GJRMI - Volume 3, Issue 3, March 2014 by Dr. Hari Venkatesh.K.Rajaraman

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ISSN 2277 â&#x20AC;&#x201C; 4289 www.gjrmi.com Editor-in-chief Dr Hari Venkatesh K Rajaraman

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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

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

105–111

Research Article ISOLATION AND CHARACTERIZATION OF PENTADECANOIC ACID ETHYL ESTER FROM THE METHANOLIC EXTRACT OF THE AERIAL PARTS OF ANISOMELES MALABARICA (L). R.BR. Ismail Shareef M1*, Leelavathi S2, Gopinath S M3 1

Assistant Professor, Department of Biotechnology, Acharya Institute of Technology, Soladevanahalli, Acharya Post (O), Bangalore-560 107, Karnataka, India 2 Professor, DOS in Botany, Manasagangotri, University of Mysore, Mysore-570 006, Karnataka, India 3 Professor 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.:08023700242

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, antiinflammatory 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;1HNMR; 13C-NMR; LC-ESI-MS

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

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

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 phytoconstituents 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.

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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. 2. 3. 4. 5.

Hexane Hexane: Ethyl acetate: 7:3 Ethyl acetate Ethyl acetate : Methanol : : 5 : 5 Methanol

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

II. Column Chromatography Purification Of Ethyl Acetate: Methanol: : 5 : 5

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.

a) Adsorption of sample on silica gel

RESULTS

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.

Spectral studies

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.

b) Loading of column (wet packing) Column was packed with silica gel slurry of mesh size 200–400 with chloroform. On top of

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

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

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 13C-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.

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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.: 13C-NMR of bio-active compound from the aerial parts of Anisomeles malabarica R.Br

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

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 phytopharmaceuticals (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 GCMS. The relative contents of the fatty acids were calculated with Area normalization.

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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 antirheumatic and immuno-modulatory properties.

REFERENCES Balandrin MJ, Klocke JA (1988): Medicinal aromatic and industrial materials from plants. Springer Verlag, Berlin, Heidelberg.,4, 1–36.

Kritikar KR, Basu BD (1935): Indian Medicinal plants. 2nd Edition, International Book Distributor, Dehradun, India.,2011–2012.

Ganeshan S (2008): Traditional oral care medicinal plants survey of Tamil Nadu. Nat. Prod. Rad. 7, ,166–172.

Madhava C (1998).: Pharmacognostic studies of Plumbago Zeylanica L. (chitreka, chitramulamu), dissertation, post graduate diploma in plantdrugs, S.V. University, Tirupati, India

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 antirheumatic 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.

Mujumdar AM, Naik DG, Dange CN, Puntambekar HM (2000): Antiinflammatory 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.

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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.

chromatography-mass Pharmacog. J. 3, 44–47.

spectrometry.

Ushir Y, Patel K (2011): Chemical composition and antibacterial activity of essential oil from Anisomeles species grown in India. Pharmacog. J. 2, 55–59.

Ushir Y, Patel K, Sheth N (2011): Analysis of fatty acid in Anisomelesspecies by gas

Source of Support:

NIL

Conflict of Interest: None Declared

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Research Article ANTIBACTERIAL ACTIVITY OF SOME INDIGENOUS MEDICINAL PLANTS Jagan Mohan Reddy P1, Ismail Shareef M2*, Gopinath S M3, Dayananda K S4, Ajay Mandal5, Sreekanth B6, Purushotham K M7 1,2,3,4

Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore- 560 107, Karnataka, India. 5 Research Scholar, Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore560 107, Karnataka, India. 6 Department 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

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): 75â&#x20AC;&#x201C;79

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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 40ÂşC 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 30ÂşC until dry mass were obtained by Buchirota vapour.

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Table 1. Plants details collected with the name of locality Serial No. 1 2 3 4 5 6

Name of plant

Plant parts

Family

locality

Leaf Leaf Leaf Leaf Leaf Leaf

Verbenaceae Anacardiaceae Moraceae Moraceae Lythraceae Apocynaceae

Kolar, Karnataka, India. Kolar, Karnataka, India. Kolar, Karnataka, India. Kolar, Karnataka, India. Kolar, Karnataka, India. Kolar, Karnataka, India.

Callicarapa arborea Lannea coromandelica Ficus racemosa Streblus asper Lawsonia inermis Holarrhena antidysenterica Mentha arvensis

Lamiaceae

Mallur, Karnataka, India.

8 9 10 11 12 13 14 15 16

Enhydra fluctuans Blumea lacera Glinus oppositifolius Chenopodium album Hemidesmus indicus Coccinea cordifolia Cuscuta reflexa Capparis zeylanica Kalanchoe pinnata

Aerial parts with flowers Leaf and root Aerial parts Leaf Leaf Root Leaf and rhizome Leaf Leaf Leaf

Asteraceae Asteraceae Molluginaceae Chenopodiaceae Periplocaceae Cucurbitaceae Convolvulaceae Capparidaceae Crasselaceae

Mallur, Karnataka, India. Mallur, Karnataka, India. Mallur, Karnataka, India. Mallur, Karnataka, India. Mallur, Karnataka, India. Mallur, Karnataka, India. Kolar, Karnataka, India. Mallur, Karnataka, India. 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.

Name of plants

Parts of plant used

Sh. dysenteriae

Salmonella typhi

Pseudomonas sp

Bacillus cereus

Salmonella paratyphi

Vibrio cholerae

Bacillus megaterium

E.coli

Bacillus subtilis

Staphylococcus aureus

Table 2. Antibacterial activity of alcoholic extracts of five plants Average zone of inhibition in diameter (mm) Bacterial test organisms

Blumea lacera

Aerial parts

Chenopodium album

Leaf

Enhydra fluctuans

Leaf and rhizome

Mentha arvensis

Aerial parts with flowers

Glinus oppositifolius

Leaf

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.

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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 gramnegative 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.

Source of Support:

NIL

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, Global J Res. Med. Plants & 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 streptozotocindiabetc 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

Conflict of Interest: None Declared

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Review Article GENE SILENCING AND ITS APPLICATIONS IN CROP IMPROVEMENT & FUNCTIONAL GENOMICS Jemal Ali 1, Pagadala Vijaya Kumari 2* 1

Department of Biotechnology, Gondar University, GONDAR, Post Box 196 – Ethiopia. Department of Biology, AMBO University, AMBO , Post Box 19 – Ethiopia. *Corresponding Author: E-mail: vkpagadala@rocketmail.com; 2

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 22kD 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

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 proteinencoding 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-toyear inconsistency of viral disease pressure. Therefore RNAi triggered by hairpin RNA (hpRNA) transcribed from the transgenic inverted-repeat sequence provides a straightforward 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 2â&#x20AC;&#x201C;3 weeks in greenhouse, and then transplanted into the field for self pollination to produce T0 seeds.

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:

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 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 bestcharacterized 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

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 αzein 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.

In most cereal grains, including maize, lysine is usually the most limiting essential 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 αzein 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 αzein 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 selfpollinated 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 (MALDITOF 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 geneticcytoplasmic) 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 PCRamplify 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 H 9-12 (resistant control) h2 13 h1 9 5 3 18-599 (nontransformed control) 21 10L 10h MoI7 (Susceptible control)

Disease incidence Disease index (%) Xinzhou Ya’an Average Xinzhou Ya’an Average 56.2 48.1 52.2 22.9 17.9 20.4a

Resistance grade R

26.0 45.3 52.2 6.5 72.0 82.9 93.7

29.5 38.2 38.6 65.2 65.6 78.4 100.0

27.8 41.8 45.4 63.4 68.8 80.7 96.9

25.4 33.3 34.1 42.7 47.8 55.1 48.6

27.5 27.1 28.8 46.0 49.3 46.7 55.8

26.5a 30.2ab 31.5ab 44.4bc 48.6c 50.9c 52.2c

I I I S S S S

88.7 100.0 100.0 100.0

83.5 95.7 100.0 100.0

86.1 97.9 100.0 100.0

60.4 85.4 86.8 85.5

57.4 72.7 81.2 83.5

58.9c 79.1d 84.0d 84.5d

S HS HS 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 H 9-21 (resistant control) h2 I3 H1 9 5 3 21 18-599 (non transformed control) 10L 10h Mo17 (susceptible control) 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-

Absorbance at 405nm 0.201 0.207 0.285 0.311 0.503 0.692 0.778 0.896 0.904 1.002 1.323 1.580

kD α-zeins. The majority of these pMON73566 events, 10 of the 14, showed reduction in 19kD 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 nonzein 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 Leaf discs no. treated (no.) 1 2 3

71 84 60

Explants regenerated on selected media* (no.) 49 51 43

Plants which developed roots (no.) 28 22 27

Positive plants for bar genes** 20 17 21

Number Seed of production up sterile on cross plants pollination 17/20 Yes 12/17 Yes 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 nontransgenic plants to produce hybrid seeds. In these plants there were non-viable pollens to fertilize their own female partners on selfpollination. 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.

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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

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Review Article CALLICARPA MACROPHYLLA: A REVIEW OF ITS PHYTO-CHEMISTRY, PHARMACOLOGY, FOLKLORE CLAIMS AND AYURVEDIC STUDIES Pandey Ajay Shankar1, Srivastava Bhavana2*, Wanjari Manish M3, Pandey Narendra Kumar4, Jadhav Ankush D5 1

Senior Research Fellow (Pharmacognosy), National Research Institute for Ayurveda-Siddha, Human Resource Development, Gwalior-474009 MP, India. 2 Research Officer, Dept. of Chemistry, National Research Institute for Ayurveda-Siddha, Human Resource Development, Amkho, Gwalior-474009, MP, India. 3 Research Officer, Dept. of Pharmacology, National Research Institute for Ayurveda-Siddha, Human Resource Development, Amkho, Gwalior-474009, MP, India. 4 Research Officer, Dept. of Botany, National Research Institute for Ayurveda-Siddha, Human Resource Development, Amkho, Gwalior-474009, MP, India. 5 Research 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 phytochemistry, 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.

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

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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 phaseHigh performance liquid chromatography (RPHPLC) 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, βsitosterol 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,17isopropylideno-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-3oxophylloladane,16-hydroxy-17-acetoxy-3oxophyllocladane, β-amyrin and β -sitosterol-3O-β-D-glucoside have been isolated from fruits. α-Amyrin, ursolic acid, 2α,3α,19αtrihydroxyurs-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

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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 invitro 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 DibruSaikhowa 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

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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 Ayurveda

properties

the

plant

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 phytoconstituents 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.

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REFERENCES Ahmad, S. A., Siddiqui, S. A., & Zaman, A. (1976). Chemical examination of Callicarpa macrophylla, Lagerstromea lanceolata, Ficus palmata, and Taxodium mucronatum. J Indian Chem Soc. 53(11):1165–1166. Anonymous. (1992). The Wealth of India Raw materials [Revised edition], New Delhi: Council of Scientific and Industrial Research. Vol. 3 (Ca-Ci), pp. 61–62. Anonymous. (1999). The Ayurvedic Pharmacopoeia of India, New Delhi: Department of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homoeopathy, Ministry of Health and Family Welfare, Government of India. Part-I, Vol. 2, pp. 151–152. Anonymous. (2008). The Ayurvedic Pharmacopoeia of India, New Delhi: Department of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homoeopathy, Ministry of Health and Family Welfare, Government of India. Part-I, Vol. 4, pp. 111–112. Arya, K. R., & Agarwal, S. C. (2008). Folk therapy for eczema, bone fracture, boils sores and gingivitis in Taragtal province of Uttaranchal. Indian J Tradit Knowle. 7(3): 443–445. Atal, C. K., Srivastava, J. B., Wali, B. K., Chakravarty, R. B., Dhawan, B. N., & Rastogi, R. P. (1978). Screening of Indian plants for biological activity. Part VIII. Indian J Exp Biol. 16: 330– 349. Bensky, D, Gamble A, Kaptchuk T. (1986). Chinese Herbal Medicine- Material Medica. Seattle: Eastland Press. Bhakuni, D. S., Dhar, M. L., Dhar, M. M., Dhawan, B. N., Gupta, B., & Srimal, R. C. (1971). Screening of Indian plants for biological activity. Part III. Indian J Exp Biol. 9: 91–102.

Bhatt, V. P., & Negi, G. C. S. (2006). Ethnomedicinal plant resources of Jaunsari tribe of Garhwal, Himalaya, Uttaranchal. Indian J Tradit Knowle. 5: 331–335. Vol. 7, pp. 353–355. Billore, K. V., Yelne, M. B., Dennis, T. J. (2005). Database of medicinal plants used in Ayurveda. New Delhi, Central Council for Research in Ayurveda and Siddha. Chatterjee, A., Desmukh, S. K., & Chandrasekharan, S. (1972). Diterpenoid constituents of Callicarpa macrophylla Vahl: the structures and stereochemistry of calliterpenone and calliterpenone monoacetate. Tetrahedron.28:4319–4323. doi:10.1016/S0040-4020(01)88954-7. Chaudhary, A., Bhattacharya, A., Mitra, S. R., & Adityachaudhary, N. (1978). Phytochemical investigation on the leaves of Callicarpa macrophylla Vahl. J Indian Chem Soc. 55:628–629. Chunekar, K. C., & Pandey, G. S. (1999). Bhavaprakasha Nighantu (Indian Materia Medica) of Shri Bhavamishra, Varanasi: Chaukhambha Bharati Academy, pp. 250. Chung, I., Upadhyaya, K., & Ahmad, A. (2005). Isolation and cytotoxic activity of acyclic triterpene callicarpenol from Callicarpa macrophylla. Asian J Chem. 17:1907–1914. Chung, I., Upadhyaya, K., & Ahmad, A. (2006). Isolation of fatty acids and other constituents from Callicarpa macrophylla fruits. Asian J Chem. 8:1751–1758. Dangol, D. R. (2008). Traditional uses of plants of commonland habitats in Western Chitwan. Nepal. J Inst Agric Anim Sci. 29: 71–78.

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Dangwal, L. R., Rana, C. S., & Sharma, A. (2011). Ethno-medicinal plants from transitional zone of Nanda Devi Biosphere Reserve, District Chamoli, Uttarakhand (India). Indian J Nat Prod Resour. 2(1):116–120. Das, T., Mishra, S. B., Saha, D., & Agarwal, S. (2012). Ethnobotanical survey of medicinal plants used by ethnic and rural people in eastern Sikkim Himalayan region. African Journal of Basic and Applied Sciences. 4(1): 16– 20. doi: 10.5829/idosi.ajbas.2012.4.1.61133. Dhar, M. L., Dhar, M. N., Dhawan, B. N., Mehrotra, B. N., Srimal, R. C., & Tandon, J. S. (1973). Screening of Indian plants for biological activity. Part IV. Indian J Exp Biol. 11:43–54. Fujita, E., Ochiai, M., Ichida, I., Chatterjee, A., & Desmukh, S. K. (1975). Confirmation of the structure of calliterpenone, a diterpene from Callicarpa macrophylla. Phytochemistry. 14(10): 2249–2251. Gautam, T. P. (2013). Indigenous uses of some medicinal plants in Panchthar district Nepal. Nepalese Journal of Biosciences. 1:125-130. doi: http://dx.doi.org/10.3126/njbs.v1i0. 7469. Goel, M. K., Kukreja, A. K., Singh, A. K., & Khanuja, S. P. S. (2007). In vitro plant growth promoting activity of phyllocladane diterpenoids isolated from Callicarpa macrophylla Vahl. In shoot cultures of Rauwolfia serpentina. Nat Prod Commun. 2: 799–802. Gupta, A. K., Tandon, N., Sharma, M. (2008). Quality standards of Indian medicinal plants, New Delhi: Indian Council for Medical Research, Vol 5, pp. 134–141. Hossan, M. S., Hanif, A., Khan, M., Bari, S., Jahan, R., & Rahmatullah, M. (2009). Ethnobotanical survey of the Tripura

tribe of Bangladesh. Am-Eurasian J Sustain Agric. 3(2):253–261. Jeyachandran, R. & Mahesh, A. (2007). Enumeration of antidiabetic herbal flora of Tamil Nadu. Res J Med Plant, 1, 144–8. Kala, C. P. (2005). Ethnomedicinal botany of the Apatani in the Eastern Himalayan region of India. J Ethnobiol Ethnomed. 1: 11. Khare, C. P. (2004). Indian Herbal Remedies: Rational Western Therapy, Ayurvedic and Other Traditional Usage, Botany. New York: Springer-Verlag. Khare, C. P. (2007). Indian Medicinal Plants: An Illustrated Dictionary. New Delhi: Springer Inc. Kumar, K. C. S., & Muller, K. (1999). Medicinal plants from Nepal: II. Evaluation as inhibitors of lipid peroxidation in biological membranes. J Ethnopharmacol. 64(2):135–139. Liu, X. R., Zhou, R. B., Tang, L. J., Tong, Q. Z., Liu, X. D., Liu, J. S., & Luo, Y. L. (2010). Research of total flovone content in Callicarpa macrophylla Vahl. in different harvest time. J Tradit Chin Med. 11:012. Manandhar, N. P. (1993). Ethnobotanical note on folklore remedies of Baglung district Nepal. Contributions to Nepalese Studies. 20(2):183–196. Manandhar, N. P. (1995). An inventory of some vegetable drug resources of Makawanpur district Nepal. Fitoterapia. 66:231–238. Mathew, L., & Shah, G. L. (1981). Observations on the structure and ontogeny of stomata in some Verbenaceae with a note on their taxonomic significance. Feddes Repert. 92 (7–8): 515–526. doi: 10.1002/fedr.19810920704

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Mehta, P. S., Negi, K. S., Ojha, S. N. (2010). Native plant genetic resources and traditional foods of Uttarakhand Himalaya for sustainable food security and livelihood. Indian J Nat Prod Resour. 1(1):89–96. Mudgal, V., Khanna, K. K., Hajra, P. K. (1997). Flora of Madhya Pradesh, Calcutta: Botanical Survey of India. Vol 2, pp. 365. Pan, P., & Sun, Q. S. (2006). Chemical constituents of Callicarpa macrophylla Vahl. Journal of Shenyang Pharmaceutical University. 9:003. Pan, P., Jia, L. Y., & Sun, Q. S. (2008). RPHPLC determination of betulinic acid in Callicarpa macrophylla. China journal of Chinese Materia Medica. 33(7):753– 755. Panda, A. K. (2007). Medicinal plants of Sikkim in Ayurvedic practice. Sikkim: Regional Research Institute (Ayurvedic). Retrieved from http://sikkimforest.gov.in/docs/Ayurved hic%20Medicines.pdf Patel, S. R. (2011). Evaluation of alcoholic extract of Callicarpa macrophylla flowers for its anti-diabetic activity (Unpublished M.Pharm thesis). Rajiv Gandhi University of Health Sciences Bangalore, Karnataka. Purkayastha, J., Nath, S. C., & Islam, M. (2005). Ethnobotany of medicinal plants from Dibru-Saikhowa biosphere reserve of Northeast India. Fitoterapia, 76(1), 121–127. Rahman, M. A., Uddin, S. B., & Wilcock, C. C. (2007). Medicinal plants used by Chakma tribe in Hill Tracts districts of Bangladesh. Indian J Tradit Knowle. 6(3):508–517.

Rahmatullah, M., Jahan, R., Azam, F. S., Hossan, S., Mollik, M. A. H., & Rahman, T. (2011). Folk medicinal uses of Verbenaceae family plants in Bangladesh. Afr J Tradit Complement Altern Med. 8(5S). doi: 10.4314/ajtcam.v8i5S.15 Rai, M. B. (2003). Medicinal plants of Tehrathum district, Eastern Nepal. Our Nature. 1(1): 42–48. doi:10.3126/on.v1i1.304. Rai, P. K., & Lalramnghinglova H. (2010). Ethnomedicinal plant resources of Mizoram, India: Implication of traditional knowledge in health care system. Ethnobotanical Leaflets. (3):6. Rai, S. K. (2004). Medicinal plants used by Meche people of Jhapa district eastern Nepal. Our Nature. 2(1): 27-32. doi:10.3126/on.v2i1.321. Samal, P. K., Shah, A., Tiwari, S. C., & Agrawal, D. K. (2004). Indigenous health care practices and their linkages with bio-resource conservation and socio-economic development in central Himalayan region of India. Indian J Tradit Knowle. 3(1):12–26. Semwal, D. P., Saradhi, P. P., Kala, C. P., & Sajwan, B. S. (2010). Medicinal plants used by local Vaidyas in Ukhimath block, Uttarakhand. Indian J Tradit Knowle. 9(3):480–485. Singh, A. K., & Agrawal, P. K. (1994). 16α, 17-Isopropylideno-3-oxophyllocladane, a diterpenoid from Callicarpa macrophylla. Phytochemistry. 37:587–588. doi: 10.1016/0031-9422(94)85106–9. Singh, A. K., Chanotiya, C. S., Yadav, A., & Kalra, A. (2010). Volatiles of Callicarpa macrophylla: a rich source of selinene isomers. Nat Prod Commun. 5(2): 269–272.

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Suffness, M., Abbott, B. J., Statz, D., Wonilowicz, E., & Spjut, R. (1988). The utility of P388 leukemia compared to B16 melanoma and colon carcinoma 38 for in vivo screening of plant extracts. Phytother Res. 2: 89–97. doi: 10.1002/ptr.2650020209. Talapatra, S. K., Polley, M., & Talapatra, B. (1994). Terpenoids and related compounds. Part 32 Calliphyllin, a new diterpene from the leaves of Callicarpa macrophylla. J Indian Chem Soc. 71:527–532. Uniyal, B. & Shiva, V. (2005). Traditional knowledge on medicinal plants among rural women of the Garhwal Himalaya Uttaranchal. Indian J Tradit Knowle. 4(3):259–266. Verma, R. K., Singh, A. K., Srivastava, P., Shanker, K., Kalra, A., & Gupta, M. M. (2009). Determination of novel plant growth promoting diterpenes in Callicarpa macrophylla by HPLC and HPTLC. J Liq Chromatogr R T. 32(16):2437–2450. doi:10.1080/10826070903188211 Yadav, V. K., Satpathy, S., & Patra, A. (2012a). Pharmacognostical studies of Callicarpa macrophylla Vahl. stem. Int J Phytother Res. 2(1): 35–41. Yadav, V., Jayalakshmi, S., Patra, A., & Singla, R. K. (2012b). Investigation of analgesic and anti-pyretic potentials of

Source of Support:

NIL

Callicarpa macrophylla Vahl. Leaves extracts. Webmed Central: Int J Mol Med. 3(6): WMC003447. Yadav, V., Jayalakshmi, S., Singla, R. K., & Patra A. (2012c). Evaluation of antibacterial activity of Callicarpa macrophylla Vahl. stem extracts. Webmed Central: Ayurvedic Medicine. 3(8):WMC003651 Yadav, V., Jayalakshmi, S., Singla, R. K., & Patra, A. (2011). Preliminary assessment of anti-inflammatory activity of Callicarpa macrophylla Vahl. leaves extracts. Indo Global Journal of Pharmaceutical Sciences. 1(3): 219–222. Yadav, V., Jayalakshmi, S., Singla, R. K., & Patra, A. (2012d). Ex vivo screening of stem extracts of Callicarpa macrophylla Vahl. for antifungal activity. Indo Global Journal of Pharmaceutical Sciences. 2(2): 103– 107. Zarkhande, O., & Mishra, U. (2004). Dhanwantari Nighantu of Dhanwantari, Varanasi: Chaukhanbha Surbharati Prakashan. pp. 125–126. Zhou, R. B., Liu, X. R., Tang, L. J., & Liu, J. S. (2011). Content determination of luteolin in Callicarpa kwangtungensis and Callicarpa macrophylla in different harvesting time by RP-HPLC. China Pharmacy. 23: 030.

Conflict of Interest: None Declared

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Short communication PHYTOCHEMICAL ANALYSIS OF SOME INDIGENOUS WOUND HEALING PLANTS Jagan Mohan Reddy P1, Ismail Shareef M2*, Gopinath S M3, Dayananda K S4, Ajay Mandal5, Purushotham K M6 1,2,3,4

Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore- 560 107, Karnataka, India. 5 Research Scholar, Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore560 107, Karnataka, India. 6 Institute 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

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): 101â&#x20AC;&#x201C;104

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INTRODUCTION

Collection of plant material

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.

The plants were collected from different regions of Bangalore, Karnataka, India.

MATERIALS AND METHODS

All the extracts were dissolved in different solvents for checking the solubility of extracts.

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).

Preparation of plant extract Plant material was kept for drying for about 2 weeks, away from direct Sun light below 45Â° 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

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.

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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).

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).

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

Consistency

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) 242.20 217.60 300.00

Calotropis procera (Leaves) Ricinus communis (Leaves) Mentha piperita (Leaves)

Weight of dry extract (gm) 24.80 49.60 43.40

Yield (%) 10.23 22.79 14.46

Table: 3. Qualitative determinations of active ingredients in crude extract of different Plants Calotropis procera Ricinus communis Mentha piperita (Leaves) (Leaves) (Leaves) + − + + + + + + + + + − − − + + + + + − + phenolic

Phyto-chemicals

Alkaloids Saponins Flavonoids Glycosides Carbohydrate Fixed oils and fats Tannins & Compound Proteins & amino Acids

−

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−

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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. Source of Support:

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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.

Conflict of Interest: None Declared

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Research Article A PHARMACEUTICAL APPROACH ON MANIKYA PISHTI TOWARDS STANDARDIZATION Wavare Ramesh1, Yadav Reena2*, Sheth Suchita3, Sawant Ranjeet4 1

Associate Professor & Head, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu Ayurved Mahavidyalaya, Charni road, Mumbai. 2 P. G Scholar, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu, Ayurved Mahavidyalaya, Charni road, Mumbai. 3 Assistant Professor, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu, Ayurved Mahavidyalaya, Charni road, Mumbai. 4 Assisitant 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.

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

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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)

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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.

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

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 Stokesâ&#x20AC;&#x201C;Einstein equation.

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, welldefined 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.

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

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 Rough Shape Uncut Cut Anisotropic Isotropic/Anisotropic 4.78cts Weight 5.00 length approx. Dimension Unmounted Mounted / Unmounted Milky pinkish brownish Colour Inert Fluorescence Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

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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 Raw Sample of Ruby

Test below 10% particles 50% particles 90% particles 100% particles

Observation 134.717 706.819 1445.421 1999.400

Figure 1: Particle size of Raw Ruby by DLS

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105â&#x20AC;&#x201C;111

Table 3: Particle Size of Manikya Pishti BY NTA Sample

Particle size

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 3.479 100 25.580 3.346 10.7 26.621 3.243 6.1 27.480 2.552 84.3 35.140 2.379 35.5 37.780 2.085 97.5 43.360 1.739 64.2 52.580 Table 5: X-ray diffraction of Manikya Pishti 2-Theta value 25.620 25.800 28.00 35.380 37.940 38.060 43.500

MP d-spacing 3.4741 3.4503 3.1840 2.5349 2.3969 2.3624 2.087

Intensity 513 3971 600 1504 1088 600 1904

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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

Al Cu Fe Cr K Na Ca Mn Mg As S

(%) mg/Kg (ppm) mg/Kg (ppm) mg/Kg (ppm) mg/Kg (ppm) mg/Kg (ppm) mg/Kg (ppm) mg/Kg (ppm) mg/Kg (ppm) mg/Kg (ppm) mg/Kg (ppm)

51.2 21.6 1600 2884 274 52 174.4 ND 1004 ND ND

46.9 74.0 25776 3928 951 3372 12092 186 5752 ND ND

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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

Source of Support:

NIL

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. Conflict of Interest: None Declared

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