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International Journal of Interdisciplinary Research Centre (IJIRC) ISSN: 2455-2275(E)

Volume III, Issue 3 March 2017

All rights are reserved 54

PHARMACOGNOSTICAL AND PHARMACOLOGICAL PROFILE OF

CRATAEGUS SONGRICA

* Rohit Mohan and * * Dr. Anil Middha

* Research Scholar, OPJS University, INDIA

* * Department of Pharmacy OPJS University, INDIA

Abstract

The Current research work showed the importance of herbal medicines in now a days, and it

also provide knowledge of standard parameters for quality control of herbal drugs. Some

parameters are given in the research are macroscopic and microscopic evaluation physic-

chemical parameters such as ash value, extractive values with different solvents, and for

evaluating efficacy of crude extracts biological acivity has been performed.

Introduction

GENERAL INTRODUCTION

History Traditional Medicines

Traditional medicines believe on the basis of knowledge and clinical experience of the

practitioners for indigenous systems of medicine. According to our history, many infectious

diseases have been treated with the help of herbals. The traditional medicine is increasingly

solicited through the traditional practitioners and herbalists in the treatment of infectious

diseases. Among the remedies used, plant drugs constitute an important part. Current status

on scientific investigations have highlighted the importance and the contribution of many

plant families i.e. Asteraceae, Liliaceae, Apocynaceae, Solanaceae, Caesalpinaceae,

Rutaceae, Piperaceae, Sapotaceae, Campanulacae, Erytrhoxylacae, Nyssaceae, Acanthacae,

Rubiaceae, Berberidaceaae, Graminae, Moraceae, Umbelliferae, Zingiberaceae,

Leguminasae, Pinaceae, Cucurbitaceae, Styraceae, Convolvulaceae, polypodiaceae. (Kokate,

C. K, 2006)




Ayurveda is the ancient Indian system of medicine for better life and longevity though

physical and mental health management. It involves a holistic approach of humans, their

health, and illness. The origin of Ayurveda has been lost in prehistoric antiquity, but its

characteristic concepts appear to have matured between 2500 B.C and 500 B.C. in India. The

word "Ayurveda" is derived from "Ayus (r)," meaning life, and "Veda," meaning knowledge;

thus, Ayurveda literally means "Science of life." It is the ancient Indian system of health care

and longevity. Ayurveda takes a holistic view of man, his health, and illness. It aims at

positive health, which has been defined as a well balanced metabolism coupled with a healthy

state of being. Disease, according to Ayurveda, can arise from the body and/or the mind

because of external factors or intrinsic causes. Ayurvedic treatment is aimed at the patient as

an organic whole, and treatment consists of salubrious use of drugs, diets, and certain

practices. Ayurveda has a vast literature in Sanskrit and various Indian languages, covering

various aspects of diseases, therapeutics, and Pharmacy. It has evolved its own theoretical

base, which is difficult to comprehend in terms of modern scientific concepts, at least at

present. However, here we are concerned only with the exploration of its materia medica

(Dev, S., 1988).

Pharmaceutics occupies an important place in Ayurveda. Medicinal preparations are complex

mixtures including plant and animal derived products, minerals, and metals. Plants form a

dominant part of Ayurvedic Pharmacopoeia. The earliest references to such plants are found

in the Rig Veda and the Atharva Veda, dating back to the second millennium B.C. The

Charaka Samhita (900 B.C.) is the first recorded treatise fully devoted to the concepts and

practice of Ayurveda; its primary focus was therapeutics. This work listed 341 plants and

plant products for use in medicine (Sharma, S. P, 1981). The next landmark in Ayurvedic

literature was the Sushruta Samhita (600 B.C.) (Krishnamurthy, K. H., 1999) which has




special emphasis on surgery. It described 395 medicinal plants, 57 drugs of animal origin,

and 64 minerals and metals as therapeutic agents. Sushruta, the father of surgery, lived and

practiced surgery in Varanasi, India, approximately 2,500 years ago. Another important

authority in Ayurveda was Vagbhatta of Sind, in present day Pakistan, who practiced around

the seventh century A.D. His work Ashtanga Hridaya is considered unrivaled for the

principles and practice of medicine (Garde, G.K., 1954 )The Madhava Nidana (800-900 A.D)

was the next important milestone; it is the most famous Ayurvedic work on the diagnosis of

diseases. The last celebrated writer on Hindu medicine was Bhava Mishra of Magadha,

whose treatise Bhava Prakashan, is held in high esteem by modern Ayurvedic practitioners

for its descriptions of approximately 470 medicinal plants. Other than these monumental

treatises, many (>70) Nighantu Granthas (Pharmacy lexicons) were written, mostly between

the seventh and sixteenth centuries. Raj Nighantu by Narhari Pandita and Madanpala

Nighantu by Madanpala are considered masterpieces on medicinal plants.

All ancient texts on Ayurveda divide medical knowledge into eight branches (Ashtanga), and

this is no different from the contemporary approach. Also, Ayurvedic descriptions of diseases

are much like the modern delineation. Plant-derived drugs have been categorized according

to their pharmacologic/therapeutic action. For example, in the Charaka Samhita, drugs were

classified into 50 groups. Ayurveda, in its prime, was a congent, scientifically organized

discipline. Ayurvedic texts were much respected in neighboring countries, as evidenced from

their translation into Greek (300 B.C.), Tibetan and Chinese (300 A.D.), Persian and Arabic

(700 A.D.), and several languages of other Asian people. Currently, Ayurveda is widely

practiced in the Hindustan peninsula (India and the neighboring countries) and, in recent

years, has attracted much attention in economically developed countries such as those in

Europe and in the United States and Japan. ( Hartzell J. F; Zysk, K. G., 1995).




Ayurvedic treatment of a disease consists of salubrious use of drugs, diets, and certain

practices. Medicinal preparations are invariably complex mixtures, based mostly on plant

products. Around 1,250 plants are currently used in various Ayurvedic formulations. Many

Indian medicinal plants have come under scientific scrutiny since the middle of the

nineteenth century, although in a sporadic fashion. The first significant contribution from

Ayurvedic materia medica came with the isolation of the hypertensive alkaloid from the

Sarpgandha plant (Rauwolfia serpentina), valued in Ayurveda for the treatment of

hypertension, insomnia, and insanity. This was the first important ancient-modem

concordance in Ayurvedic plants. With the gradual coming of age of chemistry and biology,

disciplines central to the study of biological activities of natural products, many Ayurvedic

plants have been reinvestigated. (Kokate, C. K, 2006).

PLANT INTRODUCTION

Plant Profile

Plant Profile of Crataegus Songorica

Table: 1 Nomenclature and taxonomical classification of Crataegus Songorica

Synonym Crataegus oxycantha

Family: Rosaceae

Genus: Crataegus

Species: Songorica

English name: Hawthorn

Local Ban-sangli, Ring [Kashmiri],Ban-Sangli [Punjabi]

Trade name: English Hawthorn Bansangli

Habit: Tree

Status: Common




Description of Natural Habitat

The plant grows in river valleys and rivine slopes in the-mountain zone in India, found in the

temperate Himmalayas of Kashmir and Himachal Pradesh, altitude of 1800-3000m.

[Nadkarni, 1976].

Distribution of plant

In Pakistan, the plant is found in mountains of Chitral [Boni], Swat, Chitral, Balochistan from

1300 to 2700 meters, This plant is exceptionally common in Boni [Chitral], Swat, Astor

Gilgit and Muree hilly areas with local names shetal or gain respectively; also distributed

from Afghnistan to Uttarr Pardesh 1500-2700m

Crataegus Songarica has about 200 spicesIt is cultivated in Asia, Australia, Canada. It is an

ornamental small hardy, spiny, deciduous tree or shrub up to 9 m tall, bark peeling off in long

flakes [Anwar. et al. 1979], armed with straight axillary's or terminal thorns; leaves deeply

and sharply irregularly 4¬7 lobed; the small rose- like white flowers are beautiful and

abundant [Wealth of India, 2003], in many flowered terminal corymbose cymes, emitting

spicy odour [Anwar. et al. 1979], the flowers have a foetid smell somewhat like decaying

fish; fruit red, globose, ovoid or scarlet or some times yellow, blue or black in colour,

containing 2-3 more or less united, bony Pyrenees[Nadkarnis, 1976].it is 16mmin diameter,

the flesh is mealy and dry, like that of rosehips [Anwar. et al. 1979; Shinwari .et al. 2006], the

fruit ripen in early to mid autumn [Brown, 1995].

The plant prefers light, medium and heavy soils and can grow in heavy clay soil .The plant

prefers acid, neutral and basic [alkaline] soil. It requires moist or wet soil and can tolerate

drought. The plant can tolerate strong winds but not maritime exposure. It can tolerate

atmospheric pollution

Medicinal Uses

The liquid extract of the flowers and fruits are used as heart tonic, improves blood flow, in

the coronary arteries and used in functional diseases of the heart, such as dysproea,

hypertrophy, vulvular insufficiency and heart oppression, arteriosclerosis and angina pectoris

[Shinwari, 2006; Nadkarnis, 1976; Kiritikar, 1918]. The plant is reported to be astringent,




sedative, stomachic, hypertensive and antispasmodic properties [Anwar, 1979; Nadkarnis,

1976]. The drug relaxes the uterus and intestine but constricts the bronchi and coronary

vessels. The fruits are used as a popular remedy for diarrhea or slight phlegmasia [Mehra,

1976]. The macerated oil shows antifungal activity [Nadkarknis, 1976]. It may be used as

tincture [Kirtikar, 1918]. Both flowers and berries are act as diuretic and can be used to treat

kidney problems and dropsy [Grieve 1892

Figure1: Photograph of whole plant of Crataegus Songorica

A B C

Figure2: Photographs of Plant with (A) fruits (B) Leaf and (C)Stem




EXPERIMENTAL

MATERIAL METHOD

PLANT MATERIALS

Crataegus Songrica was collected from Kheri Bawli Delhi India. The plants were identified

by Prof. Dr. Anju Pal, Horticulture department, Panthnagar university, Panthnagar,

Uttarnchal, India.

PROCESSING OF PLANT MATERIAL

The plant materials were properly dried in shade for 5-6 days then dried in hot air oven at

400C after drying, the plant materials were milled to powder and passed through the sieve

(mesh size 40), this material were used for the identification of plant metabolite.

MACROSCOPICAL FEATURES

The morphological observations of roots, stems and leaves included colour, odour, taste, size,

shape, fracture, fracture surface, texture, duration and rootlets. The study of petiole included

colour, odour, taste, size, shape, fracture, fracture surface and texture., leaf study included

colour, taste, odour, size, shape, fracture, texture, margin, venation, surface, apex, duration,

type, phyllotaxis, presence and absence of petiole and stipule (Trease and Evans, 1992).

QUANTITATIVE LEAF MICROSCOPY

Quantitative leaf microscopy to determine palisade ratio, stomata number, stomata index,

vein - islet number and vein let termination number were carried out on epidermal strip.

FLUORESCENCE ANALYSIS

To check the fluorescent property of plants were powdered leaf material obtained whish is

used for to analysis under ultra and organic reagents like alcohol, 50% nitric acid and

water.16 The florescence sulphuric acid, 10% sodium hydroxide, 50% behaviour was noted

as in table.

Solubility Behaviour of Leaf Extracts Of Plants

Solubility of plant leaf extracts has been observed with different solvents.VIZ non polar,

Polar, Polar acidic and polar basic.




PHYSICO-CHEMICAL PARAMETERS FOR THE STANDARDISATION OF CRUDE

DRUG

Determination of foreign matter:

50 g of drug sample examined was weighed and spread out a thin layer. The foreign matter

was detected by inspection with the unaided eye. Separated and weighed it and calculated the

percent present. Drug undertaken for further study were free from moulds, insects, animal

faecal matter and other contamination such as soil, stones and extraneous material. (A.O.A.C,

2000)

Determination of moisture content (Hot Air Oven Method):

To determine the amount of moisture (water drying off from the drug) for substance

appearing to contain water as the only volatile constituent, the procedure given below, was

used. 2.78 g of drug (without preliminary drying) after accurately weighing was placed in a

tare evaporating dish. After placing the above said amount of the drugs in the tared

evaporating dish, dried at 105oC for 5 hrs, and weighed, percentage was calculated with

reference to initial weight. (BP,1980).

Calculations

W1 - W2

% Moisture = ---------------------- x100 (A.O.A.C, 2000)

Wt of the sample

Determination of ASH (PASF,1987).

Determination of Total Ash: About 2.0 g of powder drug was incinerated in a redtop silica

dish at a temperature not exceeding 4500

C. until free carbon was left, cooled and final weight

was taken. The percentage of ash calculated with reference to the air-dried drug.

Determination of Acid Insoluble Ash: The ash obtained as above method was boiled for 5 minutes

with 25 ml of dilute hydrochloric acid and collected the insoluble matter on the ash-less filter




paper, washed with hot water and ignited to constant weight. The percentage of acid insoluble

ash with reference to the air dried drug was calculated.

Determination of water soluble Ash: The ash was boiled for 5 minutes with 25 ml of water,

collected insoluble matter on the ash-less filter paper, washed with hot water, and ignited for

a temperature not exceeding 4500C. The weight of the insoluble matter was subtracted from

the weight of the drug ash. The difference in weight represents the water soluble ash. Finally

percentage of water-soluble ash with reference to the air dried drug was calculated.

Procedure for Determination of Ash

The ash is an analytical term used for inorganic residue that remains after the choring of

organic matter present in the food. The ash may not be the same asthe organic matter present

in the original food. There may be loss of organic matters due to the chemical reactions

among the different constituents.

Equipment and glassware

Muffle furnace, silica dish, electric balance, desiccators, Bunsen burner

Ash contents were determined by heating sample at 550 oC in muffle furnace. The procedure

is given below.

Procedure

A clean flat bottom silica dish was taken and was held in a hot burner flame for 1 minute.

After that it was transferred to a desiccator then cooled weighed (W). A suitable quantity of

food sample was weighed in china dish (W1) and heated gently on the Bunsen burner. Then

the charred mass was transferred to a muffle furnace at 550oC.

The sample was heated until all the carbons were burned. The dish (containing sample) was

placed in desiccater, cooled weighed (W2).

Weight of the empty dish = W

Weight of the empty dish + sample = W1

Weight of the empty dish + ash = W2




Calculations

W1 - W2

% Ash = ---------------------- x 100 (A. O. A. C, 2000)

Wt of the sample

Determination of Extractable Matter

a. Method I. Hot Extraction: About 25.0 g accurately weighed air-dried drug coarse

powder was placed in thimble and refluxed with various organic solvents hexane,

chloroform, ethyl acetate and alcohol. After recovery solvents under vacuum and

drying in desiccators, the percentage extractable matter was calculated.

b. Method II. Cold Maceration: About 2.0 g of coarsely powdered air dried

material, was accurately weighed in a glass stopper conical flask and macerated

with 100 ml of solvent for 6 hrs shaking frequently, then allowed to stand for 18

hrs, filtered rapidly taking care not to lose solvent. The extracted matter was dried

at 1050C for 6 hrs, cooled in desiccators for 30 minutes and then weighed. The

percentage extractable matter was calculated. (WHO, 1980).

Determination of Swelling Index: (WHO, 1998)

About 1.0 g fine powder accurately weighed was taken into 25 ml of glass stoppered

measuring cylinder. The internal diameter of the cylinder was about 16 mm, the length of the

graduate portion about 125 mm, marked in 0.2 ml in division from 0 to 25 ml in upward

direction. 25 ml, of water was taken and the mixture thoroughly shaken every 10 minutes for

1 hrs. Kept for 3 hrs at room temperature and the volume in ml occupied by the plant




material, including any sticky mucilage was measured. The mean value of the individual

determination, related to 1.0g of plant material was calculated.

Determination of Foaming Index (WHO,1998 ).

About 1.0 g a coarse powder of drug was placed into a 500 ml conical flask containing 100

ml of boiling water. The moderate boiling was maintained for 30 minutes. Cooled and filtered

into a 100 ml volumetric flask and volume was made up to the mark with distill water.

The decoction was poured into 10 stoppered test-tubes (height 16cm, diameter 16 mm) in

successive portion of 1ml, 2ml, 3ml, etc. Up to 10ml, and adjusted the volume of the liquid in

each tube with water to 10ml. The tubes were stopper and shaken them in length wise motion

for 15 seconds, two shake per second. After 15 minutes and height of the foam was

measured. The results are assessed as follows.

If the height of the foam in every tube is less than 1 cm, the foaming index is less than

100.

If the height of the foam 1 cm is measured in any tube, the volume of the plant material

decoction in this tube (a) is used to determine the index. If this tube is the first or second

tube in a series, prepare an intermediate dilution in a similar manner to obtain a more

precise result.

If the height of the foam is more than 1 cm in every tube, the foaming index is over 1000.

In this case repeat the determination using a new series of dilution of the decoction in

order to obtain a result.

Foaming index =1000\a




Where a = the volume in ml of the decoction used for preparing dilution in the tube where

foaming to a height of 1cm is observed.

Determination of Heavy Metals

About 0.504 g air dried material was accurately weighed and placed in the test tubes for

predigestion the test tube was contain 5ml nitric acid. It was kept as such for a day.

Now digestion was performed by adding the nitric acid and perchloric acid in the ratio of

10:4 then 5ml sample for determination was made as 3.57 nitric acid 1.42 perchloric acid.

Now it kept for heating at 170-1800C for about 4 hrs. Now sample was cool and filtered and

volume was made up to 50 ml in volumetric flask with distilled water. With the help of

instrument named as I.C.P. [O.E.S.] Model optima 5300V heavy metals was determined.

Determination of heavy metals:

Reading __

Blank = y mg / L

Yx50/0.5 = Yx100 mg/kg (ppm). Where 0.5 is wt of drug taken.

Preliminary Screening of Phytochemicals

The preliminary phytochemical studies were performed for testing the different chemical

groups present the drugs 10% (w/v) solution of extract was taken unless otherwise mentioned

in the respective individual test. The chemical group test was performed and the results are

shown in tables. General screening of various extracts of the plant material was carried out

for qualitative determination of the groups of organic compounds present in them.

Alkaliods: Test for alkaloids are following:




Dragendroff’s test: Dissolve a few mg of alcoholic extract of the in 5 ml of

distilled water, add 2 M hydrochloric acid until an acid reaction occurs, then add 1

ml of Dragendroff‟s reagents, orange or orange-red ppt is produced immediately.

Hager’s test: to 1 ml of alcoholic extract of the drug taken in test tube, add a few

drops of Hager‟s reagent. Formation of yellow ppt confirms the presence of

alkaloids.

Wagner’s test: Acidify 1 ml of alcoholic extract of the drug with1.5% v/v of

hydrochloric acid and add a few drops of Wagner‟s reagent. A yellow or brown

ppt is formed.

Mayer’s reagent: Add a few drops of mayer‟s reagent to 1 ml of alcoholic

extracts of the drug. White or pale yellow ppt. is formed.

Crbohydrates: Test for crbohydratesare following:

1. Anthrone test: To 2 ml of anthrone test solution, add 0.5 ml of alcoholic extracts of

the drug. A green or blue color indicates the presence of carbohydrates.

2. Benedict’s test: To 0.5 ml of alcoholic extracts of the drug add 5 ml of Benedict‟s

solution and boil for 5 mins. Formation of a brick red coloured ppt is due to presence

of carbohydrates.

3. Fehling’s test: To 2 ml of alcoholic extracts of the drug add 1 ml of the mixture of

equal parts of fehling‟s solution „A‟ and „B‟ then boil the contents of the test tube for

few mins. A red or brick red ppt is formed.

4. Molisch’s test: In test tube containing 2 ml of alcoholic extracts of the drug add 2

drops of a freshly prepared 20% alcoholic solution of β napthol mix poured 2 ml of

conc sulphuric acid so as to form a layer below the mixture. Carbohydrates, if present,




produce a red- violet ring, which disappears on the addition of an excess of alkali

solution.

Flavonoids: Test for flavonoids are following:

1. Shinoda’s test: In a test tube containing 0.5 ml of alcoholic extract of the drug, add 5-

10 drops of dilute hydrochloric acid followed by a small piece of magnesium. In the

presence of flavonoids a pink, reddish pink or brown colour is produced.

Triterpenoids: Test for flavonoids are following:

1. Liebermann-Burchard’s test: Add 2 ml of acetic anhydride soloution to 1 ml of

alcoholic extracts drug in chloroform followed by 1 ml of con sulphuric acid. A violet

color coloured ring is formed indicating the presence of triterpenoids.

Saponins: In a test tube containing about 5 ml of an alcoholic extracts of the drug add a drop

of sodium bicarbonate solution, shake the mixture vigorously and leave for 3 mins.

Honeycomb like froth is formed.

Steroids: Test for steroids are following:

1. Liebermann-Burchard’s test: Add 2 ml of acetic anhydride soloution to 1 ml of

alcoholic extracts of the drug in chloroform followed by 1 ml of con sulphuric acid. A

greenish colour is developed which turns to blue.

2. Salkowaski reaction:Add 1ml of conc. Sulphuric acid to 2 ml of alcoholic extracts of

the drug carefully, from the side of the test tube. A red colour is produced in the

chloroform layer.

Tannins: Test for taninsare following:




1. To 1-2 ml of plant alcoholic extracts extract, add a few drops of 5% FeCl3 solution

was added. A green colour indicates the presence of gallotannins while brown colour

tannins.

Starch: Test for starch are following:

1. Dissolve 0.015g of iodine and 0.075g of potassium Iodide in 5 ml of distilled water

and add 2-3 ml of an alcoholic extracts of drug. A blue colour is produced.

Proteins: Test for proteins are following:

1. Biuret’s test: To 1 ml of alcoholic extracts extract of the drug add 5-8 drops of 10%

w/v sodium hydroxide solution followed by 1or 2 drops of 3% w/v copper sulphate

solution. A red or violet colour is obtained.

2. Millon’s test: Dissolve a small quantity of alcoholic extracts extract of the drug in 1

ml of distilled water and add 5-6 drops of millon‟s reagent. A white ppt is formed

which turns red on heating.

Pharmacological activity

Antibacterial Assay

Antibacterial assay was performed by agar well diffusion method (Atta-ur- Rehman et al,

2001). Methanolic extract and different portions dissolved in various organic solvent were

used in amount of three mg/mL of DMSO and pure compounds were used in dose of 1

mg/mL. Antibacterial activity was carried out against various human pathogens including

Escherichia coli (ATCC 25922), Bacillus subtilus (ATCC 6633),Shigellaflexenari (clinical

isolate), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa(ATCC 27853)

and Salmonella typhi (ATCC 19430).




In this bio assay, three types of media are required viz. solid medium (nutrient agar), semi-

solid medium (soft agar) and liquid medium (nutrient broth).

• Solid medium (Nutrient agar) Nutrient agar

28 g Distilled water 1 L (q.s)

Nutrient agar was dissolved in distilled water and volume was made up to 1 liter. It

was then placed in auto clave at 121 0C for 15 minutes. Media was then chilled to 40

0C and

poured in sterile petri dishes and media was then left to solidify at room temperature.

• Semi solid medium (Soft agar) Soft agar 0.8 g

Distilled water 100 mL (q.s)

Soft agar was dissolved in distilled water and volume was made up to 100 mL. It was

then dispensed in 7 mL quantity to screw capped test tubes. Then placed in auto clave at 121

0C for 15 minutes and then cooled.

Liquid medium (Nutrient broth) Nutrient broth 0.8 g

Distilled water 100 mL (q.s)

Nutrient broth was dissolved in distilled water and volume was made up to 100 mL.

Prepared broth was dispensed in 3 ml quantity to screw capped test tubes, which was placed

in auto clave at 121 0C for 15 minutes and then refrigerated.The culture of bacterial

organisms was maintained on stock culture agar. A colony of bacterial culture was implanted

in nutrient bisque and incubate at 37+1 0C for 24 hours. Next day soft agar was melted and

then cooled to 40 0C and then add 100pL of bacterial culture, shake it well and then pour it

on nutrient agar containing plate. Rotate the plate to make even distribution of culture and

allow solidifying the lawn. Hole (6 mm diameter) was made in medium in each plate using a

sterile sharp tool with centers at least 24 mm apart. Samples (3 mg/ml of DMSO) were then




added in respective well using sterilized dropping pipettes. DMSO was added other holes

and standard anti bacterial drug provided as -ve and +ve control, correspondingly. Plates

were then incubated at 37+1 0C for 24 hours. Activity was calculated by measuring diameter

of zones depicting resistance (mm). Antibacterial potential of sample was then determined as

per criteria mentioned in Table 2.5. Percent enlargement of resistance was calculated with

standard to the +ve control.

Table:2 Criteria for Determining Antibacterial Assay

Entry Diameter Activity

1 Below 9 mm No activity

2 9-12 mm Non-Significant

3 13-15 mm Low

4 16-18 mm Good

5 Above 18 mm Significant

RESULT

Macroscopic Features of Crataegus songrica fruit

The colour of Crataegus sangrica pome is reddish black with yellow pulp,

sparsely punctuate plobose, rearly ellipsid. 1.2-1. 6cm in diameter in fresh and

dark red in dry state. Odour and taste are the same in both fresh and dry forms.

Average length and diameter is 2.3 cm and 1.7 cm in fresh form and 2.0 cm and

1.5 cm in dry form, respectively. Surface is smooth and shiny in fresh form and

fluffy in dry form.




Quantitative Leaf Microscopy

Table 4 : Quantative Leaf Microscopy of Crataegus songrica

Parameter Range

Palisade Ratio 05-10

Stomatal Number Upper surface 0

Stomatal Number Lower surface 10-15

Stomatal Index Upper surface 05

Stomatal Index Lower surface 10.44-12.32

Vein islet number 12-15

Veinlet Termination Number 6-10

Fluorescence analysis

Table 5 : Fluorescence Behaviour of Leaves of Crataegus songrica

Treatment Day light UV light

Powder as

such

Green

Fluorescent green

Powder in distilled water

Light -Green

Fluorescent yellow-Green

Powder in absolute alcohol

dark-green

Fluorescent green

Powder in 10% Nitric acid brown

Fluorescent light brown

Table 3: Macroscopical features of the fruit of Crataegus songarica.

Colour Odour Size Taste Surface Material

Ave. Length Ave. Width

Reddish Pleasant 2.5cm 1.3cm Pleasant Smooth and Shiny

Fresh

Reddish black

Pleasant 2.1cm 1.5cm Pleasant Fluffy Dried




Powder in 50% Nitric acid

Light Yellow

Fluorescent brown

Powder in 50% Sulphuric acid

Dark Green

Fluorescent brown

Table : 6 Solubility Behaviour of Crataegus songrica of Extracts with Various

Solvents

Solvent Solubility

CPE CAC CME

NON POLAR

Heptane 1 2 0

Pet Ether 2 1 1

Cyclohexane 3 0 2

POLAR

Ethanol 1 0 3

Methanol 0 3 3

Water(dist) 3 2 3

POLAR ACIDIC

Formic acid 1 2 1

Acetic acid 2 2 0

Chloroform 0 2 3

POLAR BASIC

Pyridine 0 2 3

Dimethyl sulfoxide 2 1 0

Dimethylformamide 0 3 2




(0): No Presence, (1): 33% Present, (2): 66% Present, (3): 100% Present. CPE:

Petroleum ether extract, CAC: Acetone extract, CME: Methanol Extract. [C =

Crataegus songrica]

Table 7 : Physico-chemical parameters of Crataegus songrica

S.No. Parameters Observations

1.

PHYSICO-CHEMICAL

Foreign Organic matter No adulterants

Ash Values (% w/w)

(a) Total Ash Value 4.23 0.01

(b) Acid Insoluble Ash 3.33 0.01

(c) Water Soluble Ash 1.33 0.01

2.

Extractive Values (% w/w)

Cold percolation method

Petroleum ether(40-60) 3.03 0.13

Methanol (95%) 2.51 0.43

Methanol (50%) 1.20 0.12

Water 1.01 1.24

Soxhlet successive extraction

method

Hexane 3.11

Chlorofrom 4.30

Ethyl acetate 0.77

Methanol (95%) 2.14

Moisture content 9.03 0.02

Pharmacological




Swelling Index 0.26 0.15

Foaming index 136.45

Heavy metals & Minerals ( ppm)

Cadmium 3.6

Cooper 11.2

Iron 3.10

Chromium 15.4

Manganese 2.8

Nickel 10.7

Lead 3.6

Zinc 54.6

Calcium 13.02

Potassium 4.4

Sodium 5.6




Table 8: Phytochemical test of Crataegus songrica Leaves Extracts

Active constituents CCR CPE CAC CME

Alkalodids ++ ++ +++ ++

Flavonoids +++ +++ ++ ++

Saponins +++ ++ +++ ++

Tannins +++ ++ +++ ++

Steroids +++ ++ +++ +++

Cardiac Glycosides + ++ +++ +++

Proteins

++ ++ ++ ++

Resins _ _ _ _ _ _ _

Starch +++ +++ ++ ++

Triterpenoids -- _ _ _

Steroids _ _ _ _




(-) No presence, (+) Less presence, (++) Moderate Presence, (+++) High presence,

cCR: Crude powder, CPE: Petroleum ether extract, CAC: Acetone extract,

CME: Methanol Extract, Common in CPE and CME. the Constituents can be

further isolated and purified to find its potency for biological activities. [C=

Crataegus songrica ]

ANTIBACTERIAL ASSAY

The crude methanolic extract of Crataegus songrica and its different fractions thereof

were studied against human pathogens Escherichia coli ATCC 25922, Bacillus

subtilis ATCC 6633, Shigella flexeneri (clinical isolate), Staphylococcus aureus

ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Salmonella typhi ATCC

19430. They were remained on agar skeat 4o C. The strains were make actived at 37

o

C for twenty four hours on nutrient agar (NA) or Sabouraud glucose agar (SGA) for

bacteria, before screening.

Crude extract of Crataegus songrica and various segments revealed region of

inhibition in millimeters alongside various bacterial strain percent comparisons to

standard drugs i.e. Imipenum in table. The dose (3 mg/ml) was given in single

concentration. Crude methanolic extract (SC-1), n-hexane fraction (SC-2) and ethyl

acetate (SC-4) showed well to significant activity against Escherichia coli and the

zone of inhibition were 12 mm, 16 mm and 19 mm respectively. (Table ) and showed

no inhibitory activity against other test organisms. In other fractions, varied inhibitory

activity was observed against different bacterial strains. The n- hexane showed the

zone of inhibition only against Escherichia coli, with diameter of 16 mm. Chloroform

fraction (SC-3) inhibited the activity of Bacillus subtilis and Shigella flexenari 14 mm

and 18 mm each. While fraction ethyl acetate (SC-4) illustrated the inhibitory effect

only against Escherichia coli and Bacillus subtilis inhibited growth 19 mm and 15




mm respectively. Butanol fraction (SC-5) inhibited the growth of Bacillus subtilis and

S. aureus, Pseudomonas aeruginosa with the zone of inhibition 19 mm, 11 and

12mm respectively. While in case of aqueous fraction (SC-6), inhibitory activity was

observed against growth of Shigella flexenari and Pseudomonas aeruginosa by 11

mm and 10 mm each.. Crude extract of plant exhibited potential antibacterial activity

against E. coli

Table 9: Results of antibacterial assay of crude extract and fractions

Entry Sample E. coli B. subtilus S. flexenari S. aureus P. aeruginosa S. typhi

1 2 1 2 1 2 1 2 1 2 1 2

1 SC-1 12 58 9 39 15 53 - - - - - -

2 SC-2 16 58 - - - - - - - - - -

3 SC-3 - - 14 52 18 57 - - - - - -

4 SC-4 19 70 15 56 - - - - - - - -

5 SC5 - - 19 70 - - 11 40 12 - - -

6 SC-6 - - - - 11 52 - - 10 39 - -

7 STD 24 - 23 - 28 - 27 - 20 - 26 -

E. coli:Escherichia coli S. aureus: Staphylococcus

aureus

B. subtilus:Bacillus subtilus P. : Pseudomonas aeruginosa

S. flexenari: Shigella flexenari S. typhi : Salmonella typhi

SC-1: Crude Methanolic extract SC-5: n-BuOH fraction

SC-2: n-hexane fraction SC-6: H2O fraction

SC-3: CHCl3 fraction STD: Imipenum.

SC-4: EtOAc fraction

1) Zone of inhibition (mm) 2) Percent inhibition to standard drug




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