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INDEX – GJRMI - Volume 3, Issue 3, March 2014

MEDICINAL PLANTS RESEARCH

Bio-technology & Botany ISOLATION AND CHARACTERIZATION OF PENTADECANOIC ACID ETHYL ESTER FROM

THE METHANOLIC EXTRACT OF THE AERIAL PARTS OF ANISOMELES MALABARICA (L).

R.BR.

Ismail Shareef M, Leelavathi S, Gopinath S M 67–74

Bio-technology ANTIBACTERIAL ACTIVITY OF SOME INDIGENOUS MEDICINAL PLANTS

Jagan Mohan Reddy P, Ismail Shareef M, Gopinath S M, Dayananda K S, Ajay Mandal, Sreekanth B,

Purushotham K M

75–79

Review Article GENE SILENCING AND ITS APPLICATIONS IN CROP IMPROVEMENT & FUNCTIONAL

GENOMICS

Jemal Ali, Pagadala Vijaya Kumari 80–90

Review Article CALLICARPA MACROPHYLLA: A REVIEW OF ITS PHYTO-CHEMISTRY, PHARMACOLOGY,

FOLKLORE CLAIMS AND AYURVEDIC STUDIES

Pandey Ajay Shankar, Srivastava Bhavana, Wanjari Manish M, Pandey Narendra Kumar, Jadhav Ankush D

91–100

Short communication PHYTOCHEMICAL ANALYSIS OF SOME INDIGENOUS WOUND HEALING PLANTS

Jagan Mohan Reddy P, Ismail Shareef M, Gopinath S M, Dayananda K S, Ajay Mandal, Purushotham K M

101–104

INDIGENOUS MEDICINE

Ayurveda – Rasa Shastra A PHARMACEUTICAL APPROACH ON MANIKYA PISHTI TOWARDS STANDARDIZATION

Wavare Ramesh, Yadav Reena, Sheth Suchita, Sawant Ranjeet 105–111

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FLOWER OF CEIBA PENTANDRA (L.) GAERTN., OF THE

FAMILY MALVACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT,

KARNATAKA, INDIA

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

ISOLATION AND CHARACTERIZATION OF PENTADECANOIC ACID

ETHYL ESTER FROM THE METHANOLIC EXTRACT OF THE AERIAL

PARTS OF ANISOMELES MALABARICA (L). R.BR.

Ismail Shareef M

1*, Leelavathi S

2, Gopinath S M

3

1Assistant Professor, Department of Biotechnology, Acharya Institute of Technology, Soladevanahalli,

Acharya Post (O), Bangalore-560 107, Karnataka, India 2Professor, DOS in Botany, Manasagangotri, University of Mysore, Mysore-570 006, Karnataka, India

3Professor and Head, Department of Biotechnology, Acharya Institute of Technology, Soladevanahalli,

Acharya Post (O), Bangalore-560 107, Karnataka, India

*Corresponding Author: E-mail:ismailshareef@acharya.ac.in; Mobile: +91-9538833786, Fax No.:080-

23700242

Received: 09/02/2014; Revised: 22/02/2014; Accepted: 28/02/2014

ABSTRACT

Anisomeles malabarica (L). R.Br., (Lamiaceae) is distributed in major parts of India, especially

in South India it is known as a traditional medicinal plant reported to possess anti-spasmodic, anti-

inflammatory properties and is used in Rheumatoid arthritis. The preliminary phytochemical

investigation on the methanolic extract of the aerial parts of the plant revealed the presence of

carbohydrates, phytosterols and triterpenoids. The aim of the current study was to isolate and

characterize the bioactive compounds from the aerial parts of Anisomeles malabarica as the plant is

reported to possess potent anti-inflammatory properties and is also used in the treatment of

Rheumatism. For isolation purpose, the dried powder of was extracted with methanol using Soxhlet

apparatus continuously for 16 hours. The extract was dried under reduced pressure to evaporate the

solvent and the dried mass was taken for the isolation work. Pentadecanoic acid ethyl ester was

isolated by column chromatography from the methanolic extract of aerial parts of Anisomeles

malabarica. The structural elucidation of the isolated compound was on the basis of spectroscopic

analysis.

KEYWORDS: Anisomeles malabarica; pentadecanoic acid ethyl ester; rheumatoid arthritis;1H-

NMR; 13

C-NMR; LC-ESI-MS

Research Article

Cite this article:

Ismail Shareef. M, Leelavathi. S, Gopinath. S. M (2014),

ISOLATION AND CHARACTERIZATION OF PENTADECANOIC ACID ETHYL ESTER

FROM THE METHANOLIC EXTRACT OF THE AERIAL PARTS OF ANISOMELES

MALABARICA (L). R.BR., Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 67–74

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

INTRODUCTION

Today, Ayurvedic, Homeo and Unani

Physicians utilize numerous species of

medicinal plants (Mujumdar AM et al., 2000).

Many compounds used in today's medicine

have a complex structure and synthesizing

these bioactive compounds chemically at a low

price is not easy (Madhava C, 1998). The

increasing awareness about side effects of

drugs had made the western pharmaceutical

industries to turn towards the plant based

Indian and Chinese medicine (Balandrin MJ &

Klocke JA 1988). Anisolmeles malabarica (L).

R.Br., (Lamiaceae) is distributed in major parts

of India and especially in South India as a

traditional medicinal plant commonly known as

Peymarutti (Tamil), Gouzaban (Hindi),

Chodhara (Marathi), Karithumbi (Kannada)

and Malabar catmint (English)(Kritikar KR &

Basu BD, 1935). The herb is reported to

possess anti-spasmodic, anti-periodic properties

and used in Rheumatoid arthritis (Nadkarni

KM, 2006). It is used for the traditional

treatment of snakebite as antidote and plant

leaves are used as carminative, astringent,

stomachic, rheumatism and diaphoretic in

Coimbatore district and also used as dentifrice

to cure various problems (Kalyani K et al.,

1989). The methanolic extract of aerial parts of

Anisomeles malabarica (L). R.Br., (AmA)

produced significant anti-rheumatic activity in

a dose-dependent manner (200 mg/Kg and 400

mg/Kg body weight) to that of standard drug

indomethacin (10 mg/Kg). The extract

exhibited inhibitory effect in carrageenan

induced hind paw oedema in rats with all the

doses used when compared to the control

group. The data obtained indicate that the crude

extracts of the aerial parts of the plant AmA

possess potential anti-rheumatic activity by

supporting the folkloric usage of the plant to

treat various inflammatory conditions (Setty

AR, 2005).

AmA extract was tested for cytotoxicity in

RAW and L-929 cell lines and was found to be

non-toxic. Based on the results, non-toxic doses

of extracts were tested for their inhibitory

activity against LPS induced TNF-α

production. AmA showed better activity by

reducing the LPS induced TNF-α production by

38.75 % (Ismail SM et al., 2012). So based on

the various in-vivo and in-vitro studies

conducted, it can be concluded that the plant

AmA possesses potent immuno-modulatory

and anti-rheumatic properties. With the above

findings, the present work was carried to isolate

and characterize the bio-active phyto-

constituents present in AmA.

MATERIALS AND METHODS

Collection of plant material

Fresh leaves of Anisomeles malabarica free

from disease was collected from different

regions in & around Bangalore and were

authenticated by taxonomists & the Voucher

specimen was deposited in the department for

future reference.

a) Chemicals

Hexane, ethyl acetate, chloroform,

methanol and silica gel of mesh size 60–120

and 200–400 was purchased from Sd fine

chemicals, Mumbai, India. Column length was

100 cm and column diameter was 3 cm.

b) Extraction procedure

Dried powder of AmA was extracted with

methanol using Soxhlet extraction unit for 18

hours as per standard procedure (Mukherjee

PK, 2010) the extract was dried under reduced

pressure to evaporate the solvent and dried

mass (20 gm) was taken further for isolation

work.

Isolation of phytochemicals

I. Column Chromatography Purification Of

Methanolic Extract

a) Adsorption of sample on silica gel

The methanolic extract of AmA (dried mass ;15

gms) was adsorbed on dry silica and the

adsorbed sample was kept for complete drying

and later used for coloumn elution.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

b) Loading of column (wet packing)

Column was packed with silica gel slurry of

mesh size 60–120 with hexane. Column length

was 100 cm and diameter was 3 cm. On top of

silica bed activated sample was loaded and

cotton was placed on top of it to avoid any

disturbance to the sample bed.

c) Elution of the column

Initially Hexane solvent was eluted in small

quantity for correct distribution of activated

sample in the column and later eluted with two

solvent combinations with increasing order of

polarity.

Based on preliminary TLC observations,

column elution was started with hexane, ethyl

acetate and methanol combinations. Fractions

were collected in 50 ml portions.

Pattern of column elution:

1. Hexane

2. Hexane: Ethyl acetate: 7:3

3. Ethyl acetate

4. Ethyl acetate : Methanol : : 5 : 5

5. Methanol

From the above elution process, the

fractions were pooled as per their mobile phase.

From the above fractions, Ethyl acetate:

Methanol: 5:5 was further processed for

purification through column chromatography.

The other fractions were not used due to their

low yield.

II. Column Chromatography Purification Of

Ethyl Acetate: Methanol: : 5 : 5

a) Adsorption of sample on silica gel

The ethyl acetate : methanol : : 5: 5 fraction

(dried mass; 5–8 gms, brown colour powder)

was adsorbed on dry silica and the adsorbed

sample was kept for complete drying and later

used for column elution.

b) Loading of column (wet packing)

Column was packed with silica gel slurry of

mesh size 200–400 with chloroform. On top of

silica bed, activated sample was loaded and

cotton was placed on top of it to avoid any

disturbance to the sample bed.

c) Elution of the column

Initially chloroform was eluted in small

quantity for correct distribution of activated

sample in the column and later with two

solvent combinations with increasing order of

polarity.

Column elution was started with

chloroform and methanol combinations.

Fractions were collected in 15 ml portions.

d) Evaporation of fractions

Based on TLC profiles of the eluted

fractions, they were pooled and evaporated.

Chloroform: methanol : : 7 : 3 fractions were

dried under reduced pressure and then

subjected for preparative TLC for purification.

e) Preparative TLC of chloroform :

methanol : : 7 : 3 fractions

The dried fraction was purified by

preparing the TLC plate with silica gel G, a

mobile phase of Chloroform : methanol : : 7.5 :

3 was used for TLC. After TLC separation, the

plate was air dried and observed under UV

light. A yellow fluorescent band was scrapped

off and the band was eluted by mixing with

methanol and later centrifuged. The solvent

was collected, dried and later checked for

purity by TLC and the compound (under

investigation) was sent for spectral analysis i.e.,

IR, MASS, C13

NMR &1H NMR for structural

elucidation.

RESULTS

Spectral studies

The Compound in its ESI-MS (positive

mode) spectrum exhibits a peak at m/z 279 for

an ion [M+Na] +

suggesting a molecular weight

of 256.

In its 1H-NMR spectrum (Figure 1–4) it

showed peaks at δ 0.80 showing the presence

of methyl groups in the compound. The large

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

singlet at δ 1.15 and the signals at δ 1.80 were

due to the long chain methylene groups. The

signal at δ 1.90 is due to a methylene adjacent

to a carbonyl group. The signal at δ 3.40 may

be due to the protons attached to oxygen

function.

In the 13

C-NMR (Figure 5) the signals at δ

20.00 are due to methyl group, at δ 25, 28.00 to

31.00 are due to the methylene carbons. The

signal at δ 70.00 is due to the carbon attached

to the oxygen function. The signal at δ 172.00

confirms the presence of a carbonyl group. The

results of LC-ESI-MS is depicted in Figure 6.

Based on the above data the structure of the

compound is Pentadecanoic Acid Ethyl Ester

(Figure 7).

Figure 1.: 1H-NMR of bio-active compound from the aerial parts of Anisomeles malabarica

R.Br. at 279 MHz

Figure 2.: 1H-NMR of bio-active compound from the aerial parts of Anisomeles malabarica

R.Br.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Figure 3: 1H-NMR of bio-active compound from the aerial parts of Anisomeles malabarica

R.Br.

Figure 4.: 1H-NMR of bio-active compound from the aerial parts of Anisomeles malabarica

R.Br.

Figure 5.: 13

C-NMR of bio-active compound from the aerial parts of Anisomeles malabarica

R.Br

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Figure 6.: LC-ESI-MS of bio-active compound from the aerial parts of Anisomeles malabarica

R.Br.

Fig.7. Pentadecanoic Acid Ethyl Ester

CH3- (CH2)13- COO-CH2- CH3

Molecular formula: C17H34O2

Synonym: Ethyl N-Pentadecanoate

DISCUSSION

Anisomeles indica L., and Anisomeles

malabarica R. Br. Ex Sims, is found growing

wild in India. The chemical composition and

antibacterial activity of the essential oils from

Anisomeles indica L and A. malabarica were

investigated together. The aerial parts (Stem,

leaves, flowers and fruit) of hydrodistilled

essential oils were analyzed by gas

chromatography-mass spectrometry (GC-MS),

and antibacterial activity was individually

evaluated against Staphylococcus aureus,

Escherichia coli, Pseudomonas aeruginosa and

Bacillus pumilus using a paper disc diffusion

method. Collectively more than forty

compounds were identified in A. indica and A.

malabarica, representing 98.29–97.88% of the

total essential oil, respectively. The major

constituents of essential oils obtained from the

A. indica, were linalyl acetate (15.3%), and α-

thujone (11.9%). The most abundant

compounds in essential oils of A. malabarica,

were - α-thujone (17.6%), terpenyl acetate

(16.45%) and, δ-cadinene (11.5%). All tested

G+ ve& G-ve were inhibited by essential oil

samples. The GC-MS results of both plants

indicated the essential oil is rich in

monoterpenes and terpenoids, which have been

implicated antibacterial activity, comparable to

gentamycin, it was used as a positive probe.

The current findings also help to differentiate

the valuable Anisomeles species for phyto-

pharmaceuticals (Ushir Y & Patel K, 2011).

Seven fatty acids were identified from the

methanolic extract of Anisomeles indica L., and

Anisomeles malabarica L. R. Br. Ex Sims

aerial parts. The extracted fatty acids were

methyl-esterified and then analyzed by GC-

MS. The relative contents of the fatty acids

were calculated with Area normalization.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Seven fatty acids amounting to 77.778% in A.

indica and 68.027% in A. malabarica of the

total contents were detected. The major fatty

acids found in A. indica were palmitic acid

(23.334%), stearic acid (22.749%), lignoceric

acid (21.54%) and, in A. malabarica, palmitic

acid (35.252%), stearic acid (21.43%). The

results the content of fatty acids was abundant

in Anisomeles species, and it had a great range

of potential utilities and a prospect of

development in foods, medical and health care

(Ushir Y et al., 2011).

Based on the above findings by (Ushir Y et

al., 2011) it can be concluded that Anisomeles

malabarica R.Br. possesses potent bio-active

compounds, both reported in literature and yet

to be reported. So it was investigated to

evaluate the in vivo and in vitro activity of the

isolated compound namely, Pentadecanoic

Acid Ethyl Ester from the aerial parts of AmA.

CONCLUSION

The current investigation from the

methanolic extract of aerial parts of the plant

Anisomelos malabarica has revealed the

presence of Pentadecanoic Acid Ethyl Ester.

Also the plant is reported to posses’ inhibition

of invitro TNF-α production and possesses anti-

rheumatic and immuno-modulatory properties.

REFERENCES

Balandrin MJ, Klocke JA (1988): Medicinal

aromatic and industrial materials from

plants. Springer Verlag, Berlin,

Heidelberg.,4, 1–36.

Ganeshan S (2008): Traditional oral care

medicinal plants survey of Tamil Nadu.

Nat. Prod. Rad. 7, ,166–172.

Ismail SM, Leelavathi S, Anis AS (2012):

Inhibition on in vitro TNF-α production

by Anisomeles malabarica R.Br.

reinforces its anti-rheumatic and

immunomodulatory properties. Proc.

Natl. Acad. Sci., India, Sect. B Biol..83,

,187–19.

Ismail SM, Leelavathi S, Thara SKJ, Sampath

KKK (2012): Evaluation of in-vivo anti-

rheumatic activity of Anisomeles

malabarica R.Br.. Intl. J. Curr. Res.

Rev..4, ,118–125 .

Kalyani K, Lakshmanan KK, Viswanathan MB

(1989): Medico-Botanical Survey of

plants in Marudhamalai Hills of

Coimbatore district, Tamil Nadu. J.

Swamy Bot. Club.6, ,89–96.

Kritikar KR, Basu BD (1935): Indian

Medicinal plants. 2nd Edition,

International Book Distributor,

Dehradun, India.,2011–2012.

Madhava C (1998).: Pharmacognostic studies

of Plumbago Zeylanica L. (chitreka,

chitramulamu), dissertation, post

graduate diploma in plantdrugs, S.V.

University, Tirupati, India

Mujumdar AM, Naik DG, Dange CN,

Puntambekar HM (2000): Anti-

inflammatory activity of Curcuma

amadaRoxb. in albino rats. J.

pharmacol.,32, 375–377.

Mukherjee PK (2010): Quality control of

herbal drugs. 1st edition.Business

horizons pharmaceutical publishers,

New Delhi, India.,184–191.

Nadkarni KM (2006): Indian MetriaMedica. 3rd

Edition, Popular Prakashan Pvt., Ltd.,

Mumbai, India.,114–115.

Narayana R, Thammanna K. (1987): Medicinal

plants of Tirumala hills, department of

garden, tirumalatirupatidevasthanams,

Tirupati, India.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Perumalswamy R, Maung TM,

Gopalakrishnakone P, Ignacimuthu S

(2008): Ethno Botanical survey of folk

plantsfor the treatment of Snakebites

inSouthern part of Tamil Nadu, India.J

Ethnopharmacol.,115, 302–312.

Ushir Y, Patel K, Sheth N (2011): Analysis of

fatty acid in Anisomelesspecies by gas

chromatography-mass spectrometry.

Pharmacog. J. 3, 44–47.

Ushir Y, Patel K (2011): Chemical composition

and antibacterial activity of essential oil

from Anisomeles species grown in

India. Pharmacog. J. 2, 55–59.

Source of Support: NIL Conflict of Interest: None Declared

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 75–79

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

ANTIBACTERIAL ACTIVITY OF SOME INDIGENOUS MEDICINAL

PLANTS

Jagan Mohan Reddy P1, Ismail Shareef M

2*, Gopinath S M

3, Dayananda K S

4,

Ajay Mandal5, Sreekanth B

6, Purushotham K M

7

1,2,3,4

Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore- 560 107,

Karnataka, India. 5Research Scholar, Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore-

560 107, Karnataka, India. 6Department of Chemical Engineering, SDMCET, Dharward-580 002, Karnataka, India.

7 Institute of Animal health & Veterinary Biological, Hebbal, Bangalore, Karnataka 560024, India

*Corresponding author: ismailshareef@acharya.ac.in; Mobile: +91 9916836390

Received: 03/02/2014; Revised: 25/02/2014; Accepted: 05/03/2014

ABSTRACT

The evolution and spread of antibiotic resistance, as well as the evolution of new strains of

disease causing agents, is of great concern to the global health community. Our ability to effectively

treat disease is dependent on the development of new pharmaceuticals, and one potential source of

novel drugs is traditional medicine. The present study explores the antibacterial properties of plants

used in traditional medicine. We tested the hypothesis of 95% ethanol and showed the inhibitory

effect against gram-positive and gram-negative bacteria. The extracted medicinal plants used to treat

symptoms often caused by bacterial infection would show antibacterial properties in laboratory

assays, and that these extracts would be more effective against moderately virulent bacteria than less

virulent bacteria. The striking feature in most of the aromatic plants enlisted in the indigenous system

of medicine is attributed to their essential oil contents in them which exert their marked therapeutic

potency. The large volume of work accumulated so far, obviously justifies the importance of

medicinal activity of the aromatic plants; the antimicrobial activity being credited to their essential

oil fraction only.

KEYWORDS: Medicinal plants, antibacterial activity, crude ethanolic extract, Blumea lacera

Research Article

Cite this article:

Jagan Mohan Reddy P, Ismail Shareef M, Gopinath S M,

Dayananda K S, Ajay Mandal, Sreekanth B, Purushotham K M (2014),

ANTIBACTERIAL ACTIVITY OF SOME INDIGENOUS MEDICINAL PLANTS,

Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 75–79

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 75–79

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

INTRODUCTION

Indigenous plants, widely used for folk

medicinal purposes, are numerous and diverse.

In India, about 500 plant species have been

identified as medicinal plants because of their

therapeutic properties In the meantime, a large

number of industries (400 herbal factories)

have been established in this country for

producing Ayurvedic and Unani medicines.

Medicinal use of plants is the oldest form of

healthcare known to mankind. It has been

estimated that India has a market of about

rupees 100 crore worth herbal products

annually (Tyler V, 1994). The total size of the

medicinal plant market at wholesale prices was

estimated at some US$ 14 million per annum,

which corresponds to 17000 tonnes of products.

Local supply accounts for about 70 % by

volume and 40 % as value. It has been

estimated that 12,500 tonnes of dried medicinal

plant produce is sold in India. These products

are approximately worth Rs 255 per million to

rural economy. At the factory level, 5000

tonnes of imported medicinal plants cost

around 480 million rupees (Gopinath.S.M et

al., 2012). Although modern medicinal science

has developed to a greater extent, many rural

people of India still depend on plant products

and herbal remedies for treating their ailments.

Being naturally gifted by a suitable tropical

climate and fertile soil, India possesses a rich

flora of tropical plants. About 5000 species of

Phanerogams and Pteridophytes grow in its

forests, jungles, wastelands and roadsides as

indigenous, naturalized and cultivated plants.

Out of them more than a thousand have been

claimed to possess medicinal and/ or poisonous

properties, of which 546 have recently been

enumerated with their medicinal properties and

therapeutic uses (Annapoorani Chockalingam

et al., 2007). In addition of possessing various

other medicinal properties, 257 of these

medicinal plants have been identified as

efficacious remedies for diarrhoeal disease and

47 for diabetes. A large number of plants in

different locations around the world have been

extracted and semi-purified to investigate

individually their antimicrobial activity

(Dranghon, 2004). The aim of work was to

collect these indigenous plants to investigate

antibacterial activity of the leaves and

identification of particular bioactive compound

as potential drug for the medicinal applications.

MATERIAL AND METHODS

Plant materials

The Plants Callicarapa arborea,

Lanneacorom andelica, Ficus racemosa,

Streblus asper, Lawsonia inermis, Holarrhena

antidysenterica, Mentha arvensis, Enhydra

fluctuans, Blumea lacera, Glinus oppositifolius,

Chenopodium album, Hemidesmus indicus,

Coccinea cordifolia, Cuscuta reflexa, Capparis

zeylanica and Kalanchoe pinnata were

collected from in and around Bangalore district,

Karnataka, India which were used for the

treatment of various infectious diseases by

people. The plants were authenticated by

taxonomists & voucher specimen was stored in

the department for future reference. The plant

materials were oven-dried at 40ºC and then

ground into coarse powder.

Extraction

20 g of coarse powder of all plant

materials Callicarapa arborea, Lannea

coromandelica, Ficus recemosa, Streblus

asper, Lawsonia inermis, Holarrhena

antidysenterica, Mentha arvensis, Enhydra

fluctuans, Blumea lacera, Glinus oppositifolius,

Chenopodium album, Hemidesmus indicus,

Coccinea cordifolia, Cuscuta reflexa, Capparis

zeylanica and Kalanchoe pinnata were

extracted with ethanol for a week at room

temperature. The extracts were then filtered off

through Whatman filter paper number-1 and

the solvent was removed under vacuum at 30ºC

until dry mass were obtained by Buchirota

vapour.

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Table 1. Plants details collected with the name of locality

Serial

No.

Name of plant Plant parts Family locality

1 Callicarapa arborea Leaf Verbenaceae Kolar, Karnataka, India.

2 Lannea coromandelica Leaf Anacardiaceae Kolar, Karnataka, India.

3 Ficus racemosa Leaf Moraceae Kolar, Karnataka, India.

4 Streblus asper Leaf Moraceae Kolar, Karnataka, India.

5 Lawsonia inermis Leaf Lythraceae Kolar, Karnataka, India.

6 Holarrhena

antidysenterica

Leaf Apocynaceae Kolar, Karnataka, India.

7 Mentha arvensis Aerial parts with

flowers

Lamiaceae Mallur, Karnataka, India.

8 Enhydra fluctuans Leaf and root Asteraceae Mallur, Karnataka, India.

9 Blumea lacera Aerial parts Asteraceae Mallur, Karnataka, India.

10 Glinus oppositifolius Leaf Molluginaceae Mallur, Karnataka, India.

11 Chenopodium album Leaf Chenopodiaceae Mallur, Karnataka, India.

12 Hemidesmus indicus Root Periplocaceae Mallur, Karnataka, India.

13 Coccinea cordifolia Leaf and rhizome Cucurbitaceae Mallur, Karnataka, India.

14 Cuscuta reflexa Leaf Convolvulaceae Kolar, Karnataka, India.

15 Capparis zeylanica Leaf Capparidaceae Mallur, Karnataka, India.

16 Kalanchoe pinnata Leaf Crasselaceae Mallur, Karnataka, India.

Antibacterial Activity Test

Microorganisms

The bacteria used included: Shigella

dysenteriae, Salmonella typhi, Salmonella

paratyphi, Bacillus cerus, Bacillus subtilis,

Escherichia coli, Pseudomonas aeruginosa,

Staphylococcus aureus, Vibrio cholera and

Bacillus megaterium. Bacterial cultures were

maintained on Nutrient agar media. All cultures

were sub cultured monthly and subsequently

stored at 4°C (Gopinath. S. M., 2011).

Screening for Antimicrobial Activities

Disc diffusion method (Gopinath. S. M.,

2011) was used to test the antimicrobial activity

of the extractives against ten bacteria. Dried

and sterilized filter paper discs (6 mm

diameter) were then impregnated with known

amount of the test substances dissolved in

ethanol (40 μg/ml) using micropipette and the

residual solvents were completely evaporated.

Discs containing the test material (20μg/disc)

were placed on nutrient agar medium uniformly

seeded with the test microorganisms. Standard

disc of kanamycin (30μg/disc) and blank discs

(impregnated with solvents followed by

evaporation) were used as positive and negative

control, respectively. These plates were then

kept at low temperature (4°C) for 24 hours to

allow maximum diffusion of test samples. The

plates were then incubated at37°C for 24 hours

to allow maximum growth of the organisms.

The test materials having antimicrobial activity

inhibited the growth of the microorganisms and

a clear, distinct zone of inhibition was

visualized surrounding the disc. The

antimicrobial activity of the test agents was

determined by measuring the diameter of zone

of inhibition in millimetre. The experiment was

carried out in triplicate and the average zone of

inhibition was calculated (Ahmed, A.M.A.,

Rahman, M.S., and Anwar, M.N. 1999)

RESULT AND DISCUSSION

During this study, 16 plants were selected

which were used for the treatment of infectious

diseases by peoples. The aforesaid are

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summarized in Table1. From primary

Screening, it was found that only 5 plants

exhibited antibacterial activity against more

than 6 test organisms (Table 2). Blumea lacera

showed moderate to good (11–23 mm in

diameter zone of inhibition) antibacterial

activity against all organisms except

Pseudomonas aureuginosa. Enhydrus fluctuans

and Mentha arvensis showed moderate activity

(7–10 mm in diameter) against all test

organisms except Bacillus cereus in case of

Mentha arvensis. Salmonella paratyphi,

Bacillus megaterium, chenopodium album and

Glinus oppositifolius showed comparatively

better activity (9–13mm in diameter). The

largest zone of inhibition (23 mm in diameter)

was recorded against Bacillus cereus with the

leaf of Blumea lacera.

Table 2. Antibacterial activity of alcoholic extracts of five plants

Average zone of inhibition in diameter (mm)

Bacterial test organisms

Nam

e of

pla

nts

Part

s of

pla

nt

use

d

Sh

. dys

ente

riae

Salm

on

ella

typ

hi

Pse

udom

on

as

sp

Baci

llu

s ce

reu

s

Salm

on

ella

para

typh

i

Vib

rio c

hole

rae

Baci

llu

s m

egate

riu

m

E.c

oli

Baci

llu

s su

bti

lis

Sta

ph

yloco

ccu

s au

reu

s

Blumea lacera Aerial parts 11 11 _ 23 11 9 19 13 21 15

Chenopodium album Leaf 13 _ 9 10 _ 11 9 13 9 11

Enhydra fluctuans Leaf and rhizome 9 7 10 11 10 10 9 8

Mentha arvensis Aerial parts with flowers 7 9 10 _ 7 10 9 7 10 8

Glinus oppositifolius Leaf 13 9 9 10 11 11 11 9 _ 13

Similar antibacterial activity of other plant

extracts has been reported previously

(Harborne JB., 1973; Jamine.R.Daisy, 2007;

Gopinath S M. et al., 2012). The present

investigation ensures that crude extracts of 5

plants contain antibacterial properties, which

are used by local people. During the study it

was observed that gram-positive bacteria are

more sensitive than gram negative bacteria.

From our results, it appeared that the crude

extracts of some traditional medicinal plants

has good inhibitory effect against selected

bacterial strains. Among the medicinal plants

tested herein, Blumea lacera showed most

promising antibacterial properties indicating

the potential for discovery of antibacterial

principles

CONCLUSION

It was found that out of 12 different plant

materials only 5 plants exhibited antibacterial

activity against more than 6 test organisms

have potential application as therapeutic agents

and bioactive compounds. Plant extracts that

exhibits the exploitation of the pharmacological

properties involves further investigation of the

active ingredients of an implementation

technique of extraction, purification,

separation, crystallization and identification

and can further use as potential drug.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 75–79

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REFERENCES

Ahmed, A.M.A., Rahman, M.S., and Anwar,

M.N. (1999). Antimicrobial activity of

extracts and crude alkaloids

ofPolyalthia longi folia (Sonn.) Thw.

Stem bark. The Chittagong University

Journal of Science, 23(10): 53–56

Alam, M.K., Chowdhury, J.U. and Hasan,

M.A. (1996). Some folk formularies

from Bangladesh. Bangladesh Journal

of Life Science, 8(1): 49–63

Annapoorani Chockalingam, Dante S.

Zarlenga, Douglas and D. Bannerman.

(2007). Antimicrobial activity of bovine

bactericidal permeability-increasing

protein-derived peptides against gram-

negative bacteria isolated from the milk

of cows with clinical mastitis. American

Journal of Veterinary Research. 68 (11):

pp: 1151 –59

Bauer, A.W., Kirby, M.M., Sherris, J.C. and

Turck, M. (1966). Antibiotic

susceptibility testing by a standardized

single disc method. American Journal

of Clinical Pathology, 45: 493–496

Bartner, A. and Grein, E.(1994). Antibacterial

activity of plant extracts used externally

in traditional medicine. Journal of

Ethnopharmacology, 44:35–40

Dranghon, F.A. (2004). Use of botanicals as

biopreservatives in foods. Food

Technol.58:20–28.

Ghani, A. (2000).Vheshaja Oshudh (Herbal

Medicine), Bangla Academy Dhaka,

Bangladesh.

Gopinath, S.M., Suneetha, T. B., Singh, Sumer

(2012). Evaluation of effect of

methanolic and aqueous extracts of

Punica granatum against bacterial

pathogens causing bovine mastitis,

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Indigen.Med., Vol.1(10): 496–502.

Gopinath, S.M., Suneetha, T. B., Mruganka,

V.D. (2011), chemical prophylaxis and

antibacterial activity of Methanolic and

aqueous extracts of some medicinal

plants against bovine mastitis,

International journal of Advanced

Biological Research., Vol.1 (1): 93–95. Harborne JB (1973). In Phytochemical

Methods. London: Chapman and Hall;.

Methods of Plant Analysis; p. 132.

Jasmine, R and P.Daisy, (2007). Effect of crude

extract and fractions from Elephantopus

scaber on hyperglycemia in streptozotocin-

diabetc rats.Int.J.Biol.Chem., 1:111–116.

Rahman, M.S., Begum, J., Chowdhury, J.U.

and Anwar, M.N.(1998). Antimicrobial

activity of Holarrhena antidysenterica

against Salmonella typhi. The

Chittagong University Journal of

Science, 22(1): 111–112

Rojas, A., Hernandez, L., Pereda-Miranda, R.

and Mata, R.(1992).Screening for

antimicrobial activity of crude drug

extracts and pure natural products from

Mexican medicinal plants.Journal of

Ethnopharmacology, 35: 275–283

Yusuf, M., Chowdhury, J.U., Wahab, M.A. and

Begum, J. (1994). Medicinal plants of

Bangladesh. Premier enterprise,

Chittagong pp. 8–149

Source of Support: NIL Conflict of Interest: None Declared

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Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

GENE SILENCING AND ITS APPLICATIONS IN CROP IMPROVEMENT &

FUNCTIONAL GENOMICS

Jemal Ali 1, Pagadala Vijaya Kumari

2*

1Department of Biotechnology, Gondar University, GONDAR, Post Box 196 – Ethiopia.

2Department of Biology, AMBO University, AMBO , Post Box 19 – Ethiopia.

*Corresponding Author: E-mail: vkpagadala@rocketmail.com;

Received: 11/02/2014; Revised: 27/02/2014; Accepted: 01/03/2014

ABSTRACT

Gene silencing is a technique used to turn down or switch off the activity of a gene. The

mechanism of switching off a gene could be used for different purposes. Therefore the present work

was initiated with the objective to introduce some gene silencing mechanisms occurring in plants and

to introduce the applications of gene silencing in crop improvement and functional genomics. In this

review work three different articles were reviewed; In the first article: Maize dwarf mosaic virus

(MDMV), a widespread pathogenic virus of maize is targeted to be suppressed by the RNAi using a

hpRNA expression vector containing a sense arm and an antisense arm of 150 bp sequence and the

second deals with suppression of α-zein protein subfamilies of either the 19- or 22-kD using RNAi

technology to obtain moderately increased total lysine content and the third deals with inactivation of

the 587bp sized Bcp1 gene of Arabidopsis thaliana which is responsible for fertile pollen

development. The results of suppression of MDMV showed that the disease index of the transgenic

plant line h2 had no significant difference from the highly resistant control line H9-21. The

suppression of α-zein protein subfamilies displayed a reduced accumulation of both the 19- and 22-

kD α -zeins from the 26 of the 29 events for those transformed with pMON73567 construct and total

amino acid analysis showed that there is an increase in the lysine content. Finally, for the last article,

49 out of 58 Arabidopsis lines transformed with RNAi construct containing Bcp1 sequences were

male sterile. In conclusion, gene silencing is a promising science for identification of unknown genes

and for the treatment of different diseases.

KEY WORDS: RNA interference, Maize dwarf mosaic virus, Male sterility, α -zeins

Review Article

Cite this article:

Jemal Ali, Pagadala Vijaya Kumari., (2014), GENE SILENCING AND ITS

APPLICATIONS IN CROP IMPROVEMENT & FUNCTIONAL GENOMICS,

Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 80–90

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INTRODUCTION

Most plants and animal cells have unusual

forms of RNA that can naturally inactivate

gene expression. Understanding how these

RNA molecules function and how to

manipulate these novel RNA molecules is a

challenge but very essential for new discoveries

(Scanlon, 2004). Gene silencing is a technique

used to turn down or switch off the activity of

genes. It is a powerful technology for gene

discovery and determining gene function in

humans, animals, and plants. In plants RNA

mediated gene silencing, especially the

homology dependant gene silencing (HDGS) is

used to develop new crop varieties and holds

tremendous promise as a therapeutic agent to

control different diseases (Charagonda, 2008).

Now a day, the exploitation of genomic

sequences of animals, plants, fungi and

microorganisms had given us many

opportunities in predicting the genes contained

in an organism. However, this wealth of genetic

information opens new challenges in

deciphering the complete list of protein-

encoding genes (Scanlon, 2004). This is due to

a transcriptional event such as RNA splicing

and post-translational modifications that make

it difficult to predict the exact number of genes

or proteins. With this degree of complexity,

monitoring the entire proteome expression

levels as a means of elucidating the functions

of genes and proteins became significant

challenges for the biotech industry (Myers and

Ferrell, 2005).

Prior to the discovery of RNA mediated

gene silencing, scientists applied various

methods such as insertion of T-DNA elements,

and transposons, treatment with mutagens and

irradiation to generate loss-of-function

mutations in studying the functions of a gene or

gene family of interest in an organism. Apart

from being time-consuming, the above methods

did not always work satisfactorily. For

instance, transposons and T-DNA elements

were found to occasionally insert randomly in

the genome resulting in highly variable gene

expression. Furthermore, in many instances the

particular phenotype or a trait could not be

correlated with the function of a gene of

interest (Williams et al., 2004). As a result of

this the discovery of gene silencing is of

paramount importance for various areas of

genetic studies such as functional genomics,

drug discovery, for the development of plants

with virus resistance and high nutritional value.

Therefore, the present work was initiated with

the objective to introduce some gene silencing

mechanisms occurring in plants and to

introduce the applications of gene silencing in

crop improvement and functional genomics.

RNA Interference-Based Transgenic Maize

Resistant to Maize Dwarf Mosaic Virus by

Zhang et al, 2010

Maize dwarf mosaic virus (MDMV) is a

widespread, worldwide pathogenic virus that

causes chlorosis, stunting, and serious loss of

yield in maize (Zea mays). Strategies for the

management of viral diseases normally include

control of the vector population using

insecticides, adjusting seedtime, and the use of

virus-free propagating material and appropriate

cultural practices. However, these methods are

not effective because of the non-persistent

model of virus transmission by aphids. The use

of resistant germplasm is an environmentally

sustainable and effective way for controlling

viral diseases of maize but the conventional

breeding method is time consuming because

identification and development of resistant

inbred lines or hybrids needs lots of time to

respond, furthermore there will be a year-to-

year inconsistency of viral disease pressure.

Therefore RNAi triggered by hairpin RNA

(hpRNA) transcribed from the transgenic

inverted-repeat sequence provides a straight-

forward natural defense mechanism against

invasive viruses and has been proved to be

more efficient. This study was done with the

objective of: suppressing the P1 protein

(protease) of maize dwarf mosaic virus

(MDMV) using the inverted repeat of sense and

antisense arms of p1 protein.

1. MATERIALS AND METHODS

Target selection and the gene construct: A

150-bp specific fragment of the P1 protein

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(protease) gene was selected from the genomic

sequence of MDMV and amplified by PCR.

The amplified fragments were then inserted

into the pSK vector in sense and antisense

orientations, separated by introns of the maize

actin gene, to construct an hpRNA expression

vector. Then the hpRNA expression construct

was cloned into the plant expression vector

pCAMBIA1300 under the control of the

ubiquitin promoter and nos terminator,

generating the hpRNA expression vector

pASP150 (fig. 1). For selection, the

hygromycinphosphotransferase gene conferring

hygromycin B resistance was used under the

control of the cauliflower mosaic virus 35S

promoter (P-35S) and 35S terminator (T-35S).

Finally the hpRNA expression vector,

pASP150 was introduced by electroporation

into the disarmed Agrobacterium tumefaciens

strain EHA105.

LB left border, RB right border,

Hpthygromycinphosphotransferase gene, P-35S

cauliflower mosaic virus 35S promoter, T-35S

cauliflower mosaic virus 35S terminator, P-Ubi

ubiquitin promoter, T-nos terminator of

nopaline synthase, intron of maize actin gene,

P150 150-bp fragment of MDMV P1 protein

(protease) gene.

Figure 1: The T-DNA regions of hpRNA expression vector pASP50.

Transformation and Selection: Once the

Agrobacterium is ready, it was cultured for

some time, Maize immature embryos cultured

in darkness for callus isolation. Embryonic calli

were screened, sub cultured, and transformed

by co-cultivation with the transformed

Agrobacterium. After cultivation for 7 days, the

calli were transferred to selection medium

containing hygromycin B and cultured for 20

days. Then, the screened resistant calli were

transferred to regeneration medium. Plantlets

with fully grown shoots and roots were

transplanted to greenhouse and allowed to

acclimatize for 2–3 weeks in greenhouse, and

then transplanted into the field for self

pollination to produce T0 seeds.

After planting in the field, a leaf blade was

collected from each regenerated plant and used

for DNA extraction. And PCR was made to

amplify 150-bp fragment of the P1 gene for

screening the putative transgenic plants.

Southern blotting was also made with the

genomic DNA extracted from the leaf samples

of 13 T1 lines derived from the fertile T0 plants

positive in PCR to identify the stable

integration of the transgene into the maize

genome and to evaluate the transgene copy

number.

The transgenic T2 plant lines derived from

the T1 lines positive in Southern blotting,

together with non-transformed controls of a

highly resistant line, a highly susceptible line ,

and the non transformed control line 18-599

were grown in the field and mechanical

inoculation was done twice within 1 week at

the three- to four-leaf stage, using inoculums

prepared from the leaf sap of maize plants

systemically infected with MDMV. The disease

incidence and symptom scale were investigated

at the adult stage according to the standard

proposed by Lin(1989). The disease index was

calculated as:

The virus titer of the transformed lines was

quantified by DAS-ELISA using an MDMV

DAS ELISA kit (AC Diagnostics Inc., USA)

In most cereal grains, including maize,

lysine is usually the most limiting essential

amino acid for animal nutrition and lysine

supplementation is required when corn is used

as a major component of feed. Therefore, the

need for development of high lysine corn lines

is clear. During the 1960s and the 1970s, many

naturally occurring high lysine maize mutants

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were identified. Among them, opaque-2 (o2)

and floury-2 (fl2) are the two best-

characterized mutants. It is believed that the

higher lysine content in these two mutants is

caused by a general decrease in zein synthesis

and an increase in accumulation of non-zein

proteins. Zeins, which make up about 60% of

total maize seed proteins, are rich in glutamine,

proline, alanine, and leucine, but are almost

completely devoid of lysine and tryptophan.

Recently through recombinant DNA

technology, transgenic maize plants were

produced with reduced α-zein protein

subfamilies of either the 19- or 22-kD but with

moderately increased total lysine content.

These two subfamilies of α -zeins, which

comprise 40% and 20% of the total zein

fraction respectively, are almost entirely devoid

of lysine and tryptophan residues. It is possible

that by reducing both subfamilies of α-zeins,

the lysine content could be further enhanced.

This approach relies on the efficient and

concurrent suppression of multiple genes that

encode α-zeins by RNAi technology.

2. MATERIALS AND METHODS –

High lysine and high tryptophan transgenic

maize resulting from the reduction of both

19- and 22-kD α-zeins, by Huang et al., 2006

In most cereal grains, including maize,

lysine is usually the most limiting essential

amino acid for animal nutrition; Opaque-2 (o2)

and floury-2 (fl2) are the two best-characterized

mutants due in zein synthesis and an increase in

accumulation of non-zein proteins. By

Recombinant DNA technology, transgenic

maize plants were produced with reduced α-

zein protein with moderate increase of Lysine

content. Thus this approach relies on the

efficient and concurrent suppression of multiple

genes that encode α-zein by RNAi technology

(Mertz et al., 1964).

Target selection and the gene construct: Two

different constructs (pMON73567 and

pMON73566) were used in this research and

ligated on Agrobacterium based vector (fig. 2)

for making the pMON73567 construct, four

segments of 19-, 22-kD α-zeins were ligated.

The first two segments were partial cDNA

sequences of 19- & 22-kD α-zeins in antisense

orientation, which paired with the later two

segments in sense orientation driven by the

maize gamma zein endosperm specific

promoter, Z27. The 3’ untranslated region of

pea RbcS2 gene with a 643-bp fragment is used

as a terminator (E9) terminator. The selectable

marker, epsps-cp4, for glyphosate resistance is

ligated next to the right border on the

Agrobacterium based vector.

Figure 2: The T-DNA regions of the plasmids used in the transformation maize.

The plasmids contain two different designs

of α-zein reduction cassettes next to the

selectable marker, epsps-cp4, which confers the

glyphosate resistance. The designations of the

genetic elements are as follows: RB, right

border; LB, left border; pZ27, the promoter of

27-kD γ-zein gene; 19AS, a partial 19-kD α-

zein gene in antisense orientation; 22AS, a

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partial 22-kD α-zein gene in antisense

orientation; 22S, a partial 22-kD α-zein gene in

sense orientation; 19S, a partial 19-kD α-zein

gene in sense orientation; tE9, the 3¢ region of

pea RbcS2 gene.

In pMON73566, the third segment

corresponding to the sense region of 22-kD α-

zein gene was removed, leaving only the 19-kD

α-zein sequence to form dsRNA and the 22-kD

α-zein sequence as the unpaired loop region.

The selectable marker gene (epsps-cp4) and the

rest of the genetic elements in the binary vector

were similar to those previously described.

Once the constructs were made, the vectors

with the construct were electroporated into

Agrobacterium tumefaciens ABI strain and

introduced into maize embryos by

Agrobacterium mediated transformation.

To determine the transgene copy number of

resulting R0 plants, genomic DNA was

extracted from leaves, copy number determined

and single copy R0 transgenic plants were self-

pollinated to produce R1 seeds. Total RNA was

isolated from developing kernels (25 days after

pollination) and northern hybridization was

done with PCR labeled DNA fragments

corresponding to the coding sequences of 19-,

22-kD α-zeins and 27-kD γ as the probes.

Special kind of mass spectrometry called

Matrix-assisted laser desorption ionization

time-of- flight mass spectrometry (MALDI-

TOF MS) method was used for analyzing zein

proteins. To obtain free amino acid

accumulation data, extracts of ground meal

samples were filtered, diluted and analyzed by

o- phthaldialdehyde (OPA) derivatization. OPA

derivatized samples were injected onto a C18,

reverse phase HPLC column, detection with an

Agilent HPLC (model 1100) equipped with a

fluorescence detector was used. For total

tryptophan analysis, base hydrolysis was

performed prior to OPA derivatization (Huang

et al., 2006).

Male sterile mutants are of agricultural

importance for the production of hybrids and to

prevent the spread of foreign gene products via

pollen in transgenics. The emasculation of

anthers is very labor intensive, and makes it

very difficult to manage on a large scale. There

are also some natural mechanisms that breeders

can use to develop male sterile plants, (e.g.,

cytoplasmic nuclear (genetic) and genetic-

cytoplasmic) but they are not available for

many crops. Therefore engineered male

sterility is an alternate method in cases where

natural male sterility is not available. RNAi

targeted to some genes in pollen development

could be used to produce male sterile plants.

Bcp1 gene is a 587bp sized gene of

Arabidopsis thaliana responsible for fertile

pollen development and active in both diploid

tapetum and haploid microspores. Perturbation

of this gene in either tapetum or microspores

prevents production of fertile pollen. Thus,

mature anthers contain dead shriveled pollens.

Therefore the current study is initiated with the

objective of producing male-sterile lines by

specific down-regulation of the anther-specific

gene Bcp1 of Arabidopsis by RNAi.

3. MATERIALS AND METHODS –

Development of male sterility by silencing

Bcp1 gene of Arabidopsis through RNA

interference by Tehseen et al, 2010

Male sterile mutants are of agricultural

importance for the production of hybrids. It is

the alternate method to engineer these male

sterile lines. RNAi targeting is one of the

methods used; Bcp 1 gene is a 587bp

in Arabidopsis thaliana responsible for pollen

fertility. Therefore the study initiates with the

objective of producing male-sterile lines by

specific down-regulation of anther specific Bcp

1 (Tehseen et al, 2010).

Target selection and the gene construct: Total DNA was isolated by the CTAB method and both the sense and antisense copy of the Bcp1gene was amplified by PCR using primers designed to have restriction endonuclease recognition sequences for easier cloning of the amplified fragments into appropriately digested vector (Fig 3). Then BCP1 RNAi cassette was made by ligating the sense (BCP1s) and antisense (BCP1as) DNA copies of 163 bp

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region of Bcp1 gene in a reverse order on an agrobacterium based vector pFGC5941 with the help of restriction sites present in the amplified PCR products. The inverted sense

and antisense constructs were separated by a spacer region or introns of petunia Chalcone synthase gene A to increase the efficacy of PTGS.

Figure 3: Production of the Bcp1 RNAi cassette.

Two pairs of primers were used to PCR-amplify a portion of the 163 nucleotide of Bcp1 gene, yielding fragments with BamHI-XbaI and NcoI-XhoI ends. These fragments were directionally cloned into the RNAi vector pFGC5941, yielding the final pTA29RNAi cassette. Shown are the Bcp1 sense (Bcp1s) and antisense (Bcp1as) amplification fragments, the Agrobacterium tumefaciens promoter (PMAS) and polyadenylation signal (PA, the Caulifower mosaic virus 35S promoter (P35S), the petunia chalcone synthase A gene intron (C intron), the Agrobacterium tumefaciens polyadenylation signal, gene encoding BASTA (BAR), T-DNA left border (LB) and T-DNA right border (RB).

The Agrobacterium based vector, pFGC5941 has two multiple cloning sites, bar gene for resistance to the herbicide Glufosinate Ammonium which will be regulated by the Agrobacterium tumefaciens promoter (PMAS) and 35S constitutive promoter (P35s) within left and right borders of the T-DNA region. Then the gene construct Bcp1/pFGC was transformed in Agrobacterium tumefaciens strain LBA4404 by electroporation and it was cultured in LB supplemented with 50 µg/ml acetosyringon, 50 µg/ml kanamycin and 50 µg/ml rifampicin. The transformants were confirmed through PCR using forward and reverse primers.

The next step was Agrobacterium mediated transformation of the construct in to Arabidopsis thaliana, For these leaves were taken from a three weeks seedlings of Arabidopsis, cut in to discs (leaf explants)

finally immersed in to the 50 ml of diluted bacterial suspension and incubated for 5–10 min with gentle shaking, 2 days of dark incubation, washed with cefotaxime and transferred to culture jars supplemented with growth regulators and incubated at 22°C under white light conditions with a 3 weeks of subculturing interval till the shoots reach enough lengh. They were then finally transferred to rooting, acclimatized in the greenhouse. The cefotaxime kills the Agrobacterium and glufosinate ammonium (5µg/ml) that reduced glutamine and increased ammonia levels in the plant tissues and thus only leaf discs having transgene were survived.

Polymerase chain reaction (PCR) and Southern hybridization were employed for the confirmation of the putative transgenic plants through bar gene specific primers and probes and their fertility was recorded (Tehseen et al., 2010).

1. RESULTS AND DISCUSSION

From the 800 pieces of embryonic calli transformed by co-cultivation, 98 (12.3%) pieces of resistant embryonic calli were obtained after hygromicin B selection. A total of 46 (46.9%) plantlets were regenerated after the recovering subculture and the multiplication. Of the 46 regenerated plants, 18 (39.1%) were detected as positive by specific PCR amplification and certified as putative transgenic plants (Fig. 4). Out of these 18 plants, 13 (72.2%) grew to reproduce seeds of the T1 generation. Out of the 13 T1 lines, nine (69.2%) were shown by Southern blotting to have stable transgene integration.

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Figure 4: PCR detection of the regenerated plants.

Lane M is 50-bp DNA ladder marker, lane 1 is non-transformed control 18-599, lane 2 positive control of expression

vector pASP150, lanes 3–20 transformed plants 3, 5, 9, 10h, 10L, h1, h2, 12, 13, 14, m-1, 15, 16, 18, 20, 21, 24, and 25,

respectively

Virus Resistance of T2 Plant Lines: After the

pollination stage, systemic infection of MDMV

was observed in the non-transformed control

(18–599), the susceptible control (Mo17), and

in some T2 plant lines. The disease indexes of

the different T2 plant lines and the controls

matched each other in the two environments

(Table1). The non-transformed control (18–

599) was evaluated as susceptible (S) to

MDMV with a disease index between 40.1%

and 60.0%, the susceptible control (Mo17) was

evaluated as highly susceptible (HS) with a

disease index >60.1%, while the resistant

control (H9-21) was evaluated as resistant with

a disease index between 10.1% and 25.0%.

Of the nine transgenic T2 plant lines derived

from the T1 lines positive in Southern blotting,

lines h2, 13, and h1 were judged to have

intermediate resistance to MDMV with a

disease index between 25.1% and 40.0%,

showing no systemic infection. This resistance

is increased significantly when compared with

the non-transformed control line 18–599, but

was not significantly different from the highly

resistant control line H9-21.

The DAS ELISA result also showed an

increment in the viral protein as we go from a

resistant control to the highly susceptible

(Table 2).

Table 1. MDMV resistance of T2 transformed plant lines

T2 plant line and

Control

Disease incidence Disease index (%) Resistance

grade Xinzhou Ya’an Average Xinzhou Ya’an Average

H 9-12 (resistant

control)

56.2 48.1 52.2 22.9 17.9 20.4a R

h2 26.0 29.5 27.8 25.4 27.5 26.5a I

13 45.3 38.2 41.8 33.3 27.1 30.2ab I

h1 52.2 38.6 45.4 34.1 28.8 31.5ab I

9 6.5 65.2 63.4 42.7 46.0 44.4bc S

5 72.0 65.6 68.8 47.8 49.3 48.6c S

3 82.9 78.4 80.7 55.1 46.7 50.9c S

18-599 (non-

transformed control)

93.7 100.0 96.9 48.6 55.8 52.2c S

21 88.7 83.5 86.1 60.4 57.4 58.9c S

10L 100.0 95.7 97.9 85.4 72.7 79.1d HS

10h 100.0 100.0 100.0 86.8 81.2 84.0d HS

MoI7 (Susceptible

control)

100.0 100.0 100.0 85.5 83.5 84.5d HS

In the column of average disease index, the same lowercase letters indicate non-significance, and the different lowercase

letters indicate significance at possibility level of 0.05 R resistance, I intermediate, S susceptible, HS highly susceptible

to MDMV

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Table 2: Absorbance value at 405 nm in DAS-ELISA of the T2 plant lines and controls

T2 plant line and control Absorbance at 405nm

H 9-21 (resistant control) 0.201

h2 0.207

I3 0.285

H1 0.311

9 0.503

5 0.692

3 0.778

21 0.896

18-599 (non transformed control) 0.904

10L 1.002

10h 1.323

Mo17 (susceptible control) 1.580

In summary, the disease index of the

transgenic plant line h2 had no significant

difference from the highly resistant control line

H9-21. The underlying reason for the lack of

resistance in some of the transgenic lines

remains to be clarified. But the effective length

of the expressed hpRNA constructs to trigger

RNAi in transgenic plants is 300–800 bp, and

the short limit is ≈98 bp. The 150-bp hpRNA

expression construct introduced into the maize

genome might be a little too short to trigger

efficient RNAi.

2. RESULTS AND DISCUSSION

It was found that in 26 of the total 29 events

produced from pMON73567, the R1 seeds

showed reduced zein content. All 26

pMON73567 events displayed a reduced

accumulation of both 19- and 22-kD α-zeins.

Of the 14 events produced from pMON73566,

2 of the 14 had a reduction in both 19- and 22-

kD α-zeins. The majority of these pMON73566

events, 10 of the 14, showed reduction in 19-

kD α-zein accumulation.

For Conformation of zein gene suppression

Northern blot analysis was done where two

pMON73567 events, M80442 and M82186,

along with two pMON73566 events, M80780

and M80791, were chosen to advance to

subsequent generations for further analyses.

Six R2 kernels per event were analyzed and

because of the dominant nature of the transgene

these kernels should segregate 3:1 of transgenic

vs. wild-type. It was observed that apparent

reduction of both 19- and 22-kD α-zein

transcripts in M80442 kernels and the

reductions of 19-KD α-zein transcripts in

M80780 kernels (Fig 5). In M80791 kernels,

the reduction of 19-kD α-zein transcripts was

clearly observed along with some reduction in

22-kD α-zein transcripts (Fig 5).

Figure 5. Northern blot analysis of developing transgenic kernels.

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Equal amounts of RNA isolated from

individual kernels were separated through

electrophoresis on three separate agarose gels.

After blotting, each was probed with one of

three probes corresponding to the coding

sequences of 19-, 22-kD α-zeins or 27-kD γ.

Six segregating R2 kernels were used per

event. M80442 and M80791 events show

reduced accumulation of 19- and 22-kD α-zein

transcripts and M80780 event shows reduced

accumulation of 19-kD α-zein transcripts.

The result of total amino acid analysis

showed that there is an increase in the lysine

content from 2438 ppm in wild-type kernels to

4035, 5003, 4533 and 4800 ppm in kernels

from M80780, M80791, M80442 and M82186

events, respectively. Similarly, the tryptophan

content increased from 598 ppm in wild-type to

877, 1087, 940 and 1040 ppm in M80780,

M80791, M80442 and M82186,

correspondingly. Generally in this study, Up to

5.62% of lysine and 1.22% of tryptophan were

achieved in transgenic lines compared with

2.83% of lysine and 0.69% of tryptophan in

wild-type.

An important factor underlying the

significant increases in lysine and tryptophan

levels in zein reduction kernels was the

synthesis of non-zein proteins. The observation

that the average protein content across the

transgenic zein reduction lines is unchanged

relatively to wild-type suggests that the zein

proteins are being replaced with non-zein

proteins.

Figure 6. Correlations between the protein content and the total lysine or tryptophan contents

in zein reduction ears.

Linear correlations between the lysine (A) or tryptophan (B) content and the protein content among pooled kernels

of zein reduction ears were observed from the transgenic lines. The graphs indicate an incremental increase of 723-ppm

of lysine and 157-ppm of tryptophan for every percentage of protein accumulation in these ears.

Figure 7. Correlations between the protein content and the free amino acid levels in zein

reduction ears.

Linear correlations were observed between the total free amino acid (A) or asparagine (B) levels and the protein

content among pooled kernels of zein reduction ears from transgenic lines.

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The results on the above graphs confirm

that the increases in lysine and tryptophan in

the zein reduced kernels are the result of the

replacement of lysine-poor zeins with more

amino acid-balanced, lysine-containing non-

zein proteins.

The simplest explanation of the substantial

upsurge in the free amino acid level in zein

reduction kernels is that during kernel

development excess amino acids, unable to be

incorporated into zeins were either diverted to

other seed proteins or remained as free amino

acids at maturity. In such a case, one would

expect an inverse correlation between the free

amino acid level and the overall protein content

in zein reduced kernels.

3. RESULTS AND DISCUSSION

They found that the dsRNA interfere the

Bcp1 gene function in the transgenic

Arabidopsis plants and consequently male

sterile plants were obtained. About 49 out of 58

Arabidopsis lines transformed with RNAi

construct containing Bcp1 sequences were male

sterile (Table 3).

Table 3. Transformation of Arabidopsis thaliana with Agrobacterium based plasid and

evaluation of regenerated plants. Agrobacterium tumefaciens (LBA4404) bearing plasmid

containing 163 bp region of male fertility (Bcp1) gene under 35S promoter.

Batch

no.

Leaf discs

treated

(no.)

Explants

regenerated on

selected media*

(no.)

Plants which

developed

roots (no.)

Positive

plants for

bar

genes**

Number

of

sterile

plants

Seed

production up

on cross

pollination

1 71 49 28 20 17/20 Yes

2 84 51 22 17 12/17 Yes

3 60 43 27 21 19/21 Yes *Containing 10mg/l BASTA ®

** Analysed by PCR using bar gene specific pair of primers

Transgenic plants were phenotypically

indistinguishable from non-transgenic plants

except for aborted or malfunctioning pollen

grains. These transgenic plants were used as

female plants for crossing with wild type non-

transgenic plants to produce hybrid seeds. In

these plants there were non-viable pollens to

fertilize their own female partners on self-

pollination. By using the same strategy, it will

be easy to produce hybrid seeds on large scale,

for higher yield and quality of the crop. From

this study, it is concluded that silencing of

Bcp1 through RNAi is responsible for male

sterility in Arabidopsis. A sequence homology

in other crop species like Brassica (Bhalla,

P.L and Singh, 1999) of this gene also showed

that this method can be employed to obtain

hybrid seeds of commercially high valued

crops.

CONCLUSION

From the research results above, it could be

seen that the discovery of gene silencing has

great impact for various areas of genetic studies

such as functional genomics, for the

development of plants with virus resistance and

high nutritional value. Thus gene silencing is a

promising and emerging branch of Plant

Biotechnology that can be used as a tool for

identification of unknown genes and for the

diagnosing of different kinds of plant diseases.

ACKNOWLEDGEMENTS

We greatly acknowledge Department of

Biology – AMBO and Department of

Biotechnology – GONDAR ETHIOPIA, for

there constant encouragement.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 80–90

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

REFERENCES

Appasani, K. (2005). RNA Interference

Technology.From basic science to drug

development. Cambridge University

Press, United States of America, New

York.

Bhalla, P.L and Singh, M.B. (1999) Molecular

control of male fertility

in Brassica.Plant Cell, Tissue and

Organ Culture. 56: 89–95.

Charagonda, R. (2008). Antisense Suppression

of σ-Cadinene Synthase Gene in

Cotton.Unpublished Master’s Thesis,

University of Agricultural Sciences,

Dharwad.

Huang, S; Frizzi, A; Florida, C. A.; Kruger, D.

E. and Luethy, M. H. (2006).High

lysine and high tryptophan transgenic

maize resulting from the reduction of

both 19- and 22-kD α-zeins. Plant

Molecular Biology. 61:525–535.

Lin, K (1989) Studies on the resistance of corn

inbred lines and hybrids to maize dwarf

mosaic virus strain B. Sci Agric Sin

22:57–61

Mertz, E.T., Bates, L.S. and Nelson, O.E.

(1964).Mutant gene that changes

protein composition and increases

lysine content of maize endosperm.

Science 145: 279–280.

Myers, J. W and Ferrell, J.E.(2005). Dicer in

RNAi: Its roles in vivo and utility in

vitro. In: Appasani, K.RNA Interference

Technology: From Basic Science to

Drug Development. Cambridge

University Press.New York, U.S.A.

pp.20–54.

Scanlon, K. (2004). Anti-Genes: siRNA,

Ribozymes and Antisense. Current

Pharmaceutical Biotechnology. 5: 1–6

Tehseen, M; Imran, M; Hussain, M; Irum, S;

Ali, S; Mansoor, S and Zafar, Y.

(2010). Development of male sterility

by silencing Bcp1 gene of Arabidopsis

through RNA interference.African

Journal of Biotechnology. 19: 2736–

2741.

Source of Support: NIL Conflict of Interest: None Declared

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 91–100

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

CALLICARPA MACROPHYLLA: A REVIEW OF ITS PHYTO-CHEMISTRY,

PHARMACOLOGY, FOLKLORE CLAIMS AND AYURVEDIC STUDIES

Pandey Ajay Shankar1, Srivastava Bhavana

2*, Wanjari Manish M

3,

Pandey Narendra Kumar4, Jadhav Ankush D

5

1Senior Research Fellow (Pharmacognosy), National Research Institute for Ayurveda-Siddha, Human

Resource Development, Gwalior-474009 MP, India. 2Research Officer, Dept. of Chemistry, National Research Institute for Ayurveda-Siddha, Human Resource

Development, Amkho, Gwalior-474009, MP, India. 3Research Officer, Dept. of Pharmacology, National Research Institute for Ayurveda-Siddha, Human

Resource Development, Amkho, Gwalior-474009, MP, India. 4Research Officer, Dept. of Botany, National Research Institute for Ayurveda-Siddha, Human Resource

Development, Amkho, Gwalior-474009, MP, India. 5Research Officer incharge S-4 (Ayu), National Research Institute for Ayurveda-Siddha, Human Resource

Development, Amkho, Gwalior-474009, MP, India.

*Corresponding author: E-mail: bhavanakan@gmail.com; ajju4u001@gmail.com; Phone: (+91)7489814440

Received: 29/01/2014; Revised: 20/02/2014; Accepted: 05/03/2014

ABSTRACT

Callicarpa macrophylla, commonly known as Priyangu is a useful medicinal plant for the

treatment of various disorders like tumour, polydipsia, diarrhoea, dysentery, diabetes, fever, etc. In

Ayurvedic system of medicine, the plant is also known as Phalawati and used for obstetric

conditions. As the plant is very important because of its therapeutic potential, research on its phyto-

chemistry, pharmacology, folklore claims and Ayurvedic studies are reviewed in this article to

present comprehensive information on this plant, which might be helpful for scientists and

researchers to focus on the priority area of research that are yet to be discovered and to find out new

chemical entities responsible for its claimed traditional uses.

KEYWORDS:

Callicarpa macrophylla, Priyangu, Phyto-chemistry, Pharmacology, Folklore claims, Ayurvedic

studies.

Review Article

Cite this article:

Pandey Ajay S., Srivastava Bhavana, Wanjari Manish M,

Pandey Narendra K., Jadhav Ankush D (2014), CALLICARPA MACROPHYLLA: A REVIEW

OF ITS PHYTO-CHEMISTRY, PHARMACOLOGY, FOLKLORE CLAIMS, AND

AYURVEDIC STUDIES, Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 91–100

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 91–100

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

INTRODUCTION

Callicarpa macrophylla Vahl. (syn.

Callicarpa incana Roxb.) belonging to family

Verbenaceae, commonly known as Priyangu in

Sanskrit & Hindi is an important Ayurvedic

drug used in treatment of various ailments. In

Ayurvedic system of medicine, the plant is also

known as Phalawati and used for obstetric

conditions. It also forms one of the ingredients

of a compound drug - Lodhrasa; used for

gyanocological and skin diseases. There are

two varieties of the plant, described in

Samhitas, as Priyangu and Gandha Priyangu.

The second variety is a fragrant one. As a folk

medicine the plant is useful in the treatment of

various disorders viz. tumour, polydipsia,

diarrhoea, dysentery, diabetes, fever, etc

(Anonymous, 1992). An extensive review was

carried out to explore the hidden potentials and

to enumerate the benefits of the Priyangu.

Parts used

Root, Bark, Leaves, Flowers, Fruits.

Vernacular / Tribal / Common names

The plant Callicarpa macrophylla is known

as Perfumed cherry and Beauty berry in

English; Priyangu, Daya, Dhaiya and

Fulprayangi in Hindi; Priyangu, Priyanguka,

Priyaka, Gandhaphali, Gandhipriyangu,

Phalini, Vanita, Kaantaa, Shyaamaa and

Anganapriya in Sanskrit and in Ayurvedic

literature; Habb-ul-mihlb in Unani texts;

Baramala, Mathara and Mattranja in Bengali;

San-natadagidda, Kadu-edi, Priyangu and

Navane in Kannada; Gyazhalpoo and Nalal in

Tamil; Prenkhanamu in Telugu; Bonmala and

Tong-lotti in Assamese; Sumali in Punjabi;

Ghaunla and Priyango in Gujrati; Nazhal,

Kadurohini, Njazhal, Jnazhal and Chimpompil

in Malyalam; Gauhala, Gahula and Priyangu

in Marathi and Priyangu in Oriya language

(Khare, 2007; Anonymous, 2008).

GEOGRAPHICAL DISTRIBUTION

The plant is found in open and secondary

forests in upper Gangetic plains, West Bengal

Plains, Eastern and Western Himalaya region

(Gupta et al., 2008), Kashmir to Assam,

Arunachal Pradesh and northern Andhra

Pradesh, up to an altitude of 1800 m

(Anonymous, 1992). In other parts of world it

is distributed across Nepal, Bhutan, Myanmar,

South East Asia and china (Billore et al., 2005).

BOTANICAL DESCRIPTION

The plant is an erect undershrub, 1.5–2.5 m

tall. Leaves are elliptic-oblong to lanceolate or

ovate to ovate–lanceolate, 12–25 × 5–11 cm,

acute or acuminate at apex, acute or cuneate at

base, glabrescent, crenate-dentate; densely

stellate–tomentose beneath; petiole 4–12 mm

long; Inflorescence axillary, solitary or often

corymbose–cyme; Flowers purplish; Calyx 5,

4-toothed, bell-shaped, persistent

gamosepalous, covering almost half of the fruit

sometimes attached; corolla lilac or purple,

about 3 cm long; lobes 4, ovate, subacute.

Fruits globose, drupes or berries, white to

yellowish-brown with or without fruit stalk,

fresh being succulent, 1–3 mm in dia. Intact

fruits are smooth and brownish in colour and

exhibit centrally located bilocular carpel and 4

nutlets each embedded with a yellowish white

seed, in a transversely cut surface of a fruit;

Flowering & fruiting: June–Dec. Fruits taste at

first somewhat sweet, later bitterish; odour

fragrant specially after slight bruising the fruit

(Anonymous, 2008; Gupta et al., 2008; Mudgal

et al., 1997). The fruits of Callicarpa

macrophylla are edible (Dangol, 2008; Mehta

et al., 2010).

MICROSCOPY

The stomata found on the leaves of C.

macrophylla are anomocytic (Mathew & Shah,

1981). The transverse section of the dried fruits

irregularly circular in outline with undulated

margin showing a layer of epidermis, wide

parenchymatous mesocarp traversed with

vascular strands, stony endocarp and centrally

located exalbuminous seeds with

sclerenchymatous coat and oil celled layer.

Detailed transverse section of the fruit shows

thin epicarp, forms skin of fruit consisting of

outer epidermal cells covered with thin cuticle,

a few epidermal cells elongate to form short

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stalked, disc-shaped, 2–4 celled glandular hairs;

some other epidermal cells form stellate hairs;

mesocarp is parenchymatous composed of 5–8

layered, radially elongated irregular shaped

large sized, thin walled cells, traversed with

vertically and tangentially running obliquely

cut vascular strands and microcluster crystals

of calcium oxalate, the innermost few layers of

mesocarp are embedded with volatile oil

globules. Endocarp hard and stony, lignified

and shows compactly arranged 4–5 rows of

spherical small sized discontinuous thick and

thin walled stone cells, peripherally studded

with small prismatic crystals of calcium

oxalate, encircling the centrally located seed,

seeds four in each fruit; seed exhibits a thin

layer of seed coat and radially elongated thick

walled palisade like sclerenchymatous band

lying underneath it; followed by layer of oil

cells embedded with volatile oil. Endosperm 2–

6 layered consisting of isodiametric cells;

cotyledons 2, consisting of isodiametric cells

filled with fixed oil and aleurone grains

(Anonymous, 2008; Gupta et al., 2008).

Powder microscopy shows plenty of

lignified stellate and branched trichomes, their

broken fragments, sessile glandular trichomes

with one to many celled head from the pedicel

and sepals of the fruit; epidermal cells with

anomocytic stomata of sepal; fragments of

straight walled, lignified cells of seed coat; oval

to elongated, elliptical endocarp cells in surface

view; single and isolated groups of elongated,

thick and thin walled, oval to rectangular,

lignified pitted stone cells having concentric

striations, radial canal, with narrow lumen;

microcluster crystals of calcium oxalate

scattered as such or embedded in the cells of

cotyledons and mesocarp; fragments of

cotyledons embedded with oil globules and

aleurongrains (Anonymous, 2008;

Gupta et al.,

2008).

PHYTOCHEMISTRY

Qualitative/quantitative analysis

Alcoholic extract of stem showed the

presence of glycosides, flavonoid, tannins,

carbohydrates, steroids and absence of

alkaloids, saponins, proteins, and amino acids

while aqueous extract of the stem showed the

presence of glycosides, flavonoids, saponins,

carbohydrates and tannins and absence of

alkaloids, steroids, proteins, and amino acids

(Yadav et al., 2012a). In another study

ethanolic extract of plant (excluding roots)

showed the presence of tannins (Atal et al.,

1978).

The leaf and fruit oils found rich in selinene

derivatives. The fruit oil is comprised of 41.6%

beta-selinene and 6% alpha-selinene.

Dendrolasin, a potential perfumery natural

furanoidsesquiterpenoid is reported in leaf and

fruit essential oils (Singh et al., 2010). The

content of luteolin increased gradually with the

growth of plants and reached the peak at the

end of growth period (Zhou et al., 2011). Total

flavonoid accumulation of plant changed along

with the growth of the plants, i.e. the contents

increased gradually in the trunk and root,

decreased in leaves (Liu et al., 2010).

High performance thin layer

chromatography (HPTLC) and Reverse phase-

High performance liquid chromatography (RP-

HPLC) with UV detection can be used for

quantitative determination of calliterpenone

and calliterpenone monoacetate, the two major

plant growth promoters in Callicarpa

macrophylla (Verma et al., 2009). RP-HPLC

method is also suitable for the quality control

of C. macrophylla because betulinic acid can

be well separated from other compounds in the

plant by RP-HPLC method (Pan et al., 2008).

Thin layer chromatography

Thin layer Chromatography (TLC) of the

alcoholic extract of fruit on Silica gel 'G' plate

using n-butanol : acetic acid: water (4:1:5)

shows under ultra violet (UV) light (366 nm)

one conspicuous fluorescent spot at Rf 0.82

(sky blue). On exposure to iodine vapours two

spots appear at Rf 0.82 and 0.92 (both

yellowish brown). On spraying with ferric

chloride (10% aqueous solution) two spots

appear at Rf 0.82 and 0.92 (both greyish brown)

(Anonymous, 2008). TLC of methanol extract

of fruit on silica gel 60F254 plates using toluene:

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ethyl acetate (70:30) using oleanolic acid as

standard; after spraying vanillin sulphuric acid

as detecting reagent showed under visible light

five spots at Rf 0.11and 0.35 (Blue), 0.52

(Purplish grey), 0.61 (Brown) and 0.69

(Blackish blue) (Gupta et al., 2008). T.L.C. of

the alcoholic extract of the stems collected

from Tarikhet, Uttrakhand using mobile phase

hexane: chloroform: ethyl acetate (2:1:1)

showed two spots with Rf value 0.84 and 0.91

(Yadav et al., 2012a).

Phyto-chemicals

Two tetracyclic diterpenes, calliterpenone

and calliterpenone monoacetate have been

isolated from the petrol extract of the aerial

parts (Chatterjee et al., 1972). Ursolic acid, β-

sitosterol and 5,4′-dihydroxy 3'-7-3′-trimethoxy

flavone have been isolated from the petroleum

ether extract of leaves (Chaudhary et al., 1978).

Methanol extract of the deposit of the water

extract obtained after distillation of the

essential oil of the leaves yielded 16α,17-

isopropylideno-3-oxo-phyllocladane

(isopropylidenocalliterpenone) along with

calliterpenone and its monoacetate (Singh &

Agrawal, 1994). The structure and absolute

configuration of calliterpenone has been

established as 3-oxo-13β-kaurane-16α,17-diol

(Fujita et al., 1975). 16,17-dihydroxy-3-

oxophylloladane,16-hydroxy-17-acetoxy-3-

oxophyllocladane, β-amyrin and β -sitosterol-3-

O-β-D-glucoside have been isolated from

fruits.

α-Amyrin, ursolic acid, 2α,3α,19α-

trihydroxyurs-12-en-28-oic acid, betulinic acid,

β-sitosterol, daucosterol have been isolated

from plant by column chromatography on silica

gel, Sephadex LH-20

(Pan & Sun, 2006).

Chung et al., (2005) have isolated acyclic

triterpene callicarpenol from plant (Chung et

al., 2005). Phyllocladane diterpenoids

calliterpenone and calliterpenone monoacetate

have been isolated from Callicarpa

macrophylla Vahl. in shoot cultures of

Rauwolfia serpentina (Goel et al., 2007).

Identity, Purity and Strength

For Callicarpa macrophylla fruit, foreign

matter should not be more than 2 % w/w, total

ash, acid-insoluble ash should not exceed 6.5 %

w/w, 1 % w/w respectively, alcohol-soluble

extractive value should not be less than 3 %

w/w and water-soluble extractive value should

not be less than 10 % w/w (Anonymous, 2008).

For inflorescence of this plant foreign matter

should not be more than 2 % w/w, total ash,

acid-insoluble ash should not exceed 8 % w/w,

2 % w/w respectively, alcohol-soluble

extractive value should not be less than 10 %

w/w and water-soluble extractive value should

not be less than 14 % w/w (Anonymous, 1999).

Stems collected from Tarikhet, Uttrakhand

showed 3.5% total ash, 1% acid insoluble ash,

0.3% water soluble ash, 14.0% alcohol soluble

extractive, 9.8% water soluble extractive and

8.75% moisture. Inorganic elements like

potassium, phosphates, iron and sulphates are

also found in these stems (Yadav et al., 2012a).

PHARMACOLOGICAL PROPERTIES

Analgesic, digestive, diuretic (Chunekar &

Pandey, 1999), antipyretic, antiemetic,

antipoisoning, blood purifier and anti burning

(Zarkhande & Mishra, 2004).

Leaves

The ethanolic and aqueous extracts of

leaves at the dose of 200 mg/kg and 400 mg/kg

showed dose dependent anti-inflammatory

action when evaluated by carrageenan induced

rat paw edema method using diclofenac sodium

as standard drug (Yadav et al., 2011). Ethanolic

extracts of leaves at the concentration of 200

and 400 μg/disc showed significant anti-fungal

activity against Gibberella fujikoroi,

Cryptococcus neoformans, Candida albicans,

Myrothecium verrucaria, Aspergillus niger,

Neurospora crassa and Rhizopus oligosporus

fungal strains when evaluated using disk

diffusion method using fluconazole as standard

drug while aqueous extract showed no

antifungal activity

(Yadav et al., 2012d).

Aqueous extract of leaves (200 and 400 µg/ml)

showed significant analgesic activity compared

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 91–100

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to ethanolic extract (200 and 400 µg/ml) when

evaluated by tail immersion method in rats

using pentazocine as standard and also

exhibited better anti-pyretic potential at same

dosage than ethanolic extract when evaluated

by Brewer’s yeast induced pyrexia model using

paracetamol as standard (Yadav et al., 2012b).

Stem

Ethanolic extract of stem at concentration

of 200 µl/disc and 400 µl/disc exhibited in-

vitro antibacterial activity against various gram

positive bacterial strains like Streptococcus

pyogens, Bacillus cereus, Micrococcus luteus,

Staphylococcus epidermidis, Clostridium

sporogens, Streptococcus faecalis,

Staphylococcus aureus and Bacillus subtilis

and gram negative bacterial strains like

Agrobacterium tumifaciens, Klebsiella

pneumonia, Salmonella typhimurium,

Pseudomonas aeruginosa, Serratia marcesens,

Enterobacteria aerogens, Proteus vulgaris and

Escherichia coli when compared with the

standard drug ciprofloxacin. Aqueous extract

was found inactive against all the bacterial

strains (Yadav et al., 2012c).

Bark

Extract of bark was shown to inhibit lipid

peroxidation in biological membranes (Kumar

& Muller, 1999).

Flower

The alcohol extract of flowers, at the dose

of 100 and 200 mg/kg, was found to exhibit

significant dose dependent antidiabetic activity

along with reduction in hyperlipidemia in

dexmethasone induced insulin resistance and

streptozotocin induced diabetes in rats (Patel,

2011).

Whole plant

Ethanolic extracts of the plant was found to

lack cytotoxic activity against KB cells (a

subline of the ubiquitous KERATIN-forming

tumor cell line HeLa) (Bhakuni et al., 1971;

Dhar et al., 1973; Suffness et al., 1988).

Folklore claims

The plant is reported to be useful to stop

internal and external bleeding and to treat burns

(Bensky et al., 1986). According to the tribal

people of Sikkim, India, the plant is bitter in

taste and useful in blood dysentery, sweating,

burning sensation and fever due to its cold

potency. This is the best medicine for bleeding

disorders and it reduces the bad smell from

body (Panda, 2007). In Bangladesh, Tripura

tribes use this plant as a tonic, as antidote to

poison, in the treatment of dermatitis and

cancer

(Rahmatullah et al., 2011). In a

preparation the plant is used in combination

with other herbs to treat diarrhea, dysentery,

intestinal worms, and skin disorders and to

purify the blood and eliminate toxins (Khare,

2004).

Roots are useful in the treatment of

pneumonia (Gautam, 2013), stomach disorders

and rheumatic pain (Rai, 2003). Tripura tribe of

Bangladesh use the decoction of roots for the

cure of frequent diarrhea, heart palpitation and

in frequent defecation

(Rahmatullah et al.,

2011; Hossan et al., 2009). About 10 ml

decoction is drunk twice a day for fifteen days

to cure bronchitis

(Rai, 2004). In Dibru-

Saikhowa biosphere reserve of northeast India

the root powder is used for the cure of

hydrophobia (Purkayastha et al., 2005). The

people of Chamoli district of Uttarakhand India

use root powder in the treatment of urinary

complaints and for regularizing menstruation

(Dangwal et al., 2011).

The Bark is used in the treatment of

rheumatism and gonorrhoea (Das et al., 2012).

Bark extract is used orally to treat fever by

Chakma tribe of Bangladesh (Rahman et al.,

2007). Aromatic oil from the roots is reported

to be useful to treat disordered stomach

(Talapatra et al., 1994).

Leaves are used for the treatment of

stomach disorders (Rai, 2003). In the Apatani

of Ziro valley in Arunachal Pradesh, leaves are

used for treatment of headache (Kala, 2005)

and tribal people of Jaunsar area of Garhwal

region in Himalaya use leaves for the treatment

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 91–100

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of rheumatic pain (Bhatt & Negi, 2006; Uniyal

& Shiva, 2005). Tribal people of Mizoram

apply paste of leaves in bone fracture (Rai &

Lalramnghinglova, 2010). Decoction of fresh

leaves is useful as a regular mouthwash for

recovery from sores and gingivitis (Arya &

Agarwal, 2008). People of Chamoli district of

Uttarakhand India use warmed leaf infusion for

the treatment of pain in arthritis (Dangwal et

al., 2011). Leaf extract is reported to be useful

for the treatment of rheumatism (Talapatra et

al., 1994). Juice of tender leaf buds mixed with

Drumaria diandra BL., Oxalis corniculata L.

and Cheilanthus albomarginata C.B. Cl. is

reported to be given in case of acidity and

gastric troubles (Manandhar, 1993).

In Tamil Nadu, the flowers and fruits are

used to treat diabetes (Jeyachandran & Mahesh

2007). Tribal people of Jaunsar area of

Garhwal region in Himalaya use fruits for the

treatment of rheumatic pain (Bhatt & Negi,

2006). Local vaidyas in Ukhimath block,

Uttarakhand use fruit juice to treat fever

(Manandhar, 1995), while the fruit extract to

treat rheumatic pain and mouth ulcers (Semwal

et al., 2010; Samal et al., 2004). The seeds are

reported to be useful for the treatment of oral

infections and intestinal complaints (Ahmad et

al., 1976).

Medicinal properties of the plant in

Ayurveda

For fruits the taste is sweet, bitter &

astringent; physical properties are cold, heavy

and dry, potency is cold, taste of fruit after

digestion is pungent. Actions of fruit include

alleviation of vital pitta & vata and as blood

purifier (Anonymous, 2008). For inflorescence

taste is bitter and astringent, physical property

is dry, cold in potency, taste after digestion is

pungent. Actions of inflorescence include

alleviation of vital pitta & vata, refrigerant, anti

diarrheal, diuretic, jointer, wound healer and as

blood purifier (Anonymous, 1999).

Recommended dose of fruit is 1–2 g

(Anonymous, 2004) and inflorescence is 1–3 g

in powder form (Anonymous, 1999).

Therapeutic uses

Fruits are used in the therapeutics of

burning sensation in the body, fever, vomiting,

blood disorders, vertigo, nervous system and

rheumatic diseases (Anonymous, 2008) while

inflorescence are used in the therapeutics of

burning sensation in the body, fever, blood pitta

diseases, amoebic dysentery and hyperhydrosis

(Anonymous, 1999).

Safety Aspects

The drug used traditionally in prescribed

doses may be considered safe (Gupta et al.,

2008).

Important Ayurvedic formulations

Important Ayurvedic formulations of fruits

include Jirakadi Modaka, Brhatphala Ghrta,

Brhatcchagaladya Ghrta, Vyaghri Taila

(Anonymous, 2008) while Ayurvedic

formulations of inflorescence include

Khadiradi Gutika, Eladi curna, Kanaka Taila,

Kunkumadi Taila and Nilikadya Taila

(Anonymous, 1999).

CONCLUSION

The above discussion clearly indicates that

Callicarpa macrophylla is an important

medicinal plant with diverse pharmacological

spectrum. The plant shows the presence of

many chemical constituents which are

responsible for varied pharmacological and

medicinal property. The literature claims that

there is vast potential in this plant in view of

therapeutics. Chemists and pharmacologists

must explore this plant for the potent phyto-

constituents and their pharmacological

properties by which new chemical entities can

be established to meet the challenges of

pharmaceutical profession to fight the

frightening diseases of the day and future.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 91–100

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Source of Support: NIL Conflict of Interest: None Declared

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Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

PHYTOCHEMICAL ANALYSIS OF SOME INDIGENOUS WOUND

HEALING PLANTS

Jagan Mohan Reddy P1, Ismail Shareef M

2*, Gopinath S M

3, Dayananda K S

4,

Ajay Mandal5, Purushotham K M

6

1,2,3,4Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore- 560 107,

Karnataka, India. 5Research Scholar, Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore-

560 107, Karnataka, India. 6Institute of Animal health & Veterinary Biological, Hebbal, Bangalore, Karnataka 560024, India

*Corresponding author: ismailshareef@acharya.ac.in; Mobile: +91 9916836390

Received: 06/02/2014; Revised: 27/02/2014; Accepted: 03/03/2014

ABSTRACT

Present investigation has been evaluated to find out active constituents of some indigenous plants

such as Calotropis procera, Ricinus communis and Mentha piperita potent against ectoparasite. The

results revealed that the % yield of Calotropis procera, Ricinus communis and Mentha piperita was

10.23, 22.79, 14.46 respectively and physical characteristic was sticky solid, dark greenish, agreeable

in Calotropis procera, semi-solid, dark greenish, agreeable in Ricinus communis however, sticky

semi solid, dark greenish, characteristic organic in Mentha piperita. The plants having alkaloids,

saponins, flavonoids, glycosides, carbohydrates, fixed oils and fats, tannins and phenolic compounds

as main active constituents in all the three plants which is very useful for preparing drug

development against wound healing.

KEY WORDS: alkaloids, glycosides, % yields, saponins, flavonoids, Calotropis procera, Ricinus

communis, Mentha piperita

Short communication

Cite this article:

Jagan Mohan Reddy P, Ismail Shareef M, Gopinath S M, Dayananda K S, Ajay Mandal,

Purushotham K M (2014), PHYTOCHEMICAL ANALYSIS OF SOME INDIGENOUS WOUND

HEALING PLANTS, Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 101–104

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 101–104

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

INTRODUCTION

Since time immemorial plants have been

used for the treatment of various ailments.

Even today several important drug used in the

modern system of medicine are obtained from

plants. The use of medicinal plants has figured

in several ancient manuscripts like the Rigveda,

the Bible, the Iliad, the Odyssey and the history

of Herodotus. As far back as 4000 B.C. the

ancient Chinese were using drug plants. The

earliest reference to the use of medicinal plants

as a cure for disease is found in the manuscript

of Eber Papyrus written in 1600 B.C. (Abu

Hanifah Y., 1990) with the advancement of our

knowledge; such superstitions were gradually

lost. In India, earliest reference to medicinal

plants appears in the Rigveda, written between

3500 and 1600 B.C. in Artharvanaveda too,

detailed descriptions of several medicinal

plants were given. Most of the drug plants are

wild and only a few of them have been

cultivated (Aldridge K. E., 1994). Studies of

medicinal plants based on ancient literature and

its investigation in modern light is under

process. The medicinal importance of a plant is

due to the presence of some substances like

alkaloids, glycosides, resins, volatile oils,

gums, tannins etc. these active principles

usually remain concentrated in the storage

organs of the plants, viz., roots, seeds, bark,

leaves etc. Considering all these facts, the

present investigation is designed to find out

phytochemical analysis of some indigenous

plants which are potent against wound healing.

MATERIALS AND METHODS

Selection of plants

Three local plants Ricinus communis,

Calotropis procera and Mentha piperita were

selected on the basis of their medicinal

properties against ticks and lice as reported in

various literatures. These plants were identified

and verified with taxonomical studies as

reported by (Aldridge K. E., 1994).

Collection of plant material

The plants were collected from different

regions of Bangalore, Karnataka, India.

Preparation of plant extract

Plant material was kept for drying for about

2 weeks, away from direct Sun light below 45°

C (shade dried). The dried material was

crushed in an electric grinder to coarse powder

consistency. About 500 gm of the powder

material was uniformly packed into a thimble

of a soxhlet extractor. It was exhaustively

extracted with methanol for a period of about

48 hrs or 22 cycles or till the solvent in the

siphon tube of an extractor becomes colourless.

The completion of the extraction was

confirmed by taking the solvent from the

thimble and evaporated to check the absence of

residue. The extract was taken out, filtered and

distilled to concentrate to get the syrupy

consistency in rotary evaporator. The residue

was dried over anhydrous sodium sulphate to

remove traces of alcohol. The extracts were

preserved in airtight container to avoid loss of

volatile principles (Annoni, F et al., 1989).

Physical characteristic of plant

The physical characters of the extract were

noted and the percentage yield was calculated

on such basis. The extracts were preserved in

an airtight container to avoid loss of volatile

principles for further studies.

Solubility of plant extract

All the extracts were dissolved in different

solvents for checking the solubility of extracts.

Phyto-chemical analysis of plant extract

The extracts were tested for the presence of

some active chemical compounds such as

alkaloids, flavonoids, glycosides, fixed oils &

fats, proteins, tannins and phenolic compounds,

carbohydrate, saponins. The analysis was

conducted as per universal methods.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 101–104

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

RESULTS

The physical characteristics observed in the

crude extracts of all the 3 plants are depicted in

Table 1.

The percentage yield of various extracts of

Ricinus communis, Calotropis procera and

Mentha piperita was calculated as 10.23%,

22.79% and 14.46% respectively (Table 2).

The phyto-chemical analysis of methanolic

crude extract of Ricinus communis, Calotropis

procera and Mentha piperita was found

positive for saponins, flavonoids, fixed oils and

fats in common. None of the 3 plants reported

the presence of Protein & amino acids.

Carbohydrates was absent in Calotropis

procera & Glycosides were absent in Mentha

piperita whereas Alkaloids, Carbohydrates,

tannins & phenolic compound were absent in

Ricinis communis (Table 3).

Table: 1. Physical characteristics of different crude extract of plants

Plant extracts Consistency Colour Colour

Calotropis procera (Leaves) Sticky semi solid Dark white Agreeable

Ricinus communis (Leaves) Semi solid Dark greenish Agreeable

Mentha piperita (Leaves) Sticky semi solid Dark greenish Characteristics organic

Table: 2. Percentage yield of different plants extract

Plants Weight of dry

powder (gm)

Weight of dry

extract (gm)

Yield

(%)

Calotropis procera (Leaves) 242.20 24.80 10.23

Ricinus communis (Leaves) 217.60 49.60 22.79

Mentha piperita (Leaves) 300.00 43.40 14.46

Table: 3. Qualitative determinations of active ingredients in crude extract of different Plants

Phyto-chemicals Calotropis procera

(Leaves)

Ricinus communis

(Leaves)

Mentha piperita

(Leaves)

Alkaloids + − +

Saponins + + +

Flavonoids + + +

Glycosides + + −

Carbohydrate − − +

Fixed oils and fats + + +

Tannins & phenolic

Compound

+ − +

Proteins & amino Acids − − −

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 101–104

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

DISCUSSION

The search for “natural remedies” for a

common disorder such as wounds has drawn

attention to herbals From ancient times, herbals

have been routinely used to treat wounds and

in many cultures and their use in traditional

medicine. Plants are more potent healers

because they promote the repair mechanism in

the natural way. This study exposed that

traditional medicines are still used by tribal

peoples & it is established the value of a great

number of plants used in tribal medicine

especially for wound healing. Seemingly much

still unknown information about plants to treat

various disease including wounds. So far, very

few studies have been carried out on medicinal

plants which present the wound healing

activity. The aim of the review was to list out

the medicinal plants which is reported already.

However, there is a need for scientific

validation, standardization and safety

evaluation of plants of the traditional medicine

before these could be recommended for healing

of the wounds.

CONCLUSION

The study demonstrates that the crude

methanolic extracts of Ricinus communis,

Calotropis procera and Mentha piperita exhibit

phyto-chemical principles of therapeutic value

and this has provided scientific basis for its

folkloric use in the treatment of various

infectious conditions and wound healing. The

wound healing potential which was confirmed

by the in vivo experiments further supports the

ethno-medicinal uses of the plants.

REFERENCES

Abu Hanifah, Y. (1990). Post-operative

surgical wound infection. Med.

J.Malays. 45:293–297.

Aldridge, K. E. (1994). Anaerobes in

polymicrobial surgical infections:

incidence, pathogenicity and

antimicrobial resistance. Eur. J. Surg.

Suppl. 573:31–37.

American Diabetes Association. (1999).

Consensus Development Conference on

Diabetic Foot Wound Care. Diabetes

Care 22:1354.

Annoni, F., M. Rosina, D. Chiurazzi, and M.

Ceva. (1989). The effects of a

hydrocolloid dressing on bacterial

growth and the healing process of

legulcers. Int. Angiol. 8:224–228.

Armstrong, D. G., and K. A. Athanasiou.

(1998). The edge effect: how and why

wounds grow in size and depth.

Clin.Podiatr. Med. Surg. 15:105–108.

Armstrong, D. G., and L. A. Lavery. (1998).

Evidence-based options for off-loading

diabetic wounds. Clin.Podiatr. Med.

Surg. 15:95–104.

Armstrong, D. G., L. A. Lavery, and T. R.

Bushman. (1998). Peak foot pressures

influence the healing time of diabetic

foot ulcers treated with total contact

casts. J. Rehabil. Res. Dev. 35:1–5.

Source of Support: NIL Conflict of Interest: None Declared

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

A PHARMACEUTICAL APPROACH ON MANIKYA PISHTI TOWARDS

STANDARDIZATION

Wavare Ramesh1, Yadav Reena

2*, Sheth Suchita

3, Sawant Ranjeet

4

1Associate Professor & Head, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu Ayurved

Mahavidyalaya, Charni road, Mumbai. 2P. G Scholar, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu, Ayurved

Mahavidyalaya, Charni road, Mumbai. 3Assistant Professor, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu, Ayurved

Mahavidyalaya, Charni road, Mumbai. 4Assisitant Professor, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu Ayurved

Mahavidyalaya, Charni road, Mumbai.

*Corresponding Author: Email: reenasyadav007@gmail.com; Phone:+91 9922900872, +918108269466.

Received: 03/02/2014; Revised: 25/02/2014; Accepted: 05/03/2014

ABSTRACT

Throughout most of recorded history Manikya (ruby) has been the world’s most valued

gemstone, which consists of Aluminium oxide, chromium, titanium. Ruby, the rarest of gemstones is

grouped under ratna varga in Rasashastra texts. As per Ayurveda, Manikya has the properties like

such as appetizer, aphrodisiac, bhutaghna, papaghna. Also it balances vitiated vata, pitta and kapha

hence its use leads to generate mental and spiritual powers. Manikya Pishti is a unique preparation

mentioned in Rasashastra texts of Ayurveda. The objective of the study was to prepare Manikya

Pishti (dosage form) and standardize it with different physical, chemical and instrumental analysis.

The prepared Pishti was subjected to ancient as well as modern analytical tests mainly X Ray

diffraction analysis of raw ruby showed Aluminum oxide as the principal component. Mean particle

size of raw ruby was 107.58 nm and that of pishti was 52 nm. The immense decrease in particle size

of ruby in pishti form concludes it will be useful as a nanomedicine, hence it can be said that it will

have a better dissolution rate.

KEYWORDS: Manikya Pishti, bhavana, trituration, particle size, XRD.

Research Article

Cite this article:

Wavare Ramesh, Yadav Reena, Sheth Suchita, Sawant Ranjeet (2014),

A PHARMACEUTICAL APPROACH ON MANIKYA PISHTI TOWARDS

STANDARDIZATION, Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 105–111

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

INTRODUCTION

Ayurveda is not merely the science of

disease and drugs whereas it has every aspect

of life in its sphere. The main aim of Ayurveda

is to maintain good health as well as to promote

a healthy life span. Hence Rasashastra

(Iatrochemistry) had used almost all the gems

for the purpose of inducing longevity of life in

human body (Vaidyopadhyaya, 1983). Also

internal use of gems can cure several diseases

(Vaidyopadhyaya, 1983). According to

Ayurveda, Manikya has been grouped under

ratna varga. Manikya Pishti preparation from

precious gem Manikya is a famous Ayurvedic

preparation. It is a versatile drug having

properties like memory enhancing, aphrodisiac

and specially recommended in erectile

dysfunction, general debility, and it has best

rasayana (antioxidant) as well as aphrodisiac

property. Though Ruby is known for a range of

therapeutic effects, it has been observed that

practitioners donot prevalently use it due to a

non-standardized pharmaceutical process

involved in processing of Ruby. So an attempt

was made to prepare and standardize Manikya

Pishti to facilitate its use in Ayurvedic

therapeutics.

MATERIALS AND METHODS

Materials

Raw ruby (Voucher No. -

SAS/10/7188/2013) was authenticated through

microscopic examination at gems testing lab,

Mumbai. An herbal drug that is rose water was

authenticated at pharmacognosy dept. of

Nicholas Piramal Lab, ltd Mumbai.

Methods

Manikya processing was performed using

standard procedures and includes steps namely

Manikya Shodhana (purification) (Sharma,

1979; Gopalbhatt, 2006 and Kaleda, 177) and

Manikya Pishti (trituration) (Kaleda, 177).

Preparation of Manikya Pishti:

1. Purification of Manikya

a. by swedana (specialized process of

steaming) in lime juice (Sharma, 1979)

b. by swedana in Kulattha Kashaya

(Decoction of Dolichos biforus Linn.)

(Gopalbhatt, 2006)

c. by quenching of ruby in rose water

(Kaleda, 177).

2. Preparation of Manikya pishti (Kaleda, 177)

Purification of Ruby

Materials - Crude Ruby, lime juice, Kulattha

Kashaya

Method - The sample was wrapped in a cloth

and tied to an iron rod in a suspended manner,

which was kept horizontally over a steel vessel.

Lime juice was taken in the steel vessel and

Ruby was immersed in the lime juice in such a

way that, it neither touches the sides nor the

bottom of the container. The vessel was heated

for 3 hrs under medium heat. Similarly

procedure was repeated with the decoction of

Dolichos biflorus instead of lime juice.

Nirvapana (process of reducing red hot

elements in liquid media) of Ruby in Rose

water was carried out for 101 times to make it

more brittle and to convert it into a fine

powder.

Manikya Pishti preparation [MP]

Materials - Manikya, Rose water

Method - Shodhit Manikya was made into fine

powder and triturated with rose water for 15

days [3hrs/day].

Method of Analysis of Manikya & Manikya

pishti using parameters described in

Ayurveda texts

Raw Ruby - Raw Ruby was subjected to

Grahyalakshana (selection criteria mentioned

in Ayurvedic text) of Manikya mentioned in

Rasashastra text (Khare, 1992).

Prepared Pishti

Prepared Pishti was subjected to bhasma

pariksha mentioned in Rasashastra text.

(Khare, 1992)

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Methods for analysis of Ruby & Manikya

Pishti on Modern parameters

Dynamic light scattering (DLS) Instrument - Malvern Mastersizer, Ver. 2000 5.31 Place: IIT, Pawai, Mumbai. Method: A rolling table with a partly adjustable inclined piece was constructed from hardboard and mounted on a steel frame. It was varnished with ordinary wood varnish to present a smooth surface along which movement could take place. Glass jars of various sizes were filled with different masses of the various materials and rolled from a fixed position with inclined part down the rolling table. Still on the flat part of the table readings were recorded and noted. Each measurement was performed 3 times.

Nanoparticle tracking analysis (NTA) Instrument - Nanoparticle tracking analyzer. Place - Institute of Science, Churchgate, Mumbai Method: The powders were placed in a container and dispersed in liquid media. The light scattered by the particles is captured using a CCD or EMCCD camera over multiple frames. Computer software is then used to track the motion of each particle from frame to frame. The rate of particle movement is related to a sphere equivalent hydrodynamic radius as calculated through the Stokes–Einstein equation.

X-ray diffraction (XRD) Instrument: Philips Holland XRD system Place: IIT Institute, Powai, Mumbai Method: The powdered sample was spread on to a double side tape with spatula, which was then placed on an aluminum sample holder. All

the peaks were recorded on the chart, and the corresponding 2 theta values were calculated. The strongest peak identified in the sample was corundum

Elemental Analysis by (ICP-AES) Method: Inductively coupled plasma (ICP) can be generated by directing the energy of a radio frequency generator into ICP argon gas. Coupling is achieved by generating a magnetic field by passing a high frequency electric current through a cooled induction coil. This inductor generates a rapidly oscillating magnetic field oriented in the vertical plane of the coil. Ionization of the flowing argon is initiated by a spark from a Tesla coil. The resulting ions and their associated electrons from the Tesla coil then interact with the fluctuating magnetic field. This generates enough energy to ionize more argon atoms by collision excitation. The electrons generated in the magnetic field are accelerated perpendicularly to the torch. At high speeds, cations and electrons, known as eddy current, will collide with argon atoms to produce further ionization which causes a significant temperature raise. Within 2 ms, a steady state is created with a high electron density. Plasma is created in the top of the torch. A long, well-defined tail emerges from the top of the high temperature plasma on the top of the torch. This torch is the spectroscopic source. It contains all the analyte atoms and ions that have been excited by the heat of the plasma.

RESULTS

Analysis of Manikya using modern

parameters Raw Ruby sample was tested in gem lab for

its gentility showing certified natural ruby qualities [Table 1].

Table 1: Showing Certified natural ruby qualities

Shape Rough

Cut Uncut

Isotropic/Anisotropic Anisotropic

Weight 4.78cts

Dimension 5.00 length approx.

Mounted / Unmounted Unmounted

Colour Milky pinkish brownish

Fluorescence Inert

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Sample of Manikya passed all the criteria of

genuine Manikya (Ruby). Raw Ruby and MP

was also analysed using the technique like

Dynamic light scattering (DLS), Nanoparticle

tracking analysis (NTA), X Ray Diffraction

(XRD).

Prepared Pishti

The prepared pishti showed following

characters

1) Rekhapurnatva: A pinch of pishti was

taken in between the thumb and index

finger and rubbed. It was observed that the

pishti entered into the lines of the finger

and was not easily washed out from the

cleavage of the lines.

2) Nishandratva: The pishti was taken in a

petri dish and observed for any lustre in

daylight through magnifying glass. No

lustre was observed in the pishti.

3) Varitaratva: A small amount of the

prepared pishti was sprinkled over the still

water in a beaker. It was found that the

pishti particles floated over the surface of

water.

Dynamic light scattering (DLS)

DLS analysis of raw sample of Ruby for

particle size estimation showed following

results [Table 2] & [Figure 1] The average

particle size of Raw Ruby was 1071.58 nm.

Nanoparticle tracking analysis NTA:

Manikya pishti when subjected to NTA

yielded following results [Table 3] & [Figure

2].

X-ray diffraction (XRD)

XRD analysis of raw Manikya as well as its

pishti showed following results [Table 4 & 5]

[Figure 3 & 4].

Elemental Analysis by (ICP-AES)

ICP-AES analysis of raw ruby and Manikya

pishti shown following results [Table 6]

Table 2: Particle size of Raw Ruby by DLS

Sample Test below Observation

Raw Sample of

Ruby

10% particles 134.717

50% particles 706.819

90% particles 1445.421

100% particles 1999.400

Figure 1: Particle size of Raw Ruby by DLS

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Table 3: Particle Size of Manikya Pishti BY NTA

Sample Particle size

MP 52

Figure 2: Particle Size of Manikya Pishti [MP] BY NTA

Particle size of MP (Manikya Pishti) was in nano scale. Hence it can be researched for its efficacy in Nano-medicine.

Table 4: X-ray diffraction of raw ruby

2-Theta value d-spacing intensity

25.580 3.479 100

26.621 3.346 10.7

27.480 3.243 6.1

35.140 2.552 84.3

37.780 2.379 35.5

43.360 2.085 97.5

52.580 1.739 64.2

Table 5: X-ray diffraction of Manikya Pishti

MP

2-Theta value d-spacing Intensity

25.620 3.4741 513

25.800 3.4503 3971

28.00 3.1840 600

35.380 2.5349 1504

37.940 2.3969 1088

38.060 2.3624 600

43.500 2.087 1904

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Figure 3: X-ray diffraction of raw Ruby

Figure 4: X-ray diffraction of Manikya Pishti

Table 6: Showing results of elemental analysis of Raw ruby, Manikya Pishti [MP]

Elements Unit Raw Ruby MP

Al (%) 51.2 46.9

Cu mg/Kg (ppm) 21.6 74.0

Fe mg/Kg (ppm) 1600 25776

Cr mg/Kg (ppm) 2884 3928

K mg/Kg (ppm) 274 951

Na mg/Kg (ppm) 52 3372

Ca mg/Kg (ppm) 174.4 12092

Mn mg/Kg (ppm) ND 186

Mg mg/Kg (ppm) 1004 5752

As mg/Kg (ppm) ND ND

S mg/Kg (ppm) ND ND

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

DISCUSSION

The process of trituration involves

breakdown of the material by rubbing action

between two surfaces, i.e. surface phenomena,

it is also called as attrition. When stress in the

form of attrition is applied, the particle surfaces

chip and produce small particles. Bhavana

(levigation) is given by grinding with some

liquid media, so it may be considered as wet

grinding and it is observed interestingly that

finer size of particles can be achieved by wet

grinding than dry grinding. For Pishti

preparation trituration is done with rose water,

which serves as liquid media for wet grinding

of material and facilitates easy and smooth

grinding and even eliminates hazards of dust.

It is also found interestingly in practice that

the finer particle size can be achieved by wet

grinding than by dry grinding. All elements

are found in human body in trace levels, but not

to be absorbed in to body in their elemental

form, plants are having capacity to transform

them in to readily absorbable form.

Trituration may help in reduction of particle

size of Manikya as it has hardness 9 it needs

more grinding effect. Mardana (trituration

process) also helps in loosening the

molecular cohesiveness and helps the drug to

break into fine particles during the subsequent

processing (Suneeta, 2011) (Mehta, 2010).

CONCLUSION

In the present study Manikya Pishti was

successfully prepared according to the methods

mentioned in Rasashastra texts and

standardized with help of Ayurvedic as well as

modern parameters. It will be helpful for

further researchers and manufacturers to

prepare the pishti according to mentioned

standard process & parameters which will also

facilitate its usage in Ayurvedic therapeutics.

REFERENCES

Chintamani Khare, (1992).

Rasaratnasamucchaya, Sanskrit

Commentary by, 4th

ed. Chapter 4,

Verse 4, Anadashram Publications,

Pune, India. p. 67

Gopalbhatt (2006) Rasendra Sara Sangrah with

‘Rasavidyotini’ Hindi Commentary by

Indradev Tripathi, 4th

ed. Chapter 1,

Verse 378-379, Chaukhamba Orientalia

publications, Varanasi, India,. p. 94

Krishna Gopalaji kaleda (1991) Rasatantra Sara

va siddaprayoga sangraha, Vol.1 13th

ed, Krishna gopala Ayurveda bhavana.

Gujarat, India. p. 177

Mehta R. M. (2010), Pharmaceutics part 2, ed.

3rd

, Chapter 4, p.77

Ramprasad Vaidyopadhyaya (1983) Rasendra

Puran, Hindi Commentary,1st ed.

chapter 28, Verse 7, Laxmi

Venkateshwar Press, Mumbai, India, p.

391-392

Sharma S. (1979) Rasa Tarangini. Hindi &

Sanskrit Commentary by Kashinath,

11th

ed. Chapter 23,Verse 46, Motilal

Banarasidas Publication, Delhi, India, p.

609

Suneeta M. (2011) Pharmaceutico Analytical

Study of Manikya Bhasma.

Source of Support: NIL Conflict of Interest: None Declared

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