyot ―sponge-up‖ free radicals, scooping them up before they can commit

cellular sabotage (Chen and Saad, 1981).

It is an important green leafy vegetable in many tropical area including

India (Samra et al., 2007). The leaves which are also used as food vegetable (Zakaria et

al., 2006). The leaf extract of the plant is also employed in folklore medicine in the

treatment of gonorrhea, pain, fever and tumor (Ndlovuand Afolayan, 2008). The crop is

an excellent source of vitamin A and C, fiber, minerals including calcium, and iron. It is

reportedly consumed as healthy, vegetable in Japan because of its rich contents of

carotenoids, vitamin B, B2, C and E, and minerals. Its leaves and roots are eaten as

herbal medicine in South East Asia (Zeghichi et al., 2003). Recently aqueous

extracts of the seeds of C. olitorius were reported to possess peripheral and central anti

nociceptive, antiinflammatory and anti-pyretic activities (Zakaria et al., 2006). The

seeds are used as a purgative and have been found to contain cardenolide


glycosides on preliminary analysis, while the methanol extracts of the seeds have

been reported to possess a broad spectrum of antibacterial activity (Pal et al., 2006).

Pharmacology

Screening of natural products from plants in a search for a new antimicrobial

agent that would be active against organisms is the need of the hour. The leaves of C.

olitorius L. was reported to have hypoglycaemic effect and high antibacterial activity

(Adegoke and Adebayo-Tayo 2009).The seed protein enriched diet was found to

increase rats body weight (S. Laskar et al., 1986). The seeds were found to contain

reasonable percentage of biologically active cardiac principals (Sharaf and Negm 1969).

The plant stem is a source of jute fibre and folkloric uses includes, seeds for purgative,

leaves for dysentery, fever, gonorrhea and demulcent (Watt 1962).

The fact that the seed oil of this plant inhibited the growth of these

bacterial isolates showed that it could be used to treat infection caused by these bacterial

strains. The bacteria used in this study are associated with various forms of diseases, P.

aerugenosa (inflammation of the bladder), K. pneumonia (pneumonia), S. aureus (food

poisoning), S. typhimurium (typhoid fever) and B. cereus (eye infection, food spoilage

and food borne intoxication) (Nester et al., 2004). Results of the antibacterial

activity of the oil however, does not agree with the work of Burt (2004) and

Karaman et al. (2003), that Gram positive bacterial are more sensitive to plant oil and

extract, than Gram negative bacteria. This is supported by the findings of Doughari et al.

(2007), who reported that the root extracts of pawpaw showed more antibacterial

activity on Gram negative than Gram positive bacteria. The varying degrees of

susceptibilities of the bacterial isolates may be due to both the intrinsic tolerance of the

microorganisms, and the nature and combinations of phyto-compounds present in the

essential oil.

Euphorbia tirucalli L.

E. tirucalli L. belongs to family Euphorbiaceae, is widely grown as an

ornamental plant in India and is popularly known as Kalli plant. This plant was

introduced from Africa to tropical countries as a garden plant (Bhuvaneshwar et


al., 2010). It is commonly found in India, Brazil, and northeast region of Amazon and in

some coastal areas of Iran.

The plant is a large unarmed shrub or a small tree growing up to 5 m tall with

erect branches, bark is rough and cracked greenish brown, exuding a milky sap when cut,

branch lets slender, smooth, cylindrical, polished, whorled and modified into phylloclade

(Prasad et al., 2011). Crude extracts of E. tirucalli L. is reported to have antiarthritic

activity (Sarang et al., 2007). Latex is reported to possess proteolytic activity

(Cleverson de Freitas et al., 2010), anticancer activity (Ali et al., 2010), molluscidal

activity (Pedro et al., 1985) and larvicidal activity (Mwine et al., 2010). Stem of E.

tirucalli L. is reported to possess insecticidal activity (Uma and Prasanna 2009).

Taxonomy: Kingdom: Plantae, Division: Magnoliophyta, Class: Magnoliopsida,

Order: Malpighiales, Family: Euphorbiaceae, Subfamily: Euphorbioideae, Genus:

Euphorbia L., Species: E. tirucalli L.

Ethnomedicinal Imoprtance: The latex of E. tirucalli L. is traditionally used in treating

asthma, rheumatism, earache, cough and toothache (Wealth of India). The latex is used

as a folk remedy against syphilis. It is used as a laxative agent to control intestinal

parasites and also to treat verrucae, epithelioma, sarcoma and skin tumours in northeast

region of Brazil. The stem of E. tirucalli L. is used to treat whooping cough, asthma,

blood complaints and in infections of spleen. Stem is carminative, purgative, stomachic,

dyspepsia, gonorrhoea, leprosy, neuralgia and syphilis. The Bark of E. tirucalli L. is used

traditionally in healing the infections of spleen, colic, blood complaints, whooping cough

andasthma. Roots of E. tirucalli L. are used for treating colic pains (Rao and Hemadri).

In certain parts of East Africa, the leaf of E. tirucalli L. is boiled and the juice is used in

management of sterility of women traditionally (Kokwaro,). E. tirucalli L. is also

used to cure snakebites, warts, syphilis, sexual impotence and in skin parasites

extraction in Africa. It is popularly used in healing broken bones, hemorrhoids, pains,

ulcerations, swellings in Asia. In addition to this, it is used to treat scorpion bites,

asthma, cancer, spasms in Brazil (Cataluna, Rates, 1997). The plant was reported to

have abortifacient and emmenagogic effects in Ayurved ic system of medicine (Chopra,

Chopra), (Jyothi et al., 2008). .

Phytochemistry


E. tirucalli L. is reported to possess flavonoids, diterpenes, tannins, steroids and

alkaloids as major phytochemcical compounds (Fauconneau et al., 1997). The plant is

also reported to possess terpenes, alcohol eufol, alfaeuforbol (Macdonald et al., 1949),

taraxasterol, E. tirucallol, cycloeuphornol, n-hexacosanol (Rastogi and Mehrotra),

terpenic alcohol and trigliane. Whole plant has afforded to contain 7.4% citric acid with

some malonic and succinic acids. Terpenoids and sterols in plants are important

sources of vitamins, steroid compounds, insecticides and anticancer drugs industrially

(Itokowa et al., 1989, Wu et al., 1991).

The major components of E. tirucalli L. are triterpenes (Biesboer, Mahlberg

1979, Yamamoto et al., 2011). Latex contains diterpene esters of the phorbol, ingenol

and 12-deoxyphorbol esters, reported to be highly active carcinogenic and tumour

promoting agents. The fresh latex is reported to contain terpenic alcohol,

isoeuphorol, taraxasterol and tirucallol (Cataluna et al., 1999). Dried latex contains

Ketone euphorone. Resin is the principle constituent of dried latex of E. tirucalli L. The

stem is reported to posses hentriacontene, hentriacontanol, anti tumor steroid 4-

deoxyphorbol ester, beta-sitosterotchouc, casuarin, corilagin, cycloeupordenol,

cyclotirucanenol, ellagic acids, euphorbins, euphol, euphorone, ellagic acids,

euphorbins, euphol, euphorone, euphorcinol, gallic acids and glucosides (Khan,

Malik,1990). Aqueous extract of aerial parts of E. tirucalli L. was reported for

hepatoprotective activity in adult Wistar rats and Swiss albino mice against carbon

tetrachloride induced liver damage. The extract resulted in decrease of GSH depletion

and lipid peroxidation and showed effective protection of liver (Jyothi et al., 2008).

Pharmacology

The alcoholic extracts of stem bark and leaves of E. tirucalli L. were reported

to possess antimicrobial activity against clinical and lab isolates of Eschericia

coli, Proteus vulgaris, Salmonella enteritidis, Bacillus subtilis, Staphylococcus

aureus, Pseudomonas aeroginosa, Klebsiella pneumoniae, Candida albicans, C.

tropicalis, Aspergillus niger, A. fumigatus, A. flavus and Fusarium oxysporum

(Bhuvaneshwar et al., 2010).

Antibacterial activity of methanol extract and its aqueous extract of E.

tirucalli L. was reported. Antibacterial activities were performed by agar disc and agar


well diffusion methods against Staphylococcus epidermidis, Bacillus subtilis,

Pseudomonas pseudoalcaligenes, P. vulgarisand P. Typhimurium, P.

pseudoalcaligenes (Parekh et al., 2010). Acetone, hexane, methanol, chloroform and

petroleum ether extracts of the stems of E. tirucalli were reported to possess

antibacterial activity against seven bacterial species included Bacillus megaterium, B.

subtilis, Escherichia coli, Enterobacter faecalis, Proteus vulgaris, Pseudomonas

aeroginosa, Staphylococcus aureus and antifungal activity against Aspergillus niger,

A. fumigates and Candida albicans (Prasad et al., 2011).

Aerial parts of E. tirucalli L. was reported to carry antioxidant activity. Aqueous

extract of aerial parts of E. tirucalli L. shows reducing power activity, superoxide

anion scavenging activity and hydroxyl radical scavenging activity. All the doses of

the extract exhibited greater absorbance than control (Jyothi et al., 2008).

Insecticidal activity reported from Petroleum ether and ethyl alcohol extracts of

E. tirucalli L. were evaluated against larvae of diamond back moth (Plutella

xylostella) using standard leaf dip method reported by Uma and Prasanna (2009).

Mwine et al., (2010). was reported larvicidal activity using fresh latex of E.

tirucalli L. against Anopheles funestus and A. gambae in a neglected fish pond in

different dilutions. All the dilutions showed activity against the larvae, but highest

dilution (1:250) is preferable in order to minimize the excess usage and over dosage

problems of latex.

The biopolymeric fraction of E. tirucalli L. was reported for antiarthritic

activity against Mycobacterium tuberculosis induced adjuvant arthritis test in rats.

Wistar rats were injected subplantarly with 0.05 ml freshly prepared suspension of

heat killed M. tuberculosis in liquid paraffin to induce adjuvant arthritis (Newbould

BB 1963). BET showed significant inhibition of edema in theinjected paw with a

maximum effect at dose levels of 100 and 200 mg/kg orally. BET administered

groups, however, did not show significant swelling in the uninjected paw

(secondary response) of the experimental rats when compared to the vehicle contro l

group (Arrigoni, Brahm 1975).


E. tirucalli L. latex is reported by Pedro et al., (1985) to possess moluscicide

activity against Biomphalaria glabrata(snail) which is a mollusc vector of

schistosomiasis. In this study, latex was collected from this plant at large amount of

sunlight receiving sites. 10% dilution was prepared with water and was tested against

B. glabrata, eggs of the snail and fishes in comparision with Bayluscide and

copper sulphate. The extract showed molluscicidal activity with LD50at 28 ppm and

LD90at 85 ppm.

Ficus racemosa L.

Ficus racemosa L. Roxb. (Moraceae). The plant is a large deciduous tree

distributed all over India. It is a member of the four sacred trees. It is found throughout

the year, grows in evergreen forests, moist localities and bank of streams, deciduous

forests, to the elevation of 1800m above sea level, often cultivated in villages for shade

and its edible fruits.

It is commonly known as Gular fig, Cluster fig in English, Gular in Hindi and as

Udumbara in Sanskrit (Cooke 1967).

Ethnomedicinal importance

Root is used for treating dysentery, pectoral complaints, diabetes, applied in mumps,

other inflammatory glandular enlargements and hydrophobia. The bark is highly efficacious in

threatened abortionand also used in treating urological disorders, diabetes, hiccough, leprosy,

dysentery, asthma and piles.

The leaves are very much useful for wounds and ulcers. They are useful in dysentery and

diarrhea. The infusion of bark and leaves is also employed as mouth wash to spongy gums and

internally in dysentery, menorrhagia, effective remedy in glandular swelling, abscess, chronic

wounds, cervical adenitis and haemoptysis, improve skin complexion. Fruits are astringent,

stomachic, refrigerant, dry cough, loss of voice, diseases of kidney and spleen, astringent to

bowel, styptic, tonic, useful in the treatment of leucorrhoea, blood disorder, burning

sensation, fatigue, urinary discharges, leprosy, menorrhagic, epitasis, intestinal worms and

carminative.


They are useful in miscarriage, menorrhagia, spermatorrhoea, epididymitis, cancer,

myalgia, scabies, haemoptysis, intrinsic haemorrhage, excessive thirst, visceral obstructions.

Latex is aphrodisiac and administered in hemorrhoids, diarrhoea, diabetes, boils, alleviates the

edema in adenitis, parotitis, orchitis, traumatic swelling, toothache and vaginal disordersz,

(Chopra et al., 1992, Chopra et al., 1986, Prabhakar and Suresh 1990, VedavathyS and

Rao, 1995).

Phytochemistry

Very little phytochemical work has been carried out on F. racemosa L. The stem bark

showed the presence of two leucoanthocyanins: leucocyanidin-3-0-(3-glucopyranoside,

le ucope la ro go n id in- 3 - O - a- L-rhamnopyranoside, (3-sitosterol, unidentified long

than ketone, ceryl behenate, lupeol, its acetate, a-amyrin acetate.

From bark lupeol was isolated. Fruit contains glauanol, hentriacontane, (3-

sitosterol, gluanol acetate, glucose, tiglic acid, esters of taraxasterol, lupeol acetate,

friedelin, higher hydrocarbons and other phytosterol.

A new tetracyclic triterpene glauanol acetate and racemosic acid were isolated from

the leaves. An unusual thermostable aspartic protease was isolated from latex of the plant.

From stem bark and fruit glauanol acetate isolated (Sen and Chowdhary 1971, Agarwal and

Misra 1977, Joshi 1977, Shrivastava et al., 1977, Agarwal 1977, Bhatt and Agarwal

1973, Merchant 1979, Suresh 1979, Li et al., 2004, Devaraj et al., 2008).

Pharmacology

Hypoglycemic activity (Shrotri and Ranita 1960). Using ethanolic extract the

hypoglycemic activity was observed by Kar, Choudhary and Bandyopadhyay (2003).

Bhaskara et al., (2002), Baslas and Akhtar (1985), were recorded antidiabetic

activity using methanolic extract. A comparative antidiabetic activity methanolic extract and

standard was proving its folklore claim by Bhaskara et al., (2002), Baslas and Akhtar

(1985). From the stem bark an effective antihypoglycemic activity was detected using isolated

compound 3_sitosterol, when compared to other isolated compound by Akhtar and Qureshi

(1988). Methanolic powdered fruits extract was increased fecal excretion of cholesterol as

well as bile acids (Agarwal and Chauhan 1988).


The plant was shown effective antifungal activity against six species of fungi, viz.

Trichophyton mentagrophytas, Trichophyton rubrum, Trichophyton soundanense,

Candida albicans, Candida krusei and Torulopsis glabrata (Vonshak et al., 2003,

Baslas et al., 1985) Different extracts of leaves were tested for antibacterial potential

against Escherichia coli, Bacillus pumitis, Bacillus subtilis, Pseudomonas aeruginosa

and Staphylococcus aureus. Bhaskara et al., (2002) recorded effective activity in

petroleum ether extract. Khan and Sultana (2005) were reported methods using

significant recovery of renal glutathione content, antioxidant enzymes.

Ethanolic extract of leaves was evaluated for its hepatoprotective activity in rats against

carbon tetrachloride induced liver damage by Rao et al., (2002). In another report, the

methanolic extract of stem bark was evaluated for its hepatoprotective activity in rats against

carbon tetrachloride induced liver damage with silymarin as standard. It showed significant

reversal of all biochemical parameter towards normal when compared to carbon tetrachloride

treated control rats in serum, liver and kidney (Biswas, Mukherjee, 2003).

In vitro antioxidant activity was observed using ethanolic extract of F. racemosa

L., resulted in a significant decrease in the percentage of micro nucleated binuclear V79

cells suggesting its role as radio protector. The 50% ethanolic extract of fruits was studied

in different gastric ulcer models (Li et al., (2004). The extract showed dose dependent

inhibition of ulcer index in all three models of ulcers. The decoction of stem bark was

investigated for antidiuretic activity and also it increases urinary osmolarity, (Rastnasooriya

et al., 2003). Shaikh et al., (2010) performed antitussive potential activity with methanol

extract.

Ethanolic extract exhibited potent antioxidant activity against DPPH, ABTS,

hydroxyl radical, super oxide radical scavenging and inhibited lipid peroxidation (Mishra

et al., 2005). The methanolic extract of stem bark has shown potent in vitro antioxidant

activity when compared to the methanol extract of its roots (Biswas 2003).

Efficacy of a proprietary herbal preparation was evaluated on 28 cases of persistent

post prandial hyperglycemia(Basu et al., 1999). Li et al., (2003) was investigated

wound healing trails using stem bark of ethanolic extract. In results it was proved highly

efficacious in controlling Candida albicans infections and helped in quicker


epithelialization. In case of Shrotri, Ranita (1960) the burns were completely healed in

8 to 26 days of treatment.

The results were comparable with that of Phenylfiutazone, Racemosic acid isolated

from ethanolic extract of leaves by bioassay guided fractionation showed potent

inhibitory activity (Devaraj et al., 2008). Ethanolic extract of stem bark was performed

inflammatory activity by Malairajan et al., (2006), anthelmintic (Mandal et al., 1999),

Antidiarrhoeal (Veerapur et al., 2009), Antifilarkd (Channabasavaraj et al.,

2008),Analgesic (Jahan et al., 2008). Sophia, Manoharan (2007) were assayed the

larvicidal activity of crude hexane, ethyl acetate, petroleum ether, acetone and methanol

extracts of the leaf and bark.

Pongamia pinnata L.

Pongamia pinnata L. is a member of Leguminaceae, it is known as one of the

cool and green trees of India. Common names it as ‗Karanj‘ or ‗Papar‘ or ‗Kanji‘. It is

called ‗Karum Tree‘ or ‗Poonga Oil Tree‘ in English. It is an Indo -Malaysian species,

now found in India, Australia, Florida, Hawaii, Malaysia, Oceania, Philippines and

Seychelles (Edward, 2004). This plant considered as one of the most admired city trees

(Duke, 2008)

Ethno medicinal importance

The fruits and sprouts of P. pinnata L. were used in ethno remedies for

tumors (Hartwell 1971). Herbal remedies have been recommended in different

medical treatises for the cure of different diseases. It has been recognized in different

system of traditional medicines for the treatment of various diseases (Ghani 1998,

Kirtikar and Basu 1994). Seed extract used in treating hypotensive effects and produce

uterine contractions. Seed power is used in bronchitis, chronic fever, whooping

cough and chronic skin diseases and painful rheumatic joints (Ingredient guide

2006). Seed oil is used in scabies, piles, ulcers, chronic fever, leprosy, lever pain and

lumbago.

Its oil is a source of biodiesel and it is also used as fuel for cooking and lamps

(Mahli et al., 1989). It is considered as alternative source of energy, which is

renewable, safe and nonpollutant. Leaves are active against Micrococcus; their


juice is used for cold, cough, dyspepsia, flatulence, gonorrhoea diarrhoea and

leprosy. Roots are used for cleaning gums, teeth and ulcers. Bark is used internally for

bleeding piles. In the traditional system this was recorded for antiinflammatory

(Srinivasan et al., 2001), antiplasmodial, anti-nociceptive, anti-hyperglycaemics, anti-

lipidoxidative, antidiarrhoeal, anti-ulcer, antihyperammonic, CNS depressant activity

(Li et al., 2006) and antioxidant.

Phytochemistry

Numerous phytoconstituents belonging to flavonoids and fixed oils. P.pinnata

L. seeds contain six compounds (two sterols, three sterol derivatives and one

disaccharide) together with the eight fatty acids (three saturated and five unsaturated).

Their structures were elucidated with the help of physiochemical methods and

spectroscopic . The metabolites, β-sitosteryl acetate and galactoside, stigma sterol,

galactoside and sucrose are being reported for the first time.

The saturated and unsaturated fatty acids (two monoenoic, one dienoic and

two trienoic) were the next in the quantity. Karangin, pongamol, pongagalabrone and

pongapin, pinnatin and kanjone have been isolated from seeds. Immature seeds contain

a flavone derivative ‗pongol‘. The other flavonoid isolated from the seeds includes

Glabrachalcone , isopongachromene. The leaves and stem of the plant consist of several

flavone and chalcone derivatives such as Pongone, Galbone, Pongalabol, Pongagallone

A and B. Chemical investigation of stems of the mangrove plant, P. pinnata L.,

resulted in isolation and characterization of five structurally unusual flavonoid

metabolites (Tanaka1992). From Japan resulted in the isolation of 18 flavonoid

compounds including nine new ones (Goel RK et al., 1985).

Pharmacology

Anti-ulcer activity was reported by Prabha et al., (2003) using 98% methanolic

extract of P. Pinnata L. roots showed significantly protection against aspirin, but not

against ethanol- induced ulceration. It was showed tendency to decrease acetic acid-

induced (Meera et al., 2003).

Anti-microbial effect of crude decoction of dried leaves of P. Pinnata L.

(Brijesh et al., 2006) and also evaluated its effect on production and action of


enterotoxins (cholera toxin, Escherichia coli labile toxin and E.coli, stable toxin)

and adherence of enteropathogenic E. coli and invasion of enteroinvasive E. coli

and Shigella flexneri to epithelial cells. The decoction had no anti-bacterial, anti-

giardial, and anti-rotaviral activities, but reduced production of cholera toxin and

bacterial invasion to epithelial cells (Brijesh et al., 2006).

Mathias (2001), Majeed (2005) were observed that effect of P. Pinnata L. leaf

extract on circulatory lipid peroxidation and antioxidant status was evaluated in

ammonium chloride- induced hyperammonium rats. It enhanced lipid peroxidation in

the circulation of ammonium chloride-treated rats (Essa et al., 2006). Simonsen et al.,

(2001) was reported antiplasmodial activity against Plasmodium falciparum.

Ethanolic flower extract of P. Pinnata L. shows significant antihyperglycaemic

and anti- lipidperoxidative effect and enhancement in antioxidant defense system in

alloxan- induced diabetic (Punitha , Manoharan 2006). These extracts could be used as

a safe alternative antihyperglycaemic drug for diabetic patients (Kirtikar, Basu 1993).

70% ethanolic leaves extract of P. Pinnata L. has potent antiinflammatory

activity against different phases (acute, sub- acuteand chronic) of inflammation without

side effect on gastric mucosa (Nadkarni 1954, Srinivasan et al., 2001), antipyretic action

(Singh et al., 1996).

In vitro Antiviral activity (HSV-1 and HSV-2) of seeds extract of P. Pinnata L.

against was evaluated. The total inhibition growth of HSV-1 and HSV-2 at

concentrations of 1mg/ml and 20mg/ml w/v was recorded by Singh et al., (1996).

Acute and Chronic toxicological studies resulted safer (Fiala et al., 1974).

The anti-bacterial activity of leaves of P. Pinnata L. was shown potentiality.

Antibacterial compounds against enteric pathogens reported by Ahmad et al., (2004).

This plant can be used to discover bioactive natural products that may contribute as

leads for the development of new pharmaceuticals that address hither to unmet

therapeutic needs (Carcache Blanco EJ et al., 2003, Mumcuoglu 1990). Anti- lice

activity was recorded by Mumcuoglu (1999), Yang et al., (2004), Shirwaikar A et al.,

(2004) using various extracts of P. Pinnata L. leaves.


Vitex negundo L.

Vitex negundo L. (Verbenaceae) is aromatic shrub, woody, growing to a small

tree. It is grown as a economic crop in parts of Asia, Europe, West Indies and the North

America (de Padua et al., 1999). It also using as food crop (Facciola, 1990) and a very

good source of timber (Jabeen et al., 2009).

Ethnomedicinal importance

Herbal medicine, not only merely curing a particular disease bu also aims at

returning the body back to its natural state of health (Srivastava 2009). The

phytochemical components of medicinal plants frequently act individually,

synergistically in improvement of health (Schütz, 2006). V. negundo L. extensively used

in treatment of a plethora of ailments (Prajapati, et al., 2004).

Essential oil of the leaves is also effective in treatment of venereal diseases and

other syphilitic skin disorders. A leaf decoction with Piper nigrum is used in

catarrhal fever with heaviness of head and dull hearing. The root-bark provides

relief from irritability of bladder and rheumatism. Jadhav and Bhutani (2005).

Leaves along with those of Azadirachta indica, Eclipta alba, Sphaeranthus

indicus and Carum copticum in making young again Khare (2004). Whereas Patkar

(2008) refers to the formulations in cosmetology. The Chinese Pharmacopoeia

prescribes the fruit of V. negundo L. in the treatment of painful, and puffy eyes;

headache and arthritic joints (Liu, C et al., 2005).

Ethnomedicinal systems of medicine continue to serve a large segment of

population, especially those in rural and tribal areas, regardless of the advent of modern

medicine (Kosalge and Fursule 2009). The entries regarding the multifarious

applications of V. negundo L. in folk medicine have been grouped regionally to

emphasize the ethnobotanical diversity and ubiq uity of the plant.

Phytochemistry


Secondary metabolites usually occur in complex mixtures that differ from plant to

plant organs and stages (age), soil condtions, pH, climatic conditions of development

(Wink, 2004). Knowledge of the phytochemical constituents is very essential to enable

investigation of the actual effectiveness of the plant in medicine.

From the leaves of V. negundo L. hydroxy-3,6,7,3′,4′-pentamethoxyflavone

(Banerji, et al., 1969) 6′-p-hydroxybenzoyl mussaenosidic acid; 2′-p-hydroxybenzoyl

mussaenosidic acid (Sehgal et al., 1982, Sehgal et al., 1983). 5, 3′-dihydroxy-7,8,4′-

trimethoxyflavanone; 5,3′-dihydroxy-6,7,4′-trimethoxyflavanone (Achari, et al., 1984)

viridiflorol; β-caryophyllene; sabinene; 4-terpineol; gamma-terpinene; caryophyllene

oxide; 1-oceten-3-ol; globulol (Singh, et al., 1999) betulinic acid [3β-hydroxylup-20-

(29)-en-28-oic acid]; ursolic acid [2β -hydroxyurs-12-en-28-oic acid]; n-hentriacontanol;

β-sitosterol;p-hydroxybenzoic acid (Chandramu, et al., 2003) protocatechuic acid;

oleanolic acid; flavonoids (Surveswaran et al., 2007) angusid; casticin; vitamin-C;

nishindine; gluco-nonitol; p-hydroxybenzoic acid; sitosterol were isolated (Khare, 2004).

Whereas from the seeds 3β -acetoxyolean-12-en-27-oic acid; 2α, 3α-dihydroxyoleana-

5,12-dien-28-oic acid; 2β,3α diacetoxyoleana-5,12-dien-28-oic acid; 2α, 3β-diacetoxy-

18-hydroxyoleana-5,12-dien-28-oic acid (Chawla et al., 1992) vitedoin-A; vitedoin-B; a

phenylnaphthalene-type lignan alkaloid, vitedoamine-A; five other lignan derivatives

(Ono, et al., 2004) 6-hydroxy-4-(4-hydroxy-3- methoxy-phenyl)-3-hydroxymethyl-7-

methoxy-3, 4-dihydro-2-naphthaldehyde (Zheng, et al., 2009) β-sitosterol; p-

hydroxybenzoic acid; 5-oxyisophthalic acid; n-tritriacontane, nhentriacontane; n-

pentatriacontane; n-nonacosane these active compounds were isolated (Khare 2004).

negundin-A; negundin-B; (+)-diasyringaresinol; (+)- lyoniresinol; vitrofolal-E and

vitrofolal-F (Azhar-Ul-Haq et al., 2004). Essential oil of fresh leaves, flowers and dried

fruits were isolated likewise δ-guaiene; guaia-3,7-dienecaryophyllene epoxide; ethyl-

hexadecenoate; α-selinene; germacren-4-ol; caryophyllene epoxide; (E)-nerolidol; β-

selinene; α-cedrene; germacrene D; hexadecanoic acid; p-cymene and valencene

(Khokra, et al., 2008). From roots 2β, 3α-diacetoxyoleana-5,12-dien-28-oic acid; 2α,3α-

dihydroxyoleana-5,12-dien-28-oic acid; 2α,3β -diacetoxy-18-hydroxyoleana-5,12-dien-

28-oic acid; vitexin and isovitexin (Srinivas, et al., 2001). acetyl oleanolic acid;

sitosterol; 3- formyl-4.5-dimethyl-8- oxo-5H-6,7-dihydronaphtho (2,3-b)furan were

isolated (Vishnoi, et al., 1983).


Pharmacology

Demands of the scientific forum have essential experimental evidence to

further underline the medicinal importance of V. negundo L. given above. Taking cue

from these ethno and rural systems of medicine, scientifically authenticated studies have

been designed and conducted in order to pharmacologically validate these claims.

Antioxidant activity of V. negundo L. leaf extracts were determined by (Tiwari

et al., 2007). Rooban et al. (1999) evaluated the antioxidant and therapeutic potential of

V. negundo L. flavonoids in modulating solenoid- induced cataract and found it to be

effective. The extracts were played an important role in decreasing levels of

superoxide dismutase, catalase and glutathione peroxidase in Freund‘s adjuvant

induced arthritic-rats (Devi, et al., 2007). The extracts also shown the ability to combat

oxidative stress by reducing lipid peroxidation owing to the presence of flavones,

vitamin C and carotene (Vishal and Gupta 2005).

Yunos et al. (2005) and Jana et al. (1999) investigated the antiinflammatory

properties of V. negundo L. extracts in acute and subacute inflammation. Anti-

inflammatory and pain suppressing activities of fresh leaves are attributed to

prostaglandin synthesis inhibition (Telang, et al., 1999), antihistamine, membrane

stabilising and antioxidant activities (Dharmasiri, et al., 2003).

Root extracts of V. negundo L. showed inhibitory activity against enzymes such as

lipoxygenase and butyryl-cholinesterase (Azhar-Ul-Haq et al., 2004); α-chymotrypsin

(Lodhi, et al., 2008); xanthine-oxidase (Umamaheswari et al., 2007); (Azhar-Ul-Haq,

Malik, et al., 2006) and tyrosinase (Azhar-Ul-Haq, Malik, et al., 2006).

Woradulayapinij et al. (2005) reported the HIV type 1 reverse transcriptase inhibitor

activity of the water extract of the aerial parts of V. negundo L.

Bhargava, (1989) separated the rich flavonoid fraction of seeds of V. negundo L.

this was caused disruption of the latter stages of spermatogenesis in dogs and interfered

with male reproductive function in rats (Das, et al., 2004). These findings are in sharp

contrast with the ethno use of V. negundo L. as aphrodisiac (Khare, 2004). Hu et al.


(2007) investigated the estrogen-like activity and propounded its use in hormone

replacement therapy by using ethanolic extracts of V. negundo L.

Leaf extracts of V.negundo were found to possess hepato-protective activity

against liver damage induced by dgalactosamine (Yang, et al., 1987), commonly used

tubercular drugs (Tandon, et al., 2008) and carbon tetrachloride (Tasduq et al., 2008,

Raj 2008). Villasenor and Lamadrid (2006) have provided an account of the anti-

hyperglycemic activity of V.negundo leaf extracts. Laxative activity of V.negundo

leaf extracts was exhibited in rats by Adnaik et al. (2008). 98% methanolic root extracts

of V.negundo showed antagonization of the lethal activity induced by venom of

Vipera russellii and Naja kaouthia (2006). Immunomodulatory effect of V.negundo

extracts has been reported by Ravishankar and Shukla (2008). Gupta (2005 ) Gupta, et

al., (1999) were reported drug potentiating ability. Histomorphological and cytotoxic

effects was investigated by Tandon and Gupta (2004) Smit, et al., (1995). Diaz et al.

(2003), Yunos et al. (2005).

2.3 Materials and methods

2.3.1 Collection of plant material

The plant materials were collected in fresh bags from different places of

Hyderabad Karnataka region, Karnataka, India and brought to laboratory. The collected

plant materials were initially rinsed with distilled water to remove soil and other

contaminants and dried on paper towel in laboratory at 37oC for week.

2.3.2 Extraction of plant material by soxhlet apparatus

The plant materials after drying were ground in a grinding machine in the

laboratory then 25g of shade dried powder was weighed and extracted successively with

non-polar to polar method i.e., petroleum-ether, chloroform, ethyl acetate, methanol and

aqueous in soxhlet extractor for 48h. The methanol extracts were concentrated under

reduced pressure and preserved in refrigerator in airtight bottle for further use.

2.3.3 Preparation of extract dilution series

Extract stock solution: dissolved 400 mg of crude in 10 ml DMSO with glass

beads, vortex to homogenize and a two-fold serial dilution was prepared. As a precaution

not to miss trace amounts of antifungals for preliminary screening, a relatively high

concentration of 0.62 to 40 mg/ml of each extract was prepared for bioassays.


2.3.4 Test microorganisms

Five fungal cultures Trichophyton rubrum, Microsporum gypseum, Trichophyton

tonsurans, Aspergillus flavus, Candida albicans five bacterial cultures Bacillus subtilis,

Escherichia coli, S. areas, Psudomonas sps, Brevibacillus spp and Serratia marcurs

were used in the present study. All the tested strains were obtained from Department of

Microbiology M.R medical college, department of Microbiology Gulbarga University,

Gulbarga, Karnataka, MTCC of Chandighar, India. Bacterial cultures were grown in

nutrient broth (Himedia, M002) at37oC and maintained on nutrient agar slants at 4oC,

fungal cultures were grown in potato dextrose broth at 28 oC and maintained on potato

dextrose agar slants at 4oC.

2.3.5 Media for inoculation

Sabouraud’s Dextrose Agar Medium (SDA)

Peptone 10.0 g

Dextrose 40.0 g

Agar 20.0 g

Cycloheximide 0.5 g

Chloramphenicol 1 x 250 mg capsule

Distilled water 1000 ml

pH 5.6

Sabouraud’s Dextrose Medium (SDB)

Peptone 10.0 g

Dextrose 40.0 g

Cycloheximide 0.5 g

Chloramphenicol 1 x 250 mg capsule


pH 5.6

Potato Dextrose Agar (PDA)

Peeled potato 250 g

Glucose 20.0 g

Agar 20.0 g


pH 6.0-6.5


Nutrient Broth

Peptone 10.0 g

Beef extract 0.03 g

Sodium chloride 005 g


pH5.0

2.3.6 Fungal inoculum preparation

The dermatophytes were grown on SDA for a week and the spores were

collected in by flooding with 0.85% saline (Ghannoum et al.,2004). After settling the

larger particles in the test tube, the supernatant was taken and counted the number of

conidia using hemocytometer. A ten fold dilution was made. Nine ml of normal saline

solution was taken in 5 test tubes. In first test tube, 1 ml of spore suspension was poured

into test tube under aseptic conditions. The solution of first test tube was homogenized

and 1 ml of this solution was transferred to second test tube containing 9 ml of normal

saline solution. This process was repeated upto 5th test tube. In each case sterilized

pipette was used. From every test tube, (for each dilution) 0.1 ml suspension was

transferred to sterilized SDA petriplates. Triplicates of each dilution were maintained.

The SDA seeded petriplates were counted in hemocytometer. The average of 3

petriplates was taken in each case. The test inoculum were adjusted between 1.5 x105

spores/ml.

2.3.7 Antidermatophytic activity

2.3.7.1 Determination the MIC by agar well diffusion method (Magaldi S et al.,

2004)

The assay was conducted by agar well diffusion method. About 15 to 20 ml of

potato dextrose agar medium was poured in the sterilized pe tri dishes and allowed to

solidify. Fungal lawn was prepared using 5 days old culture strain. The fungal strains

were suspended in a saline solution (0.85% NaCl) and adjusted to a turbidity of 0.5 Mac

Farland standards (108 CFU/ml). 1 ml of fungal strain was spread over the medium

using a sterilized glass spreader. Using flamed sterile borer, wells of 4 mm diameter

were punctured in the culture medium. Required concentrations of serially diluted

extracts (0.6, 1.2, 2.5, 5, 10, 20 and 40mg/ml) were added to the wells. The plates thus

prepared were left for diffusion of extracts into media for one hour in the refrigerator and


then incubated at 37oC. After incubation for 48h, the plates were observed for zones of

Inhibitory. The diameter zone of Inhibitory was measured and expressed in millimetres.

Dimethyl formamide (DMSO) was used as a negative control. Ketoconazole used as

positive control (500μg/ml). The experiments were conducted in triplicates. The same

method was followed for testing antibacterial activity using nutrient agar medium

incubated at 37oC for 18h.

2.3.7.2 Determination the MIC by Broth Dilution Assay (NCCLS 1997)

The minimum inhibitory concentration of the plant extract was determined using

broth dilution assay. The medium containing different concentrations of plant extracts

viz., 100mg -1µg per ml prepared by serial dilution (10-1 dilution). After inoculation of

culture, the tubes were incubated for 72 hours at 280 C. The MIC of each sample was

determined by measuring the optical density in the spectrophotometer (Electronics India)

at 520nm and compared the result with those of the non- inoculated broth used as blank.

Control was prepared using media and inoculum without plant extract [19]. The

experiment was conducted according to NCCLS standards (now called as CLSI),(ogu-

GI-2011and shinki S A 2011)

2.3.8 Preliminary Screening Tests for Secondary Metabolites:

Preliminary tests, for the detection of secondary metabolites, were carried out for

all the extracts of selected plants by adopting standard methods (Harborne, 1998).

Preparation of Test solution: 500 mg of each extract was dissolved in 100 ml of the

respective solvent and filtered through Whatman filter paper No.1. Thus, the filtrates

obtained were used as test solutions for the following preliminary screening tests.

Tests for Alkaloids:

The stock solutions of Pet. ether, CHCI3, Et-OH, methanol and Aqueous extracts

were further mixed with the required quantity of ammonia solution followed by acidified

chloroform (0.1N Hcl) and filtered. Thus, the filtered is used as test solution for alkaloid

detection using following tests.

a) Mayer’s test: 1 ml of KI in iodine solution was added to the 2 ml of test solution.

The formation of brown precipitate indicated the presence of alkaloids.


Dragendorff’s reagent: 2 ml of Dragendorff‘s reagent and 2 ml of dilute HCI were

added to the test solution. An orange red coloured precipitate indicates the presence of

alkaloids.

Wagner’s test: 2 ml of Wagner‘s reagent was added to 2 ml of test solution. The

formation of reddish precipitate indicates the presence of alkaloids.

Tests for Flavonoids:

Lead acetate test: To the successive plant test solutions, add few drops of 10% lead

acetate solution appearance of a yellow colour precipitation indicate the presence of

flavonoids.

Shinoda test (mg/Hcl): A pinch of magnesium power and 5N Hcl were added to the test

solution and a deep red or magnets colour formation indicates the presence of flvanone

or dihydroflvanol. However, dihydrochalcone and other flavonoids did not react with

this reagent (Harborne, 1982).

NaOH test: 1 ml of 1N NaOH solution was added to the 1 ml of test solution, formation

of yellow colour indicates the presence of flavonoids.

Tests for Glycosides:

Kellar-Killiani test: 1 ml of glacial acetic acid was carefully added to 2 ml of test

solution of the extract and mixed well. Further, 2 drops of ferric chloride solution wad

added after cooling. These contents were transferred carefully to a test tube containing 2

ml of conc. H2SO4. A reddish brown ring was observed at the junction of two layers.

Conc. H2SO4 tests: 1 ml of conc. H2SO4 was added to 1 ml of test solution and is

allowed to stand for 2 minutes. The formation of reddish colour indicates the presence of

glycosides.

Molisch’s test: A mixture of Molisch‘s reagent and conc., H2SO4 (1:1) was added to the

test solution, formation of reddish coloured ring at the junction of two liquids indicates

the presence of glycosides.


Tests for phenols:

Ellagic acid test: the test solution was treated with few drops of 5% (v/v) glacial acid

and 5% (w/v) NaNO2 solution. The solution turns muddy yellow, alive brown, Niger

brown, deep chocolate colours depending on the amount of ellagic acid present.

Phenol test: When 0.5 ml of FeCl3 (w/v) solution was added to 2 ml of test solution,

formation of an intense colour indicates the presence of phenols.

Hot water test: Dip a mixture in leaf in the test tube containing hot water, warm it for

few minutes. The development of black or brown coloured ring at the junction of dipping

indicates the presence of phenols.

Test for Saponins:

Foam test; 0.1 g of crude extract was shaken vigorously in 2 ml of distilled water.

Formation of honeycomb like fourth persists for a few minutes indicate the presence of

saponins.

Tests for Sterols:

Libermann-Burchard test: A green colour was formed, when the Libermann-Burchard

reagent is added to the test solution, indicates the presence of sterols.

Salowski’s test: A wine red colour was developed when chloroform and conc. H2SO4

were added to the test solution; indicate the presence of steroidal nucleus.

Tests for Glycosides:

Kellar – killani test: 1 ml of Glacial acetic acid was carefully added to 2 ml of test

solution of the extract and mixed well and further, after cooling 2 drops of ferric chloride

solution was added. There contests were transferred carefully to a test tube containing 2

ml of concentrated H2 So4. A reddish brown ring was observed at the junction of two

layers indicates the presence of glycosides.

Conc. H2SO4 Test: 1 ml of Conc. H2

SO4 was added to 1 ml of test solution and is

allowed to stand for 2 minutes. The formation of reddish colour indicates the presence of

glycosides.


Molisch’s test: A mixture of Molisch‘s reagent and Conc. H2 So4 (1.1) was added to the

test solution, formation of reddish violet coloured ring at the junction of two layers

indicates the presence of glycosides.

Test for Triterpenoids

Salkowskis test: Few drops of Conc. H2So4 to the test solutions, shaken on standing, the

lower layer turns golden yellow colour indicates the presence of triterpenoids.

Liebermann – Bur chard test: To the test solutions add few drops of acetic anhydride

mix well and add 1 ml of Conc. H2So4 from the sides of the test tube, formation of

reddish brown ring at the junction of two layers indicates the presence of triterpenoids.

Test for Tannins (Tease and Evans, 1989)

Gelatin test: A test solution was dissolved in gelatin. The 1% gelatin was prepared in

10% Sodium chloride; the formation of white precipitate indicates the presence of

Tannins.

2.3.9 Quantitative estimations of secondary metabolites

A. Estimation of Alkaloids

The total alkaloids of selected 07 medicinal plant parts were estimated by Ikan‘s

method (1981).

Reagents required: CHcl3, NH4OH, Glacial acetic acid, n-Hexane and MeOH.

Procedure: 50 g powdered plant material was macerated with MeOH (Analytical grade)

in mortar with pestle and centrifuged (2X). The supernatant collected was condensed to

1/4th volume and dilute acetic acid was added in a separating funnel. The acid layer was

collected and 25 ml of n-hexane and chloroform (1:1) mixture was added and shaken

well (3X). The chloroform layer is collected and washed with distilled water. Its pH was

adjusted to 11-12 by the addition of NH4OH. The chloroform layer was separated and

filtered using Whatman No. 1. The filtrate was finally transferred to a clean and pre-

weighed beaker and dried under reduced pressure at 40°C for 6 h. The amount of

alkaloid was calculated using the following formula.

Weight of Alkaloid residue (X)

Total alkaloids = X 100

Weight of plant material (W)


Where,

Weight of the residue (X) = Z - Y

Y = Weight of the evaporating dish.

Z= Weight of the alkaloid containing dish.

B. Estimation of Flavonoids

The flavonoids were quantitatively estimated by Swain and Hillis (1959) method.

Reagents required

Vanillin Reagent: Freshly prepared by dissolving 1 g of re-crystallized vanillin in 100

ml of 70% (w/v) conc. H2S04.

Phloroglucinol standard: 100 mg phloroglucinol was dissolved in 100 ml distilled

water.

Procedure: 500 mg powdered selected plant material was homogenized with 10 ml

methanol using mortar and pestle. Then, the homogenate was centrifuged at 3000 rpm

for 20 min (2X). The supernatant collected was evaporated to dryness keeping in a hot

water bath (80°C). Thus, the residue obtained was redissovled in 5 ml distilled water.

From this, 0.1 and 0.2 ml extracts were taken in test tubes and d iluted to 2 ml with

distilled water. 4 ml vanillin reagent was added to each tube rapidly. After 15 min the

appeared brick red colour was read at 500 nm in the digital spectrophotometer against

blank reagent. The standard curve was plotted using different concentrations of

phloroglucinol as the standard flavonoids. The amount of flavonoids present in the

sample was calculated with the help of the standard graph.

C. Estimation of Tannin (Schanderi, 1970).

Reagents

Folin Denis reagent

Sodium carbonate solution 3.5 g of sodium carbonate was dissolved in 100 ml of

distilled water.

Standard tannic acid solution: 10 mg tannic acid was dissolved in 100 ml distilled water.

Working standard solution: 5 ml of standard solution was diluted to 10 ml by adding

distilled water. 1ml of standard solution contains 0.5 mg of tannic acid.


Procedure

500 mg of selected plant material was mixed with 75 ml distilled water in 250 ml

conical flask and was heated gently and boiled for 30 min. Centrifuged at 2,000 rpm for

20 min and collected the supernatant in 100 ml volumetric flask and made up the

volume. 1 ml of the sample extract was transferred to a 100 ml volumetric flask

containing 75 ml of water. 5 ml of Folin Denis reagent was added and 10 ml of sodium

carbonate solution and diluted to 100 ml with distilled water and shaken well. The blue

colour intensity was measured in a spectrophotometer. The absorbance was read at 700

nm after 30 min and the 30 times dilution of the sample was made with distilled water

and standard graph by using 0-100 μg tannic acid. The tannin content of the sample was

calculated as tannic acid equivalents from the standard graph expressed as mg/100mg.

D. Estimation of Phenols

Reagents

80% ethanol

20 g of Na2CO3 dissolved in 100 ml distilled water.

Folin Ciocalteau reagent (FCR) (1:1 with distilled water)

Standard- 100 mg tannic acid dissolved in 100 ml distilled water and diluted 10 times for

the working standard.

Procedure

500 mg of the selected sample was grinded with pestle and mortar in 10 times

volume of 80% ethanol. The homogenated solution was centrifuged at 10,000 rpm for 20

min. The supernant was saved and the residue was re-extracted with five times the

volume of 80% ethanol centrifuged and pooled the supernatant. The supernatant was

evaporated to dryness. Dissolved the residue in a 5 ml of distilled water. Pipette out 0.5

ml aliquots into test tubes, made the volume to 3 ml with distilled water. Added 0.5 ml

Folin Ciocalteau reagent. After 3 min, added 2 ml of 20% Na2CO3 solution to each tube

and mixed thoroughly. The test tubes were placed in boiling water for one min. cooled

and the absorbance was read at 650 nm against reagent blank. A blue coloured complex

was produced (molybdenum complex). A standard curve was prepared using different

concentrations of catechol. (Catechol was used as a standard phenol).

E. Estimation of Total Saponins


The total saponins were estimated in plant material using the method of Sanchez

et al. (1972) modified by Rishi et al. (1976).

Reagents required: 3N Hcl, NH4OH (Aqueous), chloroform, conc.H2S04 and

met ha no l .

Procedure: 500 mg selected plant material was hydrolyzed by refluxing with 25 ml of

3N HCl at 60°C for 4 h. The solid matter is retained on the Whatman No. 1 filter paper

and further washed it with half diluted aqueous NH4OH until the washings were neutral

(pH 6.8 - 7.0). Then, the residue was dried and extracted for saponins in the Soxhlet

extractor using chloroform for 6 h. From this, 1 ml extract was taken and evaporated to

dryness in vacuum. Thus, the residue obtained was re-dissolved in 4 ml H2SO4 and

methanol reagent. The absorbance was read at 405 nm in UV/VIS spectrophotometer

against a blank, after allowing the reaction to proceed for 2 min., which is

optimum time required for the chromophore to develop a stable optical density. The

amount of saponins present in the plant material was calculated.

2.3.10 Qualitative separation of secondary metabolites by thin layer chromatography

(TLC):

The following secondary metabolites were separated from the different part of selected

plants by analytical Thin Layer Chromatography (TLC) method (Stahl, 1969; Wagner

and Bladt, 1996; Harborne, 1998).

A. Preparation of thin layer chromatographic plates (Stahl, 1969)

The glass plates of size 20 x 20 cm were washed with soap water and cleaned

with acetone to remove oil stains. These were arranged on a plastic template, spreading

device was arranged on the initial plate. A thickness of 0.5 mm was adjusted on the

spreader with the help of knurled screw on the side of the spreads. The slurry of required

amount of Silica gel G (Sd Fine Chem) was prepared in distilled water to get a proper

suspension. The slurry was immediately transferred into spreader. The spreader was then

drawn smoothly over the plates up to the end. The plates were allowed to dry completely

at room temperature heated in an oven at about 105°C for 30 min for activation. These

activated plates along with the precoated ALU GRAM® SIL G/FUV254 20 x 20 plates

(Machery-Nagel GmbH, Germany) were used for the separation of following secondary

metabolites.


B. Separation of Flavonoids (Wagner and Bladt, 1996)

Extraction: 1 g powdered plat material was extracted with 10 ml methanol for 5 min.,

on a water bath at about 60°C and filtere Whatman filter paper No. 44. The extract was

evaporated to 2 ml, and to this, 1 ml distilled water and 10 ml EtOAc were added and

shaken several times. Then, the EtOAc phase was separated and reduced to 1 ml for TLC

to separate flavonoids.

Development: 20µl of each extract was loaded on a precoated Alugram® Sil G/F254

with a capillary tube. Then, the plate was kept in a saturated chromatographic chamber

containing chloroform and ethyl acetate (8:2) mixture as solvent system.

Detection: The developed chromatographic plates were observed under (UV254nm)

chamber. Thus, the colour and hRf values of fluorescent bands obtained were recorded.

C. Separation of Phenols (Harborne, 1998).

Extraction: 2 g plant material was immersed in 10 ml of methanol and allowed to stay

overnight on a rotary shaker (180 thaws/min). The filtered extract is evaporated to 1/4 th

volume and used for separation of phenols.

Development: 20 µl of the extract was loaded on the activated chromatographic plates

and kept in the saturated chromatographic chamber contain CHCl3 and MeOH (27:0.3)

mixture as solvent system.

Detection: The developed chromatograms were observed under visible light after

spraying with half diluted FCR and heated at 110°C for 10 min. Thus, the phenolic

bands obtained colour and hRf values were recorded.

Detection: The developed plate was sprayed with anisaldehyde - sulphuric acid (AS)

reagent and heated at 100°C for 6 min. The bands' colour and its hRf values were

recorded

D. Separation of Alkaloids (Nuzillard et al., 1996; Wagner and Bladt, 1996)

Extraction: 100 g plant material wetted with 50 ml of half diluted aqueous NH4OH and

lixiviated overnight with 1000 ml EtOAc. The filtered organic solution was extracted

with 2% (v/v) H2SO4. The resulted organic phase was separated using separating funnel

and basified with NH4OH (pH 11-12). This is extracted with 1000 ml CHCl3 (3X) and

was dried over Na2SO4 and evaporated to dryness at 40°C in vacuo


Development: 20 µl of redissolved alkaloid extracts were loaded with a cap illary tube

on a precoated Alugram® Sil G/UV254 plate and was allowed to dry.20µl brucine (Hi-

Media Lab.Ltd., Mumbai) was also loaded as a standard alkaloid. The plate was kept in a

saturated chromatographic chamber containing the mixture of chloroform and methanol

(15:1) as a mobile solvent phase.

Detection: The developed chromatographic plates were observed first under short

wavelength Ultra Violet light (UV254 nm) chamber, and recorded the colour and the hRf

values of bands. Later, the plates were observed under visible light after spraying with

Dragendorff‘s‘ s reagent and heated at 100°C for 5min

2.3 11 Separation of active fraction from selected plant solvent extract using column

Chromatography (CC) (Wilson and Walker, 1995).

Extraction: The successive selected solvent crude Soxhlet extract of selected plants

were extracted as mentioned earlier.

A. Column Chromatography (CC)

Preparation of column: A clean and dry 500ml capacity column (Vinsel make) of about

60 cm length with the slurry of silica gel-H of mesh size 60-120 µ ( hi-media, Mumbai)

to 45 cm portion using hexane. Due care was taken to avoid air bubbles while packing

the column with stationary phase. Then the column was run through twice with the

solvent system contains hexane (Analytical grade, Sd-fine Chem., India) to make the

column air tight and compact one.

Loading: 10 g of methanol extract of selected plant part extract was well with a small

amount of silica gel and loading on to the top of the silica gel column, which was 45 cm

in height. The column was eluted with solvents of increasing polarity (Hexane, pet ether,

chloroform, ethyl acetate, methanol and aqueous. Sd-fine, Mumbai).

Collection of fractions : Totally fractions with each 100ml were collected as they came

off column in a series of conical flasks (100ml) (Borosil, India). Thin layer

chromatography was done with these fractions. Based on the TLC results similar

fractions were pooled together and concentrated in vacuum to isolate the active principle.

All the collected fractions were tested against dermatophytic fungi. The active fraction to

be used for further isolation and purification process.


B. Isolation purification of partially purified compounds by using Preparative

Thin Layer Chromatography (PTLC)

An attempt has been made to separate / isolate and purify the active compound based on

colour intensity band width of the cc fraction, obtained through PTLC or re-column.

Preparation of PTLC plates: 1mm thick neutral Silica gel containing 20x 10 cm size

glass plates were prepared as stated elsewhere and used for separation of compound.

Development: About 500 μl of active cc collected fraction was loaded as streak with

capillary tube and allowed it to dry. These plates were kept in saturated chromatographic

chamber containing suitable developing solvent mixture.

Detection and Scraping: the developed chromatograms were observed under wave

length (UV254 and UV365 nm) and marked with a needle taking due care. Then the

marked spot was collected by scraping with a sterilized scalpel and stored in glass vials.

All the collected fractions were tested against dermatophytic fungi. In order to harvest

more amount of compound there is need to perform re-column chromatography.

C. Purification of compounds: The compound containing Silica gel-G powder thus

obtained was dissolved in a mixture of its concerned solvents system [eg. chloroform

and methanol (90:10)] and thoroughly mixed the suspension. It was then centrifuged at

5000 rpm for 10 min. to retain supernatant containing purified compound. The pellet was

once again re-suspended in the solvent mixture and centrifuged. The pooled supernatants

were condensed to dryness in vacuum (40ºC) and preserved the compound in a tight

screw cap vial of 5 ml capacity (Hi-Media Lab., Mumbai) at 4ºC in the refrigerator

further use.

The purity of the compound that isolated from the root were checked by the TLC

method on precoated Alugram® Sil G/UV254 plates developed in the chloroform and

methanol (90:10) solvent system. These plates were observed under UV light (UV254 &

UV365nm) for the occurrence of single also observed under visible light after spraying

of Anisaldehyde – Sulphuric acid reagent (ASR) and heated 110ºC for 5 min. the colour

of the band and hRf values were recorded.

D. Physico-Chemical properties of isolated compounds

The isolated single compound was subjected to the following physical and

chemical tests in order to use this data as the basis in the


process identification and structure elucidation of compounds.

Nature: The pure isolated compounds obtained recorded morphology both by naked eye

and using a magnifying lens.

Colour: The colours of the compounds were observed both under Ultra Violet (UV254

& UV365 nm) and visible light.

hRf values: The hRf value of the compounds, from the developed chromatogram in

conformed solvent system, was calculated using following formula (Bisset and

Phillipson, 1976).

hRf = Distance travelled by the solute from the origin x 100

Distance travelled by the solute from the origin

Melting point: Melting point of pure compounds were recorded in open capillary tube

using paraffin liquid bath (Kulkarni and Pathak, 1993).

Solubility: The solubility of the compounds were studied using different solvents like

water, ethanol, methanol, chloroform, ethyl acetate, DMSO etc., (Kulkarni and Patha,

1993).

Yeild: The yield of purified fraction was calculated using the following formula.

(%) = Weight of the single compound x 100

Weight of the crude compound mixture

E. Studied on Structure elucidation of isolated compounds by spectroscopic

methods

The purified compounds were subjected to UV/VIS, FT-IR, 1H NMR, 13C NMR

and LC-MS spectroscopic studies and obtained the special data, which is of immense use

in the detection of the functional groups and further to elucidate their structure (Ahmed

et al., 1985; Yamaguchi, 1970).

UV/VIS spectroscopy: The compounds were dissolved in 5 ml of chloroform

(Analytical grade) and read the absorbance in the Perkin-Elmer Lambda 15 UV/VIS

spectrophotometer in the range of 200-800 nm wavelengths against chloroform blank.

The plotted graph i.e. abscissa verses ordinate (λ max) is used in the detection of

chromophore of the compound.


FT-IR spectroscopy: The Fourier transform infrared spectra of pure compound were

obtained using KBr discs on Perkin-Elmer RX1 spectrophotometer in the wave numbers

(cm-1) in the range of 4000-450cm-1 was recorded as the inverted peaks

1H NMR & 1C NMR Spectroscopy: The pure isolated compound spectra were recorded

in JEOl Model GSx 400 spectrophotometer CDCL3 (denaturated chloroform, DMSO)

was the solvent. 1HNMR was recorded in the Bruker AMX 400 NMR

spectrophotometer using TMS (Tretraimethyl saline) as an internal standard at 400.137

(1H) and 270 c or 300 K. The chemical shift were recorded in σ (ppm) based either on

σ TMS= 0 and the coupling contestants or J in hertz.

LC-Mass Spectroscopy: LC-Mass Spectroscopy of the pure compounds was recorded.

The room temperature (27oC), M- nitrobenzyl alcohol (NBA) was used as the matrix

unless specified otherwise.

HPLC: The pure isolated compound spectra were recorded in agilent 1100 series,

Column: Agilent TC-C18 (2), 5µm, 4.6 ID, 25 cm L. Isocratic Elution: with standardized

solvent system was used for particular compound study.

F. Antidermatophytic activity and Minimum Inhibitory Concentration (MIC)

of isolated compounds:

The antidermatophytic activity (Trchophyton rubrum) Minimum Inhibitory

Concentration (MIC) of isolated compounds was performed by agar well diffusion

method, broth dilution method and the results were recorded.

2.4 Results

2.4.1. Screening of 61 ethno medicinal plants for detection of secondary metabolites and

antidermatophytic activity.

A series of 305 extracts from 61 ethno medicinal were subjected to

antidermatophytic screening against three dermatophytes namely Trichophyton rubrum,

Trichophyton tonsurans and Microsporium gypseum in Pet ether, chloroform, Ethyl

acetate, methanol and aqueous extracts using agar well diffusion method at sample

concentrations of 5 & 2.5 mg/ml. The plant extracts and their level of activity against

theses three common dermatophytes is listed in Table 2.1.


Screening against Trichophyton rubrum

The plant extracts and their level of activity against Trichophyton rubrum is

listed in Table 2.1. A series of 305 extracts from 61 ethno medicinal plants belonging to

33 different families were used in treating skin diseases at Hyderabad Karnataka region

were subjected to antidermatophytic screening against Trichophyton rubrum (MTCC

1344) in Pet ether, chloroform, Ethyl acetate, methanol and aqueous extracts of each

plant were tested for their antifungal activity using agar well diffusion method at sample

concentration of 5 & 2.5 mg/ml. Among the 61 plants, 18 exhibited very effective

antidermatophytic activity in 98% methanolic extract i.e., Allium cepa Linn., Annona

reticulata L., Annona squamosa L., Argemone mexicana L., Butea monosperma,

Ceasalpinia bonducella, Citrus medica L., Corchorus oleterius L., Emblica officinalis,

Euphorbia tirucalli L., Ficus racemosa L., Gymnosporia montana, Lawsonia inermis

Linn., Solanum nigrum L., Sterculia foetida L., Tribulus terrestris L., Vitex negundo L.

and Zingiber officinale. The effective activity was also observed in 13 plants of different

solvent extracts i.e., Coccinia indica, Datura metel, Senna auriculata, Senna tora,

Tectona grandis, Tinospora cordifolia, Thevetia nerrifolia (ethyl acetate), Achyranthes

aspera, Bergera koenigii, Celosia argentea, Tamarindus indica (chloroform) Aloe

vera, Milletia pinnata (petroleum ether). Whereas the weak activity observed in 04

plants i.e., Lantana camara, Mentha viridis, Tridax procumbens and Zizyphus jujuba.

There was no inhibition recorded from the negative control (DMSO), while the

standard drug, Ketoconazole significantly inhibited (28.66±1.15 to 12.33±1.52 mm) the

growth of the test dermatophyte. On the basis of the results obtained, it is concluded that

the 31 effective crude extracts (18 , 98% methanolic, 07 Ethyl acetate, 04 chloroform

and 02 petroleum ether) showed significant antidermatophytic activity. This probably

explains the use of these plants by the indigenous people against dermatological

infections. After this screening experiment, further work was performed to describe the

antifungal activities in more detail as well as their activity in-vivo. In addition,

phytochemical studies to isolate the active constituents and evaluate the

antidermatophytic activities against a wide range of mycotic population wise carried

out (Tables 2.1, 2, 2).


Table-2.1: Preliminary antidermatophytic screening of ethno medicinal plants of Hyderabad Karnataka region. Sl. No.

name Plant name

Fungal Strain

Zone of Inhibition in different solvents (mm) Control

Standard

P C E M A

a b a B a b a b a b DMSO Ketoconazole

01 Achyranthes aspera L.

Amarathaceae

Leaf

A 07.66±1.15 06.20±1.00 11.00±0.00 06.33±1.52 07.00±0.00 05.33±1.52 05.33±1.52 07.00±0.00 04.50±0.00 NA NA 17. 66±0.57

B 07.33±1.52 05.00±0.00 07. 33±1.52 07.66±0.57 11.66±1.15 05.00±1.00 05.00±1.00 04.33±1.52 04.00±0.00 NA NA 30. 33±1.52

C 06.66±1.15 05.33±1.52 10. 66±1.15 06.66±1.15 10.00±0.00 04.00±1.00 05.00±0.00 05.33±1.52 NA NA NA 18. 66±1.15

02 Aegle marmelos L.Corr.

Rutaceae

Leaf

A 07.00±0.00 04.00±1.00 06. 33±1.52 04.33±1.52 10.33±1.52 05.33±1.52 05.33±1.52 NA 06.33±1.52 NA NA 18. 33±1.52

B 06.00±1.00 04.33±1.52 09.90±1.00 05.33±1.52 12.33±1.52 05.00±1.00 07.33±1.52 05.33±1.52 06.80±0.00 05.00±0.00 NA 28. 66±0.57

C 07.00±0.00 05.00±1.00 07. 33±1.52 05.00±1.00 08.00±1.00 05.33±1.52 10.00±1.00 05.00±0.00 06.00±0.00 04.00±0.00 NA 21.00±1.00

03 Allium cepa L.

Liliaceae

Bulb

A 05.00±0.00 04.66±1.15 04.66±1.15 04.33±1.52 04.66±1.15 NA 10.00±0.00 05.00±0.00 06.00±1.00 05.00±0.00 NA 12. 33±1.52

B 05.33±1.52 06.00±0.00 07.00±1.00 05.00±0.00 14.00±0.00 06.33±1.52 08.00±0.00 05.00±0.00 09.00±0.00 07.00±0.00 NA 15. 66±1.15

C 05.00±1.00 NA 06.33±1.52 05.66±1.15 30.00±1.00 06.33±1.52 07.00±1.00 05.33±1.52 05.00±0.00 NA NA 15. 66±0.57

04 Allium sativam L.

Liliaceae

Bulb

A 07.33±0.57 06.66±0.57 11.33±0.57 06.66±0.57 07.00±0.00 05.66±0.57 05.00±0.00 07.00±0.00 NA NA NA 17. 33±1.52

B 07.66±1.15 05.00±0.00 07.66±1.15 07.66±0.57 11.66±1.15 05.00±0.00 05.33±0.57 04.66±0.57 NA NA NA 30. 33±1.52

C 06.66±0.57 05.33±0.57 10.66±1.15 06.00±0.00 12.00±0.00 04.66±0.57 05.66±1.15 05.33±0.57 NA NA NA 18. 66±0.57

05 Aloe vera L.

Liliaceae

Leaf

A 08.66±1.15 04.00±0.00 05.33±0.57 04.66±0.57 07.00±0.00 07.33±0.57 06.66±0.57 06.66±1.15 05. 33±0.57 NA NA 18. 33±1.52

B 10.00±0.00 06.00±1.00 07.33±1.52 06.00±0.00 09.33±0.57

06.00±0.00

07.33±0.57 07.00±0.00

06.33±1.52

05.00±0.00 NA 24.00±0.00

C 10.66±0.57 04.00±0.00 06.33±1.52 07.00±0.00 07.00±1.00

06.33±1.52

05.00±0.00 04.00±1.00 06.00±0.00 NA NA 21. 66±0.57

06 Amaranthus spinosus L.

Amarathaceae

Leaf

A 07.00±0.00 05.00±1.00 05.33±1.52 05.66±1.52 05.00±0.00 05.00±1.00 07.33±1.15 04.66±1.57 05.00±1.00 NA NA 18. 33±1.52

B 05.66±1.57 04.66±0.57 06.33±0.57 05.00±0.00 07.33±0.57 05.33±1.52 08.66±1.52 05.00±0.00 05.33±1.15 NA NA 24. 33±1.52

C 04.33±1.15 NA 07.00±0.00 05.00±0.00 05.00±1.00 05.00±0.00 06.66±1.57 05.66±0.57 05.00±0.00 NA NA 16.00±0.00

07 Annona reticulata L.

Annonaceae

Leaf

A 05.66±0.57 04.00±0.00 05.00±1.00 04.33±0.57 08.66±0.57 05.00±0.00 07.00±0.00 04.66±0.57 05.33±1.15 04.00±0.00 NA 21. 66±1.15

B 05.00±000 04.33±1.52 06.00±0.00 04.33±1.15 07.66±0.57 05.00±0.00 07.00±0.00 04.66±1.57 06.33±1.52 05.00±1.00 NA 26. 66±0.57

C 05.00±0.00 04.66±0.57 06.33±1.15 05.00±0.00 10.66±1.57 05.00±1.00 14.00±1.00 05.00±0.00 05.66±1.52 04.66±1.57 NA 22. 33±1.52

08 Annona squamosa L.

Annonaceae

A 07.00±0.00 05.00±0.00 06.66±1.52 06.33±1.15 07.00±0.00 06.66±0.57 07.00±0.00 06.33±0.57 07.66±1.57 NA NA 20. 33±1.52

B 09.33±0.57 05.33±1.52 07.00±0.00 05.00±1.00 07.66±0.57 05. 66±1.57 14. 33±0.57 08.00±0.00 07.66±1.52 NA NA 27.00±0.00


Leaf C 07.66±1.57 05. 33±1.52 06.00±1.00 05.66±0.57 14.66±0.57 06.66±1.52 12.66±1.52 06.66±0.57 06.66±1.52 NA NA 17.00±1.00

09 Argemone mexicana L.

Papaveraceae

Leaf

A 05.66±1.52 04. 00±0.00 0.66±0.57 04.66±1.52 05.33±1.52 04.33±0.57 05.66±1.52 04.00±0.00 04.66±0.57 NA NA 15. 66±0.57

B 05.00±0.00 NA

04.00±0.00 NA 06.00±0.00 NA 05.66±0.57 NA NA NA NA 28. 33±1.52

C 05.33±1.52 NA 08.00±1.52 04.66±0.57 05.66±1.52 NA 04.33±1.52 04.66±1.52 13.33±1.52 NA NA 05. 33±1.52

10 Azadirachta indica

A.Juss.

Meliaceae

Leaf

A 05.00±0.00 06. 66±0.57 05.33±0.57 07.00±0.00 08.66±0.57 05.33±1.52 07.66±0.57 10.00±0.00 04.33±0.57 NA NA 24.00±1.00

B 04.33±0.57 04. 33±0.57 05.33±0.57 04.00±1.00 06.66±1.57 06.33±1.52 05.66±1.57 08.00±1.00 06.66±1.57 05.00±0.00 NA 35. 66±1.15

C 06.00±1.57 04. 33±1.52 04.00±1.00 04.66±1.57 06.33±1.52 05.66±1.57 06.00±1.00 06.66±0.57 04.33±1.52 NA NA 13. 66±0.57

11 Bergera koenigii L.

Rutaceae

Leaf

A 05.66±0.57 04. 00±0.00 07.00±0.00 05.33±0.57 10.33±1.52 06.66±1.52 06.33±1.52 05.33±1.52 07.66±1.52 05.66±1.57 NA 18.00±1.00

B 05.33±1.52 04. 66±1.52 06.33±0.57 05.00±0.00 07.66±1.52 05.33±0.57 06.33±1.52 04.00±0.00 13.00±0.00 07.00±0.00 NA 34. 33±1.52

C 07.66±1.52 05. 66±1.52 10.00±0.00 06.66±1.52 08.33±0.57 06.66±1.52 07.00±0.00 05.33±0.57 04.66±1.52 NA NA 17. 33±1.52

12

Butea monosperma

(Lam.)

Fabaceae

Leaf

A 06.66±1.52 05. 00±0.00 07.66±1.52 06.66±1.52 08.33±1.52 05.00±0.00 08.66±1.52 08.66±1.52 04.33±0.57 NA NA 19. 66±0.57

B 07.66±1.52 NA 08.33±1.52 05.66±1.52 08.33±0.57 05.66±1.52 09.66±1.52 09.33±0.57 05.66±1.52 NA NA 34.00±0.00

C 05.33±1.15 04. 33±0.57 06.33±0.57 05.66±1.52 13.33±0.57 09.33±1.52 10.33±1.52 06.33±1.52 04.66±1.52 NA NA 22.00±0.00

13 Cajanus cajan L.(Mill.)

Fabaceae

Leaf

A 07.66±1.57 05. 00±1.00 06.00±1.00 05.00±1.00 06.00±1.00 05.66±1.57 06.66±1.57 05.00±1.00 NA NA NA 22. 33±1.52

B 07.66±0.57 06. 66±1.57 08.66±0.57 06.66±0.57 07.66±1.57 06.66±0.57 06.66±0.57 05.66±1.57 05.66±0.57 NA NA 27. 33±1.52

C 05.66±0.57 05.00.0.00 06.66±1.57 05.66±1.57 07.66±0.57 05.33±0.57 06.66±0.57 05.00±1.00 05.66±0.57 NA NA 14. 66±1.15

14 Calotropis gigantea

L.(R.Br.)

Asclepiadaceae

Leaf

A 06.66±1.57 06. 00±1.00 07.33±0.57 06.66±1.57 06.66±1.57 06.33±0.57 06.33±0.57 06.66±1.57 05.66±1.57 NA NA 19. 33±1.52

B 05.33±0.57 08. 66±1.57 12.66±1.57 09.33±0.57 10.33±0.57 06.66±0.57 07.66±1.57 05.00±1.00 05.00±1.00 NA NA 32.00±0.00

C 06.33±0.57 05. 66±1.57 07.33±0.57 06.66±0.57 07.66±1.57 06.66±0.57 07.00±1.00 05.66±1.57 06.66±1.57 NA NA 26. 66±0.57

15 Carica papaya

(L.)Roxb.Flem.

Caricaceae

Leaf

A 05.00.0.00 05.00±0.00 07.00±1.00 05.00±0.00 07.66±0.57 04.33±0.57 07.00±0.00 05.00±1.00 08.00±0.00 05.66±1.57 NA 16. 33±1.52

B 05.33±0.57 05. 66±1.52 04.33±1.15 04.66±1.52 07.33±1.15 05.66±1.52 06.66±1.52 05.33±0.57 05.33±0.57 NA NA 24. 33±1.52

C 05.33±1.15 04. 66±1.57 04.33±0.57 04.66±1.52 06.33±0.57 05.33±0.57 05.66±1.52 05.66±1.52 06.33±0.57 NA NA 13.00±0.00

16 Ceasalpinia bonduc

(L.)Roxb.

Ceasalpiniaceae

A 13.66±1.57 08. 33±1.15 13.00±0.00 07.33±1.15 12.33±1.15 07.33±1.15 10.66±1.57 07.33±1.15 05.00±0.00 NA NA 23. 66±0.57

B 09.00±0.00 07. 66±1.57 08.33±0.57 08.00±0.00 08.66±1.57 07.00±0.00 07.33±1.15 05.66±1.57 05.33±0.57 NA NA 31.50±0.00

C 08.33±0.57 08. 33±1.15 09.66±1.57 11.33±1.15 12.33±1.15 11.33±0.57 12.66±1.57 10.33±1.15 05.33±1.15 NA NA 20. 33±1.52


Seed

17 Celosia argentea L.

Amarathaceae

Seed

A 05.33±1.52 05. 33±1.15 06.33±1.52 05.66±0.57 07.33±1.52 05.33±0.57 11.33±0.57 04.33±1.52 05.33±0.57 NA NA 18. 33±1.52

B 05.33±0.57 NA 06. 00±0.00 05.33±1.15 06.00±0.00 05.66±0.57 06.33±1.52 05.33±1.15 05.66±0.57 NA NA 24. 66±0.57

C 07.00±0.00 05. 33±0.57 11.00±0.00 06.00±0.00 07.66±0.57 06.00±0.00 08.00±0.00 05.00±0.00 05.66±0.57 NA NA 12. 33±1.52

18 Coccinia grandis

(L.)Vogit. Cucurbitaceae

Leaf

A 10.00±0.00 05. 66±0.57 07. 33±1.15 05. 33±0.57 10. 33±1.15 06.66±1.52 07. 33±0.57 06. 33±1.15 13. 33±1.15 07.66±1.57 NA 18.00±0.00

B NA NA 08.00±0.00 06.33±1.15 08.00±0.00 05.00±0.00 08.33±0.57 06.33±0.57 17.33±1.15 NA NA 26. 66±1.15

C 06.66±0.57 05. 33±0.57 08.66±0.57 05.66±0.57 07.33±0.57 05.33±1.15 05. 33±1.15 04.33±0.57 12. 33±1.15 NA NA 19. 66±0.57

19 Citrus medica L.

Rutaceae

Leaf

A 08. 00±0.00 05. 33±0.57 10.00±0.00 08. 66±1.52 11.66±0.57 06.00±0.00 10. 33±0.57 07. 66±1.52 04.33±1.15 NA NA 15.00±0.00

B 14.33±1.15 04. 66±0.57 07. 66±1.52 05.66±0.57 13. 66±1.52 08.66±0.57 07.66±0.57 06.33±1.15 05.00±0.00 NA NA 28. 66±1.15

C 06. 66±1.52 05. 00±0.00 06. 33±1.15 04.33±1.15 11. 00±0.00 07. 33±1.15 08.33±1.15 07. 00±0.00 05.66±0.57 NA NA 16. 66±0.57

20 Corchorus oleterius L.

Tiliaceae

Seed

A 07.00±0.00 NA 12.66±0.57 05. 33±1.15 14.00±0.00 05. 33±0.57 08. 66±1.52 05.00±0.00 11. 33±1.15 05.66±1.57 NA 18.00±0.00

B 06. 01±0.00 05. 33±1.15 08. 00±0.00 05. 66±1.52 12.66±0.57 05. 66±1.52 07. 33±1.15 05. 33±0.57 10.66±0.57 NA NA 23. 33±1.52

C 06.66±0.57 04. 33±1.15 08.66±0.57 05. 66±1.52 17. 66±1.52 05. 33±1.15 14.66±0.57 05. 66±1.52 12. 33±1.15 07.66±1.57 NA 22. 33±1.52

21 Coriandrum sativam L.

Apiaceae

Aerial

A 05. 33±1.15 04. 00±0.00 07. 33±0.57 04. 33±1.15 09. 00±0.00 05. 33±0.57 07. 33±1.15 04. 33±1.15 06. 66±1.52 NA NA 24. 66±1.15

B 05. 00±0.00 04. 33±1.15 06±33±1.15 04. 66±1.52 07. 33±1.15 05. 66±1.52 05. 33±1.15 05. 00±0.00 05. 01±0.00 NA NA 28.00±1.00

C 05. 33±1.15 04. 00±0.00 05. 33±0.57 04. 33±1.15 06. 33±0.57 04. 00±0.00 05. 33±1.15 04. 33±0.57 04. 66±1.52 NA NA 15. 33±1.52

22 Cryptolepis buchananii

Roem&Schult.

Asclepiadaceae

Aerial part

A 07. 00±0.00 05. 66±1.52 07. 66±1.52 07. 00±0.00 07. 33±0.57 05. 66±1.52 07. 00±0.00 06. 01±0.00 05. 33±0.57 NA NA 19.00±0.00

B 05. 33±1.15 05. 33±0.57 08. 00±0.00 06. 33±1.15 07. 33±1.15 06. 33±1.15 07. 33±1.15 07. 66±1.52 05. 33±1.15 NA NA 31.00±0.00

C 05. 00±0.00 05. 33±1.15 06.33±1.15 05. 00±0.00 07. 00±0.00 06. 00±0.00 07. 66±1.52 05. 00±0.00 05. 00±0.00 NA NA 17. 33±1.52

23 Curcuma longa L.

Zingiberaceae

Rhizome

A 10. 66±1.52 09. 33±0.57 11. 33±1.15 08. 33±1.15 07. 33±0.57 06. 00±0.00 09. 33±1.15 05. 66±1.52 07. 01±0.00 05.66±1.57 NA 18.00±0.00

B 20. 33±1.15 11. 00±0.00 06. 66±1.52 06. 00±0.00 06. 66±1.52 06. 33±0.57 08. 00±0.00 04. 33±1.15 05. 66±1.52 NA NA 30. 33±1.52

C 06. 33±0.57 09. 66±1.52 06. 66±1.52 05. 33±0.57 05. 33±1.15 05. 66±1.52 08. 33±1.15 05. 33±0.57 05. 66±1.52 NA NA 19. 66±0.57

24 Dalbergia sisso Roxb.

Fabaceae

Leaf

A 05. 33±1.52 05. 66±0.57 06. 66±1.57 05. 66±1.52 07. 33±1.52 05. 66±0.57 07. 66±0.57 05. 66±1.52 06. 66±1.52 NA NA 17. 66±1.15

B 06. 33±1.52 06. 66±1.57 08. 66±0.57 06. 33±1.52 08. 66±1.57 05. 66±1.52 07. 33±1.52 06. 33±1.52 05. 66±0.57 NA NA 28.00±0.00

C 07. 66±1.52 06. 66±0.57 07. 33±1.52 06. 00±0.00 08. 66±0.57 07. 33±1.52 08. 66±0.57 06. 66±1.52 05. 00±1.00 NA NA 27. 33±1.52

25 Datura stromium L.

Solanaceae

A 05. 66±1.57 05. 66±0.57 07. 66±1.52 05. 66±0.57 06. 66±1.52 04. 66±1.57 06. 66±1.52 05. 66±0.57 06. 33±1.52 NA NA 17. 33±1.52

B 06. 00±0.00 05. 33±1.52 07. 00±0.00 04. 66±1.57 08. 66±0.57 05. 66±1.52 08. 66±0.57 05. 33±1.52 NA NA NA 26.00±0.00


Leaf C 07. 66±0.57 05. 66±1.57 07. 66±0.57 04. 00±0.00 11. 66±1.52 06. 66±0.57 07. 00±0.00 04. 66±0.57 07. 00±0.00 05. 66±0.57 NA 19. 33±1.52

26 Phyllanthus officinalis L.

Euphorbiaceae

Leaf

A 06. 33±1.52 05. 33±1.52 10. 00±0.00 05. 33±1.52 13. 66±1.57 06. 33±1.52 07. 33±1.15 05. 33±1.52 04. 66±0.57 NA NA 20.00±0.00

B 04. 33±1.52 06. 66±0.57 08. 66±1.57 06. 33±1.52 17. 00±0.00 06. 66±1.52 09. 66±0.57 07. 00±1.00 NA NA NA 28. 66±1.15

C 07. 66±0.57 05. 66±0.57 05. 00±0.00 05. 66±0.57 12. 66±0.57 05. 00±1.00 06. 33±1.15 07. 66±1.52 05. 66±0.57 NA NA 17. 66±1.15

27 Euphorbia tirucalli L.

Euphorbiaceae

Leaf

A NA NA 04. 66±0.57 04. 00±0.00 05. 66±1.52 04. 33±1.52 06. 33±1.52 05. 66±0.57 NA NA NA 18. 66±0.57

B 04. 33±1.52 04. 00±1.00 08. 00±0.00 07. 33±1.52 05. 66±0.57 04. 33±1.52 09. 00±1.00 07. 00±0.00 05. 66±0.57 NA NA 26.00±0.00

C 05. 66±1.57 05. 66±0.57 08. 33±1.52 06. 66±1.52 06. 00±1.00 05. 66±1.52 12. 33±1.52 04. 33±1.52 06. 66±1.57 NA NA 18. 33±1.52

28 Ficus racemosa L.

Moraceae

Leaf

A 05. 66±0.57 05. 66±1.57 05. 33±1.52 05. 66±0.57 09. 66±0.57 07. 33±1.15 07. 33±1.52 04. 66±1.52 07. 33±1.15 05. 33±1.52 NA 20.00±1.00

B 05. 33±1.52 05. 66±1.52 06. 66±1.52 06. 00±0.00 07. 00±1.00 05. 66±0.57 05. 33±1.15 05. 33±1.15 05. 66±0.57 NA NA 40.00±0.00

C 05. 66±0.57 05. 66±0.57 06. 66±0.57 06. 66±1.52 10. 33±1.52 08. 66±1.52 12. 66±1.57 07. 66±0.57 05. 00±0.00 NA NA 28. 33±1.52

29 Maytenus senegalensis

(Lam.)Excell. HGUG-

134Celastraceae

Leaf

A 06. 66±1.57 05. 66±1.57 08. 33±1.15 05. 66±0.57 11. 33±1.15 05. 00±0.00 11. 00±1.00 06. 00±0.00 06. 66±1.57 NA NA 16.00±0.00

B 05. 00±0.00 06. 66±1.52 08. 33±1.52 06. 66±1.52 12. 66±0.57 08. 66±1.57 08. 33±1.15 09. 66±0.57 05. 00±1.00 NA NA 30. 33±1.52

C 04. 66±0.57 04. 66±0.57 07. 00±0.00 05. 33±1.52 12. 66±1.57 06. 66±1.57 07. 66±0.57 07. 33±1.15 05. 66±0.57 NA NA 17. 66±1.15

30 Hibiscus rosa-sinensis L.

Malvaceae

Flower

A 07. 33±1.52 05. 33±1.52 06. 33±1.52 04. 66±0.57 06. 00±1.00 05. 33±1.15 08. 00±0.00 04. 00±1.00 04. 00±1.00 NA NA 19.00±0.00

B 05. 00±1.00 07. 66±0.57 08. 00±1.00 06. 33±1.15 05. 33±1.52 04. 66±0.57 07. 33±1.15 04. 66±0.57 05. 33±1.15 NA NA 30. 66±1.15

C 05. 66±0.57 04. 66±1.52 07. 66±1.57 05. 66±0.57 06. 00±0.00 05. 66±1.52 07. 66±0.57 06. 00±0.00 04. 66±0.57 NA NA 20. 66±0.57

31 Hyptis suaveolens L.

Lamiaceae

Leaf

A 06. 66±1.52 05. 66±1.57 07. 00±0.00 05. 33±1.15 07. 66±1.52 06. 33±1.15 06. 66±0.57 05. 33±1.52 04. 66±0.57 NA NA 19.00±1.00

B 06. 33±1.15 05. 33±0.57 10. 33±1.52 06. 66±0.57 10. 66±1.57 08. 33±1.52 09. 33±0.57 08. 00±0.00 05. 00±0.00 NA NA 26. 33±1.52

C 05. 33±0.57 05. 66±1.52 08. 33±1.15 06. 00±0.00 08. 33±1.15 06. 66±1.52 07. 66±1.52 06. 33±1.15 05. 33±0.57 NA NA 18.00±0.00

32 Ixora coccinea L.

Rubiaceae

Flower

A 05. 00±0.00 04. 33±1.15 06. 33±1.15 05. 33±0.57 06. 33±1.15 05. 00±0.00 07. 33±1.15 05. 66±1.52 05. 66±1.52 NA NA 16.00±0.00

B 06. 33±0.57 04. 66±1.57 06. 33±1.52 05. 66±1.52 08. 66±1.57 04. 33±1.15 07. 00±0.00 04. 33±0.57 05. 33±1.15 NA NA 28. 33±1.52

C 06. 66±1.57 04. 00±1.00 06. 00±0.00 05.66±0.57 07. 33±1.15 04. 66±0.57 06. 66±1.52 04. 66±1.57 05. 66±0.57 NA NA 21. 66±0.57

33 Jatropha glandulifera

Roxb.Euphorbiaceae

Leaf

A 05.33±1.52 04. 33±1.15 07. 66±1.52 05. 00±0.00 06. 66±0.57 05. 33±1.15 07. 66±1.57 05. 66±1.52 06. 33±0.57 NA NA 19.00±0.00

B 06. 33±1.15 04. 00±0.00 06. 66±0.57 05. 33±1.15 11. 33±1.15 05. 33±0.57 09. 33±0.57 06. 33±1.15 06. 00±0.00 NA NA 28.00±1.00

C 06. 66±1.52 06. 66±1.57 09. 33±0.57 05. 66±1.57 08. 66±0.57 06. 66±1.57 10. 66±0.57 05. 00±0.00 06. 66±1.52 NA NA 18. 66±1.15

34 Lantana camara L.

Verbenaceae

Leaf

A NA NA 66±1.52 05. 66±1.57 04. 33±1.15 NA NA 05. 66±1.52 NA NA NA NA 19. 66±1.15

B 05. 33±0.57 0433±1.15 NA 10. 66±1.52 05. 33±1.52 04. 66±1.52 04. 33±0.57 10. 33±0.57 NA NA NA 16. 33±1.52

C NA NA 08. 33±1.15 04. 00±0.00 04. 33±1.15 NA 06. 00±0.00 05. 66±0.57 05. 66±1.52 NA NA 18.00±0.00


35 Lawsonia inermis L.

Lythraceae

Leaf

A 04. 00±0.00 04. 33±1.15 13. 33±1.52 06. 33±0.57 12. 66±1.52 05. 33±1.15 09. 66±1.57 05. 33±1.15 05. 33±0.57 NA NA 24. 33±1.52

B 05. 33±1.15 04. 33±0.57 06. 66±0.57 06. 66±1.52 11. 00±1.00 04. 00±0.00 09. 66±1.52 06. 66±1.57 06. 00±0.00 NA NA 38.00±0.00

C 04. 66±1.57 NA33±1.15 05. 00±1.00 05. 33±1.52 13. 33±1.52 06. 33±0.57 NA 06. 00±0.00 04. 66±0.57 NA NA 17. 33±1.52

36 Lycopersicon esculentum

L.

Solanaceae

Leaf

A 08. 66±1.52 05. 33±1.52 09. 33±1.15 05. 00±0.00 06. 33±1.15 05. 66±1.52 09. 33±0.57 04. 33±1.15 10. 33±0.57 05.66±1.57 NA 17. 66±0.57

B 07. 33±1.15 NA 07.66±0.57 04. 66±1.52 12. 66±0.57 05. 00±0.00 07. 00±0.00 05. 66±0.57 09. 66±1.52 05. 33±1.15 NA 23.00±0.00

C 07. 00±0.00 05. 66±1.52 08. 66±1.52 05. 33±1.15 09. 66±1.52 06. 33±1.15 09. 66±1.52 05. 33±0.57 06. 00±0.00 NA NA 19. 33±1.5

37 Mangifera indica L.

Anacardiaceae

Leaf

A 05. 33±1.52 05. 33±1.15 07. 66±1.57 06. 00±0.00 06. 33±1.52 05. 66±1.57 06. 33±0.57 04. 00±0.00 05. 33±1.15 NA NA 19.00±1.00

B NA 06. 00±0.00 06. 66±0.57 07. 66±1.52 06. 66±1.52 05. 33±1.15 09. 66±1.52 07. 33±1.52 05. 66±1.52 NA NA 32. 66±1.15

C 07. 33±0.57 06. 66±1.57 06. 33±1.15 06. 66±0.57 06. 33±1.15 05. 66±1.52 06. 66±1.57 04. 33±1.15 05. 66±1.57 NA NA 25. 66±1.15

38 Mentha viridis

L.Lamiaceae

Aerial part

A NA 04. 66±1.52 04. 00±1.00 04. 66±1.52 04. 33±0.57 04. 00±1.00 04. 33±0.57 04. 66±1.57 NA NA NA 18.50±0.00

B 06. 00±1.00 NA NA NA 04. 00±1.00 NA 05. 00±1.00 NA 06. 00±1.00 NA NA 28. 33±1.52

C 04. 66±0.57 05. 66±0.57 NA 04. 33±0.57 04. 66±1.57 NA 05. 66±1.57 04. 66±1.52 06. 66±0.57 NA NA 15.50±0.00

39 Milletia pinnata L.

Fabaceae

Leaf

A 07. 00±1.00 09. 33±1.52 10. 00±1.00 07. 00±1.00 10.00±1.00 08. 66±1.52 08. 66±1.52 09. 00±1.00 05. 66±1.52 NA NA 18. 33±1.52

B 11. 33±1.52 15. 66±1.57 11. 66±1.57 11. 33±1.15 06. 33±1.15 12. 33±1.52 06. 66±0.57 11. 33±0.57 NA NA NA 16. 66±1.15

C 11. 00±1.00 06. 66±0.57 11. 33±1.52 09. 66±1.57 07. 00±1.00 05. 66±1.52 09. 66±1.57 09. 66±0.57 NA NA NA 20. 66±0.57

40 Momordica charantia L.

Cucurbitaceae

Leaf

A 07. 33±1.52 06. 33±1.52 07. 00±1.00 06. 33±0.57 06. 33±0.57 05. 00±1.00 06. 66±0.57 05. 33±1.52 NA NA NA 18. 66±0.57

B 06. 00±1.00 05. 33±0.57 07. 66±1.52 05. 00±1.00 08. 66±0.57 07. 66±1.57 08. 00±1.00 07. 66±1.57 06. 00±1.00 ± NA 28.00±0.00

C 04. 66±1.57 04. 00±1.00 05. 33±0.57 04. 33±1.52 06. 33±1.52 05. 33±0.57 05. 33±0.57 05. 00±1.00 05. 66±1.57 NA NA 16. 66±1.15

41 Nerium odorum Mill.

Apocynaceae

Leaf

A 05. 33±1.52 04. 66±1.57 04. 00±1.00 04. 66±0.57 07. 33±1.15 06. 00±1.00 06. 33±1.15 06. 33±1.52 NA NA NA 21. 33±1.52

B 04. 66±0.57 04. 00±1.00 05. 66±1.52 04. 66±1.57 07. 33±1.52 05. 33±1.15 06. 00±1.00 05. 66±0.57 04. 00±1.00 NA NA 29. 33±1.5

C 06. 33±1.15 05. 66±0.57 05. 33±1.52 05. 66±1.52 07. 00±1.00 06. 33±1.52 06. 66±1.57 05. 66±1.52 05. 66±1.52 NA NA 14.00±0.00

42 Ocimum sanctum L.

Lamiaceae

Aerial part

A 06. 00±1.00 04. 33±1.52 07. 33±0.57 NA 05. 66±1.52 04. 33±1.15 05. 66±0.57 04. 00±1.00 04. 66±0.57 NA NA 20. 66±0.57

B 08. 33±0.57 05. 66±1.52 10. 00±1.00 NA 06. 66±0.57 05. 00±1.00 06. 66±1.52 04. 66±1.57 05. 33±0.57 NA NA 29. 66±1.15

C 06. 33±1.52 05. 66±0.57 09. 66±1.57 04. 33±1.52 05. 33±0.57 04. 66±1.57 05. 33±0.57 05. 66±0.57 04. 00±1.00 NA NA 15. 33±1.52

43 Piper nigrumL.

Piperaceae

Seed

A 04.00±1.00 07. 33±0.57 NA 05. 00±1.00 06. 66±1.57 05. 33±1.52 08. 00±1.00 05. 33±0.57 04. 66±1.57 NA NA 16. 33±1.52

B 06. 66±1.57 05. 66±1.57 08. 66±1.52 06. 66±0.57 07. 33±1.15 06. 66±1.52 07. 33±1.15 05. 00±1.00 05. 33±1.15 NA NA 31.00±0.00

C 05. 66±0.57 05. 00±1.00 07. 33±1.52 05. 66±1.57 06. 00±1.00 06. 33±0.57 06. 66±0.57 04. 66±1.52 05. 00±1.00 NA NA 28. 66±1.15

44 Plumbago zeylanica L. A 07. 33±0.57 04. 66±0.57 06. 00±1.00 05. 33±0.57 08. 33±1.52 04. 00±1.00 08. 33±0.57 05. 33±1.52 08. 66±1.57 05.66±1.57 NA 22.00±1.00


Plumbaginaceae

Leaf

B 10. 00±1.00 04. 33±1.15 08. 33±0.57 04. 00±1.00 07. 66±1.52 05. 66±1.57 13. 00±1.00 04. 00±1.00 06. 66±1.52 NA NA 28.33±1.52

C 10.66±0.57 04. 00±1.00 08. 33±1.52 04. 66±1.57 07. 00±1.00 04. 33±1.52 09. 66±1.57 05. 66±1.57 06. 00±1.00 NA NA 24.00±0.00

45 Ricinus communis L.

Euphorbiaceae

Seed

A 07. 66±0.57 05. 66±1.57 06. 66±0.57 05. 66±1.52 10. 66±0.57 05. 33±1.52 08. 66±1.57 05. 66±0.57 04. 33±1.52 NA NA 18. 66±1.15

B 04. 33±1.52 04. 66±1.52 05. 33±1.52 04. 66±0.57 07. 33±1.52 05. 66±0.57 08. 66±1.52 05. 66±1.57 04. 33±1.15 NA NA 29. 66±0.57

C 05. 33±0.57 04. 66±0.57 04. 33±0.57 04. 33±0.57 07. 00±0.00 05. 66±1.57 06. 66±0.57 05. 33±1.15 04. 66±0.57 NA NA 15. 33±1.52

46 Santalum album L.

Santalaceae

Leaf

A 04. 66±1.57 04. 33±1.15 04. 66±0.57 06. 33±1.52 09. 66±0.57 05. 66±1.52 07. 33±1.52 04. 66±0.57 05. 66±1.57 NA NA 17.00±0.00

B 06. 66±0.57 05. 66±1.57 06. 66±1.57 05. 00±0.00 07. 66±1.57 04. 66±0.57 10. 66±1.57 11. 66±1.52 04. 33±1.15 NA NA 38.20±1.00

C 04. 33±1.52 04. 00±0.00 05. 33±1.52 04. 66±0.57 07. 33±1.52 06. 66±1.57 06. 00±0.00 05. 33±1.52 04. 33±1.52 NA NA 10. 33±1.52

47 Senna auriculata (L.)

Roxb.Ceasalpiniaceae

Flower

A 06. 00±0.00 04. 66±0.57 07. 66±1.52 05. 33±1.15 07. 66±1.52 05. 33±1.52 08. 33±1.15 05. 66±1.57 05. 66±0.57 NA NA 21.00±0.00

B 05. 33±1.15 05. 33±0.57 06. 00±0.00 05. 66±1.52 10. 66±0.57 05. 00±0.00 06. 33±1.52 04. 33±1.15 05. 66±1.57 NA NA 35. 33±1.52

C 06. 66±0.57 05. 33±1.52 07. 66±0.57 06. 66±1.57 10. 00±0.00 06. 66±0.57 08. 66±1.52 05. 66±0.57 05. 00±0.00 NA NA 17. 66±1.15

48 Senna tora (L.)

Roxb.Ceasalpiniaceae

Leaf

A 06. 33±0.57 05. 66±1.52 06. 66±1.57 05. 66±0.57 09. 33±0.57 05. 66±1.57 08. 66±1.57 06. 66±1.52 05. 33±1.52 NA NA 16. 66±0.57

B 06. 33±1.52 05. 66±0.57 07. 33±0.57 05. 33±1.15 09. 33±1.52 06. 33±0.57 09. 33±0.57 06. 33±1.15 05. 66±1.57 NA NA 27. 33±1.52

C 05. 66±1.57 05. 66±1.57 08. 66±1.52 06. 33±1.52 11. 66±0.57 07. 33±1.52 07. 33±1.52 06. 66±0.57 05. 66±1.52 NA NA 18.50±0.00

49 Solanum nigrum L.

Solanaceae

Leaf

A 06. 66±0.57 05. 00±0.00 08. 66±0.57 07. 00±0.00 08. 33±1.15 07. 66±0.57 08. 66±1.52 08. 66±1.57 08. 33±1.15 06. 33±1.15 NA 21. 66±1.15

B 06. 33±1.15 06. 33±0.57 09. 33±1.52 05. 66±0.57 06. 33±0.57 06. 66±1.52 06. 66±1.57 05. 33±1.52 08. 66±0.57 05.66±1.57 NA 29.00±0.00

C 06. 00±0.00 07. 66±0.57 08. 66±1.57 07. 66±1.52 12. 66±0.57 08. 66±1.57 11. 33±0.57 08. 00±0.00 10. 66±1.57 NA NA 14. 66±1.15

50 Sterculia foetida L.

Sterculaceae

Seed

A 10. 33±0.57 05. 33±1.52 08. 33±1.15 06. 66±1.57 07. 66±1.57 06. 66±0.57 09. 33±1.52 06. 66±0.57 07. 00±0.00 NA NA 21.00±0.00

B 09. 66±0.57 05. 66±1.57 07. 66±0.57 07. 33±0.57 07. 66±1.52 06. 00±0.00 08. 66±1.52 07. 66±1.52 06. 33±1.52 NA NA 27. 66±1.15

C 11. 66±1.57 05. 66±1.52 14. 00±0.00 05. 66±0.57 08. 33±1.52 05. 33±1.52 10. 66±1.57 05. 33±0.57 05. 33±1.52 NA NA 20.00±0.00

51 Semecarpus anacardium

L. Anacardiaceae

Bark

A 07. 33±1.52 05. 66±0.57 07. 66±1.57 05. 66±1.52 06. 66±0.57 05. 66±1.52 06. 33±1.52 05. 66±0.57 NA NA NA 17. 66±1.15

B 08. 00±0.00 05. 33±1.15 09. 66±0.57 05. 33±1.52 06. 00±0.00 05. 66±0.57 08. 33±1.15 05. 66±1.52 05. 66±1.57 NA NA 26. 33±1.52

C 08. 66±0.57 04. 66±1.57 08. 66±1.52 05. 66±0.57 07. 33±1.52 05. 66±1.57 06. 33±1.52 05. 66±1.57 05. 66±0.57 NA NA 16. 66±1.15

52 Tamarindus indica

Ceasalpiniaceae

Leaf

A NA 04. 66±0.57 06. 66±1.57 04. 66±0.57 08.33±1.52 07. 66±1.57 08. 33±1.52 06.00±0.00 06. 33±1.52 NA . 21.00±0.00

B NA NA NA NA 08. 66±0.57 10. 33±1.52 08. 66±0.57 07. 66±1.57 NA NA NA 18. 66±1.15

C 04. 66±0.57 NA 11. 00±0.00 06. 33±1.52 10. 66±1.57 07. 00±0.00 09. 00±0.00 06. 33±1.52 06. 33±0.57 NA NA 16. 33±1.52

53 Tectona grandis L.

Verbenaceae

Leaf

A 06. 66±1.57 05. 66±1.57 08. 66±0.57 06. 66±1.57 07. 00±0.00 04. 33±1.52 09. 00±1.00 06. 66±0.57 05. 66±1.57 NA NA 18. 66±0.57

B 06. 33±1.52 04. 00±0.00 08. 33±0.57 05. 33±0.57 10. 66±0.57 05. 33±0.57 07. 33±1.52 05. 33±0.57 05. 00±0.00 NA NA 28. 33±1.52

C 06. 00±0.00 05.00±33±0.57 08. 33±1.52 05. 66±0.57 10. 33±0.57 05. 66±0.57 10. 33±0.57 06. 66±1.57 05. 66±0.57 NA NA 18. 66±1.15


54 Tinospora cordifolia

(Willd.)J.Hook&Thoms.

Menispermaceae

Leaf

A NA NA 08. 66±1.57 05. 33±1.15 08. 00±0.00 05. 33±1.52 07. 66±0.57 06. 33±1.15 05. 33±0.57 NA NA 05. 33±1.52

B 05. 66±0.57 05. 33±0.57 10. 66±0.57 06. 66±1.57 11. 66±0.57 06. 33±1.52 06. 33±1.15 07. 33±1.15 06. 66±0.57 NA NA 24. 66±1.15

C 05. 66±1.57 04. 66±1.57 04. 00±0.00 04. 33±1.52 10. 66±1.57 06. 66±1.57 05. 33±1.52 05. 66±0.57 05. 33±1.52 NA NA 18.00±0.00

55 Tephrosia purpurea (L.)

Pers. Fabaceae

Leaf

A 05. 33±1.52 05. 66±0.57 06. 33±0.57 05. 33±0.57 08. 33±1.52 05. 66±0.57 09. 00±0.00 04. 33±0.57 04. 66±1.57 NA NA 17. 66±1.15

B 06. 00±0.00 05. 00±0.00 11. 66±1.57 06. 33±1.15 10. 00±0.00 07. 33±0.57 09. 33±0.57 06. 33±1.52 0500±0.00 NA NA 29.00±0.00

C 05. 00±0.00 04. 33±1.52 08. 66±0.57 05. 66±1.57 08. 33±1.52 05. 33±1.52 07. 66±0.57 04. 66±1.57 05. 33±1.15 NA NA 18. 66±1.15

56 Thevetia nerrifolia Juss.

Ex Steud. Apocynaceae

Leaf

A 05. 33±0.57 04. 00±0.00 07. 00±0.00 04. 66±0.57 08. 66±1.57 05. 33±0.57 06. 33±1.52 0400±0.00 05. 00±0.00 NA NA 22.00±0.00

B 05. 66±0.57 04. 33±0.57 06. 33±1.52 05. 33±0.57 10. 66±0.57 04. 66±1.57 08. 00±0.00 05. 33±0.57 05. 66±0.57 NA NA 28.00±0.00

C 06. 00±0.00 04. 66±0.57 06. 66±1.57 05. 33±1.52 10. 66±0.57 05. 33±1.52 07. 33±0.57 05. 66±0.57 04. 66±1.57 NA NA 22. 66±1.15

57 Tribulus terrestris L.

Zygophyllaceae

Aerial

A 06. 66±1.57 04. 66±1.57 09. 00±0.00 05. 66±1.57 07. 33±1.15 05. 66±0.57 07. 33±1.52 05. 66±1.57 05. 33±1.52 NA NA 20.00±0.00

B 05. 33±1.52 04. 00±0.00 07. 66±0.57 04. 33±0.57 09. 33±1.52 04. 33±0.57 07. 33±1.52 04. 00±0.00 05. 33±0.57 NA NA 23. 66±0.57

C 05. 33±0.57 04. 33±1.52 06. 66±1.57 05. 33±1.15 12. 66±1.57 05. 66±0.57 07. 33±1.15 04. 66±0.57 04. 33±1.52 NA NA 24. 66±0.57

58 Tridax procumbens L.

Asteraceae

Aerial part

A 07. 66±0.57 05. 66±1.57 12. 33±1.52 16. 66±0.57 11. 66±1.57 NA 06. 33±0.57 08. 00±0.00 NA NA NA 21. 66±1.15

B 06. 66±1.57 09. 66±0.57 10. 66±1.57 14. 33±1.52 05. 33±1.52 10. 33±1.52 04. 66±0.57 11. 33±1.52 NA NA NA 20. 66±1.15

C 04. 33±1.52 NA 04. 00±0.00 NA 04. 66±0.57 07. 66±1.57 05. 33±1.52 06. 66±1.57 NA NA NA 18.00±0.00

59 Vitex negundo L.

Verbenaceae

Leaf

A 05. 33±1.52 05. 00±0.00 05. 66±0.57 05. 00±0.00 09. 33±1.52 07. 00±0.00 07. 33±0.57 04. 00±1.00 07. 00±0.00 04. 33±1.52 NA 20.00±0.00

B 05. 66±0.57 05. 66±1.52 06. 66±1.57 06. 66±0.57 07. 33±1.15 05. 66±0.57 05. 33±1.52 05. 33±1.15 05. 00±1.00 NA NA 40. 66±1.15

C 05. 66±1.57 05. 00±1.00 06. 33±0.57 06. 66±1.52 14. 66±1.57 08. 00±0.00 12. 66±1.57 07. 66±0.57 05. 33±0.57 NA NA 28. 66±1.15

60 Zingiber officinale

Roscoe.

Zingiberaceae

Rhizome

A 08. 66±1.52 06. 33±1.52 07. 00±0.00 05. 33±1.15 10. 00±1.00 05. 00±1.00 06. 33±1.15 04. 33±1.52 05. 66±1.57 NA NA 17. 66±0.57

B 07. 66±1.57 04. 66±0.57 15. 33±1.15 06. 66±1.57 12. 33±1.15 05. 00±0.00 06. 66±1.52 07. 33±0.57 05. 66±0.57 NA NA 24. 66±1.15

C 05. 33±0.57 09.33±1.52 08. 00±1.00 06. 00±1.00 13.33±1.52 06.33±1.52 06. 00±1.00 05. 33±1.15 NA NA NA 15.00±0.00

61 Zizyphus jujuba Lam.

Rhamnaceae

Bark

A 05.33±1.52 05. 66±1.57 05.33±0.57 04.33±1.52 05.00±0.00 04.00±1.00 09.33±0.57 05.33±0.57 05. 00±1.00 NA NA 20. 66±0.57

B 07.66±1.57 06.00±0.00 05. 33±1.52 05.66±1.57 NA 06. 33±1.15 10.33±1.52 05. 00±1.00 NA NA NA 20. 66±1.15

C 05.00±1.00 04.00±66 04. 33±1.52 NA 07.00±1.00 NA 07. 33±1.15 05.33±1.52 NA NA NA 21. 33±1.52

P= Pet ether ext ract, C= Chloroform extract, E= Ethyl acetate extract, M=Methanol extract, A=Aqueous extract, C=Control (DMSO), S=Standard (Ketoconazole), NA= No

Activity, 1mg-1

, a=5 mg

-1, b=2.5

mg

-1.

A=Microsporum gypsum, B=Trichophyton tonsurans, C= Trichophyton rubrum.


Screening against Microsporum gypseum

The 61 ethno medicinal plants of Hyderabad Karnataka region belonging to 33

different families used in skin diseases were screened for their antidermatophytic

properties against Microsporum gypseum. The screening was carried out at 5 and 2.5

mg/ml concentrations of pet ether, chloroform, Ethyl acetate, methanol and aqueous

extracts of each plant by agar well diffusion technique the obtained results were given

in table 1. Out of 61 plants, 05 (Ceasalpinia bonducella, Coccinia indica, Corchorus

oleterius, Lawsonia inermis and Tridax procumbens) showed very effective

antidermatophytic activity in ethyl acetate, chloroform and in aqueous extracts,

effective activity observed in 11 plants (Achyranthes aspera, Allium sativam, Celosia

argentea, Citrus medica, Curcuma longa, Emblica officinalis, Gymnosporia montana,

Lycopersicon esculentum, Milletia pinnata, Ricinus communis, Zingiber officinale) in

different extracts, whereas 38 plants showed moderate activity, 07 plants (Euphorbia

tirucalli, Lantana camara, Mentha viridis, Tinospora cordifolia and Tridax

procumbens) showed weak activity. The negative control (DMSO) was not showed

activity, while the standard drug, Ketoconazole significantly inhibited (28. 66±1.15 to

12. 33±1.52 mm) the growth of the test dermatophyte (Tables 2.1, 2, 2).

Screening against Trichophyton tonsurans

The plant extracts and their level of activity against the Trichophyton

tonsurans was listed in Table 1. A number of 305 extracts from 61 ethno medicinal

were subjected to antidermatophytic screening against Trichophyton tonsurans in Pet

ether, chloroform, Ethyl acetate, methanol and aqueous. Extracts of each plant was

tested for its antifungal activity using agar well diffusion method at sample

concentration of 5 & 2.5 mg/ml. Out of 61 plants, 10 exhibited very effective

antidermatophytic activity in three solvent extracts, i.e., Allium sativam L., Corchorus

oleterius L., Gymnosporia montana (Roth)Benth, Milletia pinnata (L.) Panigrahi,

Lycopersicon esculentum L., (Ethyl acetate), Annona squamosa L., Plumbago

zeylanica L. (98% methanolic), Calotropis gigantea L. and Zingiber officinale Rosce.

(Chloroform). Effective activity was also observed in 14 plants of different three

solvent extracts i.e., Achyranthes aspera L., Aegle marmelos (L.), Allium sativam L.,

Citrus medica L., Lawsonia inermis Linn., Senna auriculata (L.) Roxb., Tectona


grandis L., Tinospora cordifolia (Willd.)J.Hook&Thoms, Thevetia nerrifolia Juss.,

Emblica officinalis Gaertn. (Ethyl acetate) Aloe vera L. Curcuma longa Linn.

(Petroleum ether), Tridax procumbens Linn. and Tephrosia purpurea (L.) Pers.

(Chloroform). Whereas the moderate activity observed in 34 plants. While the weak

activity observed in 03 plants i.e., Carica papaya L., Coriandrum sativam L. and

Tamarindus indica Linn. There was no inhibition recorded from the negative

control (DMSO), while the standard drug, Ketoconazole significantly inhibited (28.

66±1.15 to 12. 33±1.52 mm) the growth of the test dermatophyte (Tables 2.1, 2, 2).

Table 2.2: Antidermatophytic frequency of 61 ethno-medicinal plants.

Sl.

no

Test strain Very

effective

Effective Moderate Weak

1 Trichophyton rubrum 18 13 26 04

2 Microsporum gypseum 05 11 38 07

3 Trichophyton

tonsurans 10 14 34 03

2.4.1. B. Preliminary phytochemical screening for the detection of secondary

metabolites occurrence in 61 ethno medicinal plants.

Among the 305 extracts from 61 plants, secondary metabolites having

therapeutical importance were estimated and isolated. Further some of these were

purified from selected plant parts using biochemical and other hyphenated analytical

chromatographic and spectrophotometric methods. The results obtained are discussed

in the light of literature available hitherto.

Preliminary phytochemical screening of secondary metabolites

The crude successive extracts of 61 traditional medicinal plants were

qualitatively screened for the occurrence of various secondary metabolites such as

phenols (Lead acetate test), flavonoids (NaOH test), tannins (Ferric chloride test),

alkaloids (Dragendroff‘s test), Saponins Foam test) and glycosides (Keller-Killiani

test). The reactions with these reagents have shown the presence of metabolites and

are recorded in the Table -2.3. The Preliminary screening results and the number of

positive response of secondary metabolites were given in figure 2.1 & 2.2.


Table-2.3: Preliminary screening for the detection of secondary metabolites of traditional medicinal plant drugs used in the treatment of

skin diseases at Hyderabad Karnataka region.

Plant part

used

Plant name

and Family

Plant constituents

Phenols

Flavonoids

Tannins

Alkaloids

Saponins

Glycosides

A B C D E A B C D E A B C D E A B C D E A B C D E A B C D E

Leaf Achyranthes aspera L.

(Amarathaceae)

- - + + + - - + + + - - + + + - - + + + - - + + + - - + + +

Leaf Aegle marmelos (L.) Corr.

(Rutaceae)

- - + - + - - + - + - - + - - - - + - - - - - - + - - + - +

Bulb Allium sativam L. (Liliaceae) - - + - - - - + - - - - - - - - - + + - - - - + + + + + + +

Bulb Allium cepa L.

( Liliaceae)

- - + - - - + - + - - + + + - - + - + - - - - - + - - + + -

Leaf Aloe vera L.(Liliaceae)

- - + + + - + + + - - - + + + - - - - - - - - + + - - - - -

Leaf Amaranthus spinosus L.

(Amarathaceae)

- - - + + - - + + - - - - + + - - - - - - + - - - - - - + +

Leaf Annona reticulata L.

(Annonaceae)

- - - + - - - + + + - - + + - - - - - - - - - + + - - + - -

Leaf Annona squamosa L.

(Annonaceae)

+ - + + - - - + + + - - - + - - - - + - - - - + + - - + - -

Leaf Argemone mexicana L.

(Papaveraceae)

+ + + + - - + + + - + + + + - + - - + - - - - + - - - - - -

Leaf Azadirachta indica A.Juss. - + + + - - + + + - - - + - - - - + - - - - - - - - + - + -


(Meliaceae)

Leaf Butea monosperma (Lam)

Taub. (Fabaceae)

+ + - - - - - - + - - - - + - - - - - - - - - - + - + - + -

Leaf Cajanus cajan

L.(Mill.)(Fabaceae)

+ - + + - - - - + + + + + + - - - + - - + + + + + + - + + -

Leaf Calotropis gigantea

L.(R.Br.) (Asclepiadaceae )

- + + - - + + + + + + - + + - - + + + - + + + + + - + + + -

Leaf Carica papaya L.

(Caricaceae)

- + - + - - - + + - - + + + - + + + + - + + + + + - - + + +

Flower Cassia auriculata L.

(Ceasalpiniaceae)

- - - + - - - + + + - - + - - - - + + - - - - - - - - + + -

Leaf Cassia tora L. (Fabaceae) - - + + - - - - + + + - - + - - - - + + - - - + + - - + + +

Seed Ceasalpinia bonduc (L.)

Roxb. Flem.

(Ceasalpiniaceae)

- - - + + + - + + + - + + + + - - + - + - - - - + - - - + +

Seed Celosia argentea L.

(Amarathaceae)

- - + + - - - - + - - - - + - - - - - + - - - + + - + - + -

Leaf Coccinia grandis (L.)Vogit.

(Cucurbitaceae)

- - - + + - - - + + - - + + - - - + + + - - + + + - - + + +

Leaf Citrus limon L. (Rutaceae) - - + + + - - + + - - - + + + + + + + - - - + + + - - - - -

Seed Corchorus capsularis L.

(Tiliaceae)

- - + + - - - + + - - + - + - - - - - - - - - - - - - - - -

Aerial Coriandrum sativam L.

(Apiaceae)

- - + + + + + + + + - - - - - - - - - - - - - - - - - - - -

Aerial part Cryptolepis buchananii

Roem&Schult.(Asclepiadace

ae)

-

- + + - - + + + - - - + + + - + + + - - - - + + - - + + -

Rhizome Curcuma longa L. - - + + + - - + + + - - - - - - - - - - - - - - - - - - - -


(Zingiberaceae)

Leaf Dalbergia sisso Roxb.

(Fabaceae)

- - + + - - - - + + - + - + - - - - - - - - - - - - - - - -

Leaf Datura stromium

(Solanaceae)

- - + - + - - + + + - + - + + - + + - + - - - - + - + + + +

Leaf Phyllanthus officinalis L.

(Euphorbiaceae)

- + + - - - + + + + - - + + - + + + + - - - - - - - + + + +

Leaf Euphorbia tirucalli L.

(Euphorbiaceae)

+ - + + + + - - + + + - + + + + - + + + - - - - - - + + + +

Leaf Ficus racemosa L.(Moraceae) + - + + - - + + + + - - - + + + + + - - - - - + + - - - + -

Leaf Maytenus senegalensis (Lam.)

Excell. HGUG-134

(Celastraceae)

- + + + - - + + + - - - + + - + + + - - - - + + + - - - + -

Leaf Hyptis suaveolens (L.) Poit.

( Lamiaceae)

- - - - - - - + + - - - + + - - + - + - - - - - + - - - + -

flower Hibiscus rosa-sinensis L.

(Malveceae)

- - - - + - + + + + - - + + + + + + - - - + + + + - - - - -

flower Ixora coccinea L.

(Rubiaceae )

- - + + - + - + + + - - + + + + + + - - - - + + + - - + + -

Leaf Jatropha glandulifera Roxb.

(Euphorbiaceae)

- + + + - - - + + + - - - + + + + - - - - - - + + - - + + +

Leaf Lantana camara L.

(Verbenaceae)

- + + + - - - + + - - + + + - - + - - - + - + + + - - + + -

Leaf Lawsonia inermis L.

(Lythraceae)

- + + + + - + + + + - + - + + + + + + + - - + + + - - + + -

Leaf Lycopersicon esculentum L.

(Solanaceae)

- - + + + - - + - + - + + - + + + - - + - - - - - - - + + +

Leaf Mangifera indica L.

(Anacardiaceae)

- - + + + - + + + - - - + + - + + - + - + - - - + - + + + -

Aerial part Mentha viridis L. - - + + + - + + + + - - + + - + + + - - - - - - + - - - - -


( Lamiaceae)

Leaf Momordica charantia L.

(Cucurbitaceae)

- - - + - - - + + + - + + + - - - - + + - - - + + - + + + +

Leaf Murraya koenigii L.

(Rutaceae)

+ - - + - + + + + + - + + - - + + + + - - + + + + - - + + -

Leaf Nerium odorum Mill.

(Apocynaceae)

- - - - + - - - - + - + + + + - - - + + - - - + + - + + + +

Aerial part Ocimum sanctum L.

(Lamiaceae)

- + + + + - + + + + - + - + - + - + - - - + - + + - + + + +

seed Piper nigrum L.(Piperaceae) - + + - - - + + + + - + + + - + + + - - - - - - - - + + + -

Leaf Plumbago zeylanica

L.(Plumbaginaceae)

+ + + + + + - - + + + - - + + + - - + + - - - + + - - + + +

Leaf Pongamia pinnata L.

(Fabaceae )

- - + + - - - + + + - + + + - - + + - - - + + + + - + - + -

Seed Ricinus communis L.

(Euphorbiaceae)

- + + + - - - - - - - - + + - + + + - - - + + + + - + + + -

Leaf Santalum album L.

(Santalaceae)

- - + - - - - - - - - + - + - - + - - - - - - - - - - + + -

Leaf Solanum nigrum L.

(Solanaceae)

- - + - - - + + + + - + - - - + + + - - - - + + + - + + + +

Seed Sterculia foetida L

(Sterculaceae)

- + + + + - - + + + - + - + + + + - + - - + + + + - + + + -

Bark Semicaprpus anacardium L.

(Anacardiaceae)

- + + - + - - + + + - + + + - - + + - - - - - + + - - + + +

Leaf Tamarindus indica L.

(Fabaceae)

+ + - + - + - + + - + - - + - + + + - - - - - - + - + - + -

Leaf Tectona grandis L

(Verbenaceae)

- - + + + - - - + + + - - + - - + - - - + + + - + - - + + +

Leaf Tinospora cordifolia

(Willd.)J.Hook&Thoms.

(Menispermaceae)

- - + + - + - + + + - + + + - - + + - - - - - + + + - + + -


Leaf Tephrosia purpurea ( L.)

Pers.

(Fabaceae)

- - - - - + + - + + - + - + + - + - - - - - - + + - + - + -

Leaf Thevetia nerrifolia Juss. Ex

Steud. (Apocynaceae)

+ + - + - + + - + + + + - + - - + - + - + + + + + - + + + +

Aerial Tribulus terrestris L

(Zygophyllaceae)

- - - + - - - - + - - - + + - - + + - + - - - + + - - - + -

Aerial part Tridax procumbens

L.(Asteraceae)

- - + - + + + + + + - + + + + - - - + + - - - - - - - - + -

Leaf Vitex negudu L.

(Verbenaceae)

+ + - + + + + + + + - + + - + - - - + - - - - - - - - + + -

Rhizome Zingiber officinale Rosce.

(Zingiberaceae)

- + + + + - - + + + + - + + - + - + + - + + + + + - - - + +

Bark Zizyphus jujuba Lam.

(Rhamnaceae)

- + + - - - + + + - - + + + - - + - + - - - - + + - + - + +

A-Petroleum ether extract, B-Chloroform extract, C-Ethyl acetate extract, D-Methanol extract, E-Aqueous extract, --absent, +-Present

Preliminary screening of secondary metabolites test names: Alkaloids: Dragendorff‘s, Tannin: Ferric chloride, Phenolic: lead acetate, Glycoside:

Keller-Killiani test, Flavonoids: NaOH, Saponins: Foam test.


Phenols

Three plants viz., Argemone mexicana L., Ocimum sanctum L., Plumbago

zeylanica L. shown positive response to lead acetate test in all the solvent extracts.

Sixteen plants have shown positive response in three extracts viz., ethyl acetate,

methanol and aqueous. Whereas two plants viz., Hyptis suoveolens (L.)Poit., Tephrosia

purpurea ( L.) Pers. shown negative response in all solvent extracts

A total of 305 extracts from 61 plants were tested for phenol occurrence. The

maximum positively responded in methanol and ethyl acetate extracts (44) followed by

aqueous extracts 25 and chloroform extracts 21.the least 11 extracts shown positive

response in petroleum ether. The total number of phenols present extracts are 145.

Flavonoids

In only one plant i.e., Coriandrum sativam, all the extracts shown positive

response to NaOH test. Three extracts viz., ethyl acetate, methanol and aqueous of each

23 plants shown positive response.

A total of 305 extracts from 61 plants were tested for flavonoids occurrence. The

maximum positive responded in 55 methanol extracts, followed by ethyl acetate

extracts 45, aqueous extracts 37 and chloroform extracts 23.the least 13 extracts shown

positive response in petroleum ether. The total number of 173 extrac ts shown positive

response for NaOH test of flavonoids.

Tannins

No plant has shown all positive and negative response in all the test extracts.

Whereas 10 plants shown positive response in three extracts viz., ethyl acetate, methanol

and aqueous.

A total number of 147 extracts have shown positive response for ferric chloride test of

tannins occurrence. The maximum extracts shown positive results in methanol 51 and

followed by ethyl acetate 38, chloroform 27 and aqueous 20. The least 11 extracts

shown positive response in petroleum ether.


Alkaloids

Five extracts of Lawsonia inermis Linn. leaf shown positive response to

Dragendroff‘s test. Whereas three plants leaves viz., Achyranthes aspera L.

Cephalandra indica, Euphorbia tirucalli L. have shown positive response in three

solvent extracts viz., ethyl acetate, methanol and aqueous.

A total of 128 extracts have shown positive response for Dragendroff‘s test of

alkaloids occurrence. The maximum extracts shown positive results in chloroform 33

followed by ethyl acetate 32, methanol 27 and petroleum ether 25. The least 11 extracts

shown positive response in aqueous.

Saponins

Three plants viz., Cajanus cajan, Calotropis gigantea L. Carica papaya L., in all

the extracts has responded positively to foam test. Fifteen plants were shown positive

response in three extracts viz., ethyl acetate, methanol and aqueous. Whereas all the

extracts of 12 plants responded negatively.

A total of 123 extracts have shown positive response for foam test of saponins

occurrence. The maximum extracts shown positive results in aqueous is 46 followed by

methanol 37, ethyl acetate 19 and chloroform 13. The least 08 extracts shown positive

response in petroleum ether.

Glycosides

Allium sativam Linn of all the extracts were responded positively to Keller-

Killiani test. Seventeen plants shown positive response in three extracts viz., ethyl

acetate, methanol and aqueous. Whereas all the extracts o f 08 plants were responded

negatively.

A total of 132 extracts shown positive response for Keller-Killiani test of

glycosides occurrence. Maximum extracts shown positive results in methanol 48

followed by ethyl acetate 37, aqueous 23 and chloroform 21. The least 03 extracts shown

positive response in petroleum ether.


11 13 11

25

8

21 2327

33

13

44 45

3832

19

44

5551

27

37

25

37

20

11

46

Phenols Flavonoids Tannins Alkaloids Saponins

Screening for secondary metabolites occurance in medicinal Plants

Petrolium extract Chloroform extrct Ethyl acetate extract

Methanol extract Aqueious extract

Figure-2.1: Preliminary screening for the occurance of secondary metabolites in 61

traditional medicinal plant drugs of Hyderabad Karnataka region.

Figure-2.2: Percent secondary metabolites occurance in 305 solvent extracts of 61

traditional medicinal plant drugs used in the treatment of skin diseases.


2.4.2. Phytochemistry and pharmacology of selected seven medicinal

plants.

On the basis of preliminary screening and literature, 07 effective plants were

selected for further phytochemical and pharmacological studies.

2.4.2.1. Annona reticulata L. experimental results

Various primary and secondary metabolites having therapeutical importance were

estimated, isolated and further some of these were purified from Annona reticulata L.

leaves using biochemical and other hyphenated analytical chromatographic and

spectrophotometric methods. Further the results obtained are discussed in the light of

literature available hitherto.

Antidermatophytic activity and minimum inhibitory concentrations

In the present investigation five fungal species and six bacterial species were

tested to determine the antifungal and antibacterial activity of 98% methanol leaf extract

of A. reticulata L. The values given in tables-2.4 and 2.5, (Plate:2.1) are the mean of

three observations. The 98% methanol leaf extract showed maximum of 22.00±0.00mm

inhibition in Candida albicans at 40mg/ml followed by Trichophyton rubrum (14.

66±1.15), Microsporum gypseum (12.33±1.52mm), Aspergillus flavus (11.66±1.15mm)

and Trichophyton tonsurans (10.00±0.00mm). The minimum inhibitory concentrations

of test fungi were determined and the values are given in figure 2.3. The MIC of T.

rubrum and C. albicans are 0.62 mg/ml conc. followed by M. gypseum 1.25mg/ml conc.,

and A. flavus, T.tonsurans 2.5 mg/ml conc.

The 98% methanol leaf extract at 40 mg/ml conc. showed maximum of

21.00±1.00 mm inhibition against Escherichia coli and Serratia marcescens followed by

Bacillus subtilis 18. 66±1.15 mm, Psudomonas aeruginosa 16. 33±1.52 mm and the least

inhibition zone shows by Staphylococcus aureus, Brevibacillus brevis with 15. 00±1.00

mm. The minimum inhibitory concentrations of test bacteria were determined and the

values are given in figure 2.3. The MIC of E. coli, S. marcescens, B. subtilis, S. aureus,

B. brevis were determined as 0.6 mg/ml conc. Followed by P. aeruginosa was 1.25

mg/ml conc.


Table-2.4: Antidermatophytic activity of 98% methanolic leaf extract of Annona reticulata L. (Well diffusion technique).

T.rubrum: Trichophyton rubrum, M. gypseum: Microsporum gypseum, C .albicans: Candida albicans, T.tonsurans: Trichophyton tonsurans, A. flavus: Aspergillus flavus, Negative control: DMSO N, N- Dimethyl Formamide, Standard: Ketoconazole (Positive control).

Fungal

strains

Different conc. (mg/ml) of crude and inhibition zone in mm

40

20

10

5

2.5

1.25

0.62

Control

(DMSO)

Standard

(Ketoconazole )

T. rubrum

14. 66±1.15

12.66±0.57

09.66±1.15

08.00±1.00

07.66±1.15

06.00±0.00

06.00±1.00

-

20. 66±1.15

M .gypseum

12.33±1.52

10.00±1.00

09.00±1.00

08.66±1.15

06.00±1.00

05.33±1.52

-

-

18. 66±1.15

C .albicans

22.00±0.00

13.33±1.52

12.66±1.15

11.00±1.00

10.66±0.57

09.66±1.15

06.33±1.52

-

21. 00±0.00

T.tonsurans

10.00±0.00

09.00±1.00

08.00±1.00

07.00±0.00

05.00±1.00

-

-

-

24. 66±0.57

A. flavus

11.66±1.15

09.66±0.57

07.00±1.00

06.66±1.15

05.66±0.57

-

-

-

25. 66±1.15


Table-2.5: Antibacterial activity of 98% methanolic leaf extract of Annona reticulata L. (Well diffusion technique).

Bacterial

strains


40

20

10

05

2.5

1.25

0.62

Control

(DMSO

)

Standard

(Streptomycin)

E. coli

21.00±1.00

20. 66±1.15

17. 33±1.52

14. 00±1.00

12. 33±1.52

10. 66±1.15

08. 66±1.15

-

24. 00±1.00

B. subtilis

18. 66±1.15

15. 33±1.52

14. 00±1.00

13. 66±1.15

10. 33±1.52

09. 33±1.52

07. 00±1.00

-

20. 66±1.15

S. marcescens

21. 00±1.00

19. 33±1.52

1800±1.00

.

16. 00±1.00

12. 33±1.52

10. 33±1.52

09. 33±1.52

-

24. 33±1.52

S. aureus

15. 33±1.52

13. 00±1.00

10. 33±1.52

09. 00±1.00

07. 00±0.00

06. 66±1.15

05. 00±1.00

-

18. 00±1.00

P. aeruginosa

16. 33±1.52

14. 33±1.52

13. 66±1.15

11. 66±1.15

10. 33±1.52

08. 33±1.52

-

-

16. 66±1.15

B. brevis

15. 00±1.00

14. 33±1.52

13. 00±0.00

11. 00±0.00

09. 00±1.00

08. 00±1.00

06. 00±0.00

-

24. 33±1.52

E. coli: Escherichia coli, B. subtilis: Bacillus subtilis, S.marcescens: Serratia marcescens, S. aureus: Staphylococcus aureus, P. aeruginosa: Psudomonas aeruginosa, B. brevis: Brevibacillus brevis, Negative control: DMSO N, N- Dimethyl Formamide, Standard: Streptomycin sulphate (Positive control).


A: Trichophyton rubrum, B: Microsporum gypseum, C: Trichophyton tonsurans, D: Candida albicans, E: Escherichia coli, F:

Bacillus subtilis, G: Bacillus subtilis, H: Staphylococcus aureus, I: Psudomonas aeruginosa, J: Brevibacillus brevis, 1=40 mg/ml,

2=20 mg/ml, 3=10 mg/ml,4=5 mg/ml, 5=2.5 mg/ml, 6=1.25 mg/ml, 7=0.62 mg/ml, C=Negative control: DMF N, N- Dimethyl

Formamide, 8=Standard: Ketoconazole (Positive control against fungi),Streptomycin sulphate (Positive control against bacteria).

Plate 2.1: Antidermatophytic activi ty of 98% methanolic leaf extract of Annona reticulata L.

(Well diffusion technique).

A A B

B

C C D

G G

F F E E

D

H

I I J J

H


00.5

11.5

22.5

3

MIC

Figure -2.3: Minimum Inhibitory Concentrations of 98% methanolic leaf extract of Annona

reticulata L. against test strains.

The negative control used, DMSO could not show inhibition against all the tested

fungal and bacterial strains. Ketoconazole used as standard at conc.5mg/ml shows

antifungal activity 24.00mm whereas streptomycin used standard against bacteria shows

inhibition zone in 24.00mm.

Preliminary screening of secondary metabolites

The crude successive extract of seed viz., petroleum ether, chloroform, ethyl-

acetate and 98% methanol extracts were qualitatively screened for the occurrence of

various secondary metabolites such as alkaloids, phenol, flavonoids, tannins, triterpenes,

steroids, saponins and glycosides. The reactions with these reagents have shown the

presence of metabolites and are presented in the Table: 2.6.

Alkaloids

The chloroform and 98% methanol extracts were positive to the preliminary

alkaloids tests i.e., Meyers, Dragendorff‘s and Wagner‘s reagents. These extracts have

produced a creamy white precipitate with Meyers reagent, orange red precipitate with

Dragendorff‘s reagent and reddish brown precipitate with Wagner‘s reagent. Whereas,

the Ethyl acetate extract (accept Wagner‘s test) responded negatively. The petroleum

ether extract not responded to all the tests.


Phenols

The 98% methanolic leaf extract shown positive response to all the test viz.,

ferric chloride test, ellagic acid test and hot water test pointing out of the presence of

phenols. In hot water test, the leaf showed prominent brownish black demarcation at the

junction of dipped and undipped portion. Ethyl acetate extract shows positive response to

ferric chloride test and ellagic acid test. The Chloroform extract shown positive response

to ellagic acid test. The petroleum extract was not responded to all the tests.

Table-2.6: Preliminary screening of secondary metabolites in Annona reticulata L.

Secondary

metabolites

Name of the test

PE

CHCL3

Et OH

98%

Methan

ol

Alkaloids

Mayers test - + - +

Dragendoff‘s test - + - +

Wagner‘s test - + + +

Phenol

Hot water test - - - +

Ferric chloride test - - + +

Ellagic acid test - + + +

Flavonoids


Lead acetate test - - + +

Shinoda test - - - +

NaOH test - + + +

Tannins Gelatin test - - + +

Triterpenoi

ds

Salkowski‘s test + - + -

Libermann-

Burchard test

+ + + +

Steroids

Salkowski‘s test + - + -

Libermann-

Burchard test

+ + + +

Saponins Foam test - - - +

Glycosides

Keller-Killiani test - + - +

Conc. H2So4 test - - - -

Molisch‘s test - - - -

Glycoside test - - - -

Flavonoids

The 98% methanolic leaf extracts responded positively to flavonoids test like

ferric chloride. Lead acetate, Shinoda and NaOH test indicating the presence of

flavonoids. While ethyl acetate extract shows positive response to above tests axcept

Shinoda test. The chloroform extract positively responded to NaOH test. Whereas pet.

ether did not responded to all the tests.


Tannins

The Ethyl acetate and 98% methanolic leaf extract showed the positive result for

gelatin test. This indicates the presence of tannin in ethyl acetate and 98% methanolic

extract. All the other extracts responded negatively to gelatin test.

Triterpenes

The petroleum ether and Ethyl acetate extracts responded positively to

Salowski‘s, Libermann-Burchard imparting the presence of triterpenes. Whereas in

chloroform and 98% methanol extracts showed positive for Libermann-Burchard test.

Steroids

The petroleum ether and Ethyl acetate extracts responded positively to

Salkowski‘s, Libermann-Burchard imparting the presence of steroids. Whereas in

chloroform and 98% methanol extracts showed positive for Libermann-Burchard test.

Saponins

The 98% methanol extract responded positively to foam saponins tests indicating

the presence of saponins.

Glycosides

The chloroform and 98% methanol extract shown positive response to Kellar –

Kiliani test pointing out the presence of glycosides.

Quantitative estimations of secondary metabolites

Five important secondary metabolites were extracted from the dried powdered

material of Annona reticulata L. leaves estimated quantitatively using various methods.

(Figure.2.4).

The maximum content estimated was total flavonoids (4.93mg/100mg) followed

by total phenol (4.21 mg/100mg), total tannins (3.74 mg/100mg), total alkaloid (1.32

mg/100mg) and total saponins (0.5 mg/100mg).


0

1

2

3

4

5

Alkaloids Flavonoids Tannins Phenols Saponins

Quantitative estimations of secondary metabolites mg/100mg

A.reticulata

Fig. 2.4 Quantitative estimations of secondary metabolites in Annona reticulata L. leaf mg/100mg.

Qualitative separation of secondary metabolites by TLC method

The following secondary metabolites of therapeutic important from seed were

separated through thin layer chromatography using various solvent systems. The hRf

values and characteristic colours of the bands were recorded.

Table-2.7: Qualitative separation of secondary metabolites from Annona reticulata L.

Secondary

metabolites

No of

bands

hRf values

Colour of the bands

Phenols 6 18 Light brown

24 Light blue

36 Yellow

56 Light yellow

70 Light brown

86 Light sky blue

Flavonoids 4 39 Smoke green

52 Thick smoke green

70 Yellow

92 Green

Alkaloids 3 26 Light blue

40 Yellow

53 Light yellow

A. Separation of phenols

The 98% methanol extract of the leaf exhibited 6 distinct bands having Light brown,

Light blue, Yellow, Light yellow, Light brown, Light sky blue with hRf values

18,24,36,56,70 and 86 respectively (Table-2.7).


B. Separation of flavonoids

The developed leaf chromatogram indicated 4 distinct bands possessing smoke green

thick smoke green yellow and green with hRf values 39,52, 70 and 92 respectively.

E. Separation of Alkaloids’

The chromatogram of leaf displayed having 3 d istinct bands possessing light

yellow blue with hRf value 26, yellow colour with hRf value 40 and 53 hRf value band

colour show light yellow. (Table- 2.7).

Table-3.1.8: Separation of flavonoids fractions from A. reticulata L. by the Column

chromatography and PTLC (Preparative thin layer chromatography).

It is evident from the earlier results of qualitative and quantitative studies of

flavonoids that Annona reticulata L. is a rich source of flavonoids of pharmacological

importance further; this was supported by the literature available on Annona sps. Thus an

attempt is made here to isolate some of these flavonoids fractions from the leaf of A.

reticulata L. by column chromatography (CC) and purified with the help of preparative

thin layer chromatography (PTLC).

Column chromatography studies (CC)

The crude effective extract of A. reticulata L. leaf about 10 g was fractioned on a

Silica gel-H (60-120Mesh) column at a room temperature and pressure (26ºC. 1bar).

After discarding 200 ml dead volume from the column (Hexane), total 38 fractions of

100 ml each were collected.

The fractions 1 to 11 were obtained from the pet ether: ethyl acetate. Fractions 12

to 32 were collected from the ethyl acetate : methanol, (1) 100:00, 90:10 (2), 80:20 (3),

70:30 (4), 60:40 (5), 50:50 (6), 40:60 (7), 30:70 (8), 20:80 (9), 10:90 (10) and 00:100

(11). Similarly, 12 to 32 fractions were collected from the solvent mixture of ethyl

acetate: methanol, (12) 100:00, 90:10 (13), 80:20 (14), 70:30 (15), 60:40 (16), 50:50

(17), 40:60 (18), 30:70 (19), 20:80 (20), 10:90 (21) and 00:100 (22). While the fractions

37 to 54 were collected from the solvent mixture of chloroform methanol (90:10 (5),

80:20 (2), 60:50 (02) 40:60 (2), 20:80 (02) and 10:90 (05) finally, fractions 55 to 58 were

collected from the methanol 100% (4) mobile phase (Table-2.8).


However, the collected 38 fractions were pooled into seventeen major fractions

owing to their in colour. The concentrated solutions of these fractions had waxy nature

fractions of 8 to 9, 20 to 27 (waxy) and 40 to 49 (solid) brown. 6 to 7 (waxy) and 28 to

36 fractions shows yellow colour and fractions 44 to 58 were colourless.

Separation of flavonoids fractions by PTLC

Out of four flavonoids fractions two fractions (AR-I) was isolated by the method

of preparative thin layer finding suitable solvent and their economy and isolation of the

maximum amount of compound at a faster rate. Further, the purity of these fractions was

also checked by the TLC using various solvent systems. The appearance of single

discrete spot of effective fraction. From the effective fraction the purified compound was

isolated through chromatographic method are subjected to further detailed spectroscopic

studies.


Table-2.8: Isolation of compound fractions through column chromatography.

Sl

no

Mobile phase Ratio of mobile

phase

Number of

fractions

Colour of the extract Nature of the

extract

Weight of the

extract

Antidermatophy

tic (T.rubrum)

activity

1 Pet ether: ethyl acetate 100:00 1 Light brown Waxy - -

2 Pet ether: ethyl acetate 90:10 2 Dark brown mass Waxy 0.12 05.00

3 Pet ether: ethyl acetate 80:20 2 Brown mass Waxy 0.18 -

4 Pet ether: ethyl acetate 70:30 2 Reddish brown powder Amorphous 0.20 06.00

5 Pet ether: ethyl acetate 60:40 1 Reddish brown powder Amorphous 0.10 -

6 Pet ether: ethyl acetate 50:50 2 Brick red powder Solid -- -

7 Pet ether: ethyl acetate 40:60 2 Dark brownish red powder Solid 0.36 -

8 Pet ether: ethyl acetate 30:70 1 Dark brown powder Solid 0.38 06.00

9 Pet ether: ethyl acetate 20:80 2 Dark brown powder Solid 0.34 04.00

10 Pet ether: ethyl acetate 10:90 2 Dark brown mass Solid 0.28 -

11 Pet ether: ethyl acetate 00:100 2 Dark brown mass Semi-Solid 0.38 05.00

12 Ethyl acetate: Methanol 100:00 1 Light brown Waxy 0.41 -

13 Ethyl acetate: Methanol 90:10 2 Dark brown mass Waxy 0.18 05.00

14 Ethyl acetate: Methanol 80:20 1 Brown mass Solid 0.20 -

15 Ethyl acetate: Methanol 70:30 2 Reddish brown powder Semi-Solid 0.10 05.00

16 Ethyl acetate: Methanol 60:40 3 Reddish brown powder Solid -- 04.00

17 Ethyl acetate: Methanol 50:50 1 Brick red powder Semi-Solid 0.36 09.00

18 Ethyl acetate: Methanol 40:60 2 Dark brownish red powder Solid 0.38 12.00

19 Ethyl acetate: Methanol 30:70 2 Dark brown powder Solid 0.34 08.00


21 Ethyl acetate: Methanol 10:90 3 Dark brown mass Solid 0.36 04.00



TLC and column chromatography were most widely used techniques for the

separation and purification of many bioactive molecules. Plant materials with highly

complex profiles of phytochemicals, isocratic separation cannot achieve satisfactory

separation. Multiple mobile phases with increasing polarity are, therefore, useful for

good separation. In the present study, activity-guided fractionation of the A. ret iculata

L. chloroform and methanol extracts using silica gel column chromatography resulted in

the successful isolation and identification of the one effective antidermatophytic

compound. The antidermatophytic activity observed in isolated fractions / compo und

was more pronounced than the crude methanol extract of A. ret iculata L. This could be

due to presence of many undesirable compounds in the crude extract that got re moved

during fractionation. TLC was used for analyzing the isolated fractions and to select the

mobile phase for column chromatography. In TLC fingerprint, ethyl acetate: methanol

(4:6) was found to be the most efficient mobile phase for methanol extract o f A.

ret iculata L. leaves.

Column chromatography, using petroleum ether-ethyl acetate-methanol as

mobile phase yielded sixteen major column fractions. Bioassay results demonstrated the

promising activity of the column fraction 17, 18 and 19 against T. rubrum in agar well

diffusion assay (Table-2.8). In order to isolate the active compound from 18th active

fraction, repeated column chromatography was performed using petroleum ether-

dichloromethane-ethyl acetate-methanol as mobile phase. The thin layer chromatogram

of column fraction-18 showed one spot when eluted with different mobile phases i.e.,

hexane: ethyl acetate (3:1) (hRf value 53.4). The hRf values provide corroborative

evidence for the identity of a compound. The UV, HNMR, CNMR, FTIR and HPLC

analysis of the active fraction showed presence of one major compound polyphenols 1-

methyl-H-cyclopenta[b]naphthalene-4, 8-diol.


Fig-2.4.: UV-S pectrum of AR-1

532.

6254

3.86

569.

85

668.

57

1072

.01

1393

.07

1606

.38

2049

.83

2359

.49

2929

.08

3295

.75

68

70

72

74

76

78

80

82

84

86

88

90

92

94

96

98

%T

500 1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

Fig-2.5: Infra Red S pectrum (IR) of AR-1

Fig-2.6: NMR (NMR) of AR-1




Fig-2.9:13

C NMR (13

C -NMR) of AR-1


Fig-2.10:13

C NMR (13

C -NMR) of AR-1

Fig-2.11:13

C NMR (13

C -NMR) of AR-1

Fig: 2.12: LCMS (LCMS ) of AR-1


Detection and Scraping: diluted Sulphuric acid, Iodine chamber

Physico-Chemical properties and characterisation of isolated compound-AR-1

Nature: waxy, Colour: Dark brownish red powder, hRf values:53.4, Melting point: 189,

Solubility: Water, DMSO, DMF, 1-methyl-H-cyclopenta[b]naphthalene-4,8-diol.

UV: 511 nm.

IR: 3295 (-OH), 2929.08 cm-1 (Ar-CH Stretching).

1H NMR: δ 1.50 (s, 3H, CH3), 3.63 (d, 1H, Cyclpentyl-CH), 5.0 (s, 2H, 2xOH), 6.39-

7.73 (m, 6H, Ar-H).

13C NMR: δ16.0 (CH3), 45.7 (Cyclopentyl-CH), 110.9, 113.8, 115.2, 120.1, 125.6,

126.5, 131.3, 132.5, 141.1 (Ar-C), 152.3 (OH), 158.2 (OH).

Mass spectrum: Molecular ions M+ peaks at m/z 212 δ corresponds to the molecular

formula C12 H12 O2 (100%) or m/z 212.08 (100%), 213.09 (15.4%), 214 (1.5%).

Elemental analysis: Found: C, 79.22, H, 5.70, O, 15.08. Calculated: C, 79.24, H, 10.71,

O, 15.9.

C14H12O2Exact Mass: 212.08Mol. Wt.: 212.24

m/e: 212.08 (100.0%), 213.09 (15.4%), 214.09 (1.5%)C, 79.22; H, 5.70; O, 15.08

Physico-chemical properties of isolated compound polyphenols.

The compound was dark-brownish colour and the melting point was found to be

210-220°C. This is in accordance with the reported value of 200ºC for AR-1. It showed

hRf values of 70 in TLC in the solvent system Ethyl acetate: methanol (4:6). The spot

turned yellow on spraying with 1:1 H2So4 on heating at 110ºC for 5 min. The compound


was soluble in water, dilute acids and alkalies. Based on the physico-chemical properties

AR-1 is identified as 1-methyl-H-cyclopenta[b]naphthalene-4, 8-diol.

STANDARD 3.

434

4.628

Sl.No. Time Area Height Width Area% Symmetry

1 3.434 18585.7 1559.4 0.175 96.074 0.472

2 4.628 759.6 35.3 0.3151 3.926 0.594

AR-1

min0 2 4 6 8 10 12 14

mAU

0

1

2

3

4

5

DAD1 E, Sig=260,16 Ref =750,100 (180513_1\1FF-0701.D)

Area: 2

22.427

3.25

2

Area: 4

4.8828

5.71

2


1 3.252 222.4 5.4 0.6892 83.209 0

2 5.712 44.9 7.10E-01 1.0471 16.791 1.899

Fig: 2.13: HPLC Profile of AR-1

Table-2.9: Antidermatophytic activity & Minimum Inhibitory concentration of isolated compound

AR-1.

6

Test strain Inhibition zone in mm & in different conc. of compound

01mg-1 0.5mg-1 0.25mg-1 0.12mg-1 Control Standard(K)

01mg-1

AR-1 T.rubrum 14.00±0.00

10. 33±1.52

09. 00±0.00

06. 66±0.57

_ 17. 00±0.00

M. gypseum 15. 66±1.15

13. 66±0.57

08. 33±1.52

07. 33±1.52

_ 20. 66±1.15

MIC of the isolated components

The polyphenolic isolated compound 1-methyl-H-cyclopenta[b]naphthalene-4, 8-diol

was found to be potentially active against T. rubrum and M. gypseum. The MIC

values of the isolated component ranged in between 0.12 and 0.2 mg ml-1 whereas MIC

values of standard antifungal agents ketoconazole was 0.3 mg ml-1 against T. rubrum

and M. gypseum.


Separation of flavonoid by Column chromatography & Separation of Polyphenol from A. reticulata L. by the PTLC

(Preparative thin layer chromatography).

Antidermatophytic - Minimum Inhibitory concentration of isolated compound AR-1(Agar well diffusion method)

Antidermatophytic - Minimum Inhibitory concentration of isolated compound AR-1(Broth dilution method)

A: T. rubrum, B: M. gypseum, C: C. albicans

1=01, 2=0.5, 3=0.25, 4=0.12mg-1 conc. compound AR, C=Control, S= 01mg

-1 Ketoconazole.

Plate 2.2: Phytochemical and pharmacological profile of Annona reticulata L.

A B


2.4.2.2. Annona squamosa L. experimental results

Various primary and secondary metabolites having therapeutical importance

were estimated, isolated and further some of these were purified from Annona squamosa

L. leaves using biochemicals and other hyphenated analytical chromatographic and

spectrophotometric methods. The results obtained are discussed in the light of literature

available hitherto.




of A. Squamosa L. The values given in tables-2.10 and 2.11 are the mean of the three

observations.

The 98% methanol leaf extract showed maximum of 10.33±1.52 mm inhibition

in Trichophyton rubrum at 40mg/ml followed by 09. 66±1.15mm Microsporum

gypseum, Candida albicans Aspergillus flavus and Trichophyton tonsurans shows least

inhibition 08. 33±1.52, 08.66±1.15 mm. The minimum inhibitory conc. of test fungi was

determined and the values are given in fig-2.14. The MIC of M. gypseum recorded as

1.25mg/ml conc., whereas T. rubrum, C. albicans A. flavus and T. tonsurans are 2.5

mg/ml conc.

The 98% methanol leaf extract at 40 mg/ml conc. showed maximum of 17.

66±0.57 mm inhibition against Staphylococcus aureus and Psudomonas aeruginosa

followed by Bacillus subtilis 15. 33±1.52 mm, Brevibacillus brevis 13. 66±1.15 mm,

Escherichia coli 12. 66±0.57 mm and the least inhibition zone shows by Serratia

marcescens with 09. 66±1.15mm. The minimum inhibitory concentration of test bacteria

was determined and the values are given in figure 2.14. The MIC of S. marcescens, B.

subtilis, S. aureus, P. aeruginosa were determined as 0.6 mg/ml conc. whereas E. coli,

B. brevis were recorded at 1.25 mg/ml conc. The negative control used, DMSO could not

show inhibition against all the tested fungal and bacterial strains. Ketoconazole used as

standard at conc.5mg/ml shows antifungal activity 18. 33±1.52 mm whereas

streptomycin used standard against bacteria shows inhibition zone in 25. 66±1.15 mm.


Table-2.10: Antidermatophytic activity of 98% methanolic leaf extract of Annona squamosa L. (Well diffusion technique).

Fungal strains


40

20

10

5

2.5

1.25

0.62

Control (DMSO)

Standard

(Ketoconazole )

T. rubrum

10. 33±1.52

08. 33±1.52

07. 66±0.57

07. 66±1.15

06. 66±1.15

-

-

-

14. 33±1.52

M .gypseum

09. 66±1.15

07. 66±0.57

06. 33±1.52

05. 00±0.00

05. 66±1.15

05. 66±1.15

-

-

18. 33±1.52

C .albicans

08. 66±0.57

07. 33±1.52

06. 66±0.57

06. 00±0.00

05. 33±1.52

-

-

-

22. 66±1.15

T.tonsurans

08.66±1.15

06. 33±1.52

06. 66±1.15

05. 66±0.57

05. 66±0.57

-

-

-

17. 66±0.57

A. flavus

08. 33±1.52

07. 00±0.00

06. 66±1.15

05. 00±0.00

04. 00±0.00

-

-

-

16. 66±1.15

T. rubrum: Trichophyton rubrum, M. gypseum: Microsporum gypseum, C .albicans: Candida albicans, T.tonsurans: Trichophyton tonsurans, A. flavus: Aspergillus flavus,

Negative control: DMSO N, N- Dimethyl Formamide, Standard: Ketoconazole (Positive control).


Table-2.11: Antibacterial activi ty of 98% methanolic leaf extract of Annona squamosa L. (Well diffusion technique).

Bacterial strains


40

20

10

05

2.5

1.25

0.62

Control

(DMSO)

Standard

(Streptomycin)

E. coli

12. 66±0.57

09. 33±1.52

08. 66±0.57

07. 00±0.00

06. 33±1.52

06. 66±0.57

-

-

22. 33±1.52

B. subtilis

15. 33±1.52

11. 66±1.15

08. 66±1.15

07. 66±1.15

06. 00±0.00

05. 33±1.52

05. 66±0.57

-

25. 66±1.15

S. marcescens

09. 66±1.15

07. 00±0.00

07. 33±1.52

06. 00±0.00

06. 66±0.57

05. 66±0.57

05. 33±1.52

-

21. 66±1.15

S. aureus

17. 66±0.57

16. 66±0.57

14. 00±0.00

11. 33±1.52

08. 00±0.00

06. 00±0.00

05. 66±0.57

-

23. 66±0.57

P. aeruginosa

17. 66±0.57

15. 00±0.00

13. 33±1.52

12. 33±1.52

10. 33±1.52

09. 00±0.00

07. 66±1.15

-

28. 33±1.52

B. brevis

13. 66±1.15

11. 00±0.00

10. 33±1.52

08. 66±0.57

07. 00±0.00

06. 66±1.15

_

-

24. 33±1.52

E. coli: Escherichia coli, B. subtilis: Bacillus subtilis, S. marcescens: Serratia marcescens, S. aureus: Staphylococcus aureus, P. aeruginosa: Psudomonas aeruginosa, B. brevis: Brevibacillus brevis, Negative control: DMSO N, N- Dimethyl Formamide, Standard: Streptomycin sulphate (Positive control).


A: Trichophyton rubrum, B: Microsporum gypseum, C: Trichophyton tonsurans, D: Candida albicans, E:Escherichia coli, F: Bacillus subtilis, G: Staphylococcus aureus, H: Psudomonas aeruginosa, 1=40 mg/ml, 2=20 mg/ml, 3=10

mg/ml,4=5 mg/ml, 5=2.5 mg/ml, 6=1.25 mg/ml, 7=0.62 mg/ml, C=Negative control: DMF N, N- Dimethyl

Formamide, 8=Standard: Ketoconazole (Positive control against fungi),Streptomycin sulphate (Positive control against

bacteria).

Plate 2.3: Antidermatophytic activi ty of 98% methanolic leaf extract of Annona squamosa L.


0

1

2

3MIC

MIC

Figure-2.14: Minimum Inhibitory Concentrations of 98% methanolic leaf extract of Annona squamosa L. against test strains.

A A B B

F E E

C C D D

F

G G H H



The crude successive extracts of leaf viz., petroleum ether, chloroform, ethyl-acetate

and 98% methanol extracts were qualitatively screened for the occurrence of various

secondary metabolites such as alkaloids, phenol, flavonoids, tannins, triterpenes,

steroids, saponins and glycosides.

Table-2.12: Preliminary screening of secondary metabolites in Annona squamosa L.

Secondary

metabolites

Name of the test

PE

CHCL3

Et OH

98%

Methano

l

Alkaloids

Mayers test _ + _ +

Dragendoff‘s test _ + + +

Wagner‘s test _ + _ +

Phenol


Ferric chloride test _ _ + +

Ellagic acid test _ _ + +

Flavonoids


Leadacetate test _ + + +

Shinoda test _ + + +

NaoH test _ + + +

Tannins Gelatine Test + + _ +

Triterpenoids

Salkowski‘s test + + + +

Libermann-Burchard

test

_ + + +

Steroids


Libermann-Burchard

test

_ + + +

Saponins Foam test _ _ _ +

Glycosides

Keller-Killiani test

_ _ _ _

Alkaloids

The chloroform and 98% methanol extracts were positive to the preliminary

alkaloids tests i.e., Mayer‘s, Dragendorff‘s and Wagner‘s reagents. These extracts have

produced a creamy white precipitate with Mayer‘s reagent, orange red precipitate with

Dragendorff‘s reagent and reddish brown precipitate with Wagner‘s reagent. Whereas,

the Ethyl acetate extract responded positive to Dragendorff‘s test. The petroleum ether

extract not responded to all above tests.


Phenols

The Ethyl acetate and 98% methanolic leaf extracts shown positive response to

all the test viz., ferric chloride test, ellagic acid test and hot water test pointing out of the

presence of phenols. In hot water test, the leaf showed prominent brownish black

demarcation at the junction of dipped and undipped portion. The petroleum and

chloroform extracts were not responded to all the above tests.

Flavonoids

The Ethyl acetate and 98% methanolic leaf extracts responded positively to

flavonoids test like ferric chloride, lead acetate, Shinoda and NaOH test indicating the

presence of flavonoids. While chloroform extract shows positive response to above tests

accept ferric chloride test. Whereas pet. ether extract did not responded to all the four

tests.

Tannins

The pet.ether, chloroform and 98% methanolic leaf extracts showed the positive

result for gelatin test. This indicates the presence of tannin. While the ethyl acetate

extract responded negatively to gelatin test.

Triterpenes

The chloroform, ethyl acetate and 98% methanolic extracts responded positively

to Salkowski‘s, Libermann-Burchard imparting the presence of triterpenes. Whereas in

petroleum ether extract showed positive for salkowski‘s test.

Steroids

The chloroform, Ethyl acetate and 98% methanolic extracts responded positively

to Salkowski‘s, Libermann-Burchard imparting the presence of steroids. Whereas in

petroleum ether extract showed positive for salkowski‘s test.

Saponins




Glycosides

The petroleum ether, chloroform, ethyl acetate and 98% methanol extracts shown

negative response to Kellar – Kiliani test pointing out the absence of glycosides.



material of Annona squamosa L. leaves estimated quantitatively using various methods.

(Figure.2.15).

The maximum content estimated was total flavonoids (4.72 mg/100mg) followed

by total phenol (3.94 mg/100mg), total tannins (2.86 mg/100mg), total alkaloid (1.90

mg/100mg), total saponins (0.34 mg/100mg).

0

1

2

3

4

5

Alkaloids Flavonoids Tannin Phenols


A. squamosa

Fig. 2.15 Quantitative estimations of secondary metabolites in Annona squamosa L. leaf mg/100mg.






Table-2.13: Qualitative separation of secondary metabolites from Annona squamosa L.

Secondary

metabolites

No of

bands

hRf values

Colour of the

bands

Phenols 7 14.81 Smoke green

20.37 Smoke green

24.07 Bus green

37.03 Yellow

55.55 Pastal green

72.22 Light s moke

green

85.15 Thick smoke

green

Flavonoids 4 38.46 Smoke green

44.23 Light s moke

green

57.69 Thick smoke

green

90.38 Yellow green

Alkaloids 2 16.00 Bus green

24.05 Yellow


The 98% methanol extract of the leaf exhibited 7 distinct bands having smoke

green, smoke green, bus green, yellow, pastal green, light smoke green, thick smoke

green with hRf values 14.81, 20.37, 24.07, 37.03, 55.55, and 72.22 respectively (Table-

2.13).


The leaf chromatogram developed indicated 4 distinct bands possessing smoke

green, light smoke green, thick smoke green and yellow green with hRf values 38.46,

44.23, 57.69 and 90.38 respectively.

E. Separation of Alkaloids

The chromatogram of leaf displayed having 2 distinct bands possessing

bus green with hRf value 16 and yellow colour with hRf value 24.05.

Separation of flavonoid fractions from A. squamosa L. by the Column



flavonoids that Annona squamosa L. leaf is a rich source of flavonoids of

pharmacological importance further, this was supported by the literature available on


Annona sps. Thus an attempt is made here to isolate some of these flavonoids fractions

from the leaf of A. squamosa by column chromatography (CC) and purified with the help

of preparative thin layer chromatography (PTLC).


Ten gm crude 98% methanolic extract of A. Squamosa L. leaf was fractioned on

a Silica gel-H (60-120Mesh) column at a room temperature and pressure (26ºC. 1bar).



The fractions 1 to 11 were obtained from the pet ether: methanol. Fractions 1 to

26 were collected from the pet ether : methanol, (1) 100:00, 90:10 (2), 80:20 (3), 70:30

(4), 60:40 (5), 50:50 (6), 40:60 (7), 30:70 (8), 20:80 (9), 10:90 (10) and 00:100 (11).

However, the collected 26 fractions were pooled into seventeen major fractions

owing to their similarly in colour. The concentrated solutions of these fractions had

waxy nature fractions of 1 to 3 (waxy), 4 & 5 (waxy), 6 to 10 (solid) and 11 (semi-solid)

dark brown (table-2.14).



Sl.

no


phase

Number of

fractions

Colour of the

extract

Nature of the

extract

Weight of

the extract

Antidermatophyt

ic (T.rubrum)

activity

1 Pet ether 100 1 Light brown Waxy -- -

2 Pet ether: Methanol 95:5 2 Dark brown mass Waxy 0.13 12.00

3 Pet ether: Methanol 90:10 2 Brown mass Waxy 0.18 08.00

4 Pet ether: Methanol 85:15 3 Reddish brown

powder

Amorphous 0.60 -

5 Pet ether: Methanol 80:20 3 Reddish brown

powder

Amorphous 0.75 05.00

6 Pet ether: Methanol 75:25 2 Brick red powder Solid 0.15 06.00

7 Pet ether: Methanol 70:30 3 Dark brownish red

powder

Solid 0.18 -

8 Pet ether: Methanol 65:35 2 Dark brown

powder

Solid 0.75 -

9 Pet ether: Methanol 60:40 3 Dark brown

powder

Solid 0.17 04.00

10 Pet ether: Methanol 55:45 2 Dark brown mass Solid 0.19 11.00

11 Pet ether: Methanol 50:50 3 Dark brown mass

(120mg)

Semi-Solid 0.12 05.00


Separation of flavonoid fractions by PTLC

Among the four flavonoids fractions, two fractions (AS-I) were isolated by the

method of preparative thin layer finding suitable solvent and isolation of the maximum

amount of compound at a faster rate. Further, the purity of these fractions was also

checked by the TLC using various solvent systems was in the appearance of single


isolated using chromatographic method and subjected to further detailed spectroscopic

studies.


AS-1.

Comp

ound

code

Test strain Inhibition zone in mm & in dif ferent conc. of compound

01mg-1 0.5mg-1 0.25mg-1 0.12mg-1 Contr

ol

Standard(K)

01mg-1

AS-1 T.rubrum 16. 00±0.00

13. 33±1.52

09. 33±1.52

07. 66±1.15

_ 20. 66±1.15

M. gypseum 10. 66±1.15

09. 00±0.00

07. 00±0.00

05. 33±1.52

_ 21. 00±0.00

The flavonoid isolated compound Rutin was found to be potentially active against T.

rubrum and M. gypseum. The MIC values of the isolated components ranged in

between 0.2 mg ml-1 whereas MIC values of standard antifungal agents ketoconazole

was 0.1 mg ml-1 against T. rubrum and M. gypseum (Plate-2.6).

UV

Polyphenolic compounds reveal two characteristic UV absorption bands with

maxima in the 240 to 285 and 300 to 550 nm range. The UV spectrum of compound AS-

1 in methanol indicated the presence of chromophoric group with an extended

conjugation. (Fig-2.16.)

Infra Red Spectrum (IR)

The IR spectrum (Fig 5.4) of AS-1 indicated the presence of phenolic OH groups

and alcoholic OH by absorbing between 3391 cm-1 to 3414 cm-1. It is because of

alcoholic OH present at position-3 of the AS-1 ( tentative structure 3414 cm-1).The


presence of cyclic C=O is notices at 1651 cm-1 at its characteristic range. The number of

presence of C=C is indicated by strong absorbing peak at 1608 cm-1 and presence of

glycoside is indicated by strong absorbing peak at 1074 cm-1.

LCMS

The Mass Spectrum of AS-1, sample isolated by LC-MS indicted the molecular

ion peak at m/z 327. This corresponds to molecular weight of AS-1 isolated from the A.

Squamosa L. leaves ethanol extract.

1H NMR spectrum

The 1H NMR (Fig.2.19) recorded in DMSO exhibited strong H of CH3 peak at

3.85 δ to 3.19 δ which is an overlapped peak. Another set of OH peak are absorbed at

6.83 δ and 6.81 δ, for 5 aromatic protons which are seen as multiple from 7.01 δ to 7.37

δ.

Fig-2.16: UV- Spectrum of AS-1

573.

9659

3.40

628.

88

655.

4366

8.29

707.

23

807.

6482

6.25

879.

4691

1.59

943.

9096

8.52

1000

.34

1012

.87

1041

.36

1059

.29

1092

.46

1122

.46

1166

.96

1202

.53

1234

.70

1294

.56

1313

.7413

60.4

0

1455

.29

1503

.02

1556

.30

1573

.93

1596

.38

1651

.76

2341

.84

2359

.51

2938

.68

3340

.13

3726

.85

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

%T

500 1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)


Fig-2.17: Infra Red Spectrum (IR) of AS-1

Fig-2.18: HNMR Spectrum (HNMR) of AR-1

Fig-2.19: HNMR Spectrum (HNMR) of AS-1


Fig-2.20: LCMS Spectrum (LCMS) of AS-1

STANDARD

3.43

4

4.62

8


1 3.434 18585.7 1559.4 0.175 96.074 0.472

2 4.628 759.6 35.3 0.3151 3.926 0.594

AS-1

min0 2 4 6 8 10 12 14

mAU

0

200

400

600

800

1000

1200

1400

1600

DAD1 E, Sig=260,16 Ref =750,100 (180513_1\1FI-1001.D)

3.47

4

4.38

6


1 3.474 58062.4 1812 0.4458 95.386 0.939

2 4.386 2808.5 84.4 0.4861 4.614 0.25

Fig-2.21: HPLC Profile of AS-1

The structure assign to AS-1.

Physico-chemical properties of isolated compound AS-1

The compound was brownish colour and the melting point was found to be 190-

220°C. This is in accordance with the reported value of 200ºC for Rutine. It showed hRf

values of 61.01 in TLC in the solvent system n-hexane: chloroform (3:1). The spot

OHO

OH

O

OH

OH

O

OO

HO OHOH

O

OH

OH OH


turned yellow on spraying with 1:1 H2So4 on heating at 110ºC for 5 min. The compound

was soluble in water, dilute acids and alkalies. Based on the physico-chemical properties

AS-1 is identified as rutin.

A. UV

Polyphenolic compounds reveal two characteristic UV absorption bands with

maxima in the 420 to 450 nm range. The UV spectrum of compound AS-1 in methanol

indicated the presence of chromophore group with an extended conjugation. (Fig.2.16)

B. The IR spectrum recorded in (FT-IR (Jasco-5300) (KBR) V max/cm)

The IR spectrum of SR indicated the broad peak at 3414 cm-1 due to the –OH

stretching. The peak at 2928 cm-1 is due to asymmetric stretching of methyl group of the

sugar moiety. The peak at 2852 cm-1 might due to symmetric stretching of the methyl

group of the sugar moiety. The peak at 1738 cm-1 is due to keto group of the flavonoid.

C. LCMS

The Mass Spectrum of AS-1, sample isolated by LC-MS indicted the molecular

ion peak at m/z 611. This corresponds to molecular weight of AS-1 isolated from the A.

Squamosa L. leaves 98% methanolic extract.

1H NMR spectrum

The 1H NMR recorded in the CD3OD in 500 MHz. The H of the –OH group of

the phenol group of flavones, when shifted down field as 12.33 δ ppm as single peak.

The multiple peak when is find in between 6.22 δ to 7.68 δ ppm is due to aromatic

protons. The broad peak as 4.89 δ ppm is due to the –OH groups of the sugar moiety,

which resonate at up field. The peak representing 3.30 δ to 3.83 δ ppm is due to the –

CH2 group of the sugar moiety. The single peak at 1.13 δ is due to –CH3 group of the

sugar moiety

13C NMR spectrum.

The 13C NMR recorded in the CD3OD in 500 MHz. The peak at 178 is due to the

carbonyl carbon of the flavones. The peak at 164.56, 161.47, 148.34, and 144.40 is due

to carbon atom attached with –OH groups in the flavones of phenols. The peak at


157.94, 157.08, 134.22, 129.21, 122.22, 121.25, 116.37, 114.25, 104.26, 103.28, and

101.01 is the aromatic carbon. The peak at 72.55, 74.33, 75.77, 76.76, 70.88, 70.68. is

due to the carbon atoms attached with –OH group in the glycosidic ring. The peak at

98.62, 93.58, 70.00, 68.34, 67.18, and 29.35 is due to the –CH2 carbons of the glycoside

ring. The peak at 16.9 is due to methyl group associated to the glycosidic ring of the

Rutin.

Detection and Scraping: butanol: acetic acid: water in ratio of 7:2:1 solvent systems.

Physico-Chemical properties of isolated compound Rutin

Nature: Brownish amorphous, Colour: Dark brown mass, hRf values: 18.00, Melting

point: 220-2250C, Solubility: DMSO, Methanol.


Separation of flavonoid by column chromatography & Separation of flavonoid from A. squamosa by the PTLC



Antidermatophytic - Minimum Inhibitory concentration of isolated compound AS-1(Broth dilution method)


1=01, 2=0.5, 3=0.25, 4=0.12mg-1 conc. compound AS, C=Control, S= 01mg

-1 Ketoconazole.

Plate 2.4: Phytochemical and pharmacological profile of Annona squamosa L.

A B


2.4.2.3. Corchorus olitorius L. experimental results


were estimated; isolated and further some of these were purified from Corchorus

olitorius L. seeds using biochemicals and other hyphenated analytical chromatographic

and spectrophotometric methods. Further the results obtained were discussed in the light

of literature available hitherto.



tested to determine the antifungal and antibacterial activity of 98% methanol seed extract

of Corchorus olitorius. The values given in tables-2.16 and 2.17 are the mean of the

three observations.

The 98% methanol leaf extract showed maximum of 15.00±0.00 mm inhibition

in Candida albicans at 40mg/ml followed by Microsporum gypseum (12. 33±1.52 mm),

Trichophyton tonsurans (12.00mm). Trichophyton rubrum (11. 33±1.52 mm) and

Aspergillus flavus (10. 00±0.00mm) and The minimum inhibitory concentration of test

fungi were determined and the values are given in fig.2.22. The MIC of 0.62 mg/ml was

recorded against M. gypseum followed by 1.25mg/ml conc. for T. rubrum, C. albicans

and T.tonsurans. Whereas A. flavus MIC was determined at 2.5 mg/ml conc.

The 98% methanol leaf extract at 40 mg/ml conc. showed maximum of

19.66±1.15 mm inhibition against Serratia marcescens followed by Psudomonas

aeruginosa 17.66±1.15 mm, Bacillus subtilis 16.66±0.57 mm, Escherichia coli

16.00±0.00 mm, Staphylococcus aureus 15.66±1.15 mm and the least inhibition zone

shows by Brevibacillus brevis with 13. 66±0.57mm. The minimum inhibitory

concentration of test bacteria was determined and the values are given in fig.2.22. The

MIC of E. coli, B. subtilis, S. aureus, P. aeruginosa were determined as 0.62 mg/ml

conc. Whe reas 1.25 mg/ml conc. was detected as MIC for B. brevis and S.

marcescens. The negative control used DMSO could not show inhibition against all the

tested fungal and bacterial strains. Ketoconazole used as standard at conc.5mg/ml shows

antifungal activity 18.66±0.57mm whereas streptomycin used standard against bacteria

shows inhibition zone in 24.66±1.15mm.


Table-2.16: Antidermatophytic activities of 98% methanolic seed extract of Corchorus olitorius (Well di ffusion technique).

Fungal

strains


40

20

10

5

2.5

1.25

0.62

Control (DMSO)

Standard

(Ketoconazole )

T. rubrum

11. 33±1.52

10. 00±0.00

09. 33±1.52

07. 33±1.52

06. 33±1.52

05. 00±0.00

_

-

26. 66±0.57

M .gypseum

12. 33±1.52

10. 00±0.00

09. 66±0.57

08. 66±1.15

06. 33±1.52

05. 00±0.00

_

-

23. 66±1.15

C .albicans

15. 00±0.00

11. 33±1.52

09. 33±1.52

08. 00±0.00

07. 00±0.00

06. 66±0.57

_

-

23. 33±1.52

T.tonsurans

13. 00±0.00

10. 66±0.57

09. 66±1.15

08. 66±1.15

06. 33±1.52

05. 66±1.15

-

-

18. 00±0.00

A. flavus

10. 00±0.00

09. 66±1.15

07. 66±0.57

06. 66±1.15

05. 66±1.15

-

-

-

22. 00±0.00

T. rubrum: Trichophyton rubrum, M. gypseum: Microsporum gypseum, C .albicans: Candida albicans, T.tonsurans: Trichophyton tonsurans,

A. flavus: Aspergillus flavus, Negative control: DMSO N, N- Dimethyl Formamide, Standard: Ketoconazole (Positive control).


Table 2.17: Antibacterial activi ty of 98% methanolic seed extract of Corchorus olitorius (Well diffusion technique).

Bacterial strains


40

20

10

05

2.5

1.25

0.62

Control

(DMSO)

Standard

(Streptomycin)

E. coli

16. 00±0.00

13. 66±0.57

11. 66±1.15

10. 00±0.00

09. 66±0.57

07. 66±0.57

06. 66±0.57

-

24. 66±0.57

B. subtilis

16. 66±0.57

15. 66±0.57

14. 66±1.15

13. 66±1.15

12. 66±1.15

11. 66±0.57

10. 66±0.57

-

26. 66±0.57

S.

marcescens

19. 66±1.15

18. 66±0.57

16. 00±0.00

14. 66±0.57

12. 66±0.57

09. 66±0.57

_

-

30. 66±1.15

S. aureus

15. 66±1.15

13. 00±0.00

11. 00±0.00

10. 66±1.15

09. 00±0.00

08. 00±0.00

05. 00±0.00

-

28. 00±0.00

P. aeruginosa

17. 66±1.15

15. 66±0.57

13. 66±0.57

12. 00±0.00

10. 66±0.57

09. 66±0.57

07. 00±0.00

-

28. 00±0.00

B. brevis

13. 66±0.57

11. 66±0.57

10. 00±0.00

08. 66±0.57

07. 00±0.00

06. 00±0.00

_

-

24. 66±0.57

E. coli: Escherichia coli, B. subtilis: Bacillus subtilis, S. marcescens: Serratia marcescens, S. aureus: Staphylococcus aureus, P. aeruginosa: Psudomonas aeruginosa,

B. brevis: Brevibacillus brevis, Negative control: DMSO N, N- Dimethyl Formamide, Standard: Streptomycin sulphate (Positive control).


A: Trichophyton rubrum, B: Microsporum gypseum, C: Trichophyton tonsurans, D: Aspergillus flavus E: Candida albicans, F: Escherichia coli, G: Bacillus subtilis, H: Staphylococcus aureus, I: Psudomonas aeruginosa, 1=40 mg/ml, 2=20 mg/ml, 3=10

mg/ml,4=5 mg/ml, 5=2.5 mg/ml, 6=1.25 mg/ml, 7=0.62 mg/ml, C=Negative control: DMF N, N- Dimethyl Formamide, 8=Standard: Ketoconazole (Positive control against fungi),Streptomycin sulphate (Positive control against bacteria).

Plate 2.5: Antidermatophytic activity of 98% methanolic seed extract of Corchorus olitorius (Well

di ffusion technique).

A A B B

C C D D

E E F F

I

H H G G

I


0

0.5

1

1.5

2

2.5

3

MIC

Figure-2.22: Minimum inhibitory concentrations of 98% methanolic seed extract of Corchorus

olitorius L. against test strains.


The crude successive extract of leaf viz., petroleum ether, chloroform, ethyl-

acetate and 98% methanol extracts were qualitatively screened for the occurrence of


steroids, saponins and glycosides.

Table-2.18: Preliminary screening of secondary metabolites in Corchorus olitorius L. seed.

Secondary metabolite

s

Name of the test

PE

CHCL3

EtOH

98%

Methanol

Alkaloids

Mayers test _ _ _ + Dragendroff’s test + _ + +

Wagner’s test + _ _ _

Phenol Hot water test _ _ _ _

Ferric chloride test _ _ + + Ellagic acid test _ _ _ +

Flavonoids


Lead acetate test _ + + + Shinoda test _ _ + +

NaOH test _ + + + Tannins Gelatin test _ _ + +

Triterpen

oids

Salkowski’s test _ _ + +

Libermann-Burchard test

_ _ + +

Steroids

Salkowski’s test _ _ + +

Libermann-Burchard

test

_ _ + +


Glycosides

Keller-Killiani test _ _ + +

Conc. H2So4 test _ _ _ _ Molisch’s test _ _ _ _ Glycoside test _ _ _ -


Alkaloids

The 98% methanol extract was positive to the preliminary alkaloids tests i.e.,

Mayers, Dragendroff‘s reagents. These extracts have produced a creamy white

precipitate with Mayers reagent, orange red precipitate with Dragendroff‘s reagent.

Whereas, the petroleum ether extract responded positively to Dragendroff‘s and

Wagner‘s reagents. The ethyl acetate extract responded positive to Dragendroff‘s

reagent. Whereas the chloroform extract was not responded to all the three tests.

Phenols

The 98% methanolic leaf extract shown positive response to the entire test viz.,

ferric chloride test, ellagic acid test. The hot water test was not shown the junction of

dipped and undipped portion. Ethyl acetate extract shows positive response to ferric

chloride test. The chloroform and petroleum extracts are not shown to all the three tests.

Flavonoids

The ethyl acetate and 98% methanolic leaf extracts responded positively to

flavonoids test like ferric chloride, lead acetate, shinoda and NaOH test indicating the

presence of flavonoids. While the chloroform extract shows positive response to lead

acetate and NaOH test. Whereas as the petroleum-ether extract did not respond to all the

four tests..

Tannins

The chloroform, ethyl acetate and 98% methanolic leaf extracts showed the

positive result for gelatin test. This indicates the presence of tannin. Whereas the

petroleum-ether extract responded negatively to gelatin test.

Triterpenes

The ethyl-acetate and 98% methanol extracts responded positively to

Salkowski‘s, Libermann-Burchard imparting the presence of triterpenes. Whereas in

petroleum-ether and chloroform extracts showed negative results to both the test.


Steroids

The ethyl-acetate and 98% methanol extracts responded positively to

Salkowski‘s, Libermann-Burchard imparting the presence of steroids. Whereas in

petroleum-ether and chloroform extracts showed negative results to both the tests.

Saponins



Glycosides

The ethyl acetate and 98% methanol extract shown positive response to Kellar –

Kiliani test pointing out the presence of glycosides. While all the tests are not responded

to four extracts.



material of Corchorus olitorius seeds estimated quantitatively using various methods.

(Figure. 2.23).

The maximum content estimated was total phenol (5.43 mg/100mg) followed by

total tannins (3.88 mg/100mg), flavonoids (3.28 mg/100mg), total alkaloid (0.5


0246


mg/100mg

Corchorus olitorius

F

ig. 2.23 Quantitative estimations of secondary metabolites in seeds of Corchorus olitorius L. in mg/100mg.






Table-2.19: Qualitative separation of secondary metabolites from Corchorus olitorius

Secondary

metabolites

No of

bands

hRf values

Colour of the bands

Phenols 4 17.39 Brown

23.91 Light brown

54.35 Yellow

76.08 Smoke brown

Flavonoids 2 61.2 Brownish

73.46 Greenish brown

Alkaloids 2 14.21 Light Brownish

21.57 Slight Yellowish


The 98% methanol extract of the seed exhibited 4 distinct bands having brown,

light brown, Yellow, smoke brown with hRf values 17.39, 23.91, 54.35 and 76.08

respectively (Table-2.19).


The developed seed chromatogram indicated 2 distinct bands possessing

brownish and green brown with hRf values 61.2 and 73.46 respectively.

C. Separation of Alkaloids

The chromatogram of seed displayed having 2 distinct bands possessing light

brownish with hRf value 14.21 and slight yellowish with hRf value21.57 (Table-2.19).

Separation of glycosides fractions from Corchorus olitorius seed by the Column



Glycosides that Corchorus olitorius L. seed is a rich source of flavonoids of

pharmacological importance Further, this was supported by the literature available on

Corchorus sps. Thus an attempt was made here to isolate some of these flavonoids


fractions from the seed of Corchorus olitorius L. by column chromatography (CC) and

purified with the help of preparative thin layer chromatography (PTLC).


The crude effective extract of C. Olitorius L. leaf about 10 g was fractioned on a




The fractions 1 to 11 were obtained from the Hexane: ethyl acetate. (1) 100:00,

90:10 (2), 80:20 (3), 70:30 (4), 60:40 (5), 50:50 (6), 40:60 (7), 30:70 (8), 20:80 (9),

10:90 (10) and 00:100 (11).

However, the collected 23 fractions were pooled into eleven major fractions owing

to their similarity in colour. The concentrated solutions of these fractions had waxy

nature fractions of 2 & 3, 4 & 5 (Amorphous), 06 to 10 (solid) brown and 11 ( semi-solid)

01 to 03 fractions shows yellow colour, fractions 4 shows white precipitate slight yellow

and fractions 10 shown light green colour (Table: 2.20).

Separation of glycosides fractions by PTLC

Out of four Glycosides fractions two fractions (CR-I) were collected by the

method of preparative thin layer finding suitable solvent and their economy and isolation

of the maximum amount of compound at a faster rate. Further, the purity of these

fractions was also checked by the TLC using various solvent systems where in the

appearance of single discrete spot of effective fraction. From the effective fraction the

purified compound was isolated through chromatographic method are subjected to

further detailed spectroscopic studies.



Sl

no

Mobile phase Ratio of

mobile phase

Number of

fractions

Colour of the

extract

Nature of the

extract

Weight of

the extract

Antidermato

phytic

(T.rubrum)

activity

1 n-Hexane 100:00 1 Light Yellow Creamy 0.15 09.00

2 n-Hexane : Ethyl acetate 90:10 3 Yellow Waxy 0.33 05.00

3 n-Hexane : Ethyl acetate 80:20 2 Light yellow Waxy 0.26 07.00

4 n-Hexane : Ethyl acetate 70:30 2 White precipitate

slight yellow


5 n-Hexane : Ethyl acetate 60:40 1 Transparent Amorphous 0.18 06.00

6 n-Hexane : Ethyl acetate 50:50 2 Light brown Solid 0.36 05.00

7 n-Hexane : Ethyl acetate 40:60 2 Yellowish Solid 0.11 -

8 n-Hexane : Ethyl acetate 30:70 2 Light brownish Solid 0.39 06.00

9 n-Hexane : Ethyl acetate 20:80 4 greenish Solid 1.20 -

10 n-Hexane : Ethyl acetate 10:90 3 Light green Solid 0.83 10.00

11 n-Hexane : Ethyl acetate 00:100 1 Yellow Semi-Solid 0.66 04.00


Table-2.21: Antidermatophytic activity & minimum inhibitory concentration of isolated compound

COR-1.

Compo

und

code

Test strain Inhibition zone in mm & in dif ferent conc. of compound

01mg-1 0.5mg

-1 0.25mg

-1 0.12mg

-1 Control Standard(

K)

01mg-1

CR-1 T.rubrum 16. 00±0.00

13. 66±1.15

10. 66±1.15

08. 66±0.57

_ 18. 00±0.00

M. gypseum 14. 66±1.15

11. 00±0.00

07. 33±1.52

05. 00±1.00

_ 21. 66±1.15

The polyphenolic isolated compound Hexadecahydro-17-(2,5-dihyro-5-

oxofuran-3-yl)-3,5,14-trihydroxy-13-methyl-iH- cyclopenta[a]phenanthrene-10-

carbaldehyde was found to be potentially active against T. rubrum and M. gypseum.

The MIC values of the isolated components ranged in between 0.12 mg ml-1 whereas

MIC values of standard antifungal agents ketoconazole was 0.3 mg ml-1 against T.

rubrum and M. gypseum (Plate-2.6).

Fig-2.24: UV-S pectrum of CR-1 53

6.16

718.

14812.

9285

3.04

982.

44

1115

.34

1171

.32

1283

.52

1471

.5715

24.6

716

04.2

316

32.0

7

1711

.79

2850

.08

2916

.68

3373

.92

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

%T

500 1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

Fig-2.25: Infra Red S pectrum (IR) of CR-1


Fig-2.26: H

NMR S pectrum (H

NMR) of CR-1

Fig-2.27: C

NMR Spectrum (C

NMR) of CR-1

Fig-2.28: C

NMR Spectrum (C

NMR) of CR-1


Fig-2.29: LCMS S pectrum (LCMS) of CR-1 (PEAK 404 m/z)

STANDARD

3.43

4

4.62

8


1 3.434 18585.7 1559.4 0.175 96.074 0.472

2 4.628 759.6 35.3 0.3151 3.926 0.594

CR-1

min0 2 4 6 8 10 12 14

mAU

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

DAD1 E, Sig=260,16 Ref =750,100 (180513_1\1FE-0501.D)

Area: 8.53604

3.154

Area: 7.60021

3.242

Area: 58.1403

3.521

Area: 15.9133

5.183

Area: 10.0968

5.933

Sl. No. Time Area Height Width Area% Symmetry

1 3.154 8.5 1.2 0.1149 8.512 2.922

2 3.242 7.6 1.2 0.1016 7.578 1.349

3 3.521 58.1 1.7 0.5677 57.974 0.476

4 5.183 15.9 3.90E-01 0.676 15.868 0

5 5.933 10.1 2.20E-01 0.7756 10.068 0.506

Fig-2.30: HPLC Profile of CR-1


Physico-Chemical properties & Characterization of isolated compound CR-1

Nature: crystalline, Colour: Dark brownish, hRf values: 61.25, Solvent system: n-

hexane: ethyl acetate (7:3), Melting point: 190-200, Solubility: soluble in water, dilute

acids and alkalies. Active fraction: n-Hexane: Ethyl acetate, 70:30, White precipitate

slight yellow, Amorphous Hexadecahydro-17-(2,5-dihyro-5-oxofuran-3-yl)-3,5,14-

trihydroxy-13-methyl-iH- cyclopenta[a]phenanthrene-10-carbaldehyde.

UV: 380 nm

IR: 3373 Broad peak (-OH), 2916 (Aliphatic-CH Streching), 1711(Oxo-furanyl carboxyl

funeion), 1632 (CHO).

1H NMR: δ 1.4 (s, 3H, CH3), 1.49 (q, 1H, CH), 1.52 (q, 4H, 2xCH2), 1.56 (t, 2H, CH2),

1.63 (q, 1H, CH), 1.64 (q, 2H, CH2), 1.68 (t, 2H, CH2), 1.72 (q, 2H, CH2), 1.75 (t, 2H,

CH2), 1.83 (d, 2H, CH2), 2.08 (t, 2H, CH2), 3.17 (q, 1H, CH), 2.17 (d, 1H, Pentyl-CH),

3.58 (s, 1H, OH), 3.65 (s, 2H, 2XOH), 4.9 (s, 2H, CH2), 5.93 (S, 1H1, CH), 9.52 (s, 1H,

CHO).

13C NMR: δ 208.2 (CHO), 174.0 (C=O), 1171 (Oxa-pyranyl-CH), 66.7, 76.0, 86.0 (3X-

C-OH), 7.3.6 (Oxa-pyranyl-CH2), 16.7 (CH3), 20.7 to 49.7 (-CH2).

Mass: Molecular formula: C23 H32 O6 , Molecular weight: 404.50, m/z 404.22 (100%)

Elemental analysis: C, 68.29, H, 7.97, O, 23.73. Calculated: C, 68.31, 7.92, 23.76.

“Hexadecahydro-17-(2,5-dihyro-5-oxofuran-3-yl)-3,5,14-trihydroxy-13-methyl-iH-

cyclopenta[a]phenanthrene-10-carbaldehyde‖


Separation of flavonoid by Column chromatography & Separation of flavonoid from Corchorus olitorius L. by the PTLC



Antidermatophytic - Minimum Inhibitory concentration of isolated compound CR-1 (Broth dilution method

A: T. rubrum, B: M. gypseum, C: C. albicans, 1=01, 2=0.5, 3=0.25, 4=0.12mg-1 conc. compound CR,

C=Control, S= 01mg-1

Ketoconazole.

Plate 2.6: Phytochemical and pharmacological profile of Corchorus olitorius L.

A B C


2.4.2.4. Euphorbia tirucalli L. experimental results


were estimated; isolated and further some of these were purified from Euphorbia

tirucalli L. cladode using biochemicals and other hyphenated analytical chromatographic

and spectrophotometric methods. Further the results obtained are discussed in the light of





of E. Tirucalli L. The values given in tables- 2.22 and 2.23 are the mean of the three

observations.

The 98% methanol E. tirucalli L. cladode extract showed maximum of 14.

00±1.00 mm inhibition against Trichophyton rubrum, at 40mg/ml followed by

Trichophyton tonsurans (13. 33±1.52 mm), Aspergillus flavus (10. 66±1.15 mm)

Microsporum gypseum (09.66±1.15 mm) and Candida albicans (08. 66±1.15 mm). The

minimum inhibitory concentration of test fungi was determined and the values are given

in figure.2.31. The MIC of 0.62 mg/ml was recorded against T. rubrum followed by

1.25 mg/ml conc. for, M. gypseum and T.tonsurans. Whereas C. albicans, A. flavus MIC

was determined at 2.5mg/ml conc. The 98% methanol E. Tirucalli L. cladode extract at

40 mg/ml conc. showed maximum of 19. 00±1.00 mm inhibition against Escherichia

coli and Psudomonas aeruginosa followed by Bacillus subtilis 18.33±1.52 mm,

Staphylococcus aureus 17.66±1.15 mm and Brevibacillus brevis 16. 00±1.00. The least

inhibition zone shows by Serratia marcescens with 15.66±1.15 mm. The minimum

inhibitory concentrations of test bacteria were determined and the values are given in

figure.2.31. The MIC of S. marcescens, B. subtilis, S. aureus, B. brevis were determined

as 0.62 mg/ml conc. Followed by E. coli and P. aeruginosa were 1.25 mg/ml conc. The

negative control used, DMSO could not show inhibition against all the tested fungal and

bacterial strains. Ketoconazole used as standard at conc.5mg/ml shows antifungal

activity 24.33±1.52 mm whereas streptomycin used standard against bacteria shows

inhibition zone in 24.00±1.00 mm.


Table-2.22: Antidermatophytic activity of 98% methanolic cladode extract of Euphorbia tirucalli L (Well diffusion technique).

Fungal

strains


40

20

10

5

2.5

1.25

0.62

Control

(DMSO

)

Standard

(Ketoconazol

e )

T. rubrum

14. 00±1.00

12. 66±1.15

09. 33±1.52

08. 66±1.15

07. 66±1.15

06. 00±0.00

06. 00±1.00

-

20. 66±1.15

M .gypseum

09. 66±1.15

07. 33±1.52

06. 00±1.00

05. 66±1.15

05. 00±1.00

05. 33±1.52

---

---

18. 00±1.00

C .albicans

08. 66±1.15

07. 00±1.00

06. 66±1.15

06. 66±1.15

05. 33±1.52

---

---

---

22. 33±1.52

T.tonsurans

13. 33±1.52

10. 00±0.00

09. 00±1.00

08. 00±1.00

06. 33±1.52

05. 00±1.00

-

-

18. 66±1.15

A. flavus

10. 66±1.15

09. 33±1.52

07. 00±1.00

06. 00±0.00

05. 33±1.52

-

-

-

22. 33±1.52



Table-2.23: Antibacterial activity of 98% methanolic cladode extract of Euphorbia tirucalli L. (Well diffusion technique).


Bacterial

strains


40

20

10

05

2.5

1.25

0.62

Control

(DMSO

)

Standard

(Streptomycin

)

E. coli

19. 00±1.00

17. 66±1.15

13. 33±1.52

11. 00±0.00

07. 00±1.00

06. 33±1.52

-

_

21. 00±0.00

B. subtilis

18. 33±1.52

15. 00±1.00

12. 33±1.52

09. 00±1.00

07. 00±1.00

06. 00±1.00

06. 33±1.52

_

19. 33±1.52

S.

marcescens

15. 66±1.15

15. 66±1.15

14. 33±1.52

13. 00±0.00

12. 66±0.57

11. 66±0.57

08. 00±1.00

_

20. 00±1.00

S. aureus

17. 66±1.15

16. 00±0.00

14. 00±1.00

11. 33±1.52

08. 66±0.57

06. 33±1.52

05. 00±0.00

_

23. 66±1.15

P.

aeruginosa

19. 00±0.00

18. 00±1.00

16. 66±1.15

14. 66±1.15

12. 00±0.00

09. 66±0.57

_

-

30. 66±1.15

B. brevis

16. 00±1.00

15. 00±1.00

14. 66±1.15

13. 66±0.57

12. 33±1.52

11. 66±0.57

10. 66±0.57

-

26. 00±1.00




A: Trichophyton rubrum, B: Microsporum gypseum, C: Trichophyton tonsurans, D: Aspergillus flavus, E:Candida

albicans, F: Escherichia coli, G: Bacillus subtilis, H: Staphylococcus aureus, I: Psudomonas aeruginosa, 1=40 mg/ml, 2=20 mg/ml, 3=10 mg/ml,4=5 mg/ml, 5=2.5 mg/ml, 6=1.25 mg/ml, 7=0.62 mg/ml, C=Negative cont rol: DMF

N, N- Dimethyl Formamide, 8=Standard: Ketoconazole (Positive control against fungi),Streptomycin sulphate

(Positive control against bacteria).

Plate 2.7: Antidermatophytic activi ty of 98% methanolic leaf extract of Euphorbia tirucalli L.


A

C

G G

E E

C

F

D

F

D

B B A

I

H H

I


00.5

11.5

22.5

MIC

MIC

Figure -2.31: Minimum inhibitory concentrations of 98% methanolic cladode extract of

Euphorbia tirucalli L. against test strains.


The crude successive extract of E. Tirucalli L. cladode viz., petroleum ether,

chloroform, ethyl acetate and 98% methanol extracts were qualitatively screened for the

occurrence of various secondary metabolites such as alkaloids, phenol, Flavonoids,

tannins, triterpenes, steroids, saponins and glycosides. The reactions with these reagents

have shown the presence of metabolites and are recorded in the Table: 2.24.

Table-2.24: Preliminary screening of secondary metabolites in E tirucalli L.

Secondary metabolites

Name of the test

PE

CHCL3

EtOH

98% Methanol

Alkaloids

Mayer‘s test - - - -

Dragendroff‘s test - - - + Wagner‘s test - - + +

Phenol


Ferric chloride test - + + + Elegiac acid test - + + +

Flavonoids

Ferric chloride test - + + +

Lead acetate test - + + + Shinoda test - - + - NaOH test - - + +

Tannins Gelatin test - + + +

Triterpenoids


Libermann-Burchard

test _ + + +

Tschugajiu test

Steroids



_ + + +


Glycosides

Keller-Killiani test _ _ _ +

Conc. H2So4 test - - + + Molisch‘s test - - - - Glycoside test - - - -


Alkaloids

The 98% methanolic extract shows positive results to the preliminary alkaloids

tests i.e., Dragendorff‘s and Wagner‘s reagents. This extract has produced a orange red

precipitate with Dragendroff‘s reagent and reddish brown precipitate with Wagner‘s

reagent. Whereas, the ethyl acetate extract responded to Wagner‘s test. The petroleum

ether and chloroform extracts not responded to all the three tests.

Phenols

The 98% methanolic leaf extract shown positive response to all the phenol tests

viz., ferric chloride test, ellagic acid test and hot water test pointing out of the presence of

phenols. In hot water test, the leaf showed prominent brownish black demarcation at the

junction of dipped and undipped portion. Ethyl acetate and chloroform extracts shows

positive response to ferric chloride test and ellagic acid test. The petroleum ether extract

was not responded to all the phenol tests.

Flavonoids


flavonoids test like ferric chloride, lead acetate, Shinoda (accept 98% methanolic

extract) and NaOH test indicating the presence of flavonoids. While the chloroform

extract positively responded to lead acetate test. Whereas pet.ether did not responded to

all the flavonoid tests..

Tannins

The chloroform, ethyl acetate and 98% methanolic leaf extract showed the

positive result to gelatin test. This indicates the presence of tannin in chloroform, ethyl

acetate and 98% methanolic extract. The pet.ether extract responded negatively to

gelatin test.

Triterpenes

The chloroform, ethyl acetate and 98% methanolic leaf extracts responded

positively to Salkowski‘s, Libermann-Burchard imparting the presence of triterpenes.

Whereas the petroleum ether extract showed positive respond for Salkowski‘s test.


Steroids

The chloroform, ethyl acetate and 98% methanolic leaf extracts responded

positively to Salkowski‘s, Libermann-Burchard imparting the presence of steroids.

Whereas the petroleum ether extract showed positive respond for Salkowski‘s test.

Saponins

The 98% methanolic leaf extract responded positively to foam saponins tests

indicating the presence of saponins.

Glycosides

The 98% methanol extract shown positive response to Kellar – Kiliani and conc.

H2So4 tests pointing out the presence of glycosides. Whereas the ethyl acetate extracts

shown positive response to conc. H2SO4 test. Whereas the chloroform and petroleum

ether extracts have not shown positive results to glycosides tests.


Five important secondary metabolites extracted from the dried powdered material of

Euphorbia tirucalli L. cladode were estimated quantitatively using various methods.

(Figure.2.32).


flavonoids (3.89 mg/100mg), total tannins (2.67 mg/100mg), total alkaloid (0.7 mg/100mg), total

saponins (0.3 mg/100mg).

0

2

4

6

Alkaloids Flavonoids Tannins Phenols Saponins


Fig. 2.32 Quantitative estimations of secondary metabolites in cladode of E. tirucalli L. in mg/100mg.






Table-2.25: Qualitative separation of secondary metabolites from E. tirucalli L.

Secondary

metabolites

No of

bands

hRf values

Colour of the bands

Phenols 4 14.28 Brownish

21.42 Light yellow

30.35 Light brown

46.42 Yellow


49.15 Brownish

61.01 Yellow

94.91 Light brownish

Alkaloids 3 24.02 Light yellow

38.16 Brownish

51.00 Yellow


The 98% methanol extract of the leaf exhibited 4 distinct bands having

brownish, Light yellow, light brown, yellow with hRf values 14.28, 21.42, 30.35 and

46.42 respectively (Table-2.25).

A. Separation of Flavonoids

The developed leaf chromatogram indicated 4 distinct bands possessing

brownish, brownish, yellow and light brown with hRf values 35.59, 49.15, 61.01 and

94.91 respectively.

C. Separation of Alkaloids

The chromatogram of leaf displayed having 3 distinct bands possessing

light yellow with hRf value 24.02, brownish colour with hRf value 38.16 and 51.00 hRf

value band colour shown yellow (Table-2.25).

Separation of Triterpenes fractions from E. Tirucalli L. by the Column chromatography and PTLC



triterpenes in E. Tirucalli L. This plant is a rich source of triterpenes. Thus an attempt is


made here to isolate some of these triterpene fractions from the cladode of E. Tirucalli L.

by column chromatography (CC) and purified with the help of preparative thin layer

chromatography (PTLC).


The crude effective extract of E. tirucalli L. cladode about 10 g was fractioned

on a Silica gel-H (60-120Mesh) column at a room temperature and pressure (26ºC. 1bar).



The fractions 1 collected from n-Hexane (1) 100:00, 2 to 04 were obtained from

the pet ether: chloroform 100:00 (2), 50:50 (3), 00:100 (4), Fractions 05 to 09 were

collected from the chloroform : methanol 100:00 (5), 70:30 (6), 50:50 (7), 30:70 (8),

100:00 (9) and the tenth fraction was collected from the solvent mixture of Methanol:

Aqueous (95:05). mobile phase ( Table. 2.26).

However, the collected 15 fractions were pooled into ten major fractions owing to

their similarly in colour. The concentrated solutions of these fractions had waxy nature

fractions of 2 to 4 green, light green, light yellow, 05 to 07 (amorphous) yellow, light

brown, dark brown. 8 to 10 (solid) fractions were shown light brown, light yellow, dark

brown colours.

Separation of Triterpenes fractions by PTLC

Out of four triterpenes fractions one fraction (ET -I) was isolated by the method



also checked by the TLC using various solvent systems were in the appearance of single


isolated through chromatographic methods are subjected to further detailed spectroscopic

studies.



l

no


phase

Number of

fractions

Colour of the

extract

Nature of the

extract

Weight of the

extract

Antidermatoph

ytic (T.rubrum)

activity

1 n-Hexane 100 1 Transparent Transparent _ -

2 Pet ether: chloroform 100:00 2 Green Waxy 0.11 -

3 Pet ether: chloroform 50:50 1 Light green Waxy 0.15 07.00

4 Pet ether: chloroform 00:100 1 Light yellow Waxy 0.27 -

5 Chloroform : Methanol 100:00 2 Yellow Amorphous 0.19 07.00

6 Chloroform: Methanol 70:30 2 Light brown Amorphous 0.96 13.00

7 Chloroform: Methanol 50:50 1 Dark brownish Amorphous 1.42 05.00

8 Chloroform: Methanol 30:70 2 Light brownish Solid 0.85 -

9 Chloroform: Methanol 00:100 2 Light yellow Solid 0.89 08.00

10 Methanol: Aqueous 95:05 1 Dark brownish Solid 0.37 05.00


Physico-Chemical properties and characterization of isolated compound ET-1

Nature: waxy, Colour: dark green, hRf values: 49.15, solvent system: chloroform:

methanol (7:3), Melting point: 220-230°C, Solubility: DMSO, Methanol. Active

fraction: Chloroform: Methanol, 70:30, light brown, amorphous.

Infra Red Spectrum (IR)

The Infra Red Spetrum of AR-I showed peaks at 3298 (Amide-NH), 1662 ( C=O

), 1618 ( COOR ), 1118 cm-1 ( C-O-C ).

LCMS PEAK 251 m/z

H Nuclear magnetic resonance spectrum (H - NMR)

δ3.89 (s.34.och3) 4.19 (t, 2H, CH2), 4.30 (t. 2H, CH2), 4.40 (t, 2H, CH2), 4.34 (t,

2H, CH2), 6.12-7.18 cm, 5H, Ar-H).

“2-methoxyethyl 3-benzamideopropanoate”


Fig-2.33: UV-S pectrum of E calli-1

535.

1553

9.17

543.

04

550.

78

554.

86

566.

04

570.

31

593.

08

1040

.27

1204

.93

1355

.5716

04.1

41714

.92

2939

.92

3320

.04

35

40

45

50

55

60

65

70

75

80

85

90

95

100

%T

500 1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

Fig-2.34: Infra Red S pectrum (IR) of E calli-1

Fig-2.35: H

Nuclear magnetic resonance spectrum (H – NMR) of E.calli-1


Fig-2.36: C


Fig-2.37: C



Fig-2.38: LCMS (LCMS) of E.calli-1

STANDARD

3.43

4

4.62

8


1 3.434 18585.7 1559.4 0.175 96.074 0.472

2 4.628 759.6 35.3 0.3151 3.926 0.594

ET-1

min0 2 4 6 8 10 12 14

mAU

0

10

20

30

40

50

60

70

80

DAD1 E, Sig=260,16 Ref =750,100 (180513_1\1FB-0201.D)

3.23

4


1 3.234 3923.5 89 0.6004 100 0.42

Fig-2.39: HPLC Profile of E.calli-1


Table-2.27: Antidermatophytic activity & minimum inhibitory concentration of isolated compound

ET-1.

Compound

code

Test strain

Inhibition zone in dif ferent conc. of compound (in mm)

01mg-1 0.5mg-1 0.25mg-1 0.12mg-1 C

on

trol

Standard(K)

01mg-1

ET-1 T.rubrum 16. 66±0.57

14. 00±0.00

09. 33±1.52

06. 66±1.15

_ 21. 00±0.00

M.

gypseum

11. 33±1.52

07. 00±1.00

06. 66±0.57

04. 33±1.52

_ 20. 66±0.57

The polyphenolic isolated compound 2-methoxyethyl 3-benzamideopropanoate

was found to be potentially active against T. rubrum and M. gypseum. The MIC

values of the isolated components ranged in between 0.12 mg ml-1 whereas MIC values

of standard antifungal agents Ketoconazole was 0.1 mg ml-1 against T. rubrum and M.

gypseum (Plate-2.8).


Separation of pure compounds by column chromatography & Separation of flavonoid from Euphorbia tirucalli

L. by the PTLC (Preparative thin layer chromatography).

Antidermatophytic - Minimum Inhibitory concentration of isolated compound ET-1(Agar well diffusion method)

Antidermatophytic - Minimum Inhibitory concentration of isolated compound Ecalli -1 (Broth dilution method).


1=01, 2=0.5, 3=0.25, 4=0.12mg-1 conc. compound ET, C=Control, S= 01mg

-1 Ketoconazole.

Plate 2.8: Phytochemical and pharmacological profile of Euphorbia tirucalli L. cladode.

A B


2.4.2.5. Ficus racemosa L. experimental results


were estimated, isolated and further some of these were purified from Ficus racemosa L.

leaves using biochemicals and other hyphenated analytical chromatographic and

spectrophotometric methods. The results obtained were discussed in the light of





of Ficus racemosa L. The values given in tables-2.28 and 2.29 are the mean of three

observations.

The 98% methanolic Ficus racemosa L. leaf extract showed maximum of 15.

66±1.15mm inhibition against Trichophyton rubrum, at 40mg/ml followed by Candida

albicans 14. 66±0.57mm, Microsporum gypseum 12. 66±1.15mm, Aspergillus flavus 11.

66±0.57mm and Trichophyton tonsurans 10.33±1.52mm. The minimum inhibitory

concentrations of test fungi were determined and the values are given in fig.2.40. The

MIC of 1.25 mg/ml was recorded against T. rubrum, M. gypseum, C. albicans followed

by 2.5mg/ml conc. for T.tonsurans, A. flavus.

The 98% methanolic Ficus racemosa L. leaf extract at 40 mg/ml conc. showed

maximum of 20.33±1.52mm inhibition against Serratia marcescens followed by

Escherichia coli 19. 66±1.15mm, Bacillus subtilis and Brevibacillus brevis 17. 66±0.57

mm. whereas Staphylococcus aureus inhibited with 16.33±1.52mm zone. The least

inhibition zone showed by Psudomonas aeruginosa with 14.66±0.57 mm. The minimum

inhibitory concentrations of test bacteria were determined and the values are given in

fig.2.40. The MIC of E. coli, S. marcescens, B. subtilis, B. brevis were determined as

0.62 mg/ml conc. followed by and P. aeruginosa, S. aureus, were 1.25 mg/ml conc. The

negative control used DMSO could not show inhibition against all the tested fungal and

bacterial strains. Ketoconazole used as standard at conc.5mg/ml shows antifungal

activity 24.33±1.52 mm whereas streptomycin used standard against bacteria shows

inhibition zone in 24.00±00±0.00mm.


Table-2.28: Antidermatophytic activity of 98% methanolic leaf extract of Ficus racemosa L. (Well di ffusion technique).

Fungal strains


40

20

10

5

2.5

1.25

0.62

C

Standard (Ketoconazo

le )

T. rubrum

15. 66±1.15

13. 33±1.52

10. 66±1.15

08. 33±1.52

06. 66±0.57

05. 33±1.52

-

-

23. 66±0.57

M .gypseum

12. 66±1.15

10. 66±0.57

08. 00±0.00

07. 66±1.15

06. 66±0.57

05. 00±1.00

_

-

21. 00±0.00

C .albicans

14. 66±0.57

11. 66±1.15

09. 00±1.00

07. 33±1.52

06. 00±0.00

05. 66±0.57

_

-

23. 66±1.15

T.tonsurans

10. 33±1.52

09. 00±1.00

07. 66±0.57

06. 00±1.00

05. 33±1.52

_

-

-

18. 33±1.52

A. flavus

11. 66±0.57

09. 66±1.15

08. 00±1.00

06. 00±0.00

05. 66±0.57

-

-

-

22. 33±1.52

T. rubrum: Trichophyton rubrum, M. gypseum: Microsporum gypseum, C .albicans: Candida albicans, T.tonsurans: Trichophyton tonsurans, A. flavus: Aspergillus flavus, Negative control: DMSO N, N- Dimethyl Formamide, Standard: Ketoconazole (Positive control).


Table-2.29: Antibacterial activity of 98% methanolic leaf extract of Ficus racemosa L (Well di ffusion technique).

Bacterial

strains


40

20

10

05

2.5

1.25

0.62

C

Standard

(Streptomyci

n)

E. coli

19. 66±1.15

17. 33±1.52

14.66±0.57

13. 00±0.00

10. 33±1.52

08. 33±1.52

06. 66±0.57

-

24. 66±1.15

B. subtilis

17. 66±0.57

15. 66±1.15

11. 33±1.52

10. 33±1.52

08. 66±0.57

07. 66±0.57

06. 66±0.57

-

20. 33±1.52

S.

marcescens

20. 33±1.52

19. 66±0.57

18. 00±0.00

16. 00±0.00

13. 00±0.00

09. 66±1.15

07. 00±0.00

-

24. 66±1.15

S. aureus

16. 33±1.52

13. 66±0.57

12. 00±0.00

09. 66±1.15

07. 00±0.00

05. 66±0.57

_

-

18. 33±1.52

P.

aeruginosa

14. 66±0.57

11. 66±0.57

09. 00±0.00

07. 00±0.00

06. 66±0.57

05. 00±0.00

-

-

16. 33±1.52

B. brevis

17. 66±1.15

14. 66±0.57

12. 33±1.52

11. 00±0.00

09. 66±0.57

08. 00±0.00

06. 33±1.52

-

24. 33±1.52


B. brevis: Brevibacillus brevis, Negative control: DMF= N, N- Dimethyl Formamide, Standard: Streptomycin sulphate (Positive control).


A: Trichophyton rubrum, B: Microsporum gypseum, C: Trichophyton tonsurans, D: Aspergillus flavus, E: Candida

albicans, F: Escherichia coli, G: Bacillus subtilis, H: Staphylococcus aureus, I: Psudomonas aeruginosa, 1=40

mg/ml, 2=20 mg/ml, 3=10 mg/ml,4=5 mg/ml, 5=2.5 mg/ml, 6=1.25 mg/ml, 7=0.62 mg/ml, C=Negative control: DMF



Plate 2.9: Antidermatophytic activity of 98% methanolic leaf extract of Ficus racemosa L. (Well

di ffusion technique).

C

G

E

G

C

E F

H

F

D D

B B A A

I I

H


00.5

11.5

22.5

MIC

MIC

Fig-2.40: Minimum Inhibitory Concentrations of 98% methanolic leaf extract of Ficus racemosa L

against test strains.


The crude successive extract of F. Racemosa L. leaf viz., petroleum ether,

chloroform, ethyl-acetate and 98% methanol extracts were qualitatively screened for the

occurrence of various secondary metabolites such as alkaloids, phenol, Flavo noids,



Table-2.30: Preliminary screening of secondary metabolites in Ficus racemosa L.


Name of the test

PE

CHCL3

EtOH

98%

Methanol

Alkaloids

Mayers test - + + -

Dragendoff’s test + + + -

Wagner’s test + + + -

Phenol


Ferric chloride test + + + +

Ellagic acid test + - - -

Flavonoids

Ferric chloride test + + + +

Leadacetate test + - + -

Shinoda test + + + + NaoH test + - + +

Tannins Gelatin test - - - +

Triterpenoids

Salkowski’s test - - + - Libermann-Burchard

test

- - + -

Steroids

Salkowski’s test - + + - Libermann-Burchard

test - + + -


glycosides

Keller-Killiani test - + + +

Conc. H2So4 test - - - +

Molisch’s test - - + +


Alkaloids

The chloroform and ethyl acetate leaf extracts shows positive results to the

preliminary alkaloids tests i.e., Mayers, Dragendroff‘s and Wagner‘s reagents. This

extract has produced a creamy white precipitate with Mayers reagent, orange red

precipitate with Dragendroff‘s reagent and reddish brown precipitate with Wagner‘s

reagent. Whereas, the petroleum ether extract responded to Dragendroff‘s and Wagner‘s

test. The 98% methanolic leaf extract was not responded to all the three alkaloid tests.

Phenols

The petroleum ether, chloroform, ethyl acetate, 98% methanolic leaf extracts

shown positive response to the phenol test viz., ferric chloride test. The hot water test

pointing out of the presence of phenols. In hot water test, the leaf showed prominent

brownish black demarcation at the junction of dipped and undipped portion. The

petroleum ether extract was also positively responded to ellagic acid test.

Flavonoids

The petroleum ether and ethyl acetate leaf extracts responded positively to

flavonoids test like ferric chloride, lead acetate, shinoda and NaOH test, indicating the

presence of flavonoids. While the chloroform extract positively responded to ferric

chloride shinoda tests. Whereas the 98% methanolic extract positively responded to

ferric chloride, shinoda and NaOH test.

Tannins

The 98% methanolic leaf extract showed the positive result to gelatin test. This

indicates the presence of tannin in 98% methanolic extract.

Triterpenes

The ethyl acetate leaf extract responded positively to Salkowski‘s, Libermann-

Burchard imparting the presence of triterpenes. Whereas the petroleum ether,

chloroform, 98% methanolic extracts were not respond positive for Salkowski‘s,



Steroids

The ethyl acetate leaf extract responded positively to Salkowski‘s, Libermann-

Burchard imparting the presence of steroids. Whereas the petroleum ether, chloroform,

98% methanolic extracts were not respond positive for Salkowski‘s, Libermann-

Burchard test.

Saponins The 98% methanolic leaf extract responded positively to foam saponins tests


Glycosides

The 98% methanol extract shown positive response to Kellar – Kiliani, conc.

H2So4, and Molisch‘s tests pointing out the presence of glycosides. While the ethyl

acetate extract shown positive response to Kellar – Kiliani and Molisch‘s tests, the

chloroform extract shown positive response to Kellar – Kiliani test. Whereas the

petroleum ether extract have not shown positive results to all the glycosides tests.



leaf material of Ficus racemosa L. estimated quantitatively using various methods

(Figure.2.41). The maximum content estimated was total tannins (5.16 mg/100mg)

followed by flavonoids (4.37 mg/100mg), total alkaloid (3.90 mg/100mg), total phenol

(3.84 mg/100mg) total saponins (1.2 mg/100mg).

0246


Ficus racemosa L

Fig. 2.41 Quantitative estimations of secondary metabolites in leaves of Ficus racemosa L in

mg/100mg.






Table-2.31: Qualitative separation of secondary metabolites from Ficus racemosa L.

Secondary

metabolites

No of

bands

hRf values

Colour of the bands

Phenols 8 10 Light black

12 Light black

24 Yellow

40 Yellow green

50 Sky blue

64 Smoke gray

78 Light gray

92 Yellow

Flavonoids 5 27.27 Light green

30.90 Smoke green

48.07 Gray

63.63 Yellow

87.27 Orange-gray

Alkaloids 3 20.31 Light green

24.90 Smoke green

38.17 Gray


The 98% methanol extract of the leaf exhibited 8 distinct bands having light

black, light black, yellow, yellow green, sky blue, smoke gray, light gray, yellow with

hRf values 10, 12, 24, 40, 50, 64, 78 and 92 respectively (Table-2.31).


The developed leaf chromatogram indicated 5 distinct bands possessing light

green, smoke green, gray, yellow and orange-gray with hRf values 27.27, 30.90, 48.07,

63.63 and 87.27 respectively.


The chromatogram of leaf displayed having 3 distinct bands possessing light

green with hRf value 20.31, smoke green colour with hRf value 24.90 and 38.17 hRf

value band colour shown gray (Table-2.31).


Separation of flavonoid fractions from Ficus racemosa L. by the Column



flavonoids that F. racemosa L. is a rich source of flavonoids of pharmacological

importance. Further, this was supported by the literature available on Ficus sps. Thus an

attempt was made here to isolate some of these flavonoids fractions from the leaf of F.

racemosa by column chromatography (CC) and purified with the help of preparative thin

layer chromatography (PTLC).


The crude effective extract of F. racemosa leaf about 10 g was fractioned on a



100 ml each were collected. The fraction 1 collected from n-Hexane (1) 100:00, 2 to 05

were obtained from the pet ether: chloroform 100:00 (2), 50:50 (3), 30:70 (4), 00:100

(5), Fractions 06 to 10 were collected from the chloroform : methanol 100:00 (5), 70:30

(6), 50:50 (7), 30:70 (8), 100:00 and the tenth fraction was collected from the solvent

mixture of Methanol: Aqueous (90:10), mobile phase (Table 2.32). However, the

collected 21 fractions were pooled into eleven major fractions owing to their similarly in

colour. The concentrated solutions of these fractions had waxy nature fractions of 1 to 3,

light brown, green, light green, 04 & 05 (amorphous) Light reddish brown powder, light

brown powder. 6 and 11 (solid) fractions shows brick red powder, dark brown mass and

8 to 10 fractions shown (Waxy –Solid) brown powder, dark brown powder, dark brown

mass colours.


Out of four flavonoid fractions one fraction (FR -I) was collected by the method






studies.



Sl

no


phase

Number of

fractions

Colour of the

extract

Nature of the

extract

Weight of the

extract

Antidermatoph

ytic (T.rubrum)

activity

1 n- Hexane 100 1 Light brown Waxy 0.12 -

2 Pet ether: Chloroform 100:00 2 Green Waxy 0.18 04.00

3 Pet ether: Chloroform 50:50 4 Light green Waxy 0.20 05.00

4 Pet ether: Chloroform 30:70 2 Light reddish

brown powder


5 Pet ether: Chloroform 00:100 1 Light brown

powder

Amorphous -- -

6 Chloroform: Methanol 100:00 2 Brick red powder Solid 0.36 04,00

7 Chloroform: Methanol 70:30 2 Dark brownish

powder


8 Chloroform: Methanol 50:50 3 Brown powder Waxy -Solid 0.34 06.00

9 Chloroform: Methanol 30:70 2 Dark brown

powder

Waxy Solid 0.28 06.00

10 Chloroform Methanol 0:100 1 Dark brown mass Waxy Solid 0.38 -

11 Methanol: Aqueous 90:10 1 Dark brown mass Solid 0.41 05.00


Fig-2.42: UV-S pectrum of FR-1

532.

5753

5.91

539.

8154

3.59

555.

16

563.

0166

8.34

819.

05

1074

.7212

44.8

51367

.75

1446

.14

1518

.24

1614

.08

1714

.99

2049

.78

2342

.18

2359

.84

2853

.42

2924

.79

3354

.60

58

60

62

64

66

68

70

72

74

76

78

80

82

84

86

88

90

92

94

96

98

%T

500 1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

Fig-2.43: Infra Red S pectrum (IR) of FR-1

Fig-2.44: H

Nuclear magnetic resonance spectrum (H – NMR) of FR-1


Fig-2.45: H

Nuclear magnetic resonance spectrum (H – NMR) of FR-1

Fig-2.46: LCMS S pectrum (LCMS) of FR-1 (LCMS PEAK 412 m/z)

STANDARD

3.43

4

4.62

8


1 3.434 18585.7 1559.4 0.175 96.074 0.472

2 4.628 759.6 35.3 0.3151 3.926 0.594


FR-1

min0 2 4 6 8 10 12 14

mAU

0

20

40

60

80

100

120

DAD1 E, Sig=260,16 Ref =750,100 (180513_1\1EA-1101.D)

3.112


1 3.112 4444.3 128.9 0.4343 100 0.611

Fig-2.47: HPLC Profile of FR-1

Physico-Chemical properties and characterization of isolated compound FR-1

The compound was brownish colour and the melting po int was found to be 220-

230°C. This is in accordance with the reported value of 200ºC for. 2-(4-(3-methylbut-2-

enyloxy)-3, 5-dimethoxyphenyl)-5-hydroxy-4H-chromen-4-one, (C23H2407). It

showed hRf values of 63.63 in TLC in the solvent system chloroform: methanol (7:3).

The compound was soluble in water, dilute acids and alkalies. Based on the physico-

chemical properties FR -1 is identified as 2-(4-(3-methylbut-2-enyloxy)-3, 5-

dimethoxyphenyl)-5-hydroxy-4H-chromen-4-one, (C23H2407).

Nature: Amorphous, Colour: light yellowish, hRf values: 63.63, solvent system:

chloroform: methanol (7:3), melting point: 170-190, solubility: DMSO, methanol, water,

active fraction: Chloroform: Methanol, 70:30, dark brownish, amorphous.

UV: 379, 458

IR- 3350 (Broad OH), 1714 (C=0), 1074 (C-0-C), 2359 cm-1 (HC=C=)

1H NMR: δ 3.73(s, 9, 3x OCH3 ), 1.73 (s, 64,2xCH3), 5.0 (s, 1H, OH), 5.39 ( s, 1H, CH),

4.63 (d, 2H, CH2), 5.99-671(m, 5H, Ar-H).

FTIR: m/z (Mass): 412, Molecular formula: C23 H24 07 = 412

Elemental analysis: Found: C, 66.98, H, 5.87, O, 27.15, Calculated= 66.98, H,4, 587,

0.27.


2-(4-(3-methylbut-2-enyloxy)-3,5-dimethoxyphenyl)-5-hydroxy-4H-chromen-4-one, (C23H2407).


FR-1.

Compou

nd code

Test strain Inhibition zone in dif ferent conc. of compound (in mm)

01mg-1 0.5mg-1 0.25mg-1 0.12mg-1 Con

trol

Standard(K)

01mg-1

FR-1 T.rubrum 15.33±1.52

13. 33±1.52

10. 00±0.00

07. 66±1.15

_ 19. 33±1.52

M.

gypseum

11.66±1.15

08. 66±1.15

06. 33±1.52

04. 00±0.00

_ 21. 66±1.15

The flavonoid isolated compound 2-(4-(3-methylbut-2-enyloxy)-3,5-dimethoxyphenyl)-

5-hydroxy-4H-chromen-4-one, (C23H2407) was found to be potentially active against T.


between 0.12 and 0.2 mg ml-1 whereas MIC values of standard antifungal agents

ketoconazole was 0.3 mg ml-1 against T. rubrum and M. gypseum (Plate-2.10).


Separation of pure compounds by column chromatography & Separation of flavonoid from Ficus racemosa L.

by the PTLC(Preparative thin layer chromatography).

Antidermatophytic - Minimum Inhibitory concentration of isolated compound FR-1(Agar well diffusion method)

Antidermatophytic - Minimum Inhibitory concentration of isolated compound FR-1(Broth dilution method)



-1 Ketoconazole.

Plate 2.10: Phytochemical and pharmacological profile of Ficus racemosa L.

A B


2.4.2.6. Pongamia pinnata experimental results

Various primary and secondary metabolites having therapeutically importance

were estimated, isolated and further some of these were purified from seed using

biochemical and other hyphenated analytical chromatographic and spectrophotometric

methods. The results obtained are discussed in the light of literature available hitherto.



tested to determine the antifungal and antibacterial activity of pet-ether seed extract of P.

Pinnata L. The values given in tables-2.34 and 2.35 are the mean of the three

observations.

Pet-ether seed extract showed maximum of 16. 66±0.57 mm inhibition in

Candida albicans at 40mg/ml followed by Microsporum gypseum (14. 66±1.15 mm),

Trichophyton rubrum (13. 00±0.00 mm), Aspergillus flavus (11. 00±0.00 mm) and

Trichophyton tonsurans (09. 00±0.00 mm). The minimum inhibitory conc. of test fungi

were determined and the values are given in fig.2.48. The MIC of M. gypseum and C.

albicans are 0.62 mg/ml conc. followed by T. rubrum 1.25mg/ml conc., A. flavus 2.5

mg/ml conc. and T.tonsurans 5 mg/ml conc.

The Pet-ether seed extract at 40 mg/ml conc. showed maximum of 20.00±0.00

mm inhibition against Escherichia coli followed by Serratia marcescens 19.66±1.15

mm, Bacillus subtilis, Staphylococcus aureus, Brevibacillus brevis with 16. 66±0.57 mm

and the least activity at 40 mg/ml conc. Recorded against Psudomonas aeruginosa 14.

66±0.57mm. The minimum inhibitory concentration of test bacteria was determined and

the values are given in fig.2.48. The MIC of E. coli, S. marcescens, B. subtilis, S. aureus

were determined as 0.6 mg/ml conc. Followed by P. aeruginosa was 1.25 mg/ml conc.

The negative control used DMSO could not show inhibition against all the tested fungal

and bacterial strains. Ketoconazole used as standard at conc.5mg/ml shows antifungal

activity 24. 66±1.15 mm whereas streptomycin used standard against bacteria shows

inhibition zone in 24. 66±0.57mm.


Table-2.34: Antidermatophytic activi ty of pet-ether seed extract of Pongamia pinnata L. (Well diffusion technique).

Fungal strains


40

20

10

5

2.5

1.25

0.62

Contr

ol

(DMS

O)

Standard

(Ketoconaz

ole )

T. rubrum

13. 00±0.00

11. 66±0.57

10. 66±0.57

09.66±0.57

08. 66±0.57

06. 66±0.57

---

--

23. 00±0.00

M

.gypseum

14. 66±1.15

10. 66±0.57

09. 66±0.57

08.00±0.00

07. 66±0.57

06. 66±0.57

05. 66±0.57

---

18. 66±0.57

C .albicans

16. 66±0.57

13. 66±1.15

12. 66±0.57

09.66±0.57

08. 66±0.57

06. 00±1.00

05. 66±1.15

---

24. 66±0.57

T.tonsuran

s

09. 00±0.00

08. 66±0.57

06. 66±1.15

05.00±0.00

--

---

---

---

17. 66±1.15

A. flavus

11. 00±0.00

09. 00±0.00

07. 66±1.15

06.00±0.00

05. 00±1.00

---

---

---

16. 00±0.00




Table-2.35: Antibacterial activi ty of pet-ether seed extract of Pongamia pinnata L. (Well di ffusion technique).

Bacterial

strains


40

20

10

05

2.5

1.25

0.62

Contr

ol

(DMS

O)

Standard

(Streptomy

cin)

E. coli

20. 00±0.00

18. 66±1.15

17. 66±0.57

14. 66±0.57

11. 00±1.00

10. 66±0.57

07. 66±0.57

-

24. 66±1.15

B. subtilis

16. 66±0.57

15. 66±0.57

14. 66±1.15

13. 00±1.00

10. 66±0.57

09. 00±0.00

06. 00±0.00

-

20. 66±0.57

S.

marcescens

19. 66±1.15

18. 00±0.00

17. 00±1.00

16. 66±1.15

12. 00±1.00

10. 66±0.57

07. 33±1.52

-

24. 00±0.00

S. aureus

16. 66±0.57

12. 66±0.57

10. 00±1.00

08. 00±0.00

07. 33±1.52

06. 33±1.52

05. 00±0.00

-

19. 00±1.00

P.

aeruginosa

14. 66±0.57

12. 66±0.57

11. 00±0.00

09. 33±1.52

07. 33±1.52

05. 33±1.52

-

-

18. 66±0.57

B. brevis

16. 00±0.00

14. 66±1.15

12. 66±1.15

11. 00±0.00

09. 00±0.00

07. 66±1.15

06. 00±0.00

-

24. 66±1.15

E. coli: Escherichia coli, B. subtilis: Bacillus subtilis, S.marcescens: Serratia marcescens, S. aureus: Staphylococcus aureus, P. aeruginosa: Psudomonas aeruginosa,



A: Trichophyton rubrum, B: Microsporum gypseum, C: Trichophyton tonsurans, D:Aspergillus flavus, E:Candida

albicans, F: Escherichia coli, G: Bacillus subtilis, H: Staphylococcus aureus, I: Psudomonas aeruginosa, 1=40

mg/ml, 2=20 mg/ml, 3=10 mg/ml,4=5 mg/ml, 5=2.5 mg/ml, 6=1.25 mg/ml, 7=0.62 mg/ml, C=Negative control: DMF



Plate 2.11: Antidermatophytic activity of pet ether seed extract of Pongamia pinnata L. (Well diffusion

technique).

A A B B

G G

F F E E

C D D C

H H

I I


012345

MIC

MIC

Fig-2.48: Minimum Inhibitory Concentrations of pet-ether seed extract of Pongamia pinnata L.



The crude successive extract of seed viz., petroleum ether, chloroform, ethyl-

acetae and 98% methanol extracts were qualitatively screened for the occurrence of


steroids, saponins and glycosides. The reactions with these reagents have shown the

presence of metabolites and are recorded in the Table: 2.36.

Table-2.36: Preliminary screening of secondary metabolites in Pongamia pinnata L. seed.


Name of the test

PE

CHCL3

EtOH

98% Methanol

Alkaloids

Mayers test + - + - Dragendoff‘s test + + + +

Wagner‘s test + - + -

Phenol

Hot water test - - - - Ferric chloride test - + + +

Ellagic acid test - + + +

Flavonoids

Ferric chloride test - + + + Lead acetate test - - + +

Shinoda test - - + + Zinc/Hcl test - + + +

Tannins Gelatin test - - + -

Triterpenoids

Salkowski‘s test + + + + Libermann-Burchard

test - + + -

Tschugajiu test

Steroids

Salkowski‘s test + + + + Libermann-Burchard

test - + + -


Steroidal glycosides

Keller-Killiani test - + + + Conc. H2So4 test - - - +

Molisch‘s test - - - - Glycoside test - - - +


Alkaloids

The petroleum-ether and ethyalacetate extracts were positive to the preliminary

alkaloids tests i.e., Mayers, Dragendroff‘s and Wagner‘s reagents. These extracts have

produced a creamy white precipitate with Mayer‘s reagent, orange red precipitate with

Dragendroff‘s reagent and reddish brown precipitate with Wagner‘s reagent. Whereas,

the chloroform and methanol (accept Dragendroff‘s test) extracts responded negatively

to the all the tests.

Phenols

The chloroform, ethyl acetate and 98% methanolic seed extracts shown positive

response to all the test viz., ferric chloride test, ellagic acid test and hot water test

pointing out of the presence of phenols. In hot water test, the leaf showed prominent

brownish black demarcation at the junction of dipped and undipped portion. All the other

extracts responded negatively to aforesaid test.

Flavonoids

The ethyl acetate and 98% methanolic seed extracts responded positively to

flavonoids test like ferric chloride. Lead acetate, shinoda and NaOH test indicating the

presence of flavonoids. While other extracts of pet.ether, chloroform and extracts did

not respond to it.

Tannins

The ethyl acetate extract showed the positive result for gelatin test. This indicates

the presence of tannin in ethyl acetate extract. All the other extracts responded

negatively to gelatin test.

Triterpenes

The chloroform and ethyl acetate extracts responded positively to Salkowski‘s, Libermann-Burchard imparting the presence of triterpenes. Whereas in petroleum ether

and methanol extracts showed positive for Salkowski‘s test.


Steroids

The chloroform and ethyl acetate extracts responded positively to Salkowski‘s,

Libermann-Burchard imparting the presence of steroids. Whereas in petroleum ether and

methanol extracts showed positive for Salkowski‘s test.

Glycosides

The 98% methanol extracts shown positive response to Kellar – Kiliani,

Glycoside, and sulphuric acid tests pointing out the presence of glycosides. While the

chloroform and ethyl acetate extracts responded positively to Kellar – Kiliani test.

Saponins





seed material of Pongamia pinnata L. estimated quantitatively using various methods

(Figure.2.49).


flavonoids (2.58 mg/100mg), total alkaloid (1.9 mg/100mg), total tannins (1.68


01234


Pongamia pinnata

Fig. 2.49 Quantitative estimations of secondary metabolites in seeds of Pongamia pinnata in mg/100mg.






Table-2.37: Qualitative separation of secondary metabolites from Pongamia pinnata seed.

Secondary

metabolites

No of

bands

hRf values

Colour of the bands

Phenols 4 14.28 Brownish

21.42 Light yellow

30.35 Light brown

46.42 Yellow


49.15 Brownish

61.01 Yellow

94.91 Light brownish

Alkaloids 3 13.18 Light yellow

20.32 Light brown

28.30 Yellow


The pet-ether extract of the seed exhibited 4 distinct bands having brown

yellowish with hRf values 14.28, 21.42, 30.35 and 46.42 respectively (Table-2.37).


The seed chromatogram developed indicated 4 distinct bands possessing

brownish (I and II band) and yellow, light yellow (III-VI band) with hRf values 35.59,

49.15, 61.01 and 94.91 respectively.


The chromatogram of seed displayed having 3 distinct bands possessing light

yellow colour with hRf value 13.18, light brown colour with hRf value 20.32 and 28.30

hRf value band colour show yellow (Table-2.37).

Several workers have reported the presence of alkaloids in

Asclepiadaceae family. Bhutani et al., (1983 and 1985) reported five novel

phenanthroindolizidine alkaloids from Tylophora hirsuta. Similarly, Ali and Bhutani

(1989) have isolated seven rare and four known alkaloids from two varieties of

Tylophora indica. Shabana et al., (2005) reported a novel alkaloids from stepelia hirsute.


Separation of Furanoflavonol fractions from Pongamia pinnata L. seed by the

Column chromatography and PTLC (Preparative thin layer chromatography).


flavonol that Pongamia pinnata L. seed is a rich source of flavonols of pharmacological

importance Further, this was supported by the literature available on Pongamia sps. Thus

an attempt was made here to isolate some of these flavonol fractions from the seed of

Pongamia pinnata L. seed by column chromatography (CC) and purified with the help

of preparative thin layer chromatography (PTLC).


The crude effective extract of Pongamia pinnata L. seed about 10 g was

fractioned on a Silica gel-H (60-120Mesh) column at a room temperature and pressure

(26ºC. 1bar). After discarding 200 ml dead volume from the column (Hexane), total 21

fractions of 100 ml each were collected.

The fractions 1 to 11 were obtained from the Hexane: methanol. (1) 100:00,

90:10 (2), 80:20 (3), 70:30 (4), 60:40 (5), 50:50 (6), 40:60 (7), 30:70 (8), 20:80 (9),

10:90 (10) and 00:100 (11).


to their similarity in colour. The concentrated solutions of these fractions had waxy

nature fractions of 1 to 11 colourless, white, yellow.

Separation of flavonols fractions by PTLC

Out of four flavonols fractions two fractions (P-1) was collected by the method of

preparative thin layer finding suitable solvent and their economy and isolation of the





studies.



Sl

n

o


phase

Number of

fractions

Colour of the

extract

Nature of the

extract

Weight of the

extract

Antidermatoph

ytic (T.rubrum)

activity

1 n-Hexane 100 1 colourless Waxy 0.69 -

2 n-Hexane :Pet ether 90:10 2 colourless Waxy 0.10 -

3 n-Hexane :Pet ether 80:20 2 Light white Waxy 0.18 05.00

4 n-Hexane :Pet ether 70:30 1 White Semi- Waxy 0.50 10.00

5 n-Hexane :Pet ether 60:40 4 Light white Waxy 0.65 04.00

6 n-Hexane :Pet ether 50:50 2 Light yellow Waxy 0.15 -

7 n-Hexane :Pet ether 40:60 2 Light yellow Waxy 0.10 0

8 n-Hexane :Pet ether 30:70 3 colourless Waxy 0.75 0.5.00


10 n-Hexane :Pet ether 10:90 1 Light white Waxy 0.10 -



Fig-2.50: UV- S pectrum (UV) of P-1

52

8.1

65

43.5

05

91.2

66

36.0

26

88.4

67

00.2

37

32.2

27

55.9

77

78.7

3

79

4.7

98

38.1

78

89.8

0

95

8.1

8

10

22

.62

10

35

.88

10

52

.48

10

82

.18

11

32

.71

11

63

.79

12

27

.64

12

86

.16

13

40

.84

13

72

.06

14

07

.35

14

36

.96

14

48

.43

14

59

.14

14

93

.89

15

27

.74

15

69

.12

16

04

.43

16

23

.42

16

36

.26

19

79

.36

21

61

.53

29

30

.37

30

53

.43

31

34

.38

31

55

.11

38

50

.50

45

50

55

60

65

70

75

80

85

90

95

%T

500 1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

Fig-2.51: Infra Red S pectrum (IR) of P-1


Fig-2.52: H

Nuclear magnetic resonance s pectrum (H - NMR of P-1

Fig-2.53: H

Nuclear magnetic resonance s pectrum (H - NMR) of P-1


Fig-2.54: C

Nuclear magnetic resonance spectrum (C - NMR) of P-1

Fig-2.55: C

Nuclear magnetic resonance spectrum (C - NMR) of P-1


Fig-2.56: LCMS-s pectrum (LCMS) of P-1

STANDARD

3.434

4.628


1 3.434 18585.7 1559.4 0.175 96.074 0.472

2 4.628 759.6 35.3 0.3151 3.926 0.594

P1

min0 2 4 6 8 10 12 14

mAU

0

200

400

600

800

1000

1200

DAD1 E, Sig=260,16 Ref =750,100 (180513_1\1FH-0901.D)

3.479

4.386


1 3.479 41496.7 1284.7 0.4528 95.532 0.976

2 4.386 1940.9 58.9 0.4823 4.468 0.233

Fig-2.56: HPLC Profile of P-1

Physico-Chemical properties and charecterization of isolated P-1

Nature: crystalline, colour: yellowish, hRf values: 61.01, solvent system n-

hexane: chloroform (3:1), melting point: 200-210°C, Solubility: Hexane, methanol,


water. Active fraction: n-Hexane-chloroform, 70:30, semi waxy, colourless, white,

yellow.

The compound was colour less and the melting point was found to be 214-216°C.

This is in accordance with the reported value of 195ºC for Karanjin. It showed hRf

values of 61.01 in TLC in the solvent system n-hexane: chloroform (3:1). The spot

turned yellow on spraying with 1:1 H2SO4on heating at 110ºC for 5 min. The compound

was soluble in water, dilute acids and alkalies. Based on the physic-chemical properties

P-I is identified as furanoflavonol-Karnjin.

UV: Spectrum of P-1 peak shows at 425nm

FTIR: 3053 ( Aromatic –C-H Structure ), 16136 ( CO ), 1227 ( C-O-C ), 1163 ( C-O-C

), 1132 cm-1 ( C-O-C ).

LCMS: peak 293 m/z

Table-2.38/1: H

Nuclear magnetic resonance spectrum (H - NMR) of P-1

Table: 11-NMR SPECTRAL DATA FOR

Atom No H1 (J in Hz)

Isolated Reported

A 7.54d 7.57d

B 7.15d 7.18d

O-CH3 3.85s 3.93s

5 8.13d 8.20d

6 7.73d 7.54d

2’ 8.11m 8.15m

3’ 7.51m 7.57m

4’ 7.52m 7.58m

5’ 7.50m 7.56m

6’ 8.10m 8.13m

Note: 1H NMR, CDCL3 at 400MHz


Structure of Karanjin (Furanoflavonol)


P-1.

Compound

code

Test strain Inhibition zone in different conc. of compound (in mm)

01mg-1 0.5mg-1 0.25mg-1 0.12mg-1 Control Standard(K) 01mg-1

P-1 T.rubrum 16.

33±1.52

12.

66±0.57

08.

66±1.15

06.

00±0.00

_ 19. 33±1.52

M.gypseum 12.

66±1.15

09.

00±0.00

06.

66±0.57

05.

33±1.52

_ 18. 66±1.15

The Furanoflavonol isolated compound was found to be potentially active against

T. rubrum and M. gypseum. The MIC values of the isolated components ranged 0.2

mg ml-1 whereas MIC values of standard antifungal agents Ketoconazole was 0.3 mg ml-

1 against T. rubrum and M. gypseum (Plate-2.12).


Separation of pure compounds by column chromatography & Separation of flavonoid from Pongamia pinnata

L. by the PTLC(Preparative thin layer chromatography).

Antidermatophytic - Minimum Inhibitory concentration of isolated compound P-1(Agar well diffusion method)

Antidermatophytic - Minimum Inhibitory concentration of isolated compound P-1.


2=01, 3=0.5, 4=0.25 mg-1 conc. compound P, 5=Control, 1= 01mg

-1 Ketoconazole .

Plate 2.12: Phytochemical and pharmacological profile of Pongamia pinnata L.

A B C


2.4.3.7. Vitex negundo L. experimental results


were estimated, isolated and further some of these were purified from Vitex negundo L.

leaves using biochemicals and other hyphenated analytical chromatographic and

spectrophotometric methods. The results obtained were discussed in the light of





of V. negundo L. The values given in tables-2.40 and 2.41 are the mean of the three

observations.

The 98% methanolic V. negundo L. leaf extract showed maximum of 17.

66±1.15mm inhibition against Trichophyton rubrum, at 40mg/ml followed by

Microsporum gypseum 15.00±0.00mm, Aspergillus flavus 13.33±1.52 mm, Trichophyton

tonsurans 11.00±1.00 mm and Candida albicans 10. 66±0.57mm. The minimum

inhibitory concentrations of test fungi were determined and the values are given in

fig.2.47. The MIC of 0.62 mg/ml was recorded against T. rubrum followed by

1.25mg/ml conc. for M. gypseum, C. albicans and A. flavus and 2.5 mg/ml conc. for T.

tonsurans.

The 98% methanolic V. negundo leaf extract at 40 mg/ml conc. showed

maximum of 14. 66±1.15 mm inhibition against Serratia marcescens and Psudomonas

aeruginosa followed by Escherichia coli and Brevibacillus brevis 13. 33±1.52 mm.

Whereas 11.33±1.52 mm inhibition showed against Bacillus subtilis and Staphylococcus

aureus. The minimum inhibitory concentrations of test bacteria were determined and the

values are given in fig.2.47. The MIC of E. coli, S. marcescens, P. aeruginosa, B. brevis

was determined as 1.25 mg/ml conc. followed by S. aureus, B. subtilis, with 2.5 mg/ml

conc. each. The negative control used DMSO could not show inhibition against all the

tested fungal and bacterial strains. Ketoconazole used as standard at conc.5mg/ml shows

antifungal activity 24. 00±0.00 mm whereas streptomycin used standard against bacteria

shows inhibition zone in 24.66±1.15mm.


Table-2.40: Antidermatophytic activity of 98% methanolic leaf extract of Vitex negundo L. (Well diffusion technique).

Fungal strains


40

20

10

5

2.5

1.25

0.62

Control

(DMSO)

Standard (Ketoconaz

ole )

T. rubrum

17. 66±1.15

14. 33±1.52

12. 00±1.00

09. 66±1.15

07. 00±1.00

06. 33±1.52

05. 66±1.15

-

20. 66±1.15

M

.gypseum

15. 00±0.00

13. 66±0.57

09. 00±0.00

08. 33±1.52

06. 66±0.57

05.33±1.52

-

-

18. 33±1.52

C

.albicans

10. 66±0.57

09. 66±1.15

08. 33±1.52

07. 33±1.52

06. 66±1.15

05. 00±0.00

-

-

24. 66±1.15

T.tonsuran

s

11. 00±1.00

09. 00±1.00

07. 00±0.00

06. 66±1.15

05. 66±1.15

-

-

-

24. 00±0.00

A. flavus

13. 33±1.52

11. 66±1.15

10. 66±1.15

08. 66±1.15

06. 00±0.00

05.66±0.57

-

-

21. 66±1.15




Table-2.41: Antibacterial activity of 98% methanolic leaf extract of Vitex negundo L (Well di ffusion technique).

Bacterial strains


40

20

10

05

2.5

1.25

0.62

Contro

l

(DMSO)

Standard

(Streptomycin

)

E. coli

13. 66±1.15

11. 33±1.52

09.33±1.52

08.66±1.15

06. 66±1.15

05. 33±1.52

-

-

23. 66±1.15

B. subtilis

11. 66±1.15

09. 66±1.15

08.00±1.00

06.00±0.00

05. 00±1.00

-

-

-

24. 66±1.15

S.

marcescens

14. 66±1.15

12. 33±1.52

10.66±1.15

07.66±1.15

06. 66±1.15

05. 33±1.52

-

-

25. 66±1.15

S. aureus

11. 33±1.52

08. 00±0.00

07. 00±0.00

06.33±1.52

05. 00±0.00

-

-

-

18. 33±1.52

P. aeruginosa

14. 66±1.15

13. 00±0.00

09. 66±1.15

08.66±1.15

07. 00±0.00

05. 66±1.15

-

-

20. 66±1.15

B. brevis

13. 33±1.52

09. 33±1.52

08. 00±0.00

07.00±0.00

06. 66±1.15

05. 66±1.15

-

-

24. 00±0.00

E. coli: Escherichia coli, B. subtilis: Bacillus subtilis, S. marcescens: Serratia marcescens, S. aureus: Staphylococcus aureus, P. aeruginosa:

Psudomonas aeruginosa,B. brevis: Brevibacillus brevis, Negative control: DMSO N, N- Dimethyl Formamide, Standard: Streptomycin sulphate

(Positive control).


A: Trichophyton rubrum, B: Microsporum gypseum, C: Trichophyton tonsurans, D: Candida albicans, E: Escherichia

coli, F: Bacillus subtilis, G: Staphylococcus aureus, 1=40 mg/ml, 2=20 mg/ml, 3=10 mg/ml,4=5 mg/ml, 5=2.5

mg/ml, 6=1.25 mg/ml, 7=0.62 mg/ml, C=Negative control: DMF N, N- Dimethyl Formamide, 8=Standard:

Ketoconazole (Positive control against fungi),Streptomycin sulphate (Positive control against bacteria).

Plate 2.13: Antidermatophytic activity of 98% methanolic leaf extract of Vitex negundo L.


G

A

F F E E

C C D D

B B A

G


0

0.5

1

1.5

2

2.5

MIC

MIC

Fig -2.57: Minimum Inhibitory Concentrations of 98% methanolic leaf extract of Vitex negundo L.



The crude successive extract of Vitex negundo L. leaf viz., petroleum ether,

chloroform, ethyl-acetate and 98% methanol were qualitatively screened for the

occurrence of various secondary metabolites such as alkaloids, phenol, Flavonoids,



Table-2.42: Preliminary screening of secondary metabolites in Vitex negundo L.


Name of the test

PE

CHCL3

EtOH

98% Methanol

Alkaloids

Mayer test - + - -

Dragendroff‘s test + + + - Wagner‘s test + - + -

Phenol


Ferric chloride test - - + + Ellagic acid test + + + +

Flavonoids


Lead acetate test - - + + Shinoda test + + + + NaOH test + + + +

Tannins Gelatin test - + + +

Triterpenoids

Salkowski‘s test + + + -

Libermann-Burchard test + + + - Tschugajiu test

Steroids

Salkowski‘s test + + + - Libermann-Burchard test + + + -


glycosides

Keller-Killiani test - - + + Conc. H2So4 test - - + +

Molisch‘s test - - - - Glycoside test - - - -

Alkaloids


The petroleum ether and ethyl acetate leaf extracts shows positive results to the

preliminary alkaloids tests i.e., Dragendroff‘s and Wagner‘s reagents. This extract has

produced a orange red precipitate with Dragendroff‘s reagent and reddish brown

precipitate with Wagner‘s reagent. Whereas, the chloroform extract responded to Mayers

& Dragendorff‘s test. While the 98% methanolic extract not responded to all the three

alkaloid tests.

Phenols

The ethyl acetate and 98% methanolic leaf extracts shown positive response to

the phenol test viz., ferric chloride and ellagic acid test. . In hot water test, the leaf

showed prominent brownish black demarcation at the junction of dipped and undipped

portion. The petroleum ether and chloroform extracts were responded positively to

ellagic acid test.

Flavonoids


flavonoids tests like ferric chloride, lead acetate, shinoda and NaOH test, indicating the

presence of flavonoids. While the chloroform extract positively responded to lead

acetate, shinoda and NaOH tests. Whereas the petroleum ether extract positively

responded to shinoda and NaOH tests.

Tannins

The chloroform, ethyl acetate and 98% methanolic leaf extracts shown the

positive results to gelatin test. This indicates the presence of tannin in chloroform, ethyl

acetate and 98% methanolic extracts.

Triterpenes

The petroleum ether, chloroform and ethyl acetate leaf extracts were responded

positively to Salkowski‘s, Libermann-Burchard imparting the presence of triterpenes.

Whereas the 98% methanolic extract was not responded positively for Salkowski‘s,

Libermann-Burchard tests.


Steroids

The petroleum ether, chloroform and ethyl acetate leaf extracts were responded

positively to Salkowski‘s, Libermann-Burchard imparting the presence of steroids.

Whereas the 98% methanolic extract was not responded positively for Salkowski‘s,

Libermann-Burchard tests.

Saponins

The 98% methanolic leaf extract responded positively to foam saponins tests


Glycosides

The ethyl acetate and 98% methanolic extracts were shown positive response to

Kellar – Kiliani and conc. H2So4 test pointing out the presence of glycosides. While the

petroleum ether and chloroform extracts were not shown positive response to Kellar –

Kiliani and Molisch‘s, conc. H2So4 tests.



leaves material of Vitex negundo L. estimated quantitatively using various methods

(Figure.2.58).

The maximum content estimated was total tannins (5.24 mg/100mg) followed by

total phenol (3.43 mg/100mg), flavonoids (2.12 mg/100mg), total alkaloid (1.2

mg/100mg), total saponins (0.9 mg/100mg).


0123456


mg/100mg

Vitex negundo L

Fig. 2.58 Quantitative estimations of secondary metabolites in leaves of Vitex negundo L. in

mg/100mg.





Table-2.43: Qualitative separation of secondary metabolites from Vitex negundo L.

Secondary

metabolites

No of

bands

hRf values

Colour of the bands

Phenols 04 48.07 Light brownish

67.30 Light blackish

73.07 Yellow

92.30 Green

Flavonoids 03 40.00 Light brownish

70.00 Yellow

90.00 Light green

Alkaloids 02 34.00 green

67.00 Light green


The 98% methanol extract of the leaf exhibited 4 distinct bands having light

brownish, light blackish, yellow, green with hRf values 48.07, 67.30, 73.03 and 92.30

respectively (Table-2.43).



The leaf chromatogram developed indicated 3 distinct bands possessing light

brownish, yellow and light green with hRf values 40, 70 and 90 respectively.


The chromatogram of leaf displayed having 2 distinct bands possessing green

with hRf value 34.00 and light green colour with hRf value 67.00. (Table-2.43).

Separation of flavonoid fractions from Vitex negundo L. by the Column



flavonoids that Vitex negundo L. is a rich source of flavonoids of pharmacological

importance Further, this was supported by the literature available on Vitex sps. Thus an

attempt is made here to isolate some of these flavonoids fractions from the Vitex

negundo L. leaf of by column chromatography (CC) and purified with the help of

preparative thin layer chromatography (PTLC).


The crude effective extract of Vitex negundo L. leaf about 10 g was fractioned on

a Silica gel-H (60-120Mesh) column at a room temperature and pressure (26ºC. 1bar).


100 ml each were collected. The fraction 1 collected from n-Hexane (1) 100:00, 2 to

04 were obtained from the pet ether: chloroform 100:00 (2), 50:50 (3), 00:100 (4),

Fractions 05 to 09 were collected from the chloroform : methanol 100:00 (5), 70:30 (6),

50:50 (7), 30:70 (8), 100:00 and the 10th fraction was collected from the solvent

mixture of Methanol: Aqueous (90:10). mobile phase (Table. 2.44)


to their similarly in colour. The concentrated solutions of these fractions had waxy

nature fractions of 1 to 4, transparent, green, light green, bus green, fractions 05, 07 & 08

(amorphous) yellow, dark brownish, light brownish powder. whereas fractions 6th

(semi-solid) shows light brown colour and 9 & 10 fractions (Solid) shows light yellow,

dark brownish powder.



Sl

n

o

Mobile phase Ratio of

mobile phase

Number of

fractions

Colour of

the extract

Nature of

the extract

Weight of

the extract

Antidermat

ophytic

(T.rubrum)

activity

1 n-Hexane 100 1 Transparent Waxy _ -

2 Pet ether: chloroform 100:00 1 Green Waxy 0.78 -

3 Pet ether: chloroform 50:50 2 Bus green Waxy 0.55 -

4 Pet ether: chloroform 00:100 1 Light green Waxy 0.95 05.00

5 Chloroform : Methanol 100:00 2 Yellow Amorphous 0.86 -

6 Chloroform: Methanol 70:30 2 Light brown Semi-solid 0.77 -

7 Chloroform: Methanol 50:50 2 Dark

brownish


8 Chloroform: Methanol 30:70 2 Light

brownish


9 Chloroform: Methanol 00:100 1 Light yellow Solid 0.85 -

1

0 Methanol: Aqueous 95:05 2 Dark

brownish

Solid 0.42 05.00



Out of four flavonoid fractions, one fraction (V.NEGUNDO -I) was collected by

the method of preparative thin layer finding suitable solvent and their economy and

isolation of the maximum amount of compound at a faster rate. Further, the purity of

these fractions was also checked by the TLC using various solvent systems were in the

appearance of single discrete spot of effective fraction. From the effective fraction the

purified compound was isolated through chromatographic method are subjected to

further detailed spectroscopic studies.

Fig-2.59: UV- s pectrum of VN-1

52

7.5

8

53

1.4

3

53

5.5

2

53

9.1

7

54

3.1

6

54

6.8

4

55

4.8

3

55

8.6

5

58

1.9

3

76

8.4

0

10

43.1

6

11

65.6

3

12

70.6

3

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

%T

500 1000 1500 2000 2500 3000 3500 4000

Wavenumbers (cm-1)

Fig-2.60: Infra Red S pectrum (IR) of VN-1


Fig-2.61: H

Nuclear magnetic resonance spectrum (H – NMR) of VN-1

Fig-2.62: H

Nuclear magnetic resonance spectrum (H – NMR) of VN-1


Fig-2.63: C

Nuclear magnetic resonance spectrum (C – NMR) of VN-1

Fig-2.64: C


Fig-2.65: C



Fig-2.66: LCMS s pectrum (LCMS) of VN-1

LCMS PEAK 408 m/z

STANDARD

3.43

4

4.62

8


1 3.434 18585.7 1559.4 0.175 96.074 0.472

2 4.628 759.6 35.3 0.3151 3.926 0.594

VN-1

min0 2 4 6 8 10 12 14

mAU

0

100

200

300

400

500

DAD1 E, Sig=260,16 Ref =750,100 (180513_1\1FD-0401.D)

3.325


1 3.325 17879.6 547 0.4694 100 0.671

Fig-2.67: HPLC Profile of VN-1


Physico-chemical properties and characterization of isolated compound VN-1

Nature: amorphous, colour: brownish, hRf values: 90.00, solvent system: chloroform:

methanol 50:50 , melting point: 180, solubility: water, dmso, methanol, active fraction:

Chloroform-methanol, 50:50, dark brownish, yellow, dark brownish. Based on the

physico-chemical properties VN -1 is identified as (1R)-5a,5b,8,8,11a-pentamethyl-1-

(prop-1-en2-yl)2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13b-hexadecahydro-1H-

cyclopenta[a]chrysene-9-ol, (C29H440).

UV: 351,475

IR: 3440 (OH), 2892 (Aliphatic CH- Stretching), 2928 (CH3Streching), 1612 (C=C).

1H NMR: δ 3.58(s,1H,OH), 3.23(s,1H,CH), 1.25 (s,6H, 2XCH3), 5.37 (t, 1H, 2XCH),

2.50 (d, 2H, CH2), 1.04 (s, 3H, CH3), 1.64 (t, 1H, CH), 1.30 (s, 3H, CH3), 1.38 (t, CH,

3X CH2), 1.56 (m, 2H, CH2), 2.0H (t, 2H, CH2), 2.21 (d, 1H, CH), 2.22 (t, 1H, CH), 1.64

(t, 2H, CH2), 1.60 (t, 2H, CH2), 1.82 (s, 3H, CH3), 4.91 (s, 1H, CH) 5.11 (s, 1H, CH).

Mass: Molecular formula: C29H44O = 408

Elemental analysis: Found: C, 85.23, H, 10.85, N, 3.92. Calculated: C, 85.29, H, 10.78,

O, 3.92.

(1R)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-

en2yl)2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13b-hexadecahydro-1H-

cyclopenta[a]chrysene-9-ol, (C29H440).



VN-1.

Compound

code

Test strain Inhibition zone in different conc. of compound (in mm)

01mg-1 0.5mg-1 0.25mg-1 0.12mg-1 Control Standard(K)

01mg-1

VN--1 T.rubrum 15.66±1.15 11. 00±0.00

09. 00±0.00

07. 00±1.00

_ 21. 66±1.15

M. gypseum 12. 33±1.52

08. 00±0.00

06. 33±1.52

04. 33±1.52

_ 24. 00±1.15

The flavonoid isolated compound (1R)-5a,5b,8,8,11a-pentamethyl-1-

(prop-1-en2yl)2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13b-hexadecahydro-1H-

cyclopenta[a]chysen-9-ol, (C29H440) was found to be potentially active against T.


between 0.12 and 0.2 mg ml-1 whereas MIC values of standard antifungal agents

ketoconazole was 0.3 mg ml-1 against T. rubrum and M. gypseum (Plate-2.14).


Separation of pure compounds by column chromatography & Separation of flavonoid from Vitex negundo L. by the PTLC


Antidermatophytic - Minimum Inhibitory concentration of isolated compound VN-1(Agar well diffusion method)

Antidermatophytic - Minimum Inhibitory concentration of isolated compound VN-1



-1 Ketoconazole.

Plate 2.14: Phytochemical and pharmacological profile of Vitex negundo L.

A B


05

101520 14 14

17 16 15 16 151510

1411 11 12 12

T.rubrum M.gypsuem

Figure-2.68: Maximum Inhibition zone in mm against dermatophytic fungi with purified compounds from

selected plants.

In the present investigation two dermatophytic fungal species were screened

with purified compounds from selected medicinal plants of Hyderabad Karnataka region.

The purified compound CR-1 from Corchorus olitorius L. seeds shows maximum of

17.00mm inhibition against Trichophyton rubrum, at 01mg/ml concentration followed by

ET-1, P-1 from Euphorbia tirucalli L. leaf, Pongamia pinnata L. seed 16.00mm.

Whereas Ficus racemosa L., Vitex negundo L. compounds FR-1, v.negundo-1 shown

15.00mm, followed by AR-1 of Annona reticulata L. and AS-1 of Annona squamosa L.

showed 14.00mm zones in cup plate method.

While the Microsporum gypseum was shown maximum inhibition 15.00mm

from 01mg/ml concentration of Annona reticulata L. AR-1 compound followed by CR-

1 14.00mm, VN-1, P-1 with 12 mm. whereas 11.00 mm inhibition shows from Ficus

racemosa L., Euphorbia tirucalli L. The AS-1 compound shown 10 mm zone.

The negative control used DMSO could not show inhibition against the tested

fungal strains. Ketoconazole used as standard at conc.01mg/ml shows antifungal activity

21.00mm.


2.5 Discussion

On the basis of ethonobotanical studies 61 locally available medicinal plants were

collected from Hyderabad Karnataka region. 305 extracts were prepared by using

various solvents and plant parts (such as leaves, bark, seeds). These extracts were

screened for preliminary phytochemicals (Phenols, flavonoids, and tannins) and

antidermatophytic activity (Trichophyton rubrum, Trichophyton tonsurans and

Microsporum gypseum).

Preliminary phytochemical antidermatophytic activity of 61 ethno medicinal plants.

The preliminary phytochemical screening tests may be useful in the detection o f the

bioactive principles and subsequently may lead to the drug discovery and development.

Further, these tests facilitate their quantitative estimation and qualitative separation of

pharmacologically active chemical compounds (Varadarajan et al., 2008). The

phytochemical screening has revealed the presence of triterpenoids, steroids, glycosides,

flavonoids, alkaloids, tannins, saponins, carbohydrate and phenolic compounds in

various solvent extracts of different plant extracts.

Screening for antidermatophytic activity of 61 ethno medicinal plants.

In the present study the antidermatophytic screening was done against three

common dermatophytes namely Trichophyton rubrum, Trichophyton tonsurans and

Microsporum gypseum. The screening results were classified into four types i.e., very

effective, effective, moderate and weak. Among 61 plants 18 were showed very

effective against T. rubrum followed by T. tonsurans 10 and M. gypseum 05.

The highest antidermatophytic activity against three test fungi was displayed by

Corchorus oleterius and Lawsonia inermis.

Allium sativam showed effective activity in methanolic extract, while Seema

Bhadauria and Padma Kumar (2011) reported in aqueous extract. Because aqueous

extract highly polar solvent than methanolic extract. Lawsonia inermis leaves extract

showed very effective activity against T. rubrum and M. gypsum. Singh et al., (1989)

screened barks of 30 plant species against Microsporum gypseum and Trichophyton

mentagrophytes, only L.inermis L. bark extract showed broad fungi spectrum when


tested against 13 ring worm fungi. Whereas the moderate screening report was recorded

by Vidyasagar and Kavitha Sagar (2013).

In the present antidermatophytic screening 61 ethno medicinal plants were tested

against three Test fungi according to the reports of Vaijayanthimala et al., on anti-

dermatophytic activities of 23 South Indian medicinal plant materials have been studied

using five isolates of Trichophyton rubrum and four isolates of Trichophyton

mentagrophytes. The solvents used in both the study were different Ali-Shtayeh and

Suheil I. Abu Ghdeib was reported 21 plants in aqueous extracts.

Leaves of Annona reticulata L., Annona squamosa L. have recorded very

effective antidermatophytic activity against T. rubrum, M. gypseum. The hot soxhlet

standard extraction technique was followed so that effective antidermatophytic activity

can be observed with A. squamosa L. leaves. While Anand, et al., (2007) observed weak

activity using cold extraction of A. squamosa L. leaf against dermatophytes.

The antidermatophytic results of Corchorus olitorius L. seed extracts

demonstrated very effective with three test fungi. Semra İlahan et al., reported an

effective antifungal activity from the leaves extract. Adegoke et al., (2009) reported

phytochemical composition and antimicrobial effects of Corchorous olitorius L. leaf

extracts on four bacterial isolates. Whereas using seed extract only antibacterial activity

was reported by Pal et al., (2006).

Detection of secondary metabolites occurrence in 61 ethno medicinal plants.

High polar extracts like ethyl acetate and methanol were shown effective and

moderate activity. The secondary metabolites contribute significantly towards the

biological activities of medicinal plants such as antidermatophytic, antiinflammatory,

hypoglycemic, antidiabetic, antioxidant, anticarcinogenic, antimalarial, anticholinergic,

antileprosy activities etc. (Negi et al., 2011).

In the present report three plants viz., Argemone mexicana L., Ocimum sanctum

L., and Plumbago zeylanica L. shown positive response to phenol test in all the five

solvent extracts and a total number of phenols present in 145 extracts. The positive

response of three plants results were not correlating with the previous report of Saranya

et al., (2012).

All extracts of Coriandrum sativum shown positive response to Flavonoids test.

Three extracts viz., ethyl acetate, methanol and aqueous of high polar solvent extracts

http://www.sciencedirect.com/science/article/pii/S0367326X06001626


each with 23 plants shown positive response and also the total number of 173 extracts

were shown positive response. In previous studies the root of C. sativum (Sasi Kumar et

al., 2014), leaves extract (Nirmal pul et al., 2013) showed positive in high polar solvent

extracts.

The present study reveals a total of 147 extracts responded to tannins occurrence

test. 10 plants shown positive response in three solvent extracts viz., ethyl acetate,

methanol and aqueous. It is correlated with previous report of Vidyasagar et al., (2012).

In the present study, 128 extracts shown positive response for alkaloids

occurrence. Whereas five extracts of Lawsonia inermis Linn. leaf shown positive

response to all the alkaloids tests. Whereas three plants leaves viz., Achyranthes aspera

L. Cephalandra indic and Euphorbia tirucalli L. shown positive response in three

solvent extracts viz., ethyl acetate, methanol and aqueous. It is similar to previous reports

of Narendranath Alluri and Mala Majumdar (2014).

In the present saponins profile, 123 extracts have shown positive response.

Whereas the three plants viz., Cajanus cajan, Calotropis gigantea L. Carica papaya L.,

of five each extracts was responded positively. These results correlated with past reports

of Narendranath Alluri and Mala Majumdar (2014).

This study revealed the positive respond to glycoside from Allium sativam Linn.

In this screening a total number of 132 extracts showed positive response to glycosides

occurrence. This report is differing with previous reports of Madhumitha and Saral

(2011), Jigna Parekh and Sumitra Chanda (2007).

On the basis of screening for antidermatophytic activity, secondary metabolites

and literature survey, seven ethno medicinal plants were selected for further detailed

phytochemical and pharmacological studies. This obtained information would be helpful

as a primary platform for further phytochemical and pharmacological studies.

Annona reticulata L.

The effective molecules of many plant derived drugs are secondary metabolites.

Therefore, phytochemical analysis of the plant extract for its main bioactive components

is vital to establish scientific rationale for its use as drug.

http://www.sciencedirect.com/science/article/pii/S1995764511600679

http://www.sciencedirect.com/science/article/pii/S1995764511600679


The MIC value of methanolic leaf extract was found to be 0.62 and 1.25 mg ml-1

against all the test dermatophyte species. Owing to better efficacy, with methanolic leaf

extract was studied further to isolate the active component through chromatographic

methods. This is in accordance with the observations recorded in case of other medicinal

plants by various authors (Kaushik and Goyal, 2008; Parekh and Chanda, 2007 and

Sener, 1994; Lokhande et al., 2007).

The active components of many plant derived drugs are secondary metabolites.

Therefore, basic phytochemical analysis of the plant extract for its main bioactive

components is vital to establish scientific rationale for its use as drug. Phytochemical

analysis of the methanolic extracts of A. ret iculata L. (leaf) revealed the presence of

polyphenolic compound. However, Chang et al isolated a new cytotoxic 7-1actone

acetogenin, cis-/trans-isomurisolenin, along with six known cytotoxic acetogenins,

annoreticuin, annoreticuin - 9-one, cis-/trans-bullatacinone, bull atacin, cis-/trans-

murisolinone, and squamocin from ethyl acetate extract of seeds of Annona reticulata

(Anonymous, 1994). Terpenes like spathenelol muurolene, copaene and eudesmol were

also reported by Saad et al., (1991), a new triterpenoid annonaretin A, was isolated from

the leaves of Annona reticulata by Shung , Wu et al.

In the present report one of the novel polyphenol was isolated from the methanolic

leaf extract A. reticulata L. However, two cyclopeptides, the cycloheptapeptide

cycloreticulin C, cyclo(Pro1-Gly 2-Gln3-Pro4-Pro5-Tyr6-Val7) and the cyclohexapeptide

glabrin A, cyclo( Pro1-G ly2-L eu3-V al 4-Ile5-Tyr6) and one cyclooctapeptides

(Anonymous, 1994 and Chang et al., 1993) have been isolated from the methanolic

extract of the seeds. The findings of this work would be useful in antidermatophytic drug

designing.

Fractionation leading to isolation of active compounds should result higher

activity than the original extract. This approach forms the basis of discovery of bioactive

compounds from the naturally occurring sources and has led to discovery of many

important drugs. The main difficulty in using natural products as sources for

pharmaceutical leads is the separation of the plethora of compounds from the active

compounds in crude extracts. New compounds have been isolated from leaf parts of A.

ret iculata L. through bioassay-guided isolation using silica gel column chromatography


(Anonymous, 1994, Saad et al., 1991, Chang et al., 1993, Jirovetz et al., 1998) in the

past.

Saad et al., (1991) isolated terpenes like spathenelol muurolene, copaene and

eudesmol using leaf extract, a new triterpenoid annonaretin A, was reported by Wu et al.

Ogunwande and Ekundayo were reported the isolation of hydro distilled oil using the

leaves of Annona reticulata L. from Nigeria.

Nine antiinflammatory compounds were isolated from the leaves of A. reticulata

L. by Thang et al., ( 2013). The ethanolic roots extract was reported as an effective

anticancer drug by Yuan et al., (2003). Cytotoxicity potential against cancer cell lines

using compoundsTAR-01 (neoannonin) and AAR-02 (norushinsunine) from the root of

A. reticulata L. was reported by Suresh et al., (2012). Bhalke and Chavan (2011)

investigated the Analgesic and CNS depressant activity using leaf extract. Thang (2013)

proved antioxidant activity using leaves extracts of A. reticulata L. Most of the

compounds are isolated from the roots and other parts of A. reticulata L. There are no

reports on isolation of antidermatophytic compound from A. ret iculata L., particularly

from leaves.

In the present study methanolic leaf extract was used fo r Phytopharmacology,

whereas in previous report of antioxidant and antimicrobial activities methanolic root

extract of A. reticulata L. was used Jamkhande et al.,(2014). Phenolic compounds are

products of secondary metabolism and have strong antimicrobial agents (Mohamed et

al., 2013). Preliminary phytochemical investigation revealed that root part of A.

reticulata L. is rich in secondary metabolites (Suresh et al., 2011). The antibacterial

activity has been evaluated using A. reticulata L. root extracted compound (neoannonin)

against eight strains of bacteria using agar cup method by Jamkhande PG, et al., (2014).

A. reticulata L. aqueous leaf extract also reported against plant pathogenic fungi by

Umesh P. Mogle (2013).

Annona squamosa L.

Nature has always served as an immense source for humans as it is known that

the flavonoids have various properties like antimicrobial, fungicidal, anthelmintic,

antimalarial, anti-diabetic, leukemic, larvicidal and rutin was shown antimicrobial,


anthelmintic, larvicidal, and cytotoxic potential (Shagun Dubey et al., 2013). In the

present study rutin (flavonoid) was isolated using methanolic leaf extract of A. Squamosa

L. the antidermatophytic activity of isolated flavonoid was first recorded these were

agreement with previous reports of Santos et al. (2006), Lima et al. (2009) Sahai et al.

(1994) have done the identification by IR, 1H and 13C NMR and MS spectra of

acetogenins from seeds of Annona cornifolia. Yang et al. (2009) isolated antitumor

compound namely bis-THF acetogenins was isolated from A. Squamosa L. Rutin is one

of the plant derived flavonoid. Rutin has demonstrated cardio protective, analgesic, and

anticancer effects (Luciana, et al., 2014). The results of the current study indicated

antidermatophytic properties of rutin, there are few studies in the literature reporting the

activity of natural products against the pathogenic fungus. The natural product (R)-

goniothalamin and its synthetic enantiomer were evaluated (Fátima, et al. 2008).

The Minimum Inhibitory Concentration values of methanolic leaf extract was

found to be 0.62 and 1.25 mg ml-1 against all the test dermatophyte species. Owing to

better efficacy, with methanolic leaf extract was studied further to isolate the active

component through chromatographic methods. This is in accordance with the

observations recorded in case of other medicinal plants by various authors (Kaushik and

Goyal, 2008; Parekh and Chanda, 2007 and Sener, 1994, Lokhande et al., 2007).

Some observations recorded on this plant, post-cortical anti- fertility activity

using seed extract was reported by Chavan et al., (2010). Methanolic roots extract was

found to be effective as a drastic purgative and in acute dysentery (Mukhlesur

Rahman et al., 2005). The hot aqueous leaves extract showed hypoglycaemic, anti-

diabetic activity, astringent, chronic diarrhoea, estomatic disease and an insecticide

(Rajesh Kumar Gupta et al., 2008). Dos Santos and Sant Ana (2001). New antioxidant

annonaceous acetogenins (2,4-cis and trans)-squamolinone, (2,4-cis and trans)-9-oxo-

asimicinone, and bullacin isolated by Craig Hopp et al., (1998).

The anti-bacterial and antifungal activities were proved using compounds such

as annotemoyin-1, annotemoyin-2, squamocin and cholesteryl glucopyranoside by

Mukhlesur Rahman et al., (2005). Whereas anti-ulcer activity was reported by Dinesh,

et al., (2011). Antidiabetic and hypoglycemic activity was reported by Mujeeb, et al.,


(2009). Genotoxic effect was proved using compound isosquamocin by Paramjit

Grover, et al., (2009).

Twelve different acetogenins namely asimicin18, squamocin18, squamocin-

D18, desacetyluvaricin 18, Isodesacetyluvaricin18, squamostatin-D18, squamostatin-

E18, squamostatin B18, squamostatin-A18, 12, 15-cis-squamostatin-A19, 4-

deoxyannoreticuin20, and cis-4-deoxyannoreticuin20 were evaluated for their ability to

inhibit the growth of cancer cell lines by Haijun Yang et al., (2009).

Alcoholic and water leaf extracts of A. squamosa L. was shown

hepatoprotective role due to the antioxidative effect of the flavonoids (Mohamed

Saleem et al., 2008). Whereas Mohamed saleem et al., (2011) reported

hepatoprotective activity using methanolic extract of A. Squamosa L.

Methanolic extract is high polar phytoconstituents, it was used in this study for

isolation, whereas the low polar petroleum ether solvent extract was used for the

isolation of caryophyllene oxide from the bark of A. squamosa L. by Chavan et al.,

(2010). Recently, cyclic heptapeptides were isolated from a methanolic seeds extrac t of

Annona squamosa L. by Hiroshi Arayaa et al., (2002) such as hymenamide,

pseudostellarin, yunnanin, and segetalins and E10.Two bis-tetrahydrofuran acetogenins,

squamocin-O1 and squamocin-O2. While two more new Annonaceous acetogenins

(squamostanin-C and squamostanin-D) were isolated from the 95% ethanolic seed

extract of Annona squamosa L. by HaiJun Yang et al., (2009). From the methanolic

seed extract the effective anthelmintic activity was proven by Srilakshmi S et al., (2011).

Audrey Leatemia J and Murray B. Isman (2004) reported using compounds

―annonaceous acetogenins‖ for antiinsecticidal activity.

Annonaceous acetogenins are a group of compounds that were isolated so far

from the Annonaceae family particularly from the seeds of A. Squamosa L. but was

recently reported to be present in the family of Vitaceae (Idensi Bajin ba Ndob et al.,

2009. The acetogenins commonly used as parasiticide, insecticide and other cytotoxic

activities, parkinsonism (Idensi Bajin ba Ndob et al., 2009).


Corchorus olitorius L.


found to be 0.62 and 1.25 mg ml-1 against all the test dermatophyte species. Owing to

better efficacy, with methanolic leaf extract was studied further to isolate the active

molecule through chromatographic methods. This is in accordance with the observations

recorded in case of other medicinal plants by various authors (Kaushik and Goyal, 2008;

Parekh and Chanda, 2007 and Sener, 1994; Lokhande et al., 2007).

The rural people of Hyderabad Karnataka are using seed powder in treating skin

diseases. The other uses reported in ayurveda include ascites, pain, piles, and tumors.

Elsewhere the leaves are used for cystitis, dysuria, fever, and gonorrhoea Duke and

Wain, 1981). The effective antibacterial activity was reported by Adegoke and Adebayo-

Tayo (2009).

In the present study the secondary metabolites like Flavonoids, phenols,

saponins, alkaloids present strongly, the similar type of results reported from worldwide

i.e., flavonoids (Taoying Zhou et al., 2009, Kaku Nakagawa et al., 2004), alkaloids (Day

Cartwright 1990), saponins (George Francis et al., 2002), hypoglycaemic agents (Ahad

et al., 2011, Ocho-Anin Atchibri et al., 2010, Atangwho et al., 2009).

The potential antidermatophytic novel flavonoid ―hexadecahydro-17-(2,5-

dihyro-5-oxofuran-3-yl)-3,5,14-trihydroxy-13-methyl- iH-cyclopenta[a]phenanthrene-10

carbaldehyde‖ was isolated in this study using methanolic seed extract of C. Olitorius

L. Whereas the 17 active nutrient compounds reported in leaves of C. Olitorius L.

including protein, fat, carbohydrate, fibre, ash, Calcium, Potassium, iron, sodium,

phosphorous, beta-carotene, thiamine, riboflavin, niacin, ascorbic acid etc by Islam,

(2010), Calleja, (2010). Cardenolide glycosides isolated from seeds by Gupta et al.,

(2003). Anti- inflammatory and anti-pyretic activities were reported by Zakaria et al.,

(2006). Keiko Azuma et al., (1999) isolated six phenolic antioxidative compounds [5-

caffeoylquinic acid (chlorogenic acid), 3,5-dicaffeoylquinicacid, quercetin 3-galactoside,

quercetin 3-glucoside, quercetin 3-(6-malonylglucoside), and quercetin3-(6-

malonylgalactoside) (tentative)] from the leaves of C. olitorius L. Among six 5-

caffeoylquinic acid was a predominant phenolic antioxidant.


Chen and Saad, (1981), Duke (1983) have isolated oxydase and chlorogenic acid,

folic acid from the leaves, these molecules useful in good eyesight, healthy red blood

cells, strong bones and teeth, smooth, clear skin, strong immune cells, and fast wound -

healing.

As per the available litetrature, the scientific report on the antidermatophytic

activity of the methanolic seed extracts are lacking. So that the antidermatophytic

activity of methanolic seed extract was the first from the present study, while the

previous report of Pal et al., (2006) recorded a broad spectrum of antibacterial activity

using methanolic seed extract.

In the present report dermatophytic bacteria were effectively inhibited by

methanolic extract. Whereas in past report of Nester et al., (2004) a potential

antibacterial activity [P. aerugenosa (inflammation of the bladder), K. pneumonia

(pneumonia), S. aureus (food poisoning), S. typhimurium (typhoid fever) and B. cereus

(eye infection, food spoilage and food borne intoxication)] was recorded using seed

oil. The antidiabetic effect of ethanolic seed extract of Corchorus olitorius L. was

studied by Maxwell Osaronowen Egua et al., (2013).

Semra ilhan et al., (2007) described the antimicrobial activity of 3 extracts of C.

olitorius L. Successive petroleum ether, methanol and ethyl acetate, water extracts of C.

olitorius L. leaves were tested (in vitro) for their antibacterial and antifungal activities

by agar-well diffusion assay. All extracts displayed varied levels of antibacterial or

antifungal activity. The petroleum ether extract exhibited antibacterial effect against all

of the bacteria tested and the diameter of zones varied between 14-20 mm. The

petroleum ether extract of C. olitorius L. leaves presented a good activity against

Escherichia coli, Staphylococcus aureus and Yersinia enterocolitica, 20 mm, 19 mm and

19 mm, respectively. The ethyl acetate, water extract presented a good activity against

Geotrichum candidum and Botrytis cinerea, 20 mm and 12 mm, respectively. The results

obtained in this study appear to confirm the antibacterial and antifungal potential of C.

Olitorius L. leaves, as well as its usefulness in the treatment of diseases that may be as a

result of infection.

Euphorbia tirucalli L.

The Minimum Inhibitory Concentration of methanolic phylloclade extract was


found to be 0.62 and 1.25 mg ml-1 against all the test dermatophyte species.

Owing to better efficacy, with methanolic leaf extract was studied further to isolate the

active molecule through chromatographic methods. This is in accordance with the


Goyal, 2009; Parekh and Chanda, 2007)

Ethnic people of Hyderabad Karnataka region commonly use phylloclade of E.

tirucalli L. in treating skin diseases. Previously, the latex used in treating asthma,

rheumatism, earache, cough and toothache in India (Wealth of India), syphilis, laxative

verrucae, epithelioma, sarcoma and skin tumours in northeast region of Brazil. Whereas

the Bark is used in healing the infections of spleen, colic, blood complaints, whooping

cough and asthma. Roots are used in treating colic pains (Rao and Hemadri). Sterility of

women controlled by phylloclade (Kokwaro,). It is also frequently used for the control

of healing, broken bones, hemorrhoids, pains, ulcerations, swellings in Asia. In addition

to this, it is used to treat scorpion bites, asthma, cancer, spasms in Brazil (Cataluna,

Rates 1997). Whereas this is first report on ethnopharmacological validation.

In the present report phylloclade was used for antidermatophytic study. In past

the stem bark and leaves are reported to possess antibacterial activity. Aerial parts of

E. Tirucalli L. are reported to possess antioxidant activity (Jyothi et al., 2008). Organic

solvent exhausted material of E. Tirucalli L. is reported to have antiarthritic activity

(Sarang et al., 2007). Latex is reported to possess proteolytic activity (Cleverson de

Freitas et al., 2010), anticancer activity (Ali et al., 2010), molluscidal activity (Pedro

et al., 1985) and larvicidal activity (Mwine et al., 2010). Stem of E. tirucalli L. is

reported to possess insecticidal activity (Uma and Prasanna 2009).

In the present phytochemical report the potential and novel antidermatophytic

alkaloid ―2-methaoxy 3-benzamidopropanoate‖ was isolated. The occurrence of major

phytochemcical compounds, secondary metabolites E. tirucalli L. is reported to possess

flavonoids, diterpenes, tannins, steroids and alkaloids reported by Fauconneau B et al.,

(1997). The plant is also reported to possess terpenes, alcohol eufol, alfaeuforbol

(Macdonald et al., 1949), taraxasterol, E. tirucallol, cycloeuphornol, n-hexacosanol

(Rastogi and Mehrotra), terpenic alcohol and trigliane. Whole plant has afforded to

contain 7.4% citric acid with some malonic and succinic acids. Terpenoids and sterols


in plants are important sources of vitamins, while the steroid compounds having

very much demand in insecticides and anticancer drugs industrially (Itokowa et al.,

1989, Wu et al., 1991). Bioactive compounds extracted from natural sources can benefit

human health. The total phenolic content and antioxidant activity in extracts of

Euphorbia tirucalli L. was reported by Keline Medeiros de Araújo et al.,(2014) and they

resulted excellent antioxidant capacity and moderate antimicrobial activity.

The major components of E. tirucalli L. are triterpenes (Biesboer, Mahlberg

1979, Yamamoto et al., 2011). Latex contains diterpene esters of the phorbol, ingenol

and 12-deoxyphorbol esters, reported to be highly active carcinogenic and tumour

promoting agents.

From the fresh latex, terpenic alcohol, isoeuphorol, taraxasterol and tirucallol

were reported by Cataluna et al., (1999). Whereas from dried latex, Ketone euphorone

was reported. Resin is the principle constituent of dried latex of E. Tirucalli L. The

stem is reported to posses hentriacontene, hentriacontanol, anti tumor steroid 4-

deoxyphorbol ester, beta-sitosterotchouc, casuarin, corilagin, cycloeupordenol,

cyclotirucanenol, ellagic acids, euphorbins, euphol, euphorone, ellagic acids,

euphorbins, euphol, euphorone, euphorcinol, gallic acids and glucosides (Khan AQ,

Malik 1990). Aqueous extract of aerial parts of E. Tirucalli L. was reported for

heraoprotective activity in adult Wistar rats and Swiss albino mice against carbon

tetrachloride induced liver damage. The extract resulted in decrease of GSH depletion

and lipid peroxidation and showed effective protection of liver (Jyothi et al., 2008).

In the present study the methanolic phylloclade extract was used for

Phytopharmacology. While acetone, hexane, methanol, chloroform and petroleum ether

stems extracts of E. Tirucalli L. were used by Prasad et al., (2011). Parekh J et al.,

(2010) reported a potential antibacterial activity, using agar disc and well diffusion

techniques with methanolic extracts against S. epidermidis, B. subtilis, P.

pseudoalcaligenes, P. vulgarisand, P. typhimurium, P. pseudoalcaligenes. The

effective antioxidant activity of aerial parts was reported by Jyothi et al., (2008).

Petroleum ether and ethyl alcohol extracts of E. Tiruaclli L. were evaluated for

insecticidal property by Uma MS and Prasanna (2009). The fresh latex was shown

larvicidal activity against Anopheles funestus and A. gambae in a neglected fish pond in


different dilutions (Mwine et al., 2010). The potential antimicrobial activity alcoholic

extracts of stem bark and leaves of E. Tirucalli L. were reported by Bhuvaneshwar et al.,

(2010).

Ficus racemosa L.


found to be 0.62 mg ml-1 against all the test dermatophytes. Owing to better efficacy,

methanolic leaf extract was studied further to isolate the active molecule through

chromatographic methods. This is in accordance with the observations recorded in case

of other medicinal plants by various authors (Parekh and Chanda, 2007 and Sener, 1994;

Kaushik and Goyal, 2008; Lokhande et al., 2007).

Very little phytochemical work has been carried out on this plant. In the present

report a novel flavonoid“2-(4-(3-methylbut-2-enyloxy)-3, 5-dimethoxyphenyl)-5-hydroxy-4H-

chromen-4-one, (C23H2407)‖ was isolated using methanolic leaf extract. In past reports from stem

bark two leucoanthocyanins namely leucocyanidin-3-0-(3-glucopyranoside,

le ucope la ro go n id in- 3 - O - a- L-rhamnopyranoside, (3-sitosterol, unidentified long

than ketone, ceryl behenate, lupeol, its acetate, a-amyrin acetate. From trunk bark, lupeol,

(3-sitosterol and stigmasterol were isolated. Fruit contains glauanol, hentriacontane, (3-

sitosterol, gluanol acetate, glucose, tiglic acid, esters of taraxasterol, lupeol acetate,

friedelin, higher hydrocarbons and other phytosterol. A new tetracyclic triterpene glauanol

acetate which is characterized as 13a, 14(3, 17 PH, 20 aH-lanosta-8, 22-thane-3 a-acetate and

racemosic acid were isolated from the leaves. An unusual thermostable aspartic protease was

isolated from latex of the plant. The stem bark and fruit showed presence of glauanol acetate

(Sen and Chowdhary 1971, Agarwal and Misra1977, Joshi 1977, Shrivastava et al.,

1977, Agarwal1977, Bhatt and Agarwal 1973, Merchant 1979, Suresh 1979, Li et al.,

2004, Devaraj et al., 2008). Better hypoglycemic activity was reported by Shrotri and

Ranita (1960).

This is the first report on isolated antidermatophytic activity of methanolic novel molecule.

Whereas other activities performed using methanol extract of powdered fruits at the dose 1, 2, 3

and 4g/kg reduced the blood glucose level in normal and alloxan induced diabetic rabbits.

Dietary fibre content of fruits when fed to rats in diet induced pronounced


hypocholesterolemic effect, as it increased fecal excretion of cholesterol as well as bile

acids (Agarwal and Chauhan 1988).

Recovery of renal glutathione content, antioxidant enzyme, decrease in the enhanced

renal ornithine decarboxylase activity, DNA synthesis, blood urea nitrogen and serum

creatinine (Khan and Sultana 2005). Similar results were obtained when ferric nitrilotri

acetate was used as renal carcinogen (Khan and Sultana 2005). Both the result proves that the

extract is a very potent chemo preventive agent.

In the present report dermatophytes namely T. rubrum, T. tonsurans, M. gypseum

were effectively inhibited in methanolic extract. In past study possess potent inhibitory

activity against six species of fungi, viz. T. mentagrophytas, T. rubrum, T. soundanense,

C. albicans, C. krusei and Torulopsis glabrata (Vonshak et al., (2003) and

Deraniyagala et al.,(1998). Different extracts of leaves were tested for antibacterial

potential against Escherichia coli, Bacillus pumitis, Bacillus subtilis, Pseudomonas

aeruginosa and Staphylococcus aureus. Out of all extracts tested, petroleum ether

extract was the most effective extract against the tested microorganism (Mandal et al.,

2000). Ethanol extract of stem bark showed a potent wound healing (Biswas et al. 2003).

The decoction of stem bark was investigated for antidiuretic by Rastnasooriya et al.,The

methanolic extract of stem bark has shown potent in vitro antioxidant activity when

compared to the methanol extract of its roots (Chennabaswaraj et al., 2008).

Pongamia pinnata L.

This plant can be used to discover bioactive natural products that may serve

as leads for the development of new pharmaceuticals, therapeutics. The minimum

Inhibitory Concentration of petroleum ether seed extract of P. pinnata L. was found to

be 2.5 to 0.62 mg ml-1against all the test dermatophyte species. Owing to better efficacy,

with petroleum ether seed extract was studied further to isolate the active molecule

through chromatographic methods. This is in accordance with the observations recorded

in case of other medicinal plants by various authors (Kaushik and Goyal, 2008,

Lokhande et al., 2007, Parekh and Chanda, 2007 and Sener, 1994).

In the present ethnic study P. pinnata L. seed used in treating skin diseases. It

contains several phytoconstituents belonging to category flavonoids and fixed oils. The


fruits and sprouts of P. pinnata L. were used in folk remedies for tumours

(Hartwell 1971).

It has been recognized in different system of traditional medicines for the

treatment of different diseases and ailments of human beings (Ghani 1998, Kirtikar and

Basu 1994).

Seed extract of this plant has hypotensive effects and produce uterine

contractions. Powdered seed is used in bronchitis, chronic fever, whooping cough

and chronic skin diseases and painful rheumatic joints(Ingredient guide 2006).

Seed oil is used in scabies, leprosy, piles, ulcers, chronic fever, lever pain and

lumbago. Its oil is a source of biodiesel and it is also used as fuel for cooking and lamps

(Mahli et al., 1989).

Leaves are active against Micrococcus; their juice is used for cold, cough,

diarrhoea, dyspepsia, flatulence, gonorrhoea and leprosy. Roots are used for cleaning

gums, teeth and ulcers. Bark is used internally for bleeding piles. Juices from the plant as

well as oil are antiseptic (Kirtikar, Basu 1993).

In the traditional system of medicines, such as Ayurveda and Unani, the

Pongamia pinnata L. plant is used for antiinflammatory (Srinivasan et al., 2001),

antiplasmodial, anti-nociceptive, anti-hyperglycaemics, anti- lipidoxidative,

antidiarrhoeal, anti-ulcer, antihyperammonic, CNS depressant activity (Li et al.,

2006) and antioxidant. P. pinnata L. extract could be used as a safe alternative

antihyperglycaemic drug for diabetic patients (Kirtikar, Basu 1993).

In the present phytochemical investigation an effective antidermatophytic

molecule ―Furanoflavonol‖ was isolated from the seed extract of petroleum ether. This

molecule previously reported but there were no reports on antidermatophytic activity.

Previously ―Furanoflavonol‖ was isolated by Yin, et al., (2006), Yadav, et al., (2004)

from P. pinnata L. seeds. The seeds contain six compounds (two sterols, three sterol

derivatives and one disaccharide) together with the eight fatty acids (three saturated and

five unsaturated). Karangin, pongamol, pongagalabrone, pongapin, pinnatin and kanjone

have been isolated from seeds. Immature seeds contain a flavone derivative ‗pongol‘.

The other flavonoid isolated from the seeds includes Glabrachalcone,


isopongachromene. Anti-microbial effect of crude decoction of dried leaves of P.

Pinnata L. was determined by Brijesh et al., (2006).

Some other flavonoids isolated from leaves and stem of P. Pinnata L. were

flavone and chalcone derivatives such as Pongone, Galbone, Pongalabol, Pongagallone

A and B. Chemical investigation of stems of the mangrove plant, P. Pinnata L.,

resulted in isolation and characterization of five structurally unusual flavonoids

metabolites by Tanaka et al., (1992).

Whereas P. Pinnata L. from Japan resulted in the isolation of 18 flavonoid

compounds including nine new ones, Pongamones III-XI, from its root bark. The

new structures were determined to be;(2S)- 3‘, 4‘-dimethoxy-6‖, 6‖-dimethylpyrano

*2‘‘,3‘‘7, 8)- flavanone (III), (2S)- 6, 3‘, 4‘-3‘, 4‘-trimethoxy-6‘‘,

6‘‘dimethylpyrano [2‘‘, 3‘‘7, 8)-flavanone (IV), (2S)- 7- methoxy-6-O-

y,ydimethylallyl-3‘, 4‘-methylenedioxyflavanone (V),2‘—hydroxy-3, 4, 5‘-

trimethoxy-6‘‘, 6‘‘-dimethylpyrano *2‘‘, 3‘‘: 4‘3‘+ chalcone (VI), 2‘, 4‘-

dimethoxy- 3, 4- methylenedioxy- dioxydihydrochlcone (VII), 2‘, 5‘, β-

trimethoxy-3, 4-methylenedioxy- 6‘‘, 6‘‘- dimethylpyrano *2‘‘, 3‘‘,4‘, 3‘+

dihydrochalcone (VIII), 2, β - dimethoxy-3, 4-methylenedioxy- furano *2‘‘, 3‘‘:

4‘,3‘+-dihydrochalcone (IX), β- hydroxy- 2‘, 4‘ 6‘-trimethoxy- 3, 4-

methylenedioxychalcone (X), 3-methoxy-furane *2‘, 3‘‘: 7, 6] f; avpme

(XI),respectively, by means of spectral analysis and synthesis(Goel et al., 1985) .

In the present report antidermatophytic activity was reported, whereas other

pharmacological activities like anti-ulcer activity was reported using methanolic extract

of roots showed significantly protection against aspirin 164 h PL, but not against

ethanol- induced ulceration. It showed tendency to decrease acetic acid- induced (Prabha

et al., 2003). Anti-hyperammonemic of P. Pinnata L. leaf was reported by Mathias,

2001, Majeed, 2005, Essa, et al., 2006). Antiplasmodial activity against plasmodium

falciparum was reported by Simonsen et al., (2001). Punitha, Manoharan (2006) were

reported first time antidiabetic activity using flowers of P. Pinnata L. Nadkarni,

(1954), Srinivasan, et al., (2001) were reported potent antiinflammatory activity using

70% ethanolic leaves extract. Acute and Chronic toxicological studies conducted in

Swiss albino rats showed the safety of the P. pinnata L. seed extract by Fiala et al.,


(1974). Viral inhibition studies with the extract of P. pinnata L. seeds against HSV-1

and HSV-2 were evaluated in vitro. The most striking observation was the total

inhibition of growth of HSV-1 and HSV-2 at concentrations of 1mg/ml and

20mg/ml w/v respectively, whereas even at the highest concentrations the extract was

not toxic for Vero cells (Singh et al., 1996).

Dermatophytic bacteria were effectively inhibited by using isolated

furanoflavonoid in this study. Similar report was given by Ahmad et al., (2004)

agreement. In their results they were reported enteric pathogenic antibacterial

compounds were isolated from the leaves of P. Pinnata L.

Vitex negundo L.

Higher plants are warehouses of assorted bioactive constituents or

phytochemicals which find ample use in the pharmaceutical industry. (Namdeo

Namdeo, 2007)) states that about a quarter of all prescribed pharmaceuticals in

advanced countries contain compounds that are directly or indirectly, derived from

plants. Phytochemicals or secondary metabolites usually occur in complex mixtures that

differ among plant organs and stages of development (Wink, (2004), Banerji, et al.,

(1969). Knowledge of the phytochemical constituents is very essential to enable

investigation of the actual effectiveness of the plant in medicine.

The Minimum Inhibitory Concentration of methanolic leaves extract of V.

negundo L. was found to be 1.25 mg ml-1against all the test dermatophyte species.

Owing to better efficacy, with methanolic leaves extract was studied fur ther to isolate the

active molecule through chromatographic methods. This is in accordance with the


Goyal, 2008, Parekh and Chanda, 2007).

In the present phytochemical studies of methanolic leaf extract a noble

antidermatophytic flavonoid molecule isolated namely ―(1R)-5a,5b,8,8,11a-pentamethyl-

1-(prop-1-en2yl)2,3,3a,4,5,5a,5b,6,8,9,10,11,11a,11b,12,13b-hexadecahydro-1H-

cyclopenta[a]chrysene-9-ol, (C29H440) ― In previous a few antimicrobial compounds

were isolated, Banerji, et al., (1969) isolated a compound ―hydroxy-3,6,7,3′,4′-

pentamethoxy flavones‖ from the leaves of V. negundo L. Whereas 6′-p-hydroxybenzoyl


mussaenosidic acid; 2′-p-hydroxybenzoyl mussaenosidic acid isolated by Sehgal et al.,

(1982), Sehgal et al., (1983) 5, 3′-dihydroxy-7,8,4′-trimethoxyflavanone; 5,3′-dihydroxy-

6,7,4′-trimethoxyflavanone (Achari, et al., 1984) viridiflorol; β-caryophyllene; sabinene;

4-terpineol; gamma-terpinene; caryophyllene oxide; 1-oceten-3-ol; globulol (Singh, et

al., 1999) betulinic acid [3β-hydroxylup-20-(29)-en-28-oic acid]; ursolic acid [2β -

hydroxyurs-12-en-28-oic acid]; n-hentriacontanol; β-sitosterol;p-hydroxybenzoic acid

(Chandramu, et al., 2003) protocatechuic acid; oleanolic acid; flavonoids

(Surveswaran et al., 2007) angusid; casticin; vitamin-C; nishindine; gluco-nonitol; p-

hydroxybenzoic acid; sitosterol were isolated (Khare, 2004). Whereas from the seeds 3β

-acetoxyolean-12-en-27-oic acid; 2α, 3α-dihydroxyoleana-5,12-dien-28-oic acid; 2β,3α

diacetoxyoleana-5,12-dien-28-oic acid; 2α, 3β-diacetoxy-18-hydroxyoleana-5,12-dien-

28-oic acid (Chawla et al., 1992 (a), Chawla et al., 1992(b) ) vitedoin-A; vitedoin-B; a

phenylnaphthalene-type lignan alkaloid, vitedoamine-A; five other lignan derivatives

(Ono, et al., 2004) 6-hydroxy-4-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7-

methoxy-3,4-dihydro-2-naphthaldehyde (Zheng, et al., 2009) β-sitosterol; p-

hydroxybenzoic acid; 5-oxyisophthalic acid; n-tritriacontane, nhentriacontane; n-

pentatriacontane; n-nonacosane these active compounds were isolated (Khare, 2004)

From roots 2β, 3α-diacetoxyoleana-5,12-dien-28-oic acid; 2α,3α-dihydroxyoleana-5,12-

dien-28-oic acid; 2α,3β -diacetoxy-18-hydroxyoleana-5,12-dien-28-oic acid; vitexin and

isovitexin (Srinivas, et al., 2001) negundin-A; negundin-B; (+)-diasyringaresinol; (+)-

lyoniresinol; vitrofolal-E and vitrofolal-F (Azhar-Ul-Haq et al., 2004) acetyl oleanolic

acid; sitosterol; 3-formyl-4.5-dimethyl-8- oxo-5H-6,7-dihydronaphtho (2,3-b)furan were

isolated (Vishnoi et al., 1983). Essential oil of fresh leaves, flowers and dried fruits were

isolated likewise δ-guaiene; guaia-3,7-dienecaryophyllene epoxide; ethyl-hexadecenoate;

α-selinene; germacren-4-ol; caryophyllene epoxide; (E)-nerolidol; β-selinene; α-cedrene;

germacrene D; hexadecanoic acid; p-cymene and valencene (Khokra et al., 2008). Misra

& Subramanian (1980) isolated three new flavone glycosides which were identified

as 3,6,7,3,4-Pentamethoxy-5-Oglucopyranosyl-rhamnoside, vitexin cafeate, 4-O-methyl

myricetin-3-O-[4-O-β-D-galactosyl]-β-D-galactopyranoside.

In the present report the dermatophytic bacteria were effectively inhibited these results

correlated past results of Kumar et al., (2006) where they studied the antibacterial of

dichloromethane:methanol (1:1 v/v) extracts of Vitex negundo L. against different


bacterial strains. Their finding conclude that none of the micro organisms

including the bacterial strains like B.subtilis, S.aureus, S.epidermidis, E.coli, and

P.aeruginosa were inhibited by dichloromethane: methanol extracts.

The present results reveal that the methanolic extract was decided as potent for

antidermatophytic studies. In previous reports, the methanolic extract was used as potent

for treating different ailment i.e., Patkar (2008) refers to the formulations described in

Anubhoga Vaidya Bhaga, a compendium of formulations in cosmetology, in outlining

the use of V. negundo L. leaves along with those of Azadirachta indica, Eclipta alba,

Sphaeranthus indicus and Carum copticum in a notable rejuvenation treatment known as

Kayakalpa. Khare (2004) outlines the applications of V. negundo L. commonly

known as Nisinda in Unani medicine. Leaf extracts of V. negundo L. were

determined to possess anti-oxidant potential by (Tiwari O.P et al., 2007). Yunos et

al. (2005) and Jana et al. (1999) established antiinflammatory properties of V. negundo

L. extracts in acute and sub acute inflammation. Anti- inflammatory and pain

suppressing activities of fresh leaves of V. negundo L. are attributed to prostaglandin

synthesis inhibition (Telang, et al., 1999), antihistamine, membrane stabilising and

antioxidant activities (Dharmasiri, et al., 2003). The extracts also possess the ability to

combat oxidative stress by reducing lipid peroxidation owing to the presence of flavones,

vitamin C and carotene (Vishal and Gupta 2005), (Rooban et al.(1999).

This is novel pharmacological study using 98% methanolic extract against

dermatological pathogens. Whereas previously Renuka devi (2008) used fresh aqueous,

heated aqueous extract, chloroform and methanolic leaves extract of V. negundo L.

was tested against three bacteria Viz., Staphylococcus aureus, Escherichia coli and

Klebsiella Peumoniae. The fresh and aqueous extracts of leaves in various dilutions

were found to have antibacterial activity against the three bacteria.

The flavonoid rich fraction of seeds of V. negundo L. caused disruption of the

latter stages of spermatogenesis in dogs (Bhargava, 1989) and interfered with male

reproductive function in rats (Das, et al., 2004). It must however be noted that these

findings are in sharp contrast with the traditional use of V.negundo L. as aphrodisiac

(Khare 2004). Hu et al., (2007) determined that ethanolic extracts of V.negundo L.

showed estrogen- like activity and propounded its use in hormone replacement therapy.

phytochemistry and pharmacology 2.1...

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