GJRMI - Volume 2, Issue 7, July 2013 by Dr. Hari Venkatesh.K.Rajaraman

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An International, Peer Reviewed, Open access, Monthly E-Journal

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

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Honorary Members - Editorial Board Dr Farhad Mirzaei Mr. Harshal Ashok Pawar

INDEX – GJRMI, Vol.2, Iss. 7, July 2013 MEDICINAL PLANTS RESEARCH Bio-Chemistry IN VITRO ANTIOXIDANT AND NITRIC OXIDE SCAVENGING ACTIVITIES OF PIPER GUINEENSE SEEDS Etim Okon E, Egbuna Chibuzor F, Odo Christian E, Udo Nsikan M, Awah Francis M

475–484

Bio- Technology PHARMACOGNOSTICAL INVESTIGATIONS ON DIFFERENT PARTS OF Clerodendrum inerme: Chethana G S, Savitha H, Jyothi N, Hari Venkatesh K R, Gopinath S M

485–491

Bio-Chemistry IDENTIFICATION OF HYDROCARBON DEGRADERS IN A CRUDE OIL POLLUTED VESSEL AND POSSIBLE GROWTH INDUCING POTENTIAL OF AZADIRACHTA INDICA Etim Okon E, Udosen Christiana I, Akara Priestine O N J

492–498

Review Article ANALYSIS OF TRADITIONAL CHINESE MEDICINE INJECTIONS USED IN THE TREATMENT OF RESPIRATORY SYSTEM-RELATED DISEASES BASED ON THE CHINESE MARKET Zhi-Qiao Ma, Jin-Jian Lu, Wen-Shan Xu, Xiu-Ping Chen, Hao Hu, Yi-Tao Wang

499–508

Review Article EXOTIC MEDICINAL PLANTS GROWING IN IRAN: A SYSTEMATIC AND PHARMACOLOGICAL REVIEW Mikaili Peyman, Aghajanshakeri Shahin, Moloudizargari Milad

509–524

Review Article ETHNO MEDICINAL PRACTICES AMONG THE BINJHWAR TRIBE OF CHHATTISGARH, INDIA Shukla Rajesh, Chakravarty Moyna, Goutam M P

525–531

INDIGENOUS MEDICINE Ayurveda – Dravya Guna EVALUATION OF ANTIARTHRITIC ACTIVITY OF LEPIDIUM SATIVUM LINN SEEDS AGAINST FREUND’S ADJUVANT INDUCED ARTHRITIS IN RATS Raval Nita D, Ashok B K, Ravishankar B

532–537

Ayurveda – Dravya Guna DEVELOPMENT OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS FOR AUTHENTIFICATION OF OLAX SCANDENS ROXB. Naik Raghavendra, Borkar Sneha D, Acharya R N, Harisha C R

538–545

Ayurveda – Review Article ADVANCEMENTS IN INDIAN SYSTEM OF MEDICINE (ISM) INFORMATICS: AN OVERVIEW Samal Janmejaya

546–553

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – INFLORESCENCE OF VIRATARU [DICHROSTACHYS CINEREA (L.) WIGHT & ARN], OF THE FAMILY MIMOSACEAE PLACE – VRIDDHACHALAM, CUDDALLORE DISTRICT, TAMIL NADU, INDIA

Research article IN VITRO ANTIOXIDANT AND NITRIC OXIDE SCAVENGING ACTIVITIES OF PIPER GUINEENSE SEEDS Etim Okon E1*, Egbuna Chibuzor F2, Odo Christian E3, Udo Nsikan M4, Awah Francis M5 1, 2, 5

Department of Biochemistry, Madonna University, Elele, Rivers state, Nigeria Department of Biochemistry, University of Nigeria, Nsukka, Nigeria 4 Department of Pharmacology and Toxicology, University of Uyo, Uyo, Nigeria *Corresponding author: E-mail: okprince25@yahoo.com. 3

Received: 17/05/2013; Revised: 20/06/2013; Accepted: 28/06/2013

ABSTRACT Piper guineense is a pepper widely consumed in some parts of West Africa especially Nigeria and Ghana on account of its nutritional and medicinal properties. Indigenous people value the plant for its ethno-medical uses, as well as its food spicing capabilities. In this study, the seeds of the fruits were accessed for phyto-chemical constituents using spectro-photometric standard assays. The antioxidant activity was accessed by determining its ability to scavenge the 2, 2-diphenyl-1picrylhydrazyl (DPPH) radical, superoxide ion radical and nitric oxide radical. The results showed that the seeds were rich in flavonoids, flavonols and phenolic compounds. Total phenols were estimated at 1.16 mg gallic acid equivalents while flavonoids were 1.28 mg rutin equivalents. The seeds showed maximum inhibition of DPPH. radical of 66.4% at 500 µg/ml as compared to ascorbic acid which inhibited 77.4% at the same concentration. Superoxide anion inhibition was maximally 70% at 1000 µg/ml as compared to rutin which inhibited 99.35% at the same concentration. The seed extract was found to rapidly scavenge nitric oxide in vitro at different time intervals. The seeds could therefore be employed as a natural antioxidant booster hence its relevance in food industry and justification for its ethno-medicinal uses. KEYWORDS: Piper guineense seeds, antioxidant activity, nitric oxide scavenging

Cite this article: Etim Okon E, Egbuna Chibuzor F, Odo Christian E, Udo Nsikan M, Awah Francis M (2013), IN VITRO ANTIOXIDANT AND NITRIC OXIDE SCAVENGING ACTIVITIES OF PIPER GUINEENSE SEEDS, Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 475–484

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 7 | July 2013 | 475–484

INTRODUCTION Logically, human beings live in a highly oxidative environment and many processes involved in metabolism may result in the production of more oxidants (Rui and Boyer, 2004). It has been estimated that there are more than ten thousand oxidative hits to DNA per cell per day in humans (Amnes et al., 1993). For protection against free radicals, organisms are endowed with endogenous (catalase, SOD, gluthathione peroxidase / reductase) and exogenous (vitamin C and E, ß-carotene, uric acid) defense systems. However, in disease conditions and in situations like contamination, UV exposure etc. these systems are not sufficient. Most of the protective effects of plants on living cells have been attributed to their nonnutrient constituents e.g. carotenoid, flavonoids, isoflavonoid and phenolic acids. These and more different phyto-chemicals have been shown to possess a range of activities which may help in protecting against chronic diseases like cancer, inflammatory diseases etc. and also protects against lipid peroxidation (Hollman and Katan, 1997; Liu, 2003). The use of spice supplements in food has received global attention due to their potential use as anti-microbial, anti-helminthic, antioxidant, anti-diabetic, neuro-protective, hypocholesterolaemic, anti-hypertensive,antiinflammatory, cancer preventive and antimutagenic agents (Kong et al., 2010; Mann, 2011). Piper guineense Schumach. & Thonn, popularly known as Ashanti pepper (Uziza), is a climbing pepper belonging to the plant family called Piperaceae (Rehm and Espig, 1991; Amusan and Okorie, 2002; Asawalam, 2006). This bush pepper is widely consumed in some parts of West Africa especially in Nigeria and Ghana on account of its nutritional and medicinal properties (Negbenebor, 1999). Studies have shown that apart from the use of these plants as spices and condiments, they have several other wide applications in the local treatment and management of many

diseases. Indigenous people value the plants for their ethno-medical uses as much as for spicing foods (Stethberger, 1996). P. guineense is used as an anti-convulsant (Pei, 1983; Abila et al., 1993). Piper guineense provides oil used as aromatic in the drink industry and also medicinally (Rehm and Espig; Burkill, 1984). The fruits contain the pungent Piperine, resin and essential oils. The pungency of the pepper is due to the presence in the fruits of various resins particularly Chavicine and a yellow alkaloid, piperine that contains 5–8% of the weight of black pepper (Rehm and Espig; Lale, 1992). The fruit and leaves are used as spice for preparing soup for post-natal women. Powder from the dried fruits mixed with honey acts as a carminative and reduces stomach aches. Extract of black pepper has been reported to stimulate digestion of foods by stimulating secretion of digestive enzymes, pancreatic amylases, trypsin and chymotrypsin (Platel and Srinivasan, 2000) and is thus used for the treatment of digestive disorders. Despite these entire ethno-medicinal claims about the potential uses of P. guineense, there is little available scientific data to justify all the claims. In this study we therefore investigated the antioxidant potentials and the phytochemical composition of the seeds. MATERIAL AND METHODS Chemicals Folin-ciocalteu reagent (FCR), 2,2diphenyl-1-picrylhydrazyl (DPPH), polyvinylpolyrrolidone (PVPP), riboflavin, aluminum chloride, ferric chloride, sodium nitrite, sulfanilamide, sodium nitroprusside (SNP), methanol, L-ascorbic acid, phosphoric acid, sodium bicarbonate, N-naphthylethylenediamine dihydrochloride, disodium hydrogen phosphate, sodium chloride, potassium chloride, nitroblue tetrazolium (NBT), ethanol, sodium hydroxide, potassium dihydrogen phosphate, sodium carbonate, acetic acid, sodium acetate, methionine and ethylene diamine tetraacetate (EDTA) were all purchased from Sigma Aldrich, USA.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 7 | July 2013 | 475–484

Preparation of Crude Extract Seeds of Piper guineense were purchased from a local market in Elele, Nigeria. The plant was authenticated by Effiom O. Etim of Akwa Ibom Agricultural Development Project (AKADEP), Uyo, Nigeria. The seeds were airdried in ambient temperature. Dried seeds were crushed to powder with mortar and pestle and reduced to fine powder with a manual blender. 186.8 g of the resulting powder was subjected to extraction with 80% ethanol, concentrated using a rotator evaporator and stored at 4oC until used. Antioxidant Activity Assays Quantitative Assay

DPPH

Radical-scavenging

Scavenging activity on DPPH free radicals by the extract were assessed for according to the method reported by Awah et al., (Awah et al., 2012). Briefly, a 2.0 ml solution of extract at different concentration diluted in two fold in methanol was mixed with 0.5 ml of 0.3 mM DPPH in methanol. The mixtures were shaken vigorously and allowed to stand at room temperature in the dark for 25 min. Blank solutions were prepared with each test sample solutions (2.0 ml) and 0.5 ml of 0.3 mM DPPH solutions plus 2.0 ml of methanol. L-ascorbic acid was used as the positive control, thereafter, the absorbance of the assay mixture were to be measured at 517 nm against each blank with a UV-visible spectrophotometer. DPPH radical was calculated using the equation:

where (as in Awah et al., 2010) A0 is the absorbance of the control, and As is the absorbance of the tested sample. The IC50 value represented the concentration of the extract that caused 50% inhibition of DPPH radical and was calculated by linear regression of plots, where the abscissa represented the concentration of tested sample and the ordinate

the average percent of inhibitory activity from three replicates. Superoxide Anion Radical (O2−)-Scavenging Assay This assay was based on the capacity of the extract to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) as described by Martinez et al., (Martinez et al., 2001) with slight modifications (Awah et al., 2012). Briefly, each 3.0 ml reaction mixture contained 0.1 M phosphate-buffered saline (PBS) (pH 7.8), 0.1M methionine, 1mM riboflavin, 1mM EDTA, NBT (1mM) and 1.0 ml of test sample solutions. The tubes were kept in front of a fluorescent light and absorbance was read at 560 nm after 30 min. The entire reaction assembly was enclosed in a box lined with aluminum foil. Identical tubes containing reaction mixture were kept in the dark and served as blanks. The percentage inhibition of superoxide generation was estimated by comparing the absorbance of the control (rutin) and those of the reaction mixture containing test sample as per the equation:

Where A0 is the absorbance of the control, and As is the absorbance of the tested sample (Awah et al., 2010). In vitro Nitric Scavenging Assay

Oxide

Radical

(NO)

NO generated from sodium nitroprusside (SNP) was measured according to the method modified by Awah (Awah et al., 2012). Briefly, the reaction mixture (5.0 ml) containing SNP (5 mM) in phosphate buffered saline. (pH 7.3), with or without the plant extract at different concentrations, was incubated at 25ºC for 180 min in front of a visible polychromatic light source (25W tungsten lamp). The NO. radical thus generated interacted with oxygen to produce the nitrite ion (NO.) which was assayed at 30 min intervals by mixing 1.0 ml of incubation mixture with an equal amount of Griess reagent (1% sulfanilamide in 5%

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 7 | July 2013 | 475–484

phosphoric acid and 0.1% xiv. N-naphtyl ethylenediamine dihydrochloride). The absorbance of the chromophore (purple azo dye) formed during the diazotization of nitrite ions with sulphanilamide and subsequent coupling with N-napthyl ethylenediamine dihydrochloride was measured at 546 nm. The nitrite generated in the presence or absence of the plant was estimated using a standard curve based on sodium nitrite solutions of known concentrations. Each experiment was carried out in triplicates and the data presented as an average of three independent determinations. Phytochemical Analysis Determination of total phenolic contents Total phenolics were determined using Folin-Coicalteu reagent (FCR) as described by Velioglu et al., (Velioglu, 1998), with slight modifications (Awah et al., 2012). FCR consists of a yellow acidic solution containing complex polymeric ions formed from phosphomolybdic and phosphor tungistic heteropoly acids. Dissociation of a phenolic proton in a basic medium leads to a phenolate anion which reduces FCR forming a blue colored molybdenum oxide whose colour intensity is directly proportional to the phenolic contents. Briefly, 100 µl of the extract dissolved in methanol (1 mg/ml) was mixed with 750 µl of FCR (diluted 10-fold in dH2O) and allowed to stand at 22ºC for 5 min; 750 µl of Na2CO3 (60 g/l) solution was then added to the mixture. Shaken to mix. After 90 minutes, the absorbance was measured at 725 nm. Determination of Tannins Tannins content in each sample was determined using insoluble polyvinylpolypirrolidone (PVPP), which binds tannins (Makkar et al., 1993). Briefly, 1 ml of extract dissolved in methanol (1 mg/ml), in which the total phenolics were determined, was mixed with 100 mg PVPP, vortexed, left for 15 minat 4ºC and then centrifuged for 10 min at 3000 rpm. In the clear supernatant, the non-tannin phenolics were determined the same way as the total phenolics (Velioglu et al., 1998). Tannin

content was calculated as difference between total and non-tannin phenolic content. Determination of Flavonoids and Flavonols The flavonoids content was determined according to the method described by Kumaran and Karunakaran (Kumaran and Karunakaran, 2006) with slight modifications (Awah, 2012). This method is based on the formation of a flavonoid-aluminium complex which absorbs maximally at 415 nm. Briefly, 100 µl of plant extracts in methanol (10 mg/ml) was mixed 100 µl of 20% aluminium trichloride in methanol and a drop of acetic acid, and then diluted with methanol to 5 ml. The absorbance at 415 nm was read after 40 min. Blank samples were prepared from 100 µl of plant extract and a drop of acetic acid, and then diluted to 5 ml with methanol. The absorption of standard rutin solution (0.5 mg/ml) in methanol was measured under the same conditions. The amount of flavonoids in the plant extract in rutin equivalents (RE) was calculated by the following formula:

where A is the absorption of plant extract solution, Ao is the absorption of standard rutin solution, m is the weight of plant extract, mg and mo is the weight of rutin in the solution, mg. The flavonoid content is expressed in mg rutin equivalents/mg plant extract. The content of flavonols was also determined as described by Kumaran and Karunakaran (Kumaran and Karunakaran, 2006) with slight modifications (Awah, 2012). Briefly, 1 ml of methanolic extract (10 mg/ml) was mixed with 1 ml aluminium trichloride (20 mg/ml) and 3 ml sodium acetate (50 mg/ml). The absorbance at 440 nm was read after 2.5 h. The absorbance of standard rutin solution (0.5 mg/ml) in methanol was also measured under the same conditions. The amount of flavanols in the extract was calculated by the same formula for flavonoids.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 7 | July 2013 | 475–484

In vitro Free Radical Scavenging Activity

manner. As shown in Figure 2, the extract showed the maximal O2.- anion inhibitory activity of 70.0 % at the concentration of 1000 μg/ml compared to the standard antioxidant rutin 99.35% at 1000 μg/ml. The O2.scavenging effect of the extracts could culminate in the prevention of .OH radical formation since O2.- and H2O2 are required for . OH radical generation.

Effect of extract on DPPH. Radicals

IC50 for free radical inhibition

The Piper guineense seed extracts showed significant dose-dependent DPPH radical scavenging capacity (Figure 1). The extract was most efficient at 500 μg/ml, inhibiting 66.4 % of DPPH radical compared to ascorbic acid which inhibited 77.4% at the same concentration.

The concentration of the Piper guineense seed extracts that inhibited 50% of the free radicals and lipid peroxidation (IC50) was used to determine the potency of the extracts. The lower the IC50 value the better the extract potency. As shown in Table 1 below, the Piper guineense seed extracts were efficient inhibitors of different free radicals compared to standard anti-oxidants. The IC50 values for DPPH radical was 227.66 μg/ml and that for O2- anion 83.04 μg/ml.

Statistical analysis Linear regression plots were done using Microsoft Excel for Windows Vista. All the results are to be expressed as mean ± standard error of the mean (SEM) (n = 3). RESULTS

Effect of extract on superoxide (O2-) anion radical The Piper guineense seed extracts inhibited the formation of reduced NBT in a dose-related

Figure 1: DPPH radical scavenging activity of Piper guineense seed extract Data represented as mean ± SEM (n = 3)

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 7 | July 2013 | 475–484

Figure 2: Superoxide anion radical (O2-) inhibition by Piper guineense seed extract. Data represented as mean ± SEM (n = 3)

Table 1: Free radical and lipid peroxidation inhibitory potency (IC50) IC50 value for inhibitory potential (μg/ml) Extract

DPPH. radical

Superoxide anion (O2.-)

Piper guineense seed

227.66

83.04

Standard anti-oxidant

18.638 *

22.2535 β

Data represented as mean ± SEM (n = 3) * compared to ascorbic acid β compared to rutin.

Effect of extracts on nitric oxide (NO) radical production Nitric oxide (NO.) released from sodium nitroprusside (SNP) has a strong NO character which can alter the structure and function of many cellular components. This study showed that the Piper guineense extract in SNP solution decreased levels of nitrite, a stable oxidation product of NO. liberated from SNP (Figure 3). The Piper guineense extract exhibited strong NO. radical scavenging activity leading to the reduction of the nitrite concentration in the assay medium, a possible protective effect against oxidative damage. The

NO. Scavenging capacity was concentration dependent with 1000 µg/ml of the extracts scavenging most efficiently. Quantitative Analysis on Phyto-chemical Constituents of Piper guineense seed extract. Phenolic compounds were a major class of bioactive components in the extracts. The amount of total phenolics was 1.16 ± 0.17 mg gallic acid equivalent (GAE)/mg of dry plant extract and flavonoid contents was 1.28 ± 0.47 mg rutin equivalents / g dry weight plant extract (Table 2).

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

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 7 | July 2013 | 475–484

Figure 3: Effect of Piper guineense seed extract on nitric oxide (NO.) radical production.

Table 2: Quantitative phytochemical constituents Extract

Phenolic contents * Total Phenols

Piper guineense seed

1.16 ± 0.17

Nontannins 0.95 ± 0.14

Total flavonols ‡ Tannins 0.21 ± 0.04

0.53 ± 0.05

Total flavonoids

‡

1.28 0.47

Data represented as Mean ± SD (n = 3) * Expressed as mg gallic acid equivalents (GAE) / mg dry weight Piper guineense seed extract ‡ Expressed as mg rutin equivalents (RE) / g dry weight Piper guineense seed extract

DISCUSSION Free radicals are organic molecules responsible for aging, tissue damage, and implicated in a wide variety of diseases. These molecules are very unstable, therefore they look to bond with other molecules due to the presence of unpaired electrons in their outer shell,thereby causing mitochondria malfunction,cell membrane damage and eventually apoptosis. Antioxidants, present in many foods, are molecules that prevent free radicals from harming healthy tissue. The free radicals are also involved in the normal

physiology of living organisms. In this study the antioxidant activity of Piper guineense extract was investigated using its DPPH radical and nitric oxide scavenging potentials. DPPH radicals are stable free radicals. This method depends on the reduction of the purple DPPH by accepting electrons from antioxidants to stable coloured (yellow-coloured) DPPH. The degree of colour change from purple to yellow at different concentration was measured at 517 nm. The extract showed a potent DPPH radical scavenging potential inhibiting 66.4% of DPPH

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at 500 Îźg/ml compared to the standard ascorbic acid 77.4%. The antioxidant in the extract neutralized the free radical character of DPPH by transferring either electrons or hydrogen atoms to DPPH (Naik et al., 2003), thereby giving rise to the colour change. This interaction depends on the structural conformation of the bioactive compounds present in the plant extract of which the hydroxyl groups of flavonoids are highly favorable (El-sayed, 2009). The degree of discoloration indicated the scavenging potential of the extract in terms of hydrogen donating ability (Mosquera et al., 2007).

reduce the risk of various diseases. Flavonoids extend the activity of vitamin C, acts as antioxidant, protects LDL cholesterol from oxidation to unsafe cholesterol, reduce the risk of cancer and also have anti-inflammatory action (Ajali, 2004).

Though having its beneficial effect, nitric oxide (NO) contribution to oxidative damage is increasingly becoming evident. Excess production of NO has been associated with several ailments such as carcinomas, juvenile diabetes, multiple sclerosis, arthritis and ulcerative colitis (Hazra., et al., 2008). The Piper guineense extract scavenged nitric oxide in a time dependent manner figure 3, the nitrite oxide levels were higher in the SNP only tubes compared to SNP and extract mediums at the different time interval. This suggests that SNP generated nitric oxide, but the extract with its potent nitric oxide scavenging activity, was able to mop up the radicals.

This study therefore portrays that ethanol extract of piper guineense seeds exhibited high antioxidant and free radical scavenging activities. Some reactive oxygen species (ROS) are associated with the pathogenesis of inflammatory diseases as such the free radical inhibitory effect of the extract justifies its ethno-medical use in the treatment of different disease conditions.

Phenolic compounds, flavonoids and flavonols which are known to possess good medicinal values (Desta, 1993), were assayed for in this extract. These phyto-chemicals have a lot of pharmacological properties as proved by earlier studies (Ajali, 2004). The observed presence of tannins could be of great medicinal importance since tannins serve as a good antioxidant (Gulein, 2005). Therefore Piper guineense extracts are good source of antioxidants, which are widely believed to be an important line defense against oxidative stress leading to a lot of diseases like insomnia, diabetes etc. Flavonoids were also observed to be present in the plant which could at the same time contribute extensively to some biological properties that promote human health and

Plant phenolics present have received considerable attention because of the potential antioxidant activity. Phenolic compounds are effective hydrogen donors which make them good antioxidants. As shown in Table 1 the total phenolic content of the extract was moderately high.

CONCLUSION Piper guineense being a plant used as spices and condiments, have several other wide applications in the local treatment and management of many diseases. It has significant antioxidant activity owing to its free radical scavenging potential as shown in the results. Therefore, this plant could be relevant in the prevention and treatment of diseases whose pathogenesis could implicate the oxidative stress as well as in the food industry as a good preservative owing to its antioxidative potential. ACKNOWLEDGEMENTS The authors acknowledge the chancellor; Madonna University, Elele, Very Rev. Prof. E. M. P. Edeh for his support and Prof. A. A. Uwakwe of Department of Biochemistry, University of Port Harcourt, Nigeria, for his useful suggestions.

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REFERENCES Abila B, Richens J, Davies D, Anti-convulsant effect of extracts of West African black pepper (P. guineensis), J Enthno Pharmacol, 39, 1993, 113–117. Ajali U, Chemistry of Bio-compounds, Ryce Kerex publishers, Enugu, 2004, pp 81– 161. Amnes B, Shigenaga M, Hagen T, Oxidants, anti-oxidant and the degenerative diseases of aging, Proc Nat Acad Sci, 90, 1993, 7915–7922. Amusan A, Okorie T, The use of Piper guineense fruit oil (PFO) as protectant of dried fish against Dermestes maculates (Degeer) infestation, G J P Appl Sci, 8, 2002, 197–201. Asawalam EF, Insecticidal and repellent properties of Piper guineense seed oil extract for the control of maize weevil, Sitophilus zeamais, Electron J Environ Agri Food Chem, 5, 2006, 1389–1394. Awah FM, Offor, NN, Ndunaka, AC, Okafor, FU, Enyabine, CO, Free Radical Scavenging Activities and Phenolic Contents of the Spices Thymus vulgaris (Thyme), Helichrysum italicum (Curry leaf) and Laurus nobilis (Bay leaf) Extracts, J Pharm Res, 5(6), 2012, 3417–3421. Awah FM, Uzoegwu PN, Oyugi JO, Rutherford J, Ifeonu P, Yao X, Fowke KR, Eze MO, Free radical scavenging activity and immunomodulatory effect of Stachytarpheta angustifolia leaf extract. Food Chem, 119, 2010, 1409–1416. Burkill HM, The useful plants of West Tropical Africa, Vol 3. Families J-L, Royal Botanical Garden kew, 1984, pp 52. Desta B, Ethiopian traditional herbal drugs part II: Antimicrobial activity of 63 medicinal plants, J Ethno pharmacol 39, 1993, 129–139.

El-sayed SA, Total phenolic contents and free radical scavenging activity of certain Egyptian Ficus species leaf samples, Food Chemistry, 114, 2009, 1271– 1277. Gulcin I, Berashvilli D, Gepdiremen A, Antiradical and antioxidant activity of total anthocyanins from Perilla pankinrensis decne, J Ethnopharamcol, 101, 2005, 287. Hazra B, Santana B, Nripendranath M, Antioxidant and free radicals scavenging activity of Spondias pinnata, JBMC, 8, 2008, 63. Hollman PCH, Katan MB, Absorption, metabolism and health effects of dietary flavonoids in man, Biomedicine Pharmaco-therapy, 51, 1997, 305–310 Kong B, Zhang H, Xiong YL, Antioxidant activity of spice extracts in a liposome system and in cooked pork patties and the possible mode of action, Meat Sci, 85(4), 2010, 772–778. Kumaran A, Karunakaran RJ, Antioxidant and free radical scavenging activity of an aqueous extract of Coleus Aromaticus, Food Chem, 97, 2006, 109–114. Lale NES, A Laboratory study of the comparative toxicity of products from three spices to the maize weevil, Postharvest Biol Technol, 2, 1992, 61– 64. Liu RH, Health benefits of fruits and vegetables are fron additive and synergistic combinations of phytochemicals, Am J Clin Nutr, 78(3), 2003, 517–525. Makkar HPS, Bluemmel M, Borowy NK, Becker K, Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods, J Sci Food Agric, 61, 1993, 161–165.

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Mann A.,(2011). Biopotency role of culinary spices and herbs and their chemical constituents in health and commonly used spices in Nigerian dishes and snacks, Afr J Food Sci.; 5(3): 111–124. Martinez AC, Marcelo EL, Marco AO, Moacyr M, Differential responses of superoxide dismutase in freezing resistant Solanum curtibolum and freezing sensitive Solanum tuberosum subjected to oxidative and water stress, Plant Science, 160, 2001, 505–515. Mosquera OM, Correa YM, Buitrago DC, Niö J, Antioxidant activity of twenty five plants from Colombian biodiversity, Memorias do Instituto Oswaldo Cruz, 102, 2007, 631–634. NaikGH, Priyadarsini KI, Satav JG, Banavalikar MM, Sohoni DP, Biyani MK, Mohan H, Comparative antioxidant activity of individual herbal components used in Ayurvedic medicine. Phytochemistry, 63, 2003, 97–104. Negbenebor CA, Godiya AA, Igene JO, Evaluation of Clarias anguillains treated with spice (Piper guinnense) for washed mice and kama book type product, Food Composit Anal, 2, 1999, 312–315.

Source of Support: Nil

Pei YQ, A review of pharmacology and clinical use of piperine and its derivatives. Epilepsia, 24, 1983, 177–182. Platel K, Srinivasan K, Influence of dietary spices and their activetheir active principles on pancreatic digestive enzymes in albino rats. Nahrung, 44, 2000, 42–46. Rehm S, Espig G, The Cultivated Plants of the tropics and subtropics. Cultivation, Economic value, Utilization. Verlag Josef, Margraf Scientific books, CTA, 1991, pp552. Rui H, Boyer J, Apple phytochemicals and their health benefits, Rev Nutr J, 3, 2004, 5 Stethberger S, Bomme U, Rothenburger W, Economics of medicinal and condiment plants. Germuse-Muchen, 32, 1996, 117–118. Velioglu YS, Mazza G, Gao L, Oomah BD, Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J Agric Food Chem, 46, 1998, 4113–4117.

Conflict of Interest: None Declared

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Research article PHARMACOGNOSTICAL INVESTIGATIONS ON DIFFERENT PARTS OF CLERODENDRUM INERME Chethana G S1, Savitha H2, Jyothi N3, Hari Venkatesh K R4, Gopinath S M5* 1

Research Associate, Sri Sri Ayurveda Trust, Bangalore PG Scholar, ALN Rao Memorial Ayurvedic Medical College, Koppa-577126, Chikkamagalur District, Karnataka, India 3 Research Assistant, ALN Rao Memorial Ayurvedic Medical College, Koppa-577126, Chikkamagalur District, Karnataka, India 4 Head, R & D, Sri Sri Ayurveda Trust, Udayapura, Bangalore District, Karnataka, India 5 HOD, Department of Biotechnology, Acharya Institute of Technology, Bangalore District, Karnataka, India *Corresponding Author; Email Id:drsmgnath@gmail.com 2

Received: 17/05/2013; Revised: 20/06/2013; Accepted: 28/06/2013

ABSTRACT Clerodendrum inerme is a hedge plant belongs to the Verbenaceae family, traditionally used for ornamental purpose in home gardens. Clerodendrum inerme is used in many places in landscaping, as a ground cover or a hedge plant, especially near the sea, as it tolerates the salt spray. The different parts of the plant are used as folk medicines against many diseases. The plant has many active components like alkaloids, flavonoids, terpenes etc. having a wide range of application in the field of medicine. Being a weed, with innumerable therapeutically useful components is a boon to the plant to be explored & established for its medicinal potential. The present study was undertaken with an aim to explore the pharmacognostic aspect of the plant which forms the very basic part of a drug evaluation. The microscopic and macroscopic observations made have been documented which might be an eye opener to future researches on this plant. KEYWORDS: Clerodendrum inerme, leaf, stem, root, pharmacognosy

Cite this article: Chethana G S, Savitha H, Jyothi N, Hari Venkatesh K R, Gopinath S M (2013), PHARMACOGNOSTICAL INVESTIGATIONS ON DIFFERENT PARTS OF CLERODENDRUM INERME, Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 485â&#x20AC;&#x201C;491

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INTRODUCTION Clerodendrum inerme is valued in landscaping as a groundcover or hedge plant. It has attractive evergreen foliage and fragrant white flowers that form in clusters and are accented by delicate red protruding stamens. Seaside Clerodendrum, as its name suggests, grows well along the beach tolerating the salt spray of the ocean and the harsh rays of the sun. It is a versatile plant and can be grown as topiary or as a bonsai (S.R. Harish and K. Murugan, 2011), (Forest Starr et al., 2003). Clerodendrum inerme belongs to the family Verbenaceae. The genus Clerodendrum includes over 450 species of tropical regions. The name Clerodendrum is derived from the Greek kleros, meaning chance or fate, and dendron, meaning tree, in reference to the uncertain medicinal qualities of some of the plants (Forest Starr et al., 2003). Evergreen sprawling shrub, 1–1.8 meter tall. Stems (Fig 1 & 2) woody, smooth. Leaves (Fig 1 & 2) ovate to elliptical (5–10 cm) long, acute to acuminate tip, green, smooth, slightly shiny upper surface, pinnate venation, margins entire, leaves opposite, simple. Cyme or umbel usually comprised of 3 flowers joined at a common base point; corolla white, fused, with 5 lobes; stamens 4, reddish to purple and upwardly curved. Fruit green turning black, 1– Fig. 1 Clerodendrum inerme in its habitat

1.5 cm long, obovoid (S.R. Harish and K. Murugan, 2011), (Forest Starr et al., 2003). The different parts of the plant have shown therapeutically significant activities like antinemtidalcidal activity, antimicrobial activity, and anti-hepatotoxic activity. It has also been used for curing skin diseases, rheumatism etc. The methanolic extract of root contains verbanoside, which possess analgesic and anti microbial activities which is prescribed conforming the traditional use of this plant as medicine. The crude extract of the leaf also have shown the anti microbial activity. (Chethana G.S et al., 2013). This plant Clerodendrum inerme is known to have many active principles like alkaloids, flavanoids, terpenes etc (Chethana G.S et al., 2013) It is an important medicinal plant reported to be used in the treatment of skin diseases, venereal infections, elephantiasis, asthma, topical burns and for rheumatism. It is also used as a substitute of quinine. In Siddha medicine, it is used under the names of ‘Chankan kuppi’ and ‘Pechagnan’. A glycoside ester namely Verbascoside has been isolated from the root of this plant, which has analgesic and antimicrobial properties. The antioxidant activity of the plant extract may be due to the presence of polyphenols which are reported as strong antioxidants (Forest Starr et al., 2003).

Fig. 2 Clerodendrum inerme in its habitat

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The leaves and stems contain a number of triterpenes, diterpenoids, sterols and flavones. The leaves yielded the flavanolid, friedelin, salvigenin (5-hydroxy-6, 7, 4’- methoxy flavones), acacetin, cirisimaritin, pectolinarigenin, apigenin (5, 7-dihydroxy-4’ mathoxy flavaone) and amethyl flavones, cleroflavone (7-hydroxy 5, 4’ dimethoxy-6methyl flavanone). The leaves also yielded diterpenes clerodendrin B. the leaves exhibited growth inhibition and anti-feedant activities in house flies and mosquitoes. (KS Krishnan Marg, 2010). Clerodendron inerme as a febrifugal and uterine stimulant, a pest control agent and antiseptic, to arrest bleeding, treatment of asthma, hepatitis, ringworm, stomach pains (Chellaiah Muthu et al., 2006). The aim of the present study was to explore the plant pharmacognostically by the study of the macroscopic and microscopic observation of internal and external parts of the plant. MATERIALS AND METHODS The whole plant, Clerodendrum inerme was collected from Harihar TQ, Davangere dist, Karnataka, India during the month of April 2013. The botanical identity was confirmed by the Taxonomical experts in the R&D wing, Sri Sri Ayurveda Trust, Udayapura, Bangalore-82, Karnataka, India. The voucher no. of the specimen is 129 maintained in Acharya College of Technology, Bangalore, Karnataka, India. Macroscopical evaluation The sample was cleaned and macroscopic evaluation of root, leaf, and stem was carried out. The leaf, stem and root were then separated and individual macroscopic characters like size, shape, texture were noted in detail. Microscopical evaluation Free hand sections of leaf, stem and root were taken and washed with chloral hydrate solution. Sections were first observed in distilled water then stained with Saffranin red. Photomicrographs were taken by Carl zeiss trinocular microscope.

RESULTS AND DISCUSSION Macroscopic characters of root: Roots are cylindrical in shape, woody; cut pieces 10 cm in length, 2–3 cm breadth. Externally dark brown and internally brownish cream in colour. Surface is rough and at places longitudinal striations and wrinkles seen, at some place it is exfoliated. Fracture short and splintery. Odour is slight aromatic, bland in taste. Fibrous in Texture. Microscopic characters of root: The transverse section of root shows, 10–12 rows of tangentially elongated and radially arranged cork cells [Fig 4.1] followed by cortex formed with 8-10 layers of oval to round shaped parenchyma cells which are compactly arranged with prisms of calcium oxalate [Fig 4.2]. The xylem vessels are of varying size, lignified, found isolated or in the group of 2–3 [Fig 4.3]. Medullary rays are 2–3 seriate and the cells are pitted and lignified [Fig 4.4]. Starch is found in wood only. Macroscopic characters of stem: Stem is cylindrical in shape, woody; cut pieces 15 cm in length, 0.2–0.3 mm in breadth. Externally light brown in colour & internally whitish to light green in colour. Surface is nearly smooth, pubescent with white oval shaped lenticels present on it. Fracture short and splintery. Odour is disagreeable with bland taste. Fibrous in Texture. Microscopic characters of stem: The transverse section of stem shows, single layer of epidermis with 2–3 celled covering trichomes followed by 8–10 layers of cortex consisting of oval shaped parenchymatous cells which are compactly arranged with prisms of calcium oxalate. Patches of lignified fibers are scattered at places in the outer few layers of cortex. Within the cortex, there is an interrupted layer of lignified fibers followed by phloem and xylem vessels. Pith is very large consisting of oval shaped parenchymatous cells.

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Fig 3: T.S. of Root of Clerodendrum inerme

Fig 4: Microscopy of different root parts of Clerodendrum inerme

4.1 Cork, 4.2 Cortex, 4.3 Vascular bundle, 4.4 Medullary rays, 4.5 Absence of Pith

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Fig 5: T.S of Stem of of Clerodendrum inerme

Macroscopic characters of leaf: Leaves are simple, opposite or ternate, elliptic or obovate in shape, size is 5–6 cm long and 3–3.8 cm broad, apex is obtuse or mucronate, slightly attenuate at base, margin entire, reticulate venation, glabrous. Odour disagreeable, coriaceous in texture. Microscopic characters of leaf: Cross section of midrib shows thick walled circular cells of upper and lower epidermis with cuticle [Fig 7.1 & Fig 7.6]. Just below the upper epidermis we can see two types of parenchyma cells i.e., 2–3 layer oval shape parenchyma cells followed by elongated cells. Above the lower epidermis 4–6 layer of oval shaped parenchyma cells can be seen. The

vascular bundle present in middle of midrib is semicircular [Fig 7.4]. Just below the vascular bundle, lignified fibers can be seen [Fig 7.5]. Cross section showed both the epidermis (Upper and Lower) in the lamina consisting of fairly thick walled circular cells with thick cuticle layer. In both epidermises we can observe glandular trichomes which are sub sessile and situated in a shallow cavity. The mesophyll consists of two to three layered palisade cells [Fig 7.2] just below the upper epidermis and four or five layers of spongy parenchyma cells with arenchymatous cells [Fig 7.3] at places seen below the palisade cells which extend up to lower epidermis. The lateral veins occur in the median position and has small collateral vascular bundle.

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Fig 6: T.S of Leaf of Clerodendrum inerme

Fig 7: Microscopy of different leaf parts of Clerodendrum inerme

7.1 Upper epidermis with cuticle, 7.2 Palisade parenchyma, 7.3 Spongy parenchyma with aerenchyma, 7.4 Vascular bundle, 7.5 Lignified fibres, 7.6 Lower epidermis with cuticle

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CONCLUSION From this work on Clerodendrum inerme ,it is helpful in the study of physiology of each cell structure of different parts of the plant. It is very important to understand the physiology of a plant cell like the presence of lignified cell which supports the plant and many more. The reason for undertaking this study is, it reported that this plant has a significance usage in

treatment of fever, wounds, skin disease etc. It is experimentally proven to show the activity of some of the properties of the Clerodendrum inerme like antimicrobial activity which correlates the usage as folk medicines like applying for wounds etc. Much more work has to be carried out regarding the medicinal value of this plant available in the treatment of the diseases.

REFERENCES S.R.

Harish and K. Murugan., (2011). Biochemical and Genetical Variation in the Mangrove Associate (L) Gaertn. Under Different Habitats of Kerala. Asian J. Exp. Biol. Sci. 2(4): 553–561.

Forest Starr, Kim Starr, and Lloyd Loope., (2003). Clerodendrum inerme Seaside Clerodendrum Verbenaceae. United States Geological Survey--Biological Resources Division Haleakala Field Station, Maui, Hawai'i January.1–3.

Chethana G.S., Hari Venktatesh K.R., S.M Gopinath., (2013). Review on Clerodendrum inerme. JPSI. 2(2):38–40 Chethana G.S., Hari Venktatesh K.R., S.M Gopinath (2013). Phyto Chemical Analysis of Clerodendrum inerme. IRJP. 4(5):208–209 KS Krishnan Marg (2001). The Wealth of India. National Institute of Science Communication, CSIR, New Delhi. 2:67–68.

Chellaiah Muthu, Muniappan Ayyanar, Nagappan Raja and Savarimuthu Ignacimuthu., (2006). Medicinal plants used by traditional healers in Kancheepuram District of Tamil Nadu. India Journal of Ethnobiology and Ethnomedicine.2:43. Source of Support: Nil

Conflict of Interest: None Declared

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Research article IDENTIFICATION OF HYDROCARBON DEGRADERS IN A CRUDE OIL POLLUTED VESSEL AND POSSIBLE GROWTH INDUCING POTENTIAL OF AZADIRACHTA INDICA Etim Okon E1*, Udosen Christiana I2, Akara Priestine O N J3 1, 3

Department of Biochemistry, Madonna University, Elele, Nigeria Department of Microbiology, University of Uyo, Uyo, Nigeria. *Corresponding author: E-mail: okprince25@yahoo.com 2

Received: 31/05/2013; Revised: 27/06/2013; Accepted: 30/06/2013

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds that have accumulated in the natural environment mainly as a result of anthropogenic activities such as the combustion of fossil fuels. Some microorganisms, mainly from the genera Pseudomonas and Mycobacteria were found to be capable of transforming and degrading PAHs. Bioremediation is one approach that has been used to remediate contaminated soil and waters, and at the same time promotes the natural attenuation of the contaminants using microbial community of the site. However, the aim of this study was to investigate the possible effect of Azadirachta indica leaves on hydrocarbon degraders in crude oil polluted cotton wool vessel. The study was carried out using pumpkin seeds, Azadirachta indica, and crude oil polluted cotton wool vessel. The result from this study showed that the vessel with the highest quantity of Azadirachta indica leaves had the highest growth of hydrocarbon degrading microorganism. KEYWORDS: Azadirachta indica, Hydrocarbon degraders, polycyclic aromatic hydrocarbons.

Cite this article: Etim Okon E, Udosen Christiana I, Akara Priestine O N J (2013), IDENTIFICATION OF HYDROCARBON DEGRADERS IN A CRUDE OIL POLLUTED VESSEL AND POSSIBLE GROWTH INDUCING POTENTIAL OF AZADIRACHTA INDICA, Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 492–498

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INTRODUCTION Petrochemical industries and petroleum refineries generate large amounts of priority pollutants. The major pollutants found in these industries are petroleum hydrocarbons (Chavan and Mukherji, 2008). Petroleum is a complex mixture of hydrocarbons derived from the geologic transformation and decomposition of plants and animals that lived hundreds of millions of years ago. Petroleum consists mostly of hydrocarbon molecules. Crude oil and natural gas are the most important primary fossil fuels. Polycyclic aromatic hydrocarbons (PAHs), also known as poly-aromatic hydrocarbons or polynuclear aromatic hydrocarbons, are potent atmospheric pollutants that consist of fused aromatic rings and do not contain hetero-atoms or carry substituent (Fetzer, 2000). Bioremediation is the process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state. The main principle of this technique is to remove pollutants from the natural environment or convert the pollutants to a less harmful product using the indigenous microbiological community of the contaminated environment (Mueller et al., 1997). Interest in the microbial biodegradation of pollutants has intensified in recent years as mankind strives to find sustainable ways to clean up contaminated environments. These bioremediation and biotransformation methods use the naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), pharmaceutical substances, radionuclide’s and metals. The concentration of PAHs in the environment varies widely depending on the level of industrial development, proximity of the contaminated sites to the production source and the mode of PAHs transport. Reported soil and sediment PAHs contaminations range from 1 µg/kg to over 300 g/ kg (Kanaly, 2000).

Anaerobic metabolism of PAHs is thought to occur via the hydrogenation of the aromatic ring. The basis of this mechanism is the oxidation of the aromatic ring, followed by the systematic breakdown of the compound to PAH metabolites or carbon dioxide. PAH-degrading microorganisms are ubiquitously distributed in the natural environment, such as in soils (bacteria and nonligninolytic fungi) and woody materials (ligninolytic fungi). Many PAH contaminated soils and sediments host active populations of PAH-degrading bacteria (Tam et al., 2002). Medicinal plants are part and parcel of human society to combat diseases, from the dawn of civilization. Azadirachta indica A. Juss (syn. Melia azadirachta) is well known in India and its neighbouring countries for more than 2000 years as one of the most versatile medicinal plant showing a wide spectrum of biological activity. A. indica A. Juss and M. azedarach are two closely related species of the family Meliaceae. The former is popularly known as Indian neem (margosa tree) or Indian lilac, and the latter is known as Persian lilac. Neem is an evergreen tree, cultivated in various parts of the Indian subcontinent. Every part of the tree has been used as traditional medicine for household remedy against various human ailments, from antiquity (Chatterjee and Pakrashi, 1994). This study is therefore aimed at using grounded neem tree leaves to remediate crude oil polluted vessel. The result will ascertain the effectiveness of these leaves in bioremediation process. MATERIALS AND METHODS Chemicals Crystal violet, Lugol iodine, Ethanol, Hydrogen peroxide, Urea solution, K2HPO4, Methyl red, α –naphtol, KOH, Methyl-Pphenylene diamine hydrochloride, Mineral salt medium, Sabouraud dextrose agar, Nutrient agar, Urea agar, Citrate agar, Nutrient broth, and Peptone were all purchased from Sigma Aldrich, USA.

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Experimental design: Vessel 1 ------- Pumpkin seeds + water only Vessel 2 -------- Pumpkin seeds + 200 ml of crude oil + water Vessel 3 ------- Pumpkin seeds + 200 ml of crude oil + 150 g of grounded Azadirachta indica Leaves + water Vessel 4 ------- Pumpkin seeds + 200 ml of crude oil + 300 g of grounded Azadirachta indica Leaves + water The pumpkin seeds were planted and monitored for eight weeks using sterilized cotton wool as soil. After which the cotton wool polluted with crude oil was collected and used for the microbiological assay. Isolation of hydrocarbon utilizing organisms from cotton wool polluted with crude oil. 30 g of each polluted cotton wool was soaked in saline water (100 ml) for 1 hour, after which 1 ml from each of the soaked container was pipette and mixed with 9 ml of sterile water in a test tube and the dilution were prepared up to 10-5 dilution. Enumeration of crude oil utilizing fungi This was done using the surface spreading technique (Mbagwu, 1992) 1 ml from 10-5 of each sample was used to seed each sterile plate in triplicates. 20 ml of the mineral salt agar medium at 45oC supplemented with 1% Lactic acid was poured into the seeded plates and swirled. Enumeration of crude oil utilizing bacteria 20 ml of mineral salt medium was supplemented with nystatin was poured in the seeded plates and swirled. These plates were left on the bench to set. Filter sterile paper were soaked in sterile crude oil and placed on the lids of the cultured plates, after which the plates were incubate for 14 days at room temperature. Colony that was developed on the plates was counted with colony counter and was recorded

as colony forming unit per gram (Okpowasili et al., 1988). Identification of bacteria The procedure of Hamamura et al., (2006) was used. Pure culture that was obtained was subcultured from the primary culture on Nutrient agar plates and was incubated at room temperature for 24 hours. Discrete colonies were stocked in nutrient agar slants and labeled accordingly. Further characterization and identification were carried out base on microscopic examination, gram staining and some biochemical assays. A smear of the culture was placed on a clean grease-free slide, which was air-dried and fixed. The slide was then stained with crystal violet solution (primary stain) for 1 minute, after which it was washed with tap water and well drained. Iodine solution (mordant) was used to flood the slide for 30seconds, after which the slide was washed with tap water. Decolourization was done using acetone and washed with water. The slide was flooded with safranin (counter staining) for 60 secs, after which the slide was washed and allowed to dry. It was examined under oil immersion objective. Gram positive organisms retained the colour of the primary stain (purple) while the gram negative retained the secondary stain (pink) (Fowole and Oso, 1988). Biochemical test The biochemical test for identification of bacteria isolates were carried out as described by Prescott et al., (2005) Catalase test A drop of 30% H2O2 was placed on a glass slide using a wire loop, a little inoculums was removed and mixed with the H2O2 on the slide. A positive test was indicated by bubbling; the enzyme present on catalase positive organisms degrades hydrogen peroxide and releases O2 which is detected as effervescence.

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Motility test This test was used to determine the presence or absence of Flagella. The motility medium in tubes were inoculated by making a fine stab of the isolates with an inoculating needle to the depth of about 1–2 cm short of the tubes bottom and were incubated for 24 hours at 37oC. Motile organisms grow outside the line of stabbing, while the non-motile organisms grow on the line of stabbing. Urease test This test is used to study the ability of an organism to utilize Citrate present in Simmon’s media as a sole source of carbon for growth. In order to identify an organism that is able to use citrate as carbon source, the test organisms were inoculated into Simmon citrate agar slant and incubated for 24–72 hours. The development of a blue colour indicates a positive test. Triple sugar iron test (TSI) This test is based on the fermentation of the sugars, lactose, dextrose and the production of gas and hydrogen sulphide. A sterilized wire loop was used to pick the inoculums and stab the butt of the triple sugar iron agar medium with the test organism; these tubes were inoculated for 24 hour. Oxidase test The test indicates the presence of cytochrome oxidase which catalyses the oxidation of reduced cytochrome by oxygen. It indicates the ability of microbes to oxidize amine. A freshly prepared 1% solution of oxidase reagent was soaked on a piece of filter paper and with a sterile loop the test organisms were smeared on the area impregnated with the oxidase reagent. Deep purple coloration after a few seconds shows a positive test. Screen test of isolates for ability to utilize hydrocarbons Bacterial and fungal isolates were tested for their ability to utilize hydrocarbon using

turbidity method. Bacterial isolates were cultured on Nutrient broth and incubated for 24 hours at 28oC. Fungal isolates were inoculated into malt extract broth and incubated at room temperature (Nweke and Okpowasili, 2011). 1 ml and 1 g each from bacterial and fungal isolates were inoculated into mineral salt broth and 1ml of sterile crude oil was added into the inoculated tubes .The control tubes were incubated at room temperature under stationary condition for 7 days. The growths of the inoculates were determined by visual observation of the mineral salt broth medium turbidity as compared with the un-inoculated control tubes. RESULTS Effect of Azadirachta indica Leaves on the Levels of Bacterial Hydrocarbon Degraders in Crude Oil Polluted Cotton Wool Vessel. As shown in Figure 1 below, the bacterial counts of the crude oil polluted cotton wool medium was significantly affected by bioremediation with the A. indica leaves in a concentration dependent manner. At all concentrations of A. indica the bacterial total hydrocarbon degraders levels were significantly higher (p<0.05) compared to the levels in the control. However, at 150 g of neem leaves, there was no significant difference (p > 0.05) in the bacterial counts (2.0 × 105cfu/g) compared to the vessel with plant and crude oil only (2.1 × 105cfu/g). Effect of Azadirachta indica Leaves on the Levels of Fungal Hydrocarbon Degraders in Crude Oil Polluted Cotton Wool Vessel. Figure 2 shows that the fungal counts of the crude oil polluted cotton wool was significantly improved by bioremediation with the leaves of A. indica in a concentration dependent manner. At all concentrations the fungal total hydrocarbon degraders levels were significantly higher (p < 0.05) in the A. indica treated medium compared to the levels in the control (1.0 × 104cfu/g).

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Figure 1: Effect of Azadirachta indica leaves on the Levels of Bacteria Hydrocarbon Degraders in Crude oil Polluted Cotton Wool Vessel. 3.5

Bacterial Counts (x 105 cfu/g)

2.5

1.5

0.5

0 Control

Plant + Crude only

Plant + Crude + Neem (150g)

Plant + Crude + Neem (300g)

Figure 2: Effect of Azadirachta indica Leaves on the Levels of Fungal Hydrocarbon Degraders in Crude oil Polluted Cotton Wool Vessel.

Fungal Counts (x 105 cfu/g)

2.5

1.5

0.5

0 Control

Plant + Crude Plant + Crude + Plant + Crude + only Neem (150g) Neem (300g)

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DISCUSSION The results of the present study confirmed the matter that many of bacterial strains, especially gram-negative bacteria were found to degrade poly aromatic hydrocarbons (PAHs) compounds at various extents (Frick et al., 1999; Cerniglia 1992 ‘Sutherland et al., 1995).this explains the significant growth of these degraders as seen in vessels rich in crude oil compared to control that received normal water. All of PAHs degrading bacterial strains which have, been identified in the present study were gram negative, which agreed with the results indicated that most efficient of the PAHs degrading bacteria were belong to the genus Pseudomonas (Frick et al., 1999). Microorganisms and plants have complementary roles in phyto-remediation of the polluted soil. Phyto-remediation refers to the use of plants to clean contaminated soil. Increase in biodegradation of organic contaminants in the rhizosphere soil, the zone of soil directly adjacent to and under the influence of plant roots has been reported (Frick et al., 1999). For successful phyto-remediation, both plants and microorganisms must survive and grow in crude oil contaminated soil. Phytoremediation can be applied at moderate contamination levels or after the application of other remediation measures as a polishing step to further degrade residual hydrocarbons and improve soil quality (Frick et al., 1999). From the result in figure 1 and 2, leaves of Azadirachta indica has been able to enhance the growth of hydrocarbon degraders according to the work of Frick et al., (1999) showing their

complementary role in bioremediation. There was high microbial growth in vessel 4 with 300 g of Azadirachta leaves compared to other vessels. The biostimulation potential of Azadirachta Indica leaves in increasing the microbial population in crude oil contaminated vessel maybe due to the nitrogen and phosphorus content of the leaves. Okpkwasilli (1994), observed that the use of NPK fertilizer, urea fertilizer and poultry droppings effectively stimulated bacterial growth into utilization of crude oil. The high total hydrocarbon degraders (THD) bacteria in vessel 4 with 300 g of grounded leaves (3-2 × 105cfu/g) and that of total fungal count(2.0 × 105cfu/g) compared to control (1.0 × 104cfu/g) respectively suggests its effectiveness in bioremediation process. CONCLUSION The biodegradation of polycyclic aromatic hydrocarbons in the environment is a complex process, microorganisms such as bacteria and fungi are the key agents of bioremediation, with bacteria assuming the dominant role in marine ecosystems and fungi becoming more important in freshwater and terrestrial environments (Leahy and Colwell, 1990). The result from this study shows that the vessel with the highest quantity of Azadirachta indica leaves in it had the highest growth of hydrocarbon degraders in it, which means that the presence of A. Indica leaves enhanced the growth of the hydrocarbon degraders. It therefore suggests that A. indica leaves could act as growth inducers to enhance the growth of hydrocarbon degraders (bacteria and fungi) for effective bioremediation process in a hydrocarbon polluted environment.

REFERENCES Cerniglia, C.E.,(1992). Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation, 3: 351–368. Chatterjee, A. and Pakrashi, S. (1994). The Treatise on Indian Medicinal Plants, vol. 3,p. 76 (eds)

Chavan,A.and Mukherji.(2008).Treatment of hydrocarbon rich wastewater using oil degrading bacteria and phototropic microorganisms in rotating biological contactor; Effect of N:P ratio.J.Hazardous Materials.154(1–3) 63–72.

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Fetzer, J.C. (2000) The Chemistry and analysis of the large Polycyclic hydrocarbons. Polycyclic Aromatic Compounds (New York: Wiley) 27: 2–143. Fowole M.O, and Oso, B.A (1988) Laboratory Manual of Microbiology. Frick, C.M., R.E. Farrell and J.J. Germida (1999). Assessment of phytoremediation as in-situ technique for cleaning oil-contaminated sites, Pet. Tech. All. Can. Calgary, AB. Hamamum.N., S.H.Olson., D.M.Ward and W.P.Inskeep. (2006). Appl. Environ. Microbiol. 72:6316–6324. Kanaly, R.A. and S. Harayama, 2000. Biodegradation of high-molecularweight polycyclic aromatic hydrocarbons by bacteria. J. Bacteriol., 182:2059–2067. Kausik Biswas, Ishita Chattopadhyay, Ranajit K. Banerjee and Uday Bandyopadhyay (2002). Biological activities and medicinal properties of neem (Azadirachta indica) Current Science, 82 (11). Leahy, J.G and Colwell, R.R (1990) Microbial degradation of hydrocarbons in environment Microbiol.Rev.3:305. Mbagwu J.S.C. (1992). technology.42:167–175.

Source of Support: Nil

Bioresource

Mueller JG, Devereux R, Santavy DL, Lantz SE,Willis SG and Pritchard PH, (1997) Phylogenetic and physiological comparisons of PAH-degrading bacteria from physiologically diverse soils.Antonie Leeuwenhoek 71:329–343. Nweke C. O. and Okpowasili G.C, (2011) Inhibition of β-galactosidase and αglucosidase bact. Of zinc and cadmium Journal of Env.chem & ecotoxicology. Okpokwassilli,G.C, (1994). Pollution control: The increasing role of bioremediation: In:R.A. Borofice (ed.) Biotechnology in National Development : National Agency for science and Engineering infrastructure (NASENI) Prescott,L.M.,J.P.Harley and D.A.Klein.(2005). Microbiology;Mcgraw-Hill Higher Education. Sutherland, J.B., F. Rafii, A.A. Khan and C.E.Cerniglia, (1995). Mechanisms of polycyclic aromatic hydrocarbon degradation. Microbial transformation and degradation of toxic organic chemicals. L.Y. Young and C.E. Cerniglia (Eds.), Wiley-Liss,New York, N.Y., pp: 169–306. Tam NFY, Guo CL, Yau WY and Wong YS, (2002) Preliminary study on biodegradation of phenanthrene by bacteria isolated from mangrove sediments in Hong Kong. Mar Pollut Bull 45:316–324.

Conflict of Interest: None Declared

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Review article ANALYSIS OF TRADITIONAL CHINESE MEDICINE INJECTIONS USED IN THE TREATMENT OF RESPIRATORY SYSTEM-RELATED DISEASES BASED ON THE CHINESE MARKET Zhi-Qiao Ma1, Jin-Jian Lu2, Wen-Shan Xu3, Xiu-Ping Chen4, Hao Hu5, Yi-Tao Wang6* 1, 2, 3, 4, 5, 6

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China *Corresponding Author: Email: ytwang@umac.mo

Received: 21/05/2013; Revised: 20/06/2013; Accepted: 27/06/2013

ABSTRACT Traditional Chinese medicine (TCM) injections (TCMIs) comprise TCM formulations with vital functions in the treatment of cancer, cardiovascular diseases, respiratory system-related diseases (RSRDs) and so on. This article analyzes TCMIs used in the treatment of RSRDs in the Chinese market with special emphasis on the Xiyanping, Tanreqing, Xuebijing, Yanhuning, Reduning, Chuankezhi, Chuanhuning, and Shuanghuanglian (Chinese names of these TCMIs) injections, which present excellent market performances. Analysis of the clinical applications, herbal and chemical compositions, and pharmacological activities of these TCMIs were also conducted. TCMIs have vital functions in the treatment of RSRDs. This article aims to explore the market prospects and development of TCMIs used to treat RSRDs. KEYWORDS: Traditional Chinese medicine injections (TCMIs), related-diseases (RSRDs), Qingre, adverse reactions, Chinese market

Respiratory

Cite this article: Zhi-Qiao Ma, Jin-Jian Lu, Wen-Shan Xu, Xiu-Ping Chen, Hao Hu, Yi-Tao Wang (2013), ANALYSIS OF TRADITIONAL CHINESE MEDICINE INJECTIONS USED IN THE TREATMENT OF RESPIRATORY SYSTEM-RELATED DISEASES BASED ON THE CHINESE MARKET, Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 499–508

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system

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INTRODUCTION

development of TCMIs used to treat RSRDs.

Many respiratory system-related diseases (RSRDs), such as asthma, chronic obstructive pulmonary disease and pneumonia are common diseases. According to a data from the World Health Organization (WHO), at least 3000 million people have died because of chronic respiratory diseases and more than 90% of the patients with such diseases come from low- or middle-income countries (WHO, 2012a). Lower respiratory tract infections are ranked third in the top ten global death reasons and the first in low-income countries. Almost 14 million children below five years old are killed every year by pneumonia, a classic lower respiratory tract infection (WHO, 2012b). According to data from the 2010 Chinese health statistical yearbook, the morbidity of RSRDs in China is about 6.94%, totaling over 80 million people, and RSRDs rank as the fourth leading cause of death in urban and rural areas (Ministry of Health of the People’s Republic of China, 2010).

MATERIAL AND METHODS

Besides chemical drugs used for standard treatment of RSRDs, traditional Chinese medicine (TCM) use is increasing in China because of its excellent clinical effects (Wang et al., 2006). TCMs have important functions in preventing serious infections, such as SARS and H1N1, as they have multiple functions, which include relieving cough, diminishing inflammation, eliminating phlegm, and relieving asthma (Chang et al., 2011a). TCM injections (TCMIs) are sterile formulations (emulsion, powder, or thick liquid) prepared for injecting into the body and are made after extraction and purification (National Pharmacopoeia Committee, 2005). TCMIs are becoming increasingly valued in China due to their positive clinical effects (Yao, 2007). This article aims to explore the market prospects and

Data sources Data on the sales of TCMIs in the whole Chinese market were obtained from the Chinese Clinical Medicine Terminal Competition Pattern Database of the State Food and Drug Administration (SFDA), Southern Medicine Economic Research Institute (SMERI), (China Medicine Economic Information Network, 2012), which is calculated by the TCM purchasing practices of 150 sample hospitals from nine cities (i.e., Beijing, Guangzhou, Nanjing, Chongqing, Chengdu, Xian, Haerbin, Shenyang, and Zhengzhou) in China. Thirteen large categories and 75 small categories of drugs are listed based on the WHO therapeutic classification, Anatomical Therapeutic Chemical Code, and national essential drugs list classification for TCM. Data on clinical applications, herbal and chemical compositions and pharmacological activities were searched from China National Knowledge Infrastructure (CNKI) database and/or Pubmed database. Data analysis The annual sales and sales growth rate of TCMIs were analyzed: For market share calculations, the formula SN/Stotal × 100% was used; for sales growth rate, the formula (SN-SN-1)/SN × 100% was used, where SN is the sales of one injection at the year N and Stotal are the total sales of the drugs, including the injections. The top eight selling TCMIs used in the treatment of RSRDs in the Chinese market were chosen for analysis. The names of these injections were used as key words to search for

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their clinical applications, herbal and chemical compositions, and pharmacological activities in the CNKI database. The chemical names of the injections were further used as key words to perform searches in Pubmed database. All of the studies included in this work were published from 1994 to 2012. RESULTS TCM for RSRDs treatment in the Chinese market The sales of chemical drugs and TCMs accounted for 82.6% and 17.4% of all drugs

sales in 2010, respectively, based on the data from SFDA SMERI (China Medicine Economic Information Network, 2012). The sales of chemical drugs and TCMs for the treatment of RSRDs accounted for 54.5% and 45.5% of all sales, respectively, in 2010 (excluding systemic anti-infective drugs, which are mostly treated by antibiotics in China), indicating the importance of TCMs in treating RSRDs in China. The sales of TCMs for RSRDs increased from 2006 to 2011, with an average growth rate of 34% (Figure 1), the annual growth rates from 2007 to 2011 are 24%, 17%, 29%, 16% and 25% respectively.

Figure 1. The sales of TCM for the treatment of RSRDs from 2006 to 2011

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TCMIs for RSRDs treatment in the Chinese market At present, 1,269 TCMIs formulations are registered in SFDA, including 524 species (41.3%) for RSRDs treatment (China Medicine Economic Information Network, 2012). Among these injections, 365 species were listed in the National Health Insurance Directory and 135 species were listed in the national essential drugs list. These injections account for 70% and 26% of the total number of TCMIs used in the treatment of RSRDs, respectively. The brand concentration of TCMIs for RSRDs treatment is high. In 2011, five species of injections were observed in the top ten sales of TCMs for RSRDs treatment and their sales accounted for 38% of the total sales of TCMs

for RSRDs. Among these injections, the sales of three species added up to one billion (China Medicine Economic Information Network, 2012). The top five selling TCMIs for RSRDs treatment are Xiyanping, Tanreqing, Xuebijing, Yanhuning, and Reduning injections (Chinese names of some TCMIs) (see in Table 1). All of the injections maintained a positive growth trend, except for the Yanhuning injection, the growth rate of which declined by 1% (Figure 2). Xiyanping, Tanreqing, and Reduning injections are all listed in the national health insurance directory. The sales of Xiyanping injection increased to 0.32 billion yuan (RMB) in 2011, almost twice that of Bailing capsules, the best seller of other TCM formulations for RSRDs treatment (China Medicine Economic Information Network, 2012).

Figure 2. The change of TCMIsâ&#x20AC;&#x2122; sales for the treatment of RSRDs from 2006 to 2011

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Table 1. The rank of TCMIsâ&#x20AC;&#x2122; sales for the treatment of RSRDs in 2011 Rank

Injections

Chinese herbal medicine compositions

Main chemical compositions

Xiyanping injection Tanreqing injection

Herba Andrographisis Radix Scutellariae, Bear bile powder, Goat horn, Flos Lonicerae Japonicae, Fructus Forsythiae Flos Carthami, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Radix Angelica Sinensis, Radix Salviae Miltiorrhizae Herba Andrographisis Herba Artemisiae Annuae, Flos Lonicerae Japonicae, Fructus Gardeniae Herba Epimedii, Radix Morinda Officinalis

Andrographolide, etc.

Xuebijing injection

Yanhuning injection Reduning injection

Market share of RSRDs medicine 14.7%

Sales (million, RMB)

Growth rate (%)

323

Baicalin, Chlorogenic acid, Ursodeoxycholic acid etc.

9.3%

204

Danshensu, Safflower yellow pigment A, Tetramethylpyrazine, Ferulic acid, Paeoniflorin, Protocatechuic aldehyde etc. Andrographolide, etc.

6.5%

142

4.5%

98.2

-1

Chlorogenicacid, Geniposide, Artemisinin etc.

2.2%

47.5

0.4%

9.6

150

0.4%

8.1

-32

0.1%

2.6

-17

Chuankezhi injection

Chuanhuning injection

Herba Andrographisis

Epimedium polysaccharide, Icariin,Epimedium flavonoids etc. Andrographolide , etc.

Shuanghuanglian injection

Flos Lonicerae Japonicae, Radix Scutellariae, Fructus Forsythiae

Caffeic acid, Chlorogenic acid, Baicalin, Forsythin etc.

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Clinical applications of TCMIs for RSRDs Both Xiyanping and Reduning injections are used to treat infectious diseases and mainly focus on children (Liu et al., 2007; Zhang & Yang, 2012). These injections are also used to treat hand, foot, and mouth disease in babies, infantile diarrhoea, acute icteric model hepatitis, chronic prostatitis, and old herpes zoster, among others, in addition to the treatment of RSRDs (Liu et al., 2007; Zhang & Yang, 2012). Tanreqing, Yanhuning, and Chuankezhi injections are mostly used to treat bronchitis and various types of pneumonia (Feng, 2007; He, 2008; Liang, 2006). Besides treating bronchitis and the upper respiratory tract infections, both Chuanhuning and Shuanghuanglian injections are used to treat gastrointestinal tract inflammation, urinary tract infection, and myocarditis (Li, 2005; Liu & Xu, 2005). Xuebijing injection is significantly different from the aforementioned injections, as it is generally used for emergency and critical care, such as systemic inflammatory response syndrome caused by acute respiratory distress syndrome and sepsis (Zhang, 2008). Therefore, TCMIs have wide clinical applications and excellent performance in the market for RSRDs treatment. Herbal compositions of TCMIs used for RSRDs treatment Among the TCMIs mentioned in Table 1, most belong to the Qingre area (clearing heat) in TCM. For example, the major compositions of Xiyanping, Yanhuning, and Chuanhuning injections are herbs of Andrographis (Ma & Kuang, 2010; Zhong, Zeng, & Guo, 2010). Both Tanreqing and Shuanghuanglian injections contain Flos Lonicerae Japonicae, Radix Scutellariae and Fructus Forsythiae (Li & Li, 2011; Tu & Huang, 2008). Reduning injection contains Herba Artemisiae Annuae,

Flos Lonicerae Japonicae and Fructus Gardeniae (Jiang, et al.,2008; Liang, Huang, & Cai, 2008). The main herbs that compose Xuebijing injection are Flos Carthami, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Radix Angelica Sinensis and Radix Salviae Miltiorrhizae, all of which have the effect of Huoxue (promoting blood circulation) (Chang et al., 2011b; Yu, 2011). The main herbs in Chuankezhi injection are Herba Epimedii and Radix Morinda Officinalis, which have the effect of Buyi (tonic) (Li et al., 2009). No herb belongs to the Huatan Zhike Pingchuan class (preventing phlegm from forming, stopping coughing and relieving asthma) of TCM, which is the most-represented TCM for RSRDs treatment among the top eight ranking injections. The absence of an herbal component is due to the dosage form, as syrup is the main form for the Huatan Zhike Pingchuan class of TCM. Chemical compositions and pharmacological activities of TCMIs used for RSRDs treatment We further analyzed the main chemical compositions and pharmacological activities of the top eight injections listed in Table 1. Three injections contained andrographolide or its derivatives. Andrographolide presents excellent antibacterial, antiviral, anti-inflammatory, and anticancer effects (Chen et al., 2009; Ji, 2011; Lim et al., 2012). Chlorogenic acid and baicalin have also emerged in numerous injections. Chlorogenic acid has anti-inflammatory, antioxidative, antibacterial, cardiovascular protective effects, and so on (Ji, 2011; Wang et al., 2011). Baicalin has antibacterial, antiviral, antioxidative, anti-inflammatory, sedation, and immune system regulation effects (Ji, 2011; Srinivas, 2010; Zhu et al., 2012). Several injections contain geniposide, danshensu, safflower

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yellow A, phillyrin, and icariin, among others, all of which have numerous pharmacological activities (Ji, 2011). DISCUSSION Chemical drugs continue to enjoy several advantages in the Chinese market. However, the markets for TCM and chemical drugs are similar in RSRDs treatment if the influence of antibiotics is excluded. The TCM market in RSRDs treatment has continuously grown in recent years. If the Chinese government continues to enforce numerous policies to reduce antibiotic abuse, the advantages of TCM will significantly increase. TCMIs can enter human tissues, blood, or organs directly, and can be absorbed faster than other forms of TCM. Thus, TCMIs overcome several disadvantages of TCM dosing, such as dose inaccuracy, slow effect and low bioavailability. Compared with anti-neoplastic TCMIs, which are mostly used for adjuvant therapy (Lai et al., 2012), TCMIs have more important functions in RSRDs treatment. From the herbal and chemical composition analysis, most TCMIs for RSRDs treatment have Qingre effects (clearing heat). Classifying TCM against the standards of Western medicine is difficult due to the different theories behind Chinese and Western medicine. For example, Xuebijing injection is also used to treat cardiovascular diseases. Clinical applications of cross treatments could result in greater potential for TCMIs. With more and more TCMIs used in the treatment of RSRDs are into the national health insurance directory, along with the new medical reform policy is carried out constantly, the TCMIs market will continue to expand in China.

Although the TCMIs market for RSRDs treatment is increasing significantly, the entire market scale remains small. The market concentration is extremely high such that few products occupy most of the market. The increased market is mainly attributed to these products, which are almost exclusively manufactured or monopolized by a few companies. Thus, the medicine market and patients are cautious about the TCMIs used in RSRDs treatment. Besides, the credibility of TCMIs has decreased because numerous patients have reported adverse reactions (Liang & Shi, 2012; Ma & Kuang, 2010). Chemical compositions, clinical applications, and individual differences are the three main reasons for the adverse reactions of TCMIs. TCMIs have several features, such as complex compositions, herb quality unsteadiness, and residual impurity, and these factors are the main sources of adverse events (Ma & Kuang, 2010; Wu et al., 2012). Irrational clinical applications also result in adverse reactions (Ma & Kuang, 2010; Wu et al., 2012). For such adverse events, legal and administrative regulations should be enhanced. The SFDA carried out a series of specialized policies for the quality of TCMIs and safety evaluation from 2007 to 2009. These policies have increased the threshold of TCMIs research and development and advanced efforts for the safety evaluation of TCMIs. Identifying and controlling the effective and toxic compositions of the injections are necessary. Technological studies and clinical compatibility studies for TCMIs should be given more attention. CONCLUSION TCMIs have highly important functions in RSRDs treatment in China. From the marketâ&#x20AC;&#x2122;s perspective, TCMIs for RSRDs treatment show a positive trend and great potential, with rapid

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increasing sales and expanding market demand. In clinical view, TCMIs show the predominant and effective feature, by combining some advantages between TCM and Western medicine. Development of TCMIs is a complicated issue with traditional theory and advanced technology, so research and development institutes, manufacturers, hospitals, and regulation departments must cooperate with one another to establish a

positive outlook for the development of TCMIs. ACKNOWLEDGMENTS: This study is supported by the research funding of University of Macau (UL016/09Y4/CMS/WYT01/ICMS, MYRG208(Y2-L4)-ICMS11-WYT, and MYRG160(Y2-L2)-ICMS11-HH).

REFERENCES Chang, L., Xu, B., & Gao, L. (2011a). Determination of Safflor Yellow A in Xuebijing Injection by HPLC. Chinese Pharmaceutical Affairs. 25(2): 160–165. Chang, T.-T., Sun, M.-F., Chen, H.-Y., Tsai, F.-J., Fisher, M., Lin, J.-G., & Chen, C. Y.-C. (2011b). Screening from the world’s largest TCM database against H1N1 virus. Journal of biomolecular structure & dynamics. 28(5): 773–786. Chen, J., Xue, H., Ye, W., & Fang, B. (2009). Activity of andrographolide and its derivatives against influenza virus in vivo and in vitro. Biol Pharm Bull. 32(8): 1385–1391. China

Medicine Economic Information Network. (2012). Chinese clinical medicineterminal competition pattern database (Chinese patent drug). Retrieved September 21, 2012, from http://www.menet.com.cn/

Feng, B. (2007). The clinical appplication of Yanhuning injection. China Pharmacy. 18(12): 944–946.

He, H.-P. (2008). The immune modulation and cliniacal application of Chuankezhi injucetion. Zhongguo Xiandai Yiyao Zazhi. 10(10): 143–146. Ji,

Y.-B. (2011). Active ingredients of traditional Chinese medicine: Pharmacology and application. People’s Medical Publishing Hourse Cp., LTD.

Jiang, L., Liang, J.-C., Huang, Y.-K., & Cai, Z. (2008). Determ ination of chlorogenic acid and geniposide in Reduning injection by HPLC. Chinese Journal of New Drugs. 17(17): 1522–1524. Lai, Y.-F., Lu, J.-Ji., Chen, X.-P., & Hu, H. (2012). Analysis of anti-cancer traditional Chinese medicine injections based on the market performance. Modern TCM Materia Medica-World Sci Tec. Li,

C.-G. (2005). The application of Chuanhuning injection in paediatrics. Journal of Practical Traditional Chinese Medicine. 21(7): 444–445.

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Li, X.-L., & Li, X.-Y. (2011). Determination of baicalin and forsythin content in Shuanghuanglian injection by HPLC. Journal of China Traditional Chinese Medicine Information. 3(18): 65–66. Li, Y.-X., Xiao, G.-N., Cheng, Z.-H., & Li, J.-C. (2009). Study on the antitussive and antiasthmatic efects of Chuankezhi Injection. China Medical Herald. 6(30): 27–29. Liang, H.-T., & Shi, X.-J. (2012). Quality and safety on traditional Chinese medicine injection. Shanghai Yiyao. 33(13): 24–26. Liang, J.-C. (2006). The clinical appplication of Yanhuning injection. Zhongguo Yixue Wenzhai. 27(5): 435–437. Lim, J. C. W., Chan, T. K., Ng, D. S. W., Sagineedu, S. R., Stanslas, J., & Wong, W. S. F. (2012). Andrographolide and its analogues: versatile bioactive molecules for combating inflammation and cancer. Clinical and experimental pharmacology & physiology. 39(3): 300–310. Liu, J., & Xu, Y. (2005). The application of Shuanghuanglian injection. Xinjiang Zhongyiyao. 23(5): 78–81. Liu, J.-H., Zhang, W.-D., Wang, G.-T., & Xiao, Z. (2007). The clinacal application of Xiyanping injection. Northwest Pharmaceutical Journal. 22(2): 93–95. Ma,

G.-Q., & Kuang, J.-J. (2010). Retrospective Study of 109 cases of Adverse Drug Reactions of Xiyanping Injection. Chinese Journal of Pharmacovigilance. 7(9): 558–561.

Ministry of Health of the People’s Republic of China. (2010). Chinese health statistical yearbook 2010. Beijing: Beijing Union Medical University Press. National Pharmacopoeia Committee. (2005). Pharmacopoeia of the People’s Republic of China. Beijing: China Medical Science Press. Srinivas, N. (2010). Baicalin, an emerging multi-therapeutic agent: pharmacodynamics, pharmacokinetics, and considerations from drug development perspectives. Xenobiotica. 40(5): 357–367. Tu,

W.-S., & Huang, Q.-C. (2008). Determination of Baicalin in Tanreqing Injection by HPLC. China Pharmacy. 19(30): 2388–2389.

Wang, L.-P., Guo, D., Wang, G., & Zhou, H.-Y. (2011). Advancement of Chlorogenic Acid in Traditional Chinese Medicine. Lishizhen Medicine and Materia Medica Reseach. 22(4): 961–963. Wang, X., Jia, W., Zhao, A.-H., & Wang, X.-R. (2006). Anti-influenza agents from plants and traditional Chinese medicine. Phytotherapy research. 20(5): 335–341. WHO. (2012a). About chronic respiratory diseases. World Health Organization. Retrieved September 21, 2012, from http://www.who.int/respiratory/about_t opic/en/index.html WHO. (2012b). The top 10 causes of death. World Health Organization. Retrieved September 21, 2012, from http://who.int/mediacentre/factsheets/fs 310/en/

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Wu, J.-R., Zhang, B., Dong, L., & Zheng, J. (2012). System analysis and evaluation for the adverse reactions of traditional Chinese medicine injections. Guide of China Medicine. 10(20): 274–276. Yao, Z.-Y. (2007). Market prospect of TCM injections. World Clinical Drugs. 28(2): 126–128. Yu, S.-L. (2011). Content Determination of Danshensu in Xuebijing Injeetion by HPLC. Research of Integrated Traditional Chinese and Western Medicin. 3(4): 179–181. Zhang, C.-J., & Yang, J. (2012). The clinical application of Reduning injection. Haerbin Medicine. 32(4): 310–312.

Source of Support: University of Macau, China

Zhang, S.-F. (2008). The clinical application of Xuebijing injection. Modern Journal of Integrated Traditional Chinese and Western Medicine. 17(15): 2418–2419. Zhong, H., Zeng, L.-Q., & Guo, L. (2010). Determination of Potassium Dehydroandrograpolide Succinate Injection by HPLC. Heilongjiang Medicine. 23(3): 323–325. Zhu, J., Wang, J., Sheng, Y., Zou, Y., Bo, L., & Wang, F. (2012). Baicalin improves survival in a murine model of polymicrobial sepsis via suppressing inflammatory response and lymphocyte apoptosis. PLoS One. 7(5): 8.

Conflict of Interest: None Declared

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Review article EXOTIC MEDICINAL PLANTS GROWING IN IRAN: A SYSTEMATIC AND PHARMACOLOGICAL REVIEW Mikaili Peyman1, Aghajanshakeri Shahin2*, Moloudizargari Milad3, Javaherypour Soheil4 1

Department of Pharmacology, Faculty of Pharmacy, Urmia University of Medical Sciences, Urmia, Iran Student of Veterinary Medicine, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran *Corresponding Author: E-mail: shahin.aghajanshakeri@yahoo.com 2, 3, 4

Received: 15/05/2013; Revised: 30/06/2013; Accepted: 02/07/2013

ABSTRACT Medicinal plants are being used as desirable natural sources of various preparations made in traditional herbal therapy worldwide. Since ancient times, traditional remedies have been trusted by Iranian people. Plants of various families can be found in the prescription of rural healers in all regions of Iran. Some of these are native to Iran but many others are known as exotic plants which only grow in especial regions worldwide. This study for the first time illustrates the presence and the growth of several families of these plants in the climate of Iran along with some of their most important pharmacological effects and their active constituents.

KEYWORDS: Exotic medicinal plants; Iran; systematic study; pharmacological study

Cite this article: Mikaili. P., Aghajanshakeri. S., Moloudizargari. M., Javaherypour. S (2013), EXOTIC MEDICINAL PLANTS GROWING IN IRAN: A SYSTEMATIC AND PHARMACOLOGICAL REVIEW, Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 509â&#x20AC;&#x201C;524

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INTRODUCTION

RESULTS

The use of phytomedicines in the Iranian traditional medicine has a long history. Since ancient times, rural native-healers and drug sellers in different parts of Iran have used different herbs in their prescriptions that grow in Iran. Present article consists of 15 exotic medicinal plants that grow in Iran along with some of their important pharmacological properties. These species include: Buxushyrcana, Cercissiliquastrum, Cestrum nocturnum, Chamaerops humilis, Cotinus coggygria, Cupressus arizonica, Cupressus sempervirens, Diospyros khaki, Eriobotrya japonica, Citrus aurantium, Citrus limon, Citrus paradise, Citrus reticulata, Citrus sinensis and Citrus unshiu. The use of these plants in traditional medicine has been proved and their pharmacological properties have been already confirmed in different in vitro studies by many researchers. This study illustrates the potential of these plants to be employed as novel medicinal sources for the development of new drugs in Iran and other countries. Present work is the pioneer study that shows the growth of these exotic species in Iran.

[1] Buxus hyrcana

MATERIALS AND METHODS In order to gather the required information, the authors of the study traveled to Mazandaran province located in North of Iran and documented images of the exotic species which grow in that region. Systematic review of literature was performed and a summary of the important botanical and pharmacological properties of each plant was presented. Figure 1. Buxus hyrcana

Description B. hyrcana (common box) exists as tree and is distributed throughout the world (M. Mesaik et al., 2010). B. hyrcana (Fig. 1) is locally known as "šemšād-jangalī" in Iran and the medicinal parts of the plant including its dried leaves and the woody aerial parts have been traditionally used among Iranians (Mikaili et al., 2012; Esmaeili et al., 2009). The main compounds of B. hyrcana responsible for a variety of pharmacological effects of this plant are steroidal alkaloids such as Buxidin and EBuxenone (Mesaik et al., 2010) and triterpenoidal alkaloids such as 17-oxo-3benzoylbuxadine and buxhyrcamine (Ata et al., 2010). Pharmacological effects B. hyrcana possesses hypotensive effect (Mikaili et al., 2012), antiplasmodial activity (Esmaeili et al., 2009), acetylcholinesterase, butyrylcholinesterase and glutathione Stransferase inhibitory activity (Ata et al., 2010; Bahar, Ata, & Meshkatalsadat, 2006; Chouhary et al., 2003). Inhibitory effect on the growth of Mycobacterium tuberculosis attributable to the cycloprotobuxine present in the plant (Mikaili et al., 2012). Immunosuppressive activity due to the inhibition of IL-2 by steroidal alkaloids (M. A. Mesaik et al., 2010). Some triterpenoidal alkaloids from this plant are shown to have weak anti-leishmanial and modest anti-fungal effects (Ata et al., 2010). Figure 2. Cercis siliquastrum

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Figure No. 3. Chamaerops humilis

Figure 4. Cotinus coggygria

Figure 5. Cupressus arizonica

Figure 6. Diospyros khaki

Figure 7. Eriobotrya japonica

Figure 8. Citrus aurantium

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[2] Cercis siliquastrum

Pharmacological effects

Description

Its active substances possess analgesic activity provided through a peripheral action mechanism (Perez-Saad & Buznego, 2008). Its Mosquito larvicidal activity has been established (Patil et al., 2011). The n-butanol and polysaccharides extracts of C. nocturnum inhibited tumor growth in tumor-bearing mice in a dose dependent manner (Zhong et al., 2008). Steroidal saponins from this plant have been shown to be cytotoxic against squamous cell carcinoma-(HSC-2) cells and normal human gingival fibroblasts (Mimaki et al., 2001).

C. siliquastrum (Fabaceae) commonly known as Judas tree is a small deciduous tree from Southern Europe and Western Asia which is noted for its prolific display of deep-pink flowers in spring. It is one of the main components of a Unani herbal tea called "Zahraa" (Carmona, Llorach, Obon, & Rivera, 2005). According to a study three flavonoids including 3-O-monoglycosides of kaempferol, quercetin and myricetin were obtained from the genus Cercis (Salatino et al., 2000).

[4] Chamaerops humilis Pharmacological effects It shows anti-malarial activity (Kaiser et al., 2007). Leaf extracts of C. siliquastrum (Fig. 2) strongly inhibit 2C-methyl-D-erythritol 4phosphate synthase (IspC), an enzyme catalyzing the first step of the non-mevalonate pathway of isoprenoid biosynthesis. Since this pathway serves as the unique source of terpenoids in numerous pathogenic eubacteria and in apicoplast-type protozoa which is absent in mammalian cells, therefore it can be an attractive target for anti-infective chemotherapy (Kaiser et al., 2007). [3] Cestrum nocturnum Description C. nocturnum is a garden shrub from the family Solanaceae, commonly known as "lady of the night" which is used as a remedy for different health disorders. (Perez-Saad & Buznego, 2008). The crude extract of C. nocturtum has bioactive phytochemicals with predominance of saponins(Patil, Patil, Salunke, & Salunkhe, 2011). Spirostanolsaponin, furostanolsaponin, pseudo-furostanolsaponin, pregnane glycosides, cholestane glycosides, pregnane-carboxylic acid gamma-lactone glycoside, and spirostanol glycosides have been isolated from the leaves of C. nocturnum (Mimaki, Watanabe, Sakagami, & Sashida, 2002).

Description C. humilis (Fig. 3) family Arecaceae (Palm family) is known as the Mediterranean Fan Palm native to the Mediterranean coast (Mayoral, Torres, Munoz, Bartolome, & Blanca, 2006). Fruits are eaten in Morocco; heart "palmito" is consumed in Spain and its young suckers are cooked and eaten in Italy (Haynes & McLaughlin, 2000). It has been used for ornamental purposes in Japan and as a medicinal plant to treat diabetes in Morocco (Gaamoussi et al., 2010). It is also used to make brooms in Italy (vernacular name: Palma nana) (Nedelcheva et al., 2007). Some of its main constituents include: procyanidins (fruits), leucoanthocyanidin, diosgenin, flavone Cglycoside and tricin (leaves and root), and methyl proto-dioscin (stem) (Hirai et al., 1985). Pharmacological effects This plant is probably an uncommon cause of allergy because it is only consumed in its natural form in a few rural areas of the Mediterranean region (Mayoral et al., 2006). Its extract inhibited the growth of calcium oxalate monohydrate crystals in vitro (Beghalia et al., 2007). The aqueous leaf extract decreases total cholesterol and triglycerides (Gaamoussi et al., 2010). It was suggested in one study that the plant may become a good source of antidiabetic medication and may also be useful in the management of secondary complications of diabetes (dyslipidemia) (Gaamoussi et al., 2010).

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[5] Cotinus coggygria Description C. coggygria (syn. Rhus cotinus, family Anacardiaceae) is a shrub that extends from southern Europe, the Mediterranean, Moldova and the Caucasus, to central China and the Himalayas. It is also known as the "smoke tree". Plants of the family Anacardiaceae have a long history of use by various peoples for medicinal and other purposes such as its usage as a dyestuff. In folk medicine, C. coggygria (Fig. 4) is routinely used as an antiseptic, antiinflammatory, antimicrobial and antihaemorrhagic agent in wound-healing, as well as for countering diarrhea, paradontosis and gastric and duodenal ulcers (Matic et al., 2011; Valianou et al., 2009). Pharmacological effects Alcoholic extract of the plant has high antioxidant (313 and 231 mg AA/g dry extract), as well as DPPH free-radical scavenging property (IC (50) =9 and 99 μg/ml), inhibitory activity toward lipid peroxidation (IC (50) =3 and 17 μg/ml) and reducing power (Niciforovic et al., 2010; Savikin et al., 2009). Isolated compounds mainly responsible for its antioxidant activity include: disulfuretin, sulfuretin, sulfurein, gallic acid, methyl gallate, and pentagalloyl glucose (Westenburg et al., 2000). It has in vitro cytotoxic activity towards HeLa and LS174 human cancer cell lines (Savikin et al., 2009). In one study, the methanol extract of C. coggygria in a concentration of 5% induced recessive lethal mutations on X-chromosome on Drosophila melanogaster in all broods indicating its mutagenesity (Stanic et al., 2011). [6] Cupressus arizonica Description C. arizonica (Fig. 5) (family Cupressaceae) is an aromatic evergreen coniferous plant with great importance in urban horticulture and in the pharmaceutical and fragrance industries (Hassanpouraghdam, 2011; Rehfeldt, 1997). It is native to North and Central America and is

widespread in Mediterranean countries because of their optimal conditions for growth. It was introduced to Iran in 1954 and has been commonly cultivated in many parts of the country. This plant is becoming an increasingly frequent cause of allergic diseases (SanchezLopez, Asturias, Enrique, Suarez-Cervera, & Bartra, 2011; Sedaghat et al., 2011). α-pinene, limonene and umbellulone has been determined as major components of the oil of C. arizonica (Sedaghat et al., 2011). Pharmacological effects The plant has shown larvicidal activity against malaria vector Anopheles stephensi (with the highest dose of 160 ppm essential oil) (Sedaghat et al., 2011), in vivo and in vitro allergenic activity (Ariano et al., 2006), sensitization (symptoms include: allergic rhinitis and asthma) (Sanchez-Morillas et al., 2005). [7] Cupressus semperviren Description C. sempervirens (family Cupressaceae) is a tree that grows up to 30 meter height. The plant is indigenous to Turkey and is cultivated throughout the Mediterranean region. Chief compounds include: alpha-pinene, Dcamphene, D-silvestrene, p-cymene, Lcadinene, cedrol, terpinenol-4, terpineol, acetyl- and isovalerianyl esters of monoterpene alcohols. The drug is used externally for head cold, cough and bronchitis (PDR for Herbal Medicines, 2000; Rehfeldt, 1997). Pharmacological effects Cytotoxic activity against amelanotic melanoma C32 in vitro (IC (50) value of 104.90 microg/mL) (Loizzo et al., 2008), inhibition of glucose-6-phosphatase glycogen phosphorylase enzymes (Rawat et al., 2010), and sensitisation (symptoms: rhinitis and asthma) (Sposato & Scalese, 2011). Cypress also acts as an expectorant (PDR for Herbal Medicines, 2000).

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[8] Diospyros khaki Description D. kaki (Fig. 6) (family Ebenaceae) known as young persimmon is widely distributed in East Asia and its leaves are traditionally used for the treatment of hypertension, angina and internal haemorrhage. Flavonoids are the main therapeutic components of D. khaki (Bei et al., 2009). Triterpeneaglycone is also an important constituent of this plant that has drawn attentions of many researchers (Chen et al., 2009; Matsumoto et al., 2010). It has good antioxidant activity due to its phenolic content (Celep et al., 2012). Main compounds include: triterpenoids (Chen et al., 2009). Pharmacological effects D. kaki possesses neuroprotective effects attributable to its flavonoids (Bei et al., 2009) and hypolipidemic activity through an acidbinding (bile acid) mechanism (Matsumoto et al., 2010). In one study, the acetone extract of the peel of persimmon inhibited melanin biosynthesis in mouse B16 melanoma cells. The inhibitory effects were found to be mediated by suppression of tyrosinase expression. (Fukai et al., 2009; Ohguchi et al., 2010). The presence of 2-methoxy-4vinylphenol has led to high antioxidant and free radical scavenging activity of the plant (Fukai et al., 2009; Sun et al., 2011). It was shown in an in vitro study that the oral administration of starch with polyphenol concentrate of persimmon leaf tea can dose-dependently decrease the blood glucose level in Wistar rats due to inhibition of pancreas alpha-amylase (Kawakami et al., 2010). [9] Eriobotrya japonica Description Loquat (family Rosaceae) is a perennial subtropical fruit tree. The fruits can be consumed fresh or processed into jam, juice, wine, syrup, or candied fruits. The flowers (inflorescences) and leaves have been widely used as Traditional Chinese Medicine for treatment of cold, cough, gastro-enteric

disorders, diabetes mellitus, chronic bronchitis and asthma. Many studies demonstrated that large amounts of flavonoids and phenolics were found in the fruit and leaf of loquat (Alshaker et al., 2011; Zhou et al., 2011). Pharmacological effects In one study, E. japonica (Fig. 7) showed potent inhibitory effect on the inflammatory mediators including nitric oxide, iNOS, COX2, TNF-α and IL-6 via the attenuation of NFκB translocation to the nucleus (Cha et al., 2011). The plant has antinociceptive activity via both central and peripheral mechanisms as a weak opioid agonist (Cha et al., 2011). It also possesses antioxidant activity due to its flavonoids and phenolics (Xu and Chen, 2011; Zhou et al., 2011). Moreover, the plant is a potent anti-metastatic agent (Zhou et al., 2011). [10] Citrus aurantium Description Bitter orange (family Rutaceae) is indigenous to tropical Asia but is widely cultivated in other regions nowadays, such as the Mediterranean. Preparations of Bitter Orange flower and flower oil are used as a sedative and a preventive measure for gastric and nervous complaints, gout, sore throat, anxiety and sleeplessness. Fruit peel is used for the treatment of pain in the epigastrum, vomiting and anorexia as folk medicine in China. Limonoids and Flavonoids are the chief constituents of the plant (PDR for Herbal Medicines, 2000). Pharmacological effects C. aurantium (Fig. 8) is used as an alternative medicine in some countries to treat anxiety, and recently the anxiolytic role of this medicinal plant has been already established in an animal model study. Blossoms of the plant may also be effective in terms of reduction in preoperative anxiety before minor operation (Akhlaghi et al., 2011). Since ephedracontaining dietary supplements (herbal weightloss products) were banned from the US market, manufacturers changed their

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formulations by eliminating ephedra and replacing with other botanicals, including bitter orange. It contains, among other compounds, synephrine (Oxedrine), a chemical that is chemically similar to ephedrine. It was shown in a study that this compound has noticeably less effect than ephedrine on the blood pressure and heart rate (Han et al., 2012). It was shown in an study that crude methanol extracts of the peels of C. aurantium L. may induce caspasedependent apoptosis at least in part through Akt inhibition, providing evidence that CMEs have anticancer activity on human leukemia cells (Fugh-Berman & Myers, 2004). Although seville orange (C. aurantium) extracts are being marketed as a safe alternative to ephedra in herbal weight-loss products, it may also have the potential to cause adverse health effects. C. aurantium contains 69, 79dihydroxybergamottin and bergapten, both of which inhibit cytochrome P450-3A, and would be expected to increase serum levels of many drugs. Synephrine has lipolytic effects in human fat cells only at high doses (Hansen et al., 2012). Flavonoids isolated from C. aurantium L. induced G2/M phase arrest through the modulation of cell cycle related proteins and apoptosis through activation caspase. These finding suggest flavonoids isolated from C. aurantium L. were useful agent for the chemoprevention of gastric cancer (Hansen, Juliar, White, & Pellicore, 2011). Doses of up to 100 mg synephrine/kg body weight did not produce developmental toxicity in Sprague-Dawley rats (Lee et al., 2012).

Pharmacological effects

[11] Citrus limon

Grapefruit (C. paradise Macf., family Rutacaeae) (Fig. 10) is a popular plant worldwide, not only because of its taste and nutritive value, but it is also considered to be a functional food that promotes good health (Owira & Ojewole, 2010). Grapefruit's peel flour contains high levels of ascorbic acid and carotenoid. It is also a good source of dietary fiber and phenolic compounds. Possessing all these properties it could be useful in the formulations of functional food, taking advantage of the presence of dietary fiber and antioxidant compounds in one ingredient (Rincon et al., 2005).

Description C. limon (family Rutaceae) is a tree indigenous to northern India, cultivated in Mediterranean regions and in subtropical regions of the world. In folk medicine, lemon juice is recommended as a drink in fever, as a remedy for acute rheumatism and as an antidote to intoxicants, particularly opium. Flavonoids such as hesperidin, rutin and ericitrim are the chief constituents of the plant (PDR for Herbal Medicines, 2000).

C. limon (Fig. 9) essential oil (EO) possesses a strong antioxidant potential. Moreover, it presented scavenger activity against all in vitro tests. Oral EO (50, 100, and 150 mg/kg) significantly reduced the number of writhes, and at highest doses, it reduced the number of paw licks whereas naloxone antagonized the antinociceptive action of EO (highest doses). This suggested the participation of the opioid system (Campelo et al., 2011). Essential oil of C. limon may suppress the growth of Acinetobacter (a multidrug-resistant) species and could be a source of metabolites with antibacterial modifying activity (Guerra et al., 2011). The essential oil is also a safe and effective penetration enhancer for topical administration of lipid- and water-soluble vitamins (especially vitamin E). Since topical bioavailability of lipid- and water-soluble vitamins is a critical issue for protection or anti-ageing formulations. It might be useful to enhance the permeability of the skin to different vitamins (Valgimigli et al., 2012). There is an evidence of sedative and anxiolytic effects of the essential oil of the plant that might involve in action on benzodiazepine-type receptors, and also an antidepressant effect where noradrenergic and serotoninergic mechanisms will probably play a role (C et al., 2011). [12]Citrus paradise Description

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Pharmacological effects

It was found in different studies that this plant has got significant cardiovascular effects. In an in vitro study C. paradise peel extract decreased coronary vascular resistance and mean arterial pressure in dogâ&#x20AC;&#x2122;s isolated heart. This effect of the plant was blocked when the isolated hearts were pre-treated with L-NAME. Cardiovascular effects of this plant have also been shown in human cases. C. paradise juice decreases diastolic and systolic arterial pressure both in normotensive and hypertensive participants (Diaz-Juarez et al., 2009). Alcohol decoction of C. paradise seed is reputed for the local management of array of human diseases including, anemia diabetes mellitus and obesity. The results of various in vitro studies also support these effects of C. paradise. For example, in a study on alloxan-induced diabetic rats, oral treatment with 100 - 600 mg/kg/day of methanolic extract of the plant, for 30 days, resulted in significant (p < 0.05, p < 0.01, p < 0.001) reductions in FPG, TG, TC, LDL-c VLDL-c in the diabetic rats, effects which were comparable to that of metformin. The extract also caused significant (p < 0.05, p < 0.01) rise in HDL-c values in the alloxan diabetic rats (Adeneye, 2008). Oral treatment with the seeds of C. paradise for two weeks (5 to 6 seeds every 8 hours) significantly reduced the profuse growth of the bacteria in patients with urinary infections. The isolated bacterial species from the blood samples of the patients include Pseudomonas aeruginosa, Klebsiella species, Staphylococcus aureus, and Escherichia. Moreover, ingestion of the seeds of the plant altered the antibiotic resistance of P. aeruginosa in one of the patients (Oyelami et al., 2005). Glyceric extract of the seeds of C. paradise is also shown to have good antioxidant activity specially when utilized as aqueous solutions (Giamperi, Fraternale, Bucchini, & Ricci, 2004).

Since Tangerine's peel is rich in magnesium, carotenoid and extractable polyphenols, it may be suitable, to reduce risk of some diseases such as cardiovascular and some associated to lipid oxidation (Rincon, Vasquez, & Padilla, 2005). The hexane and chloroform extracts of C. reticulate (specially the alcohol-soluble fraction) are shown to have significant antibacterial activity against both gram positive and gram negative bacteria. The antibacterial effect of the plant is said to be due to the presence of three polymethoxylated flavones, namely desmethylnobiletin, nobiletin and tangeretin (Jayaprakasha et al., 2000). It was shown in a study that the essential oil of C. reticulate has inhibitory activity on proliferation of human embryonic lung fibroblasts. It also showed preventive effects on bleomycininduced pulmonary fibrosis in rats. The mechanism may be via adjusting the unbalance of oxidation and antioxidation, down-regulating CTGF protein and mRNA expressions and reducing collagen deposition and fibrosis (Zhou et al., 2012). This plant is also reported to have anaphylactic effects. The lipid transfer protein allergen from mandarin fruit was isolated and recognized as the cause to an IgE-mediated allergy in a patient with anaphylaxis from mandarin (Ebo et al., 2007).

[13] Citrus reticulata

[14] Citrus sinensis Description C. sinensis (family Rutaceae) (Fig. 12) is indigenous to Asia and is cultivated in the Mediterranean and other subtropical regions in many parts of the world. The fragrant flowers are arranged single or in short, limp racemes. The medicinal parts of the plant are the fresh and dried peel as well as the oil extracted from the peel. Flavonoids are the main chemical compounds of this plant (PDR for Herbal Medicines, 2000).

Description

Pharmacological effects

C. reticulata (Tanegrine) (Fig. 11) family Rutaceae is well known for its various pharmacological effects (Zhou et al., 2012).

Proximate analysis of sweet orange (C. sinensis) seed flour showed a composition of 54.2% fat, 28.5% carbohydrate, 5.5% crude

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fiber, 3.1% crude protein and 2.5% ash for the dehulled orange seed flour (dry weight) (Akpata & Akubor, 1999). The peel extract of C. sinensis dose-dependently inhibited H(2)O(2)-induced lipid peroxidation in red blood cells of rats, in an in vitro study. In the in vivo investigation 25 mg/kg C of the extract maximally inhibited hepatic LPO. Besides this inhibitory effect, the extract exhibited antithyroidal, hypoglycemic, and insulin stimulatory properties, which suggest its potential to ameliorate both hyperthyroidism and diabetes mellitus (Parmar & Kar, 2008). Sweet orange is also said to have insecticidal activity. One study showed significant activity of C. sinensis essential oil against larvae and pupae of housefly (Muscadomestica) (Kumar, Mishra, Malik, & Satya, 2012). It also promotes gastric juice secretion (PDR for Herbal Medicines, 2000). [15] Citrus unshiu Description C. unshiu (Fig. 13) known as Satsuma Mandarin belongs to the Rutaceae family and is well known all over the world especially in Japan. It has been long used as a traditional medicine in eastern Asian countries (Ozaki et al., 2000). Figure 9. Citrus limon

Pharmacological effects This plant is a good source of antioxidants (Ma et al., 2008). In an study on antitumor activity of the extract of C. unshiu in tumorbearing murine models. It was shown that this plant possesses significant antitumor activity probably via a mechanism of boosting cytokines such as IFN-γ and TNF-α and enhancing immune-mediated anti-tumor properties (Lee et al., 2011). C. unshiu has antiallergic activity. It was shown in a study that fifty percent methanol extract of C. unshiu powder showed potent inhibitory activity against histamine release from basophils of patients suffering from seasonal allergic rhinitis to ceder pollen. The most potent flavonoid responsible for this effect of the plant was hesperetin. Suppression of IgE-mediated stimulation of basophils through PI3-K pathway was assumed as the possible mechanism by which flavonoids inhibited the degranulation process (Kobayashi and Tanabe, 2006). It has been also shown that water and ethyl acetate extracts of C. unshiu peel decreases hepatitis C virus absorption in MOLT-4 cells. The active ingredient that markedly inhibited HCV infection was nobiletin (Suzuki et al., 2005).

Figure 10. Citrus paradise

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Figure 11. Citrus reticulata

Figure 12. Citrus sinensis

Figure 13. Citrus unshiu

CONCLUSION Present review study reveals that the mentioned species possess important pharmacological effects of the current interest in pharmaceutical sciences. Since they can grow in the climate of Iran, they have the potential to be further studied and be employed

for their various medicinal pharmacological properties in Iran elsewhere.

and and

ACKNOWLEDGMENT Hereby we express our gratitude to Mr. Ali Aghajanshakeri whose help and guidance made the preparation of this manuscript possible.

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and Cestrum nocturnum (Solanaceae) plant extracts against Aedes aegypti (Diptera: Culicide) and nontarget fish Poecilia reticulata. [Research Support, Non-U.S. Gov't]. Parasitol Res, 108(5), 1253–1263. doi: 10.1007/s00436-0102174-6

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recessive lethal test. [Research Support, Non-U.S. Gov't]. Genetika, 47(7), 874– 878. Sun, L., Zhang, J., Lu, X., Zhang, L., & Zhang, Y. (2011). Evaluation to the antioxidant activity of total flavonoids extract from persimmon (Diospyros kaki L.) leaves. [Research Support, Non-U.S. Gov't]. Food Chem Toxicol, 49(10), 2689– 2696. doi: 10.1016/j.fct.2011.07.042 Suzuki, M., Sasaki, K., Yoshizaki, F., Oguchi, K., Fujisawa, M., & Cyong, J. C. (2005). Anti-hepatitis C virus effect of citrus unshiu peel and its active ingredient nobiletin. [Research Support, Non-U.S. Gov't]. Am J Chin Med, 33(1), 87–94. Valgimigli, L., Gabbanini, S., Berlini, E., Lucchi, E., Beltramini, C., & Bertarelli, Y. L. (2012). Lemon (Citrus limon, Burm.f.) essential oil enhances the trans-epidermal release of lipid- (A, E) and water- (B(6), C) soluble vitamins from topical emulsions in reconstructed human epidermis. Int J Cosmet Sci, 34(4), 347–356. doi: 10.1111/j.14682494.2012.00725.x Valianou, L., Stathopoulou, K., Karapanagiotis, I., Magiatis, P., Pavlidou, E., Skaltsounis, A. L., & Chryssoulakis, Y. (2009). Phytochemical analysis of young fustic (Cotinus coggygria heartwood) and identification of isolated colourants in historical textiles. [Historical Article Research Support, Non-U.S. Gov't]. Anal Bioanal Chem,

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Conflict of Interest: None Declared

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Review article ETHNO MEDICINAL PRACTICES AMONG THE BINJHWAR TRIBE OF CHHATTISGARH, INDIA Shukla Rajesh1*, Chakravarty Moyna2, Goutam M P3 1

Senior Executive Technical - Head, Chhattisgarh State Minor Forest Produce (T&D) Cooperative Federation Limited, A 25 VIP Estate Khamardeeh, Shankar nagar, Raipur - 429007 C.G., India 2 Reader, School of Studies in Anthropology, Pt. Ravishankar Shukla University, Raipur (C.G.)., India 3 Retd. Director, State Forensic Science Laboratory, Penshion bada Raipur (C.G.), India *Corresponding Author: Email- rds.anth@gmail.com, rajks_anth@hotmail.com

Received: 08/06/2013; Revised: 02/06/2013; Accepted: 04/07/2013

ABSTRACT The present paper deals with the Ethno medicinal practices among the Binjhwar tribe of Raipur division of Chhattisgarh State, India. Objective of the present paper was to document the oral tradition of medicine, mode of treatment, awareness towards modern medicinal system and toxic effect of herbs used by the Binjhwar tribe. The information were collected by using various anthropological techniques of data collection. The most frequently occurring diseases among the tribe were skin infections, fever, eye infection etc. which is being treated by using medicinal plants as well as magico-religious performances. Traditional healers use herbs, shrubs, climbers, trees, animal remains and minerals for the preparations of medicines for common ailments. KEYWORDS: Traditional medicine, Magico-religious practices, Binjhwar, Chhattisgarh

Cite this article: Shukla Rajesh, Chakravarty. M., Goutam.M. P., (2013), ETHNO MEDICINAL PRACTICES AMONG THE BINJHWAR TRIBE OF CHHATTISGARH, INDIA, Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 525–531

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INTRODUCTION The tribal of India have preserved a huge knowledge of traditional medicinal uses of plants growing around them. Since the vedic times, importance of medicinal plants always has the same graph. Medicinal plants play a vital role in traditional as well as modern health care system. Ethno medicine (traditional) is the mother of all other system of medicines. Ethno medicinal study consists of evaluation of health outcomes and results from the exercise of traditional beliefs and behaviors. Traditional herbal medicine is practiced in several parts of the world especially in Australia, Africa, Bangladesh, Brazil, China, Caribbean states, Europe, Spain; North and South America, Russia, Pacific islands where large ethnic communities still live. The World Health Organization (WHO), 1978 has estimated that 80% of the populations of developing countries rely on traditional medicines, mostly plant drugs, for their primary health care needs. In India 65 % of the population relies on ethno medicine which is the only source of their primary health care needs (Rajasekharan et al., 1996).. The Indian traditional medicine can be categorized into two streams; I. The Classical Health Traditions like Ayurveda and Siddha which are highly organized, classified and codified and has a sophisticated conceptual and theoretical foundations and philosophical explanations; II. The oral health tradition which is very rich and diverse, but not codified or organized (Rajasekharan et al.,1996). The present study on Ethno medicinal practices among the Binjhwar tribe, conceptually comes under the second stream of Indian Traditional Medicine. Some sporadic studies are available on the Indian tribes such as; the Abors (Dunbar, 1915), Folk medicine of Bastar has been carried out by (Hemadri et al., 1975), The Vaidus of Maharasthra (Kurian et al., 1980), Oraon and Korwa tribes of Sarguja and Raigarh district, Madhya Pradesh in central India was carried out by (Maheshwari et al., 1990), Khairwars of Siddhi district Madhya Pradesh (Pandey et al., 1999), The Birhors of Madhya Pradesh (Pandey et al., 2000), The Munda of Bangladesh (Sharmeen, 2005), Traditional

Health Practices of Raj-Gond (Shukla et al., 2006), Health seeking behaviour among Santhal of Orissa (Sonowal et al., 2007), Indigenous medicine for Gynecological Disorders by the tribal of Chhattisgarh (Shukla et al., 2008). Binjhwar is a civilized Dravidian tribe found in Raipur and Mahasamund district of Chhattisgarh and adjoining Orissa state. They are landholding sections of Baigas, like RajGonds among the Gonds. Binjhwar is derived from the Vindhya hills; the tribes still worship the goddess Vindhyabasini. They have four sub-divisions; Sonjharas, Birjhias, Binjhias and Binjhwar proper. Binjhwar have separate Jyaati Panchayat headed by Jyaati Panchayat President at Rajadeori, governed by number of rules to solve disputes and problems within the community. The total population of Binjhwar in the state ranges from 1,00,692 to 1,04,718 (Naik, 1972). The present study aims to document the oral traditions of tribal health, attitude towards modern or allopathic medical system, identify and document plants, animals and minerals used for medicinal purposes and identify the plants having toxic and harmful effects used as folk medicine. MATERIALS AND METHODS For the present study survey was conducted in 13 tribal villages during September 2003 to January 2005. During the course of the study regular field visits were carried out in the study area. Various methods of sampling were used for area selection and primary data collection. Purposive sampling method was used for village selection from Kasdol, Sankra and Pithora blocks of Raipur and Mahasamund District of Chhattisgarh, India. Binjhwar dominating villages of three blocks were selected for the present study and 275 households were surveyed randomly. Interview schedule was used for household survey to collect information related with education, food habit, health, occupation and social structure of Binjhwars. Information about the use of

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medicinal plants, mode of administration, dosage and technique of diagnosing the diseases were collected through interview from the traditional healers (Baiga, Vaidhraj and priests). Secondary data were collected from journals, books, reports and government offices to verify the health infrastructure facilities provided by the government. RESULTS Ethno-medicinal practice among the Binjhwar is complex containing different treatment patterns i.e. herbal medicine, rituals, magico-religious treatment and allopathic medicines. They have their own concepts for a disease, cause of illness, diagnosis of disease and treatment of ailments. The most frequently occurring disease among the Binjhwar is cough and cold i.e. 21.82%, skin disease 14.91 %, fever and eye infection 10.55 % and tuberculosis and stone (0.73 %) each and other diseases like jaundice, dysentery etc were reported. shown in fig. 01. Specialists like herbalists, bone setters, mid wives and pujari / priest are ethno-medicinal service providers among the Binjhwars. Traditional healers are expert in diagnosing the disease by calculating pulse from various body parts, observation of eye colour, tongue and neck. Local traditional healers may be categorized into two types according to their nature of work; first, the people performing

magico-religious acts for diagnosing the disease who are called pujari and second, use organoleptic observation and also they may or may not use some magical formulae for diagnosing the disease. Treatment among Binjhwar consists of herbal remedies, magico-religious acts and modern medical facilities available nearby. A variety of medical specialists co-exists whose services may be availed by the Binjhwar. These healers are expert in treating various ailments like fever, tuberculosis, asthma, jaundice, gynaecological disorder and so on by using different plant species given in Table 01. The healers collect various parts like root, tuber, rhizome, stem, bark, leaf, flower, seed and whole plant for preparation of medicine. Healers use 34 % roots, 13 % seeds and 12 % barks and some animal remains or body parts and minerals in trace quantity i.e. 05 % in addition to herbs. For preparing of the medicines, 52 Plant species belonging to 35 families are used by Binjhwars. Some species are used in multiple medicinal preparations, Keo-kand (Costus speciosus) in 7 ailments, Arjun (Terminalia arjuna) in 5 ailments, Mahua (Madhuca latifolia) in 6 ailments, Dhaora (Anogeissus latifolia) in 3 ailments and tendu (Diospyros melanoxylon) in 2 ailments. Besides these wild plants several spices are also used by them such as; Elaichi, Bade-elaichi, Fenu greek leaves (Methi), Black pepper, Garlic etc. are also mixed in trace quantities.

Fig 1. Person Suffering from Disease in One Calendar Year 25 21.82

14.91 10.91

10.55

9.45 6.91

3.27

2.18

1.09

0.73

In di g

Co ug h

an d

co ld

es tio Fev e n /A r cid Jo ity in tP D i a in ar rh Gy oe ne a co Dys lo e gic nte al r di y so rd er Ja un d Tu be ice r Ey culo eI s nf is e St c om tio ac n h a Sk in che Di se as e

6.91

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0.73

St on e

Percentage

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Table 1. Botanical Name, Family and Uses of Plant Species used by Binjhwar Tribes Sl. Plant Name 1 Adusa 2

Agusti

Akarkara

4 5 6

Amaltas Amarbel Ami haldi

Arjun, Kahua

Bach, Devnasan Ban tulsi

10 Banrakash (Doodh Kochai) 11 Behra 12 Bhelwa 13 Bhui aonla 14 Bhui neem 15 Chitawar 16 Dahiman 17 Dhaora 18 19 20 21

Doodhi Girola Gular Gurmar

22 Gursukhri 23 Gurud 24 Harra 25 Harsinghar 26 Hinglaj

Botanical Name Family Adhatoda vasica Acanthaceae Nees. Sesbania grandiflora Fabaceae Pers. Spilanthes acmella Asteraceae

Disease Itching, Fever

Cassia Fistula Linn. Cuscuta reflexa Roxb. Curcuma amada Roxb. Terminalia arjuna

Caesalpiniaceae Convolvulaceae Zingiberaceae

Tuberculosis Jaundice Painful menses / Blood discharge

Combertaceae

Acorus calamus Linn.

Araceae

Acidity, Weakness, Chest pain, Arthritis, Diabetes Epilepsy, Joint Pain

Loose Motion Paralysis, Tooth ache, Epilepsy

Ocimum gratissimum Lamiaceae Linn. Alocasia indica Araceae (Roxb.) Schott.

Joint Pain

Terminalia bellirica Roxb. Semicarpus anacardium Linn. Phyllanthus niruri Andrographis paniculata Nees. Plumbago zeylanica Linn. Cordia macloidii Anogissus latifolia Wall. Euphorbia hirta Indigofera cassioides Ficus racemosa Gymnema sylvestre R. Br. Grewia hirsute Vahli. Stereospermum suaveolens DC. Terminalia chebula Retz. Nyctanthes arbortristis Linn. Balanites aegyptiaca

Combertaceae

Acidity, Loose Motion, Piles

Anacardiaceae

Tuberculosis

Euphorbiaceae Acanthaceae

Diarrhoea Fever and Malaria

Plumbaginaceae

Moti jhira, Itching

Boraginaceae Combertaceae Euphorbiaceae Fabaceae Moraceae Asclepiadaceae

Snake Bite Diabetes, Dysentery, Motion Adequate milk Secretion Vitality after Delivery Diabetes Diabetes

Asclepiadaceae Bignoniaceae

Fracture, Wound Fever and Malaria

Combertaceae

Acidity, Piles

Oleaceae

Fracture

Balanitaceae

Typhoid

Arthritis (Baat)

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Loose

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27 Kalimusli

Curculigo orchrides Gaertn. Pongamia pinnata Pierre. Strychnos potatorum Linn. Costus speciosus (Koeing.) Sm.

Hypoxidaceae

34 Lajwanti

Dolichos biflorus Leea aspera Schleichera oleosa (Lour) Oken. Mimosa pudica Linn.

35 Maha neem 36 Mahua

Melia azedarach Linn. Meliaceae Madhuca latifolia Sapotaceae

28 Karanj 29 Kaya 30 Keo-kand

31 Kulthi 32 Kurma 33 Kusum

Loganiaceae

Eye Infection

Zingiberaceae

Indigestion, Arthritis, Joint Pain, Head-ache, Strengthening, Leucorrhoea, Piles

Fabaceae Leeaceae Sapinadaceae

Body ache, Stone Chest pain / Swelling Wound

Mimosaceae

Eye Infection and redness, Fever

Lauraceae

Aristolochiaceae Piles

40 41 42

43 Rasna 44 Saan 45 Sarphoonka 46 Semhar 47 Tejraj 48 Tendu 49 Thelkajari 51 Tilai 52 Tinsa

Blepharispermum subsessile Crotalaria orixensis Tephrosia purpurea Pers. Samalia malabarica Schott & Endl. Peucedanum nagpurense Diospyros melanoxylon Alangium salviifolium (Linn.f.) Wang. Wendlandia heynei Ougeinia oojeinensis (Roxb.) Hochr.

desire

and

Fever, Jaundice Arthritis, Dysentery, Jaundice, Piles, Snake Bite Fracture

37 Maida

Litsea glutinosa (Lour.) C. B. Robins Panarijadi Aristolochia indica Linn. Pat koria Cissampelos pareira (Patha) Linn. Patal Kumrah, Pueraria tuberosa Bhui tumba DC. Pipli Piper longum Linn. Ramdatoon Smilex zeylanica

Fabaceae

Promote sexual strengthening Itching, Dog bite

Menispermaceae Fever and Malaria Fabaceae

Bemchi, Weakness

Piperaceae Liliaceae

Tuberculosis, Asthma Leucorrhoea,White discharge

Asteraceae

Arthritis (Baat)

Fabaceae Fabaceae

Jaundice Stone, Jaundice

Sterculiaceae

Irregular Menses

Apiaceae Ebenaceae

Promote sexual desire, Male Impotency Painful menses / Blood discharge

Alangiaceae

Male Impotency

Asteraceae Fabaceae

Piles, Body ache Dysentery, Loose Motion

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Fig 2. First Preference for the Health Care needs

percentage

47.64

40 30

25.82

20 10

10.18

9.45

CONCLUSION It can be concluded that the Ethnomedicinal practices of the Binjhwars are more affordable, acceptable, culturally appropriate and available as compared to modern medicines. Even today Binjhwars accept traditional medicines in remote or inaccessible villages due to their dependence on and confidence in traditional medicine men. Therefore, proper education and mass communication might be helpful in creating an awareness of the Binjhwars towards modern health care system. The Binjhwars have a pluralistic medical situation in which allopathic, ayurvedic, homoeopathic and

th ic Al lo pa

th ic/ Tr ad

ph dJh a

3.64

Al lo pa

ph dJh a

Binjhwar first approach the traditional medicine men for primary health care i.e. 47.64 % followed by 25.82 % people approach either traditional or allopathic medicine and only 10.18 % people access allopathic treatment for primary health care. The reasons for non utilization of modern health care system are low literacy, poor economic status, and lack of awareness, cultural factors and distance of health centers, doctor - patient relationship and health facilities available from the administrative side.

oo k

oo k/ Al lo

pa th

at h lo p Al l/

di tio na Tr a

Tr a

di tio na

lT re

at m en

iti on al

3.27

indigenous system of medicine exist side by side. In the present study some plants have been selected which have ethno botanical significance. During the course of field work the therapeutic and toxic effects on the users were recorded. It could be concluded that users of mentioned plants for treatment of various kinds are not aware of their toxicity and toxic chemical constituents. Terminalia bellarica Lam., Phyllanthus niruri Lam., Melia azedarach L., Madhuca latifolia Koenig., Plumbago zeylanica L., Semecarpus anacardium L. were considered to know their toxic effects. Community based awareness programme should be organized to protect this community with the over dosage, accidental poisoning and chance contamination of these drugs. Local government officers should also establish a team of subject experts including local vaidhya, medical practitioners, botanist and anthropologist so that they can prepare a list of such plants giving details regarding their vernacular names, botanical names, toxicity of the particular plant part, method of reducing toxic effect (Sodhan) and dosages. Authors would recommend that a bridge should be

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developed between Binjhwars traditional medicine and Modern medical system, which will help us to protect and conserve the traditional medical heritage as well as improve the utilization of modern medical facilities. Phyto-chemical or pharmacological investigation, nutritional analysis and clinical trials should be carried out to validate the claims. These informations may help the policy

makers for adopting the proper healthcare measures and may provide a lead in the development of new drugs.

REFERENCES

Rajasekharan S, Pushpangadan P,Biju S D (1996). Folk Medicines of Kerala – A Study on Native Traditional Folk Healing Art and its Practitioners. New Delhi: Deep Publications, New Delhi (ed. S. K. Jain).

Dunbar D S G (1915). Abors and Garllongs. The Asiatic society, Culcutta, 5: 1–19. Hemadri K, Rao S S (1975). Folk medicine of Bastar. Journal of Ethnobotany, 1: 61– 66. Kurian

Joy C, B V Bhanu (1980). Ethnomedicine: A Study of the Nomadic Vaidus of Maharastra. The Eastern Anthropologist, 33(1): 71–78.

Maheshawari J K, Painuli R M, Dwivedi R P (1990). Notes on Ethnobotany of Oraon and Korwa Tribes of M.P. Contribution to Indian Ethnobotany: 75–90. Naik, T. B. (1972): “Barah Bhai Binjhwar”: Madhya Pradesh Hindi Granth Academy, Bhopal (M.P.), India. Pandey G D, Roy J (1999). A Study of Health Seeking Behavior among the Khairwars and Non-Khairwars of Sidhi District. Tribal Health Bulletin, 5(1): 11–16. Pandey G D, Tirkey V R, Tiwary R S (2000). Some aspects of Health Seeking Behavior in Birhors - A Primitive Tribe of M.P. Man in India, 79(3&4): 291–299.

Source of Support: Nil

Note: This paper is presented in the International Conferences on “Conservation, Marketing and Patenting of Medicinal Plants” organized by Chhattisgarh State Medicinal Plant Board Raipur, India. 14–15 March 2010.

Sharmeen S, Sharmeen T (2005). Traditional Health Practices of Munda Women: Continuity and Change. Man and life, 31 (1&2): 23–36. Shukla

R, Chakravarty M (2006). Anthropological Study on Traditional Health Practices of Raj-Gonds, Tribal Health Bulletin, Vol.12 (1&2), 2006: 61–66.

Shukla R, Chakravarty M, Gautam M P (2008). Indigenous medicine used for the treatment of Gynecological Disorders by the tribal of Chhattisgarh, India. Journal of Medicinal Plant Research, 2 (12): 356–360. Sonowal C J, Praharaj P (2007). Tradition Vs Transition: Acceptance of Health Care Systems among the Santhals of Orissa. Studies on Ethno-medicine, 1(2):135– 146. World Health Organization (1978): “The Promotion and Development of Traditional Medicine”: Technical Report Series 622, Geneva. Conflict of Interest: None Declared

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Research article EVALUATION OF ANTIARTHRITIC ACTIVITY OF LEPIDIUM SATIVUM LINN SEEDS AGAINST FREUND’S ADJUVANT INDUCED ARTHRITIS IN RATS

Raval Nita D1*, Ashok B K2, Ravishankar B3 1

Lecturer, Department of Dravyaguna, Government Ayurved College, Junagadh, Drug discovery lab, R & D, Himalaya drug company, Bangalore, Karnataka, India 3 Director, SDM Research Centre for Ayurveda and Allied Sciences, Kuthpady, Udupi. Karnataka – 574118, India *Corresponding Author: Email: drnitadraval@yahoo.in; Mobile: +919898340450 2

Received: 10/06/2013; Revised: 20/06/2013; Accepted: 28/06/2013

ABSTRACT Garden cress (Lepidium sativum Linn.) leaves and seeds are used in India as food supplement and also as medicine. In traditional system of medicine, its seeds were used for various ailments like inflammation, joint pain, backache etc. the present study was carried out to explore the folkloric use of the seeds on Freund's adjuvant induced arthritic rats. The present study concludes the effect of the seed powder on the Freund's complete adjuvant induced arthritic rat paw oedema in both developing and developed phases of arthritis. Histopathology of proximal inter-phalangeal joints and radiology of hind legs were studied. Significant changes were observed in drug treated group in radiological study. In histo-pathological study bone and cartilage degeneration with bone erosion and inflammatory changes were observed in the affected joint. These changes were not significantly improved by the administration of test drugs. Result data suggested that at a lower dose, moderate anti-arthritic effect was produced by the test drug while at a higher dose it had a tendency to produce inconsistent effect. KEY WORDS: anti arthritic, Fruend’s adjuvant, paw oedema, Lepidium sativum Linn.

Cite this article: Raval Nita. D., Ashok. B.K,, Ravishankar. B., (2013) EVALUATION OF ANTIARTHRITIC ACTIVITY OF LEPIDIUM SATIVUM LINN SEEDS AGAINST FREUND’S ADJUVANT INDUCED ARTHRITIS IN RATS, Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 532–537

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INTRODUCTION: Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown aetiology. The disease is characterized by articular inflammation and by the formation of an inflammatory and invasive tissue, rheumatoid pannus that eventually leads to the destruction of joints (Stephen J. 2008). Analgesics and NSAIDs are used to suppress the symptoms while disease-modifying anti-rheumatic drugs (DMARDs) and newer therapies such as antitumour necrosis factor (TNF)-α therapy (etanercept, infliximab and adalimumab), antiCD20 therapy (rituximab) and abatacept are often required to inhibit or halt the underlying immune process (Anthony S. Fauci. et al., 2008). NSAIDs are widely prescribed all over the world to the patients with Rheumatoid arthritis, Osteoarthritis etc. However the prolonged use of these drugs has its own drawbacks. Research works on NSAIDs suggest that these drugs can increase the risk of chronic renal disease (P Ejaz et al., 2004). Garden cress is also known as Asalio or Chandrashura in local languages and it is an important medicinal crop in India (Tiwari et al, 2004). Garden cress (Lepidium sativum Linn.) is an annual fast growing edible herb, belongs to the family Brassicaceae. Seeds, leaves and roots of it are of economic importance; however, the crop is mainly cultivated for seeds. It can be grown at all elevations, throughout the year, but the best crop is obtained in the winter season (Anonymous, 1962, CSIR, New Delhi.). Garden cress seeds and leaves are used in food preparations (Datta PK et al., 2011). Leaves are diuretic and gently stimulant (Maghrani et al., 2005). To investigate the folkloric use of the seeds the present study was carried out on Freund’s adjuvant induced arthritic rats.

University, Jamnagar in the month of April 2004 and it was authenticated by the expert of botany from the Department of Pharmacognosy, Gujarat Ayurved University, Jamnagar, Gujarat, India Experimental models: Twenty four Charles Foster strain albino rats weighing 180 ± 10g were obtained from animal house attached to Pharmacology laboratory, Gujarat Ayurved University, Jamnagar Gujarat. Six rats were housed in each cage made up of poly-propylene with stainless steel top grill. The dry wheat (post hulled) waste was used as bedding material and was changed every morning. The animals were acclimatized for seven days before commencement of the experiment in standard laboratory conditions 12 ± 01 hour day and night rhythm, maintained at 25 ± 3oC and 50– 70% humidity as per CPCSEA guidelines. Animals were provided with balanced food (Amrut brand rat pellet feed supplied by Pranav Agro Mills Pvt. Limited) and water ad libitum. Protocol used in this study for the use of animals was approved by the institutional animal ethics committee (IAEC 0405/01/PhD.03). Dose selection: The dose fixation for the experimental animals was done on the basis of body surface area ratio by referring to the standard table of (Paget and Barnes, 1964). The adult human dose (6 g per day) (Chunekar K.C, 1999) was converted to animal dose. On this basis the calculated dose was fixed to be 550 mg/kg for rats. The suspension of Lepidium seed powder was made with sufficient quantity of distilled water according to the required dose and administered orally with the help of gastric catheter sleeved to syringe.

MATERIALS AND METHODS:

Experimental study (Rosenthale, 1970):

Collection of sample (Lepidium sativum Linn seeds)

The selected animals were grouped into three groups of 6 rats each. To the first group only water was administered and treated as normal control. To second and third group, test drug was administered in the dose of 550

The seeds for the proposed study were collected from the campus of Gujarat Ayurved

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mg/kg (TED) and 1100 mg/kg (TED × 02) respectively. The drug was administered for 30 consecutive days. On day one, the complete Fruend's adjuvant was made into fine emulsion with the help of a syringe and 0.1 ml of it was injected beneath the plantar aponeurosis in the left hind paw and 0.05 ml subcutaneously into the root of the tail. The volumes of both the hind paws were measured with the help of Plethysmometer. The paw volume of left hind limb was measured at 2nd, 3rd, 5th, 10th and 15th days, while of right hind limb on 15th, 20th, 25th and 30th days. Paw volume of the 0 (initial) days were taken as the reference value for determining the increase in paw volume on the subsequent days. On 31st day animals were weighed and sacrificed by over dose of ether anesthesia and both right and left synovial joints were dissected out and the histopathological slides were prepared by referring to standard procedure of (Raghuramulu et al., 1983). The slides were viewed under trinocular research Carl-Zeiss’s microscope at various magnifications to note down the changes in the microscopic features. Statistical analysis: The data were expressed as mean ± standard error mean (SEM). The Significance of differences among the test drugs and control groups was assessed using one way analysis of

variance (ANOVA) and the test followed by Dunnet’s test. The difference was considered significant when p < 0.05. RESULTS AND DISCUSSION: The data pertaining to the effect of test drug on Friend’s adjuvant induced arthritis in rats have been tabulated in Table 1 & Table 2 A moderate decrease in both primary and secondary edema was observed in therapeutic dose, and double dose group in comparison to control group. The suppression of primary edema in therapeutic dose was as follows. After 48 hrs 14%, 5th day 34.32%, 10th day 49.01%, 15th day 45.40%, 20th day 33.21%, 25th day 82.32%, 30th day 33.84%. Whereas marginal increase was observed after 24 hrs. In double dose group the suppression of primary edema was after 24 hrs 15.88%, 48hr 4.96%, 5th day 29.15%, 10th day 32.64%, 15th day 51.82%, 30th day 12.34%. Here the increase was observed on 20th and 25th day. In secondary edema the suppression observed in therapeutic dose group was as follows on 15th day 40.07%, 20th day 41.21%, 25th day 45.33%, 30th day 40.45%. In double dose group 15th day 6.92%, 25th day 2.86%, 30th day 28.57% suppression of edema was observed where as on 20th day marginal increase was observed in double dose group.

Table – 1: Effect on primary paw oedema (Oedema of left hind paw) Percentage increase in paw oedema when compared to initial paw volume 2nd day 48 hrs 5th day 10th day 15th day Control 68.49  9.30 45.33  7.58 39.86  6.73 20.77  6.40 11.21  2.36 TED 70.55  11.57 38.98  6.43 26.18  5.43 10.59  3.67** 06.12  2.12 TED×02 57.61  6.34 43.08  7.23 28.24  7.67 13.99  3.96** 05.4  2.44 Groups

Data: Mean  SEM, **ANOVA test (F = 35.45, P < 0.01; df (2, 15) (F = 7.01; P < 0.01 df, (2, 15)

Groups Control TED TED×02

Table – 2: Effect on secondary paw oedema (Oedema of right hind paw) 15th day 20th day 25th day 30th day 29.17  7.09 20.6  4.60 23.05  10.04 20.44  7.78 17.48  4.51 12.11  5.72* 12.60  4.71 12.17  5.28 31.19  7.43 31.82  7.38* 22.39  8.23 14.60  5.74 Data: Mean  SEM, *ANOVA F=3.48; P < 0.10; df (12, 15)

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Figure 1: Images depicting Radiology of hind legs in adjuvant induced arthritic rats

Figure 2: Images depicting histopathology of proximal interphalangeal joint in adjuvant induced arthritic rats (1X 50 magnifications)

Radiology of hind legs in adjuvant induced arthritic rats: In arthritic disease radiographic changes are useful diagnostic measures which indicate the severity of the disease. In the early stage soft tissue swelling can be seen, whereas severe radiological changes like bony erosion and joint space narrowing can be observed in developed stage of arthritis. In adjuvant induced arthritic rats soft tissue swelling and cartilage erosions were observed. The degrees of degenerative changes were less in test drug treated group in comparison to control group.

The radiographic features of the rat joints in adjuvant induced arthritic rats are shown in Figure 1. Effect on histopathology of joints: Figure 2 shows representative sections of inter-phalangeal joint from different groups. Bone and cartilage degeneration with bone erosion and inflammatory changes were observed in the knee joint. These changes were not significantly improved by the administration of test drugs.

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The main aim of the present study was to find out the scientific basis for the reported efficacy of test drug in arthritis. Hence it was evaluated against Fruend's adjuvant induced arthritis in rats. Injection of adjuvant elicits immune response of cell-mediated type and produces an arthritic syndrome. Arthritic rats show swelling of the soft tissue around ankle joint during initial phases of arthritis. This is mainly due to edema of periarticular tissues such as ligaments and joint capsules (Sanmugapriya et al., 2010). Like human RA, it is also characterized by synovitis, infiltration of neutrophils and proliferative response leading to synovial fibroplasias and periosteal bone deposition (Hazeena et al., 1980). In the present study a persistent moderate suppression of primary oedema ranging between 14 to 49% was observed at lower dose up to 20th day of observation. The secondary oedema represents oedema of immunological origin (Yoshikawa et al., 1985). In the secondary oedema a consistent 40 to 45% suppression was observed at lower dose indicating that the test drug at this dose has moderate suppression effect on oedema of immunological origin. At higher dose inconsistent effect was observed. The suppression ranged between 5.74% to 54%.

Radiographic changes in RA conditions are useful diagnostic measures which indicate the severity of the disease. Soft tissue swelling is the earlier radiographic sign, whereas prominent radiographic changes like bony erosions and narrowing of joint spaces can be observed only in the developed stages (final stages) of arthritis (Harris ED, 1990). The radiographic features of the rat joints in adjuvant induced arthritic model are shown in plate 2. The degrees of degenerative changes were less in test drug treated group in comparison to control group. CONCLUSION At lower dose level moderate anti-arthritic activity was observed in FA rats. Higher dose level produced inconsistent effect. Hence it would be necessary to identify suitable dose. This indicates the complex nature of the seed powder. The study confirms the antiinflammatory and anti-arthritic activity potential of the plant material however; further refinement in the form in which it is administered along with optimum dose fixation is required for optimum therapeutic application of this plant. It would also be interesting to study the other parts of the plant. It is possible that they may also express significant antiinflammatory and anti-arthritic activity.

REFERENCES Anonymous. (1962). The Wealth of India, Raw Materials. VI. Publication and information Directorate, CSIR, New Delhi. India, p.71–73. Anthony S. Fauci. and Dr. Longo(2008) Harrison’s Principles of Internal Medicine, Section 2, 17th edition, part 14, section 2 chapters 314. Chunekar K.C., (1999) G.S.Pandey (eds) Bhavprakash nighantu, haritakyadi Verga, Chaukhambha orientalia publication.pp 39.

Datta PK, Diwakar BT, Viswanatha S, Murthy KN, Naidu KA, (Mar-April 2011). Safety evaluation studies on Garden cress Lepidium sativum L. seeds in Wistar rats. IJARNP-HS Publications Vol. 4 (1), pp. 37–43. Dr. Yograj Sharma, (2003–2004). Efficacy of sowing methods, fertilizer application and growth regulations in cultivation of Lepidium sativum Linn. Published thesis (MSc), Gujarat Ayurved University, Jamnagar.

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Harris

ED (1990). Rheumatoid arthritis. Pathophysiology and implications for therapy. N Eng J Med, 322(18):1277– 1289.

Rosenthale, M.E. (1970). Arch. Int. Pharmacodyn. 188. In: Dahanukar, S., Sharma, S., Karandikar, S.M.(1983). Indian Drugs, 20 (10), 405.

Hazeena Begum, V., Sadique, J. (1980). Ind. J.exp. Biol., 26,877.

Sanmugapriya Ekambram, Senthamil Selvan Perumal, and Venkataraman Subramanian, (2010). Evaluation of antiarthritic activity of Strychnos potatorum Linn seeds in Freund’s adjuvant induced arthritic rat model.BMC, Complementry and Alternative medicine, vol.10.

Maghrani M, Zeggwagh NA, Michel JB. Eddonks M. (2005). Antihypertensive effect of Lepidium sativum L. In spontaneously hypertensive rats. J Ethnopharmacol 100: 193–197. P Ejaz, K Bhojani, VR Joshi, (2004). NSAIDs and kidney, JAPI, vol.52, pp-632. Paget, G.E., Barnes, J.M. (1969). In Evaluation of drug activity; Pharmacometrics (Laurence, D.R., Bacharacha, A.L.) Vol. 1, Academic Press, New York Raghuramulu N, Nair KM and Kalyanasundaram S (1983). A manual of laboratory techniques, (National Institute of Nutrition, Hyderabad).pp. 246–53.

Source of Support: Nil

Stephen J. Mc Phee, (2008). Current Medical diagnosis & treatment, Maxine papadakis publication. Mc Graw hill Lange Chapter Musculo skeletal disorder. Tiwari PN, Kulmi GS. (2004). Performance of Chandrasur (Lepidium sativum) under different levels of nitrogen and phosphorus. J Med Arom Pl Sci 26: 479–481. Yoshikawa T, Tanaka H, Kondo M (1985). The increase in lipid peroxidation in rat adjuvant arthritis and its inhibition by Superoxide dismutase. Biochem. Med, 33:320–326.

Conflict of Interest: None Declared

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Research article DEVELOPMENT OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS FOR AUTHENTICATION OF OLAX SCANDENS ROXB. Naik Raghavendra1*, Borkar Sneha D2, Acharya R N3, Harisha C R4 1,2

P G Scholar, Dravyaguna Department, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat, India Associate Professor, Dravyaguna Department, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat, India 4 Head, Pharmacognosy Laboratory, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat, India *Corresponding Author: Email: ayuraghu@gmail.com; Mobile: +918866679088 3

Received: 17/05/2013; Revised: 30/06/2013; Accepted: 04/07/2013

ABSTRACT Olax scandens Roxb. (Olacaceae), commonly known as “Badru” is a shrub or small tree. Fruits and leaves of this plant are used for medicinal and food purpose. The present study deals with the pharmacognostical study of stem and molecular characterization of young leaves by random amplified polymorphic DNA markers. Microscopic study of stem shows the presence of single layer of epidermis with unicellular uniseriate, horn shaped trichomes, single layered, radially arranged hypodermis, 6–7 layers of cortex, uniseriate to triseriate medullary rays, parenchyma cells with oil globules, prismatic and rhomboidal crystals. All the primers gave good band patterns. Prominent band of ~0.4 kb was obtained with OPA-02 and ~0.9 Kb prominent band was obtained with OPC-06 primer. These microscopic observations and the unique bright and light bands obtained in polymerase chain reaction (PCR) amplification could serve as a measure for authentication and standardization of the plant. KEY WORDS: Olax scandens, Pharmacognosy, RAPD

Cite this article: Naik Raghavendra, Borkar Sneha. D, Acharya. R.N., Harisha. C.R., (2013) DEVELOPMENT OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS FOR AUTHENTICATION OF OLAX SCANDENS ROXB., Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 532–537

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INTRODUCTION Historical background of traditional medical systems explains how the herbal drugs have made a great contribution in maintaining human health. Even today, in most of the developing countries a large proportion of the population relies heavily on traditional practitioners and medicinal plants to meet their health care needs (Patel H et al., 2013). Olax scandens Roxb. (Olacaceae), an ethno medicinal plant, commonly known as “Badru”, used for food and medicinal purposes (Rekha Sinha et al., 2005), (Anonymous, 1996), decoction of stem bark is used to cure fever and cough (Veeramuthu et al., 2006), (Kirtikar & Basu, 2003), leaves applied externally to cure headache (Anonymous, 1990). Though the plant has been reported for many biological activities, till now no detailed study has been reported regarding its pharmacognostical characters except on leaves (Naik R. et al., 2013). Documentation and standardization of such biological resources is important for their identification, authentication and utilization. Identification of biological resources through their molecular characterization is one of the authentic methods of their standardization. Recently certain plants have been reported for their molecular characterization through Random amplified polymorphic DNA markers (Borkar S D et al., 2013). Hence the present study was undertaken to study the microscopic characters of stem and establish the molecular characterization of Olax scandens Roxb. by random amplified polymorphic DNA markers. MATERIALS AND METHODS Collection and preservation of the sample Olax scandens Roxb. was collected from its natural habitat Balangir, Odisha, during September 2012 identified and authenticated by local taxonomist with the help of botanical flora (Saxena H.O, 1995). A sample specimen was preserved in Pharmacognosy lab of IPGT & RA Jamnagar (SPECIMEN NO- PHM 6062/21/09/2012) and the stem was preserved in a solution prepared from 70% ethyl alcohol:

glacial acetic acid: formalin (AAF) in the ratio of 90:5:5 (Johnson Alexander Donald, 1940). Pharmacognostic studies Detailed microscopic characters were studied by taking free hand thin transverse section. Sections were stained with Phloroglucinol and Hydrochloric acid to notice the lignified elements like fibers, vessels etc (Khandelwal K R, 2008). Photographs of the section were taken with the help of Canon digital camera attached to Zeiss microscope. Powder characters were studied as per the guidelines of Ayurvedic Pharmacopoeia of India (Anonymous, 1999). The histo-chemical tests were carried out according to the standard guidelines of practical pharmacognosy (Krishnamurty K V, 1988). Molecular fingerprints)

characterization

(DNA

Fresh leaves were used for molecular characterization and DNA fingerprints, by standard and most convenient RAPD method (Baum BR, Mechanda S, 2001). The RAPD reaction was performed following standard procedures at Aristogene Biosciences Pvt. Ltd, Bangalore. DNA isolation: Young leaves were selected, cut into small pieces without cutting the veins. They were washed with distilled water and ethanol. Frozen with dry ice and crushed. To that, 2 ml of plant DNA extraction buffer was added. The samples were ground thoroughly, transferred into centrifuge tube and added 10 ml plant DNA extraction buffer. 50 μl of BME added to each tube mixed well. Incubated at 65ºC for 1 hour with intermittent mixing and centrifuged for 15 minutes at 10 K (10000 rpm). Supernatant was transferred carefully into fresh tube and added equal volume of chloroform and mixed well. Centrifuged for 15 minutes at 10 K (10000 rpm). Aqueous layer was carefully pipetted into fresh tube and precipitated with isopropanol. DNA pellet suspended in 300 μl of TE and subjected to column purification.

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Column purification Silica spin columns and buffers were from Qiagen The column was placed in collection tube, 400 μl of equilibration buffer was added to the column and centrifuged at 10000 rpm for 1min. Collected buffer was discarded. 400 μl of equilibration buffer was added to the DNA samples, mixed and loaded into the column (This step was repeated till the DNA sample was completed). Flow through was collected. 500 μl of wash buffer 1 was added, centrifuged at 10000 rpm for 1minute and buffer was collected. 500 μl of wash buffer 2 was added, centrifuged at 10000 rpm for 1minute and buffer was collected. The empty column was centrifuged with collection tube to completely remove the wash buffer for 2 minute. 50 μl of

elution buffer was added to the column placed in new collection tube. Incubated at room temperature for 2 minutes and centrifuge at 10000 rpm for 1minute and eluted sample was saved (elution 1). Previous step was repeated (elution 2). Quantization of eluted DNA samples was done by loading into the agars gel (Table 1–2). PCR Conditions 38 µl of amplified DNA was aliquot into 2 different labeled vials and to this 2 µl of respective template DNA was added and PCR was set. One cycle given At 94˚C for 2 minutes, again 40 cycles given at 94˚C, 45˚C and 72˚C for 30 sec, 60 sec. and 90 sec. respectively and finally one cycle given at 72˚C for 7 min (Table 3).

RAPD PCR Table 1: Sequences of primers used OPA-02 TGCCGAGCTG OPB-10 CTGCTGGGAC OPC-06 GAACGGACTC

Table 2: Reactions were set up with PCR master mix and respective Random primer OPC-06 18 µl

OPB-10 18 µl

Notes

Double distilled water

OPA-02 18 µl

2X PCR master mix

20 µl

1X Contains 100µM each of dATP, dGTP, dCTP and dTTP. Assay buffer with 15mM MgCl2, 3U/reaction Taq Polymerase.

DNA sample

1 µl

10 pM used for each reaction

Random primer

1 µl

Total volume

40 µl

Temperature 94˚C 94˚C 45˚C 72˚C 72˚C

Table 3: PCR Conditions. Time No. of cycles 2 minutes 1 30 seconds 40 1 minute 1min 30 sec 7 minutes 1

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RESULTS AND DISCUSSION Microscopic examination of stem T. S of the young stem was circular in outline with uneven surface and showed the following characters (Plate A1). Epidermis T.S showed outer most, single layered, compactly arranged epidermis with somewhat barrel shaped cells covered with cuticle without any intercellular spaces (Plate A2). Epidermal cells were interrupted by unicellular horn shaped trichomes (Plate A3). Beneath the epidermis, compactly arranged, single layered angular cells, forming hypodermis (Plate A4). Inner to the hypodermis 6â&#x20AC;&#x201C;7 layers of parenchyma cells forming cortex, loaded with oil globules and prismatic crystals of calcium

oxalate (Plate A5, 6, 9). Pericyclic fibers are lignified, arranged circularly forming an arch shaped bundles (Plate A8, 12). Vascular bundles open and collateral, arranged radially. Metaxylem facing towards periphery and protoxylem facing towards pith region (Plate A7, 10). Xylem consists xylem parenchyma and fibers. Phloem situated above the xylem and consists phloem fibers and sieve elements. Xylem vessels were separated by uniseriate to triseriate medullary rays (Plate A11). Cells were somewhat elongated barrel shaped with starch grains, prismatic crystals and oil globules. Pith occupies the central part of the stem. Pith consist parenchyma cells. Cells were thick, lignified and pitted near the tail of vascular bundles. Most of the parenchyma cells consists oil globules, prismatic crystals and rhomboidal crystals.

PLATE A

1.Olax scandens Roxb.

4. Epidermis and hypodermis.

2. T. S of stem in outline.

5. Rhomboidal crystal.

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3. Trichomes.

6. Prismatic crystals.

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7. vascular bundles and pith.

10. T. S after staining

8. Pericyclic fibers.

11.Medullary rays

9. Crystals and oil globules.

12. Lignified pericyclic fibers.

lignified pericyclic fibers and oil globules (PLATE B1â&#x20AC;&#x201C; B7).

Powder microscopy Organoleptic characters showed that powder was straw colored, with characteristic odour and bitter taste. Diagnostic character of stem powder showed unicellular horn shaped trichomes of epidermis, prismatic and rhomboidal crystals of calcium oxalate, tannin content, cork, annular and border pitted vessels of vascular bundles,

Histochemical test To confirm the presence and absence of the chemical constituents the material was subjected to various tests. Lignified cells, starch, calcium oxalate crystals, tannin, oil globules were present in the stem (Table 4).

PLATE B

1. Trichome

2. Annular vessels

3. Crystals and tannin content

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4. Prismatic crystals

5. Lignified fiber.

6. Border pitted vessels.

7. Cork

8. DNA Fingerprints

Table 4: Histochemical tests for Olax scandens Roxb fruit. Sr. No.

Reagents

Phloroglucinol+ Conc. HCl Iodine Phloroglucinol+ Conc. HCl Fecl3 solution

Sudan III

1. 2. 3.

Observation

Characteristics

Results

Red

Lignified cells

Blue Dissolved

Starch Calcium oxalate crystals Tannin cells

++ ++

Oil globules

Dark blue to black Red

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DNA finger printing Marker used was mid range ruler with 10 bands of 100, 200. 300. 600, 1000, 1500, 2000, 2500, 3000 and 3500 bp. In the gel photograph, R-marker, lanes 1to 3- patterns obtained from random primers. All the primers gave good band pattern. Prominent band of ~0.4 kb was obtained with OPA-02 and ~0.9 Kb prominent band was obtained with OPC-06 primer (PLATE B8). CONCLUSION Presence of microscopic characters like unicellular, horn shaped trichomes, prismatic and rhomboidal crystals of calcium oxalate, oil globules are the key identification characters of Olax scandens Roxb stem. The unique bands obtained in Polymerase Chain Reaction (PCR) amplification are clearly discriminated having,

many bright and light bands indicating the genuinity of the plant. The observed pharmacognostical characters and DNA finger prints may be useful to establish the botanical standards for identification and standardization of Olax scandens Roxb. stem. ACKNOWLEDGEMENT The authors like to acknowledge the administrative authorities of the institute IPGT & RA, Jamnagar for providing facilities during work. Authors also express their sincere thanks to Mr. Pareshvar Sahoo, Taxonomist, SSN Ayurvedic college Paikmal Odisha, Mr. B. N. Hota, Rtd. DFO, Govt. of Odisha who helped us during drug collection and Dr. Sudha, Director, Aristogene Biosciences Pvt Ltd, Bangalore for their co-operation for DNA RAPD study of the plant.

REFERENCES Anonymous, (1990), Glimpses of medicobotany of Bastar district, Madhya Pradesh, CCRAS publication, pp. 114. Anonymous, (1996), Botanical exploration of Phulbani and Korapat district of Orissa, CCRAS publication, pp. 132. Anonymous, (1999), The Ayurvedic Pharmacopoeia of India, Govt. of India publication New Delhi, 1st edition, Vol. I, Appendix 2. Baum BR Mechanda S, Livesey JF, Binns SE, Arnason JT. (2001) Predicting quantitative phytochemical markers in single Echinacea plants or clones from their DNA fingerprints. Phytochemistry. 56 (6):543–9

Borkar S D, Naik R, Harisha C R, Acharya R N (2013), Development of Random Amplified Polymorphic DNA markers for authentification of Rivea hypocrateriformis (Desr.) Choisy, Global J Res. Med. Plants & Indigen. Med.,Volume 2(5): pp. 348–356 Johnson Alexander Donald, (1940), Plant Micro techniques, Macgrow Hill Book Company, New York, London. pp. 105. Khandelwal K.R. (2008), Practical Pharmacognosy Techniques and Experiments, 19th Ed, Nirali Prakashan; 2008. pp . 15–18. Krishnamurty K.V. (1988). Methods in the plant histochemistry, Vishwanadhan Pvt Limited, Madras, pp.1–77.

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Naik Raghavendra, Borkar Sneha D, Harisha C R, Acharya R N, (2013), A detailed pharmacognostical evaluation on leaf of Olax scandens Roxb, Global J Res. Med. Plants & Indigen. Med, Volume 2, (4): 189–203 Patel H, Ingalhalli R, (2013), A brief review on Ginkgo biloba L. (maidenhair tree) a rare multipurpose medicinal plant, Global J Res. Med. Plants & Indigen. Med., Volume 2(6): 418–427. Rekha Sinha, Valeria Lakra, (2005), Wild tribal food plants of Orissa, Indian journal of traditional knowledge, Vol. 4(3), pp. 246–252.Saxena H.O, (1995), The Flora

Source of Support: Nil

of Orissa, Regional Research Laboratory, Bhubaneshwar, 1st edition, pp. 288. Veeramuthu Duraipandiyan, Muniappan Ayyanar, Savarimuthu Ignacimuthu, (2006), Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India, BMC Complementary and Alternative Medicine, 6:35 doi:10.1186/1472-68826-35 Wallis

TE, (1985), Textbook of Pharmacognosy, London Churchill Publication, pp. 572–82.

Conflict of Interest: None Declared

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Review article ADVANCEMENTS IN INDIAN SYSTEM OF MEDICINE (ISM) INFORMATICS: AN OVERVIEW Samal Janmejaya1* 1

District Epidemiologist, Dist. Health Office, Gadchiroli, Maharashtra, India *Corresponding Author: Email: janmejaya_samal@yahoo.com; jaytheworld@gmail.com; Mob: +919438323843, +919901316384

Received: 17/05/2013; Revised: 13/06/2013; Accepted: 20/06/2013

ABSTRACT Indian system of medicine has its root in India, evolved through a continuous process of transformation from its original Vedic form to the modern day Indian System of Medicine. This system of medicine has crossed the Indian Territory and made its presence in different parts of the globe. More often the system remains unnoticed owing to its aboriginal facets. Digitalization is one way to go about the problem which makes it accessible to those who need to know about it. This is the need of the hour as computers can store and retrieve large pool of data which is not the case with manual approach. This can be used for several purposes be it present or future. Digitalization or computerization could be mere online web resources or software for information and decision making. The entire gamut of these developments could be clubbed together in to a domain known as ISM informatics. This can be considered as one of the tributaries of the broader umbrella of health informatics and the same will help to elucidate the former in a better way. The term Indian System of Medicine embraces many different systems of medicine practiced in India that includes Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homoeopathy. The present article tries to make a review of the recent developments in the field of ISM informatics with a special focus on Ayurvedainformatics. KEYWORDS: Ayurveda, Indian Systems of Medicine, Informatics

Cite this article: Samal Janmejaya (2013), ADVANCEMENTS IN INDIAN SYSTEM OF MEDICINE (ISM) INFORMATICS: AN OVERVIEW, Global J Res. Med. Plants & Indigen. Med., Volume 2(7): 546â&#x20AC;&#x201C;553

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INTRODUCTION Indian System of Medicine is the ethnic bequest deeply buried in the traditional believes of the people that are evolved through a continuous process of transformation from its original Vedic form to the modern day Indian System of Medicine. It has witnessed a paradigm shift from its oldest version of “Guru-Sishya-Ashram” tradition (a way of learning, by a disciple, from a Spiritual Mentor in an Ashram) to the modern day medical education system, formally taught in school of Ayurveda with use of all modern amenities. The present day Ayurveda has undergone many changes as per the need of the day but important principles have remained unchanged (Janmejaya Samal, 2013). This system of medicine has crossed the Indian Territory and made its presence in different parts of the globe. But the biggest problem in its accessibility is its aboriginal facets. Owing to its growing popularity it’s high time to digitalize this system to place it before the global audience. Several endeavors have been made from different segments to develop some of the user friendly digital versions/software of this oldest and classical system of medicine but still it has to traverse a long way to meet the growing pace of time. This is highly essential because the world is getting digital day by day and the capacity of computers to store and retrieve a large pool of data easily which is not the case with manual approach. Computers have changed human approach in every field and health care sector is also a prey to it. ISM informatics can be considered as one of the tributaries of the broader umbrella of health informatics and the same will help to elucidate the former in a better way. Health Informatics is as much a result of evolution as planned philosophy, having its roots in the histories of information technology and medicine (Cesnik B, 2010). ISM informatics is a judicious integration of Information Technology and Ayurveda, and other allied disciplines of Indian medicine. Broadly the development of health informatics in India can bring three different benefits; firstly as an instrument in continuing education, they enable health workers to be

informed and trained in advanced medical and health sciences; secondly, they deliver health services to the poor at rural and remote locations; thirdly, they can increase the transparency and efficiency of governance, which should in turn improve the availability and delivery of publicly provided health services (C P Chandrasekhar, 2001). Again the adoption of ICT (Information and Communication Technology) in Ayurveda will enhance the interaction between modern medicine and Ayurveda (Sushant Sud, 2013). In this context a diligent effort has been made to review the recent developments in Indian System of Medicine (ISM) informatics under the broad umbrella of health informatics. METHODOLOGY Be it pure health informatics or ISM informatics, the discipline becomes purely technical in nature which is a judicious blend of information and communication technology and health or in the present context Indian System of Medicine. Practically speaking this goes beyond the scope of this article hence the same has not been dealt with. This article delineates a snapshot or an overview of the developments in India System of Medicine informatics based on systematic literature review. Various literatures such as published articles, books and monographs in the said domain have been reviewed systematically and the same has also been applied to web related resources for the purpose of this study. DISCUSSION ISM informatics is somehow similar to other advanced form of medical informatics but the domain is very limited, as it cannot include the advanced areas such as computer assisted surgery or robotic surgery, neuro-computers and Artificial neural networks etc. Different organizations are working towards this direction and more recently the Institute of Ayurveda and Integrative Medicine, Bangalore, India has opened up a center known as Center for ISM informatics and Theoretical Foundations which was started in 1995 to give increased focus for the modernization of Indian

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systems of medicine in order to improve access for variety of research purposes (Institute of Ayurveda and Integrative Medicine, 2013). The major achievements of this center are as follows: 



CD on Medicinal plants on Siddha System of medicine: This CD contains 33,350 Tamil names of plants recorded in 26 well known texts pertaining to the Siddha system of medicine. These Tamil names have been correlated to 1600 botanical entities. The nomenclature section also incorporates correlation of these plant species with around 66,000 vernacular names in different Indian languages. Detailed information has been compiled and incorporated for 500 plants recommended for BSMS curriculum of Siddha system. It was released in 2008 (Institute of Ayurveda and Integrative Medicine, 2013). CD on Medicinal plants on Unani System of medicine: This CD contains information on 307 plants (including the 275 plants recommended for BUMS curriculum) in Unani system of medicine. Five texts of Unani medicine have been used for this literary research. A total of 380 plants have been identified as sources of Unani medicine from these texts. Around 500 plant entities have been estimated in Unani system of medicine. Five Classical text of Unani medicine have been used for this literary research. This was released in 2008 (Institute of Ayurveda and Integrative Medicine, 2013). CD on Medicinal plants on Homoeopathy: A thorough review and data compilation from available literature (in India) relating to plants used in Homoeopathy has generated a master list of 550 botanical names. After establishing proper synonym linkages, this number has got reduced to 506 plant species. Each of these 506 species has been analyzed for its geographical distribution. This has generated a list of 326 species which have been identified as “recorded in India”. It incorporates such species, which are wild in



India including naturalized ones and/ or cultivated/ planted in India. The Materia Medica section deals with the effect of 375 plant drugs (263 Indian & 112 exotic) on various body organs including the mind. Data has been derived from more than 23 published works dealing with Materia Medica of Homoeopathy. It was released in 2008 (Institute of Ayurveda and Integrative Medicine, 2013). CD on Medicinal plants on Ayurveda: “Dravya guna shastra” (Medicinal Plant Science) or Ayurvedic Materia Medica on plants is the complete science on pharmacognosy of the plants used in Indian medical heritage. This CD contains comprehensive information about 370 plants recommended for BAMS syllabus. It includes around 2300 Sanskrit slokas (Hymns) with their translation in English. It contains 800 plant images, botanical correlation of Sanskrit names based on accepted studies and current understanding. Source of data embedded in this CD are compiled from around 20 Ayurvedic classical sources starting from 1500 BC to 1986 AD. It includes a glossary of around 3000 Sanskrit technical terms and popular articles. It was released in 2005. Besides this institute has also released some of other CDs such as neighborhood plants of Bangalore City in 2008, CDs on Indian medicinal plants in Trade in 2005, CD on plants of Charaka Samhita in 2003, CD on clinically important plants of Ayurveda in 2002, CD on Prakriti (How to analyze body constitution) in 2003, CD on medicinal plants of Karnataka, Kerala and Tamil Nadu in 2000. (Institute of Ayurveda and Integrative Medicine, 2013).

Similarly another organization contributing to this endeavor is the center for development of advanced computing, Pune, India which is a premier Research & Development (R&D) organization of the Department of Electronics and Information Technology, Ministry of Communications & Information Technology (MCIT) for carrying out R&D activities in IT, Electronics and associated areas. This

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organization has created innovative software on various functionalities of Ayurveda known as AyuSoft (Center for Development of Advanced Computing, 2013).



AyuSoft: This interactive software has been developed in collaboration with C-DAC, Pune; Interdisciplinary School of Health Sciences and Department of Ayurveda, University of Pune; Jnana Prabodhini, NGO, Pune, India. It is a pioneering multidimensional effort for Indian traditional medical system that provides end-toend medical solutions based on traditional medicines and helps in making health decisions that are expected to be more informed, more accurate and quicker. The target end user for this software may include hospitals, practitioners and researchers. It has wide range of applications including disease diagnostics and treatment, diet and life style advice, personal management information system, multimedia based encyclopedia, and textual and analytical report tool. It has the following components: (Center for Development of Advanced Computing, 2013) 



Vaidya Sanmitra: - This application covers most of the clinical requirements. It includes Maanusha Vritta (Personal management information system), case analysis, Master data management, Vyadhi Nidaana (Disease Diagnosis) (Center for Development of Advanced Computing, 2013). Prakriti Vichaya: - Prakriti (constitution) is a unique concept of Ayurveda that seeks to explain the element of individuality by expressing the unique trait of an individual that is defined by the specific and permanent composition of their Dosha (Humour) at conception. Prakriti plays an important role in the prevention, diagnosis and treatment of diseases. This module of AyuSoft covers Dosha Prakriti, Maanas Prakriti (Psychic Constitution), and Dhaatusaarataa (Tissue System) (Center for Development of Advanced Computing, 2013).



AayurVidnyaana: - A knowledge base encompassing all the aspects of Ayurveda. It includes articles, research papers of the stalwarts from all over India; Collection of information related to different aspects of Ayurveda; Digitalized Brihattrayee (Three major treatises of Ayurveda constituting Charaka Samhita, Sushrut Samhita, Astanga Hridaya) in Devanaagaree Script; Video Clips of Therapeutic Procedures like Medicated emesis, Medicated massage, Blood Letting etc.; Photographs of different herbs, Instruments and Diseases; Audio Files of Dhanvantaree Stavana (Chants on God Dhantwantari) and other Mantra (Hymns)(Center for Development of Advanced Computing, 2013). Anveshaka: - Anveshaka is an interactive and high-speed data-mining tool useful for analyzing Ayurveda related data. It is useful to researchers and students for carrying out precise analytical search of classical data. It comprises of the common data report, Vyaadhi (Disease) diagnosis report, and Vyaadhi (Disease) treatment report (Center for Development of Advanced Computing, 2013). Vyaadhinidaan: - Provides decision support for probable diagnosis and treatment. It is based on comprehensive diagnostic and treatment data. In includes two important features i.e. Diagnostic features and Treatment features (Center for Development of Advanced Computing, 2013).

EasyAYURVEDA: This has been developed by VHCA herbals which portray an excellent blend of Ayurveda and Information Technology. There is a whole unlimited bunch of literature in Ayurveda and the demand for such a tool that can make reading, searching easier is very high. Suppose a researcher is interested in herbs with sheeta virya (Potency) from the ancient texts of Ayurveda, the process may be so tedious and slow that output of the exercise shall fade away with the passage of time. But software like EasyAYURVEDA can make the process

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relatively easier and faster. EasyAYURVEDA is a Easy to use Ayurvedic software; contains database of more than 500 Medicinal Plants formulations; helps in advanced searching of herbs & formulations and herb’s name in all Indian languages; Multiple string searching & single string searching, Indications on the basis both Ayurveda & modern diagnostic principles and terminologies. This software is the first of its kind in the history of Ayurveda and is the largest information portal of Ayurveda on internet (VHCA Herbals, 2013).

diagnosis in a faster and organized way (Shajahan, M.A, 1993; VHCA Herbals, 2013).

PRAKES:

PILEX:

This is one of the innovative software developed by Resource Center for Indian Language Technology Solutions- Malayalam, Center for Development of Advanced Computing, Thiruvanantapuram, Kerala, India and is available in both English and Malayalam version. It is an interactive menu driven interface. It helps in examining the Lakshanas (Symptomatology) and assessing the dominance of Tridoshas (Three Humors). Advices on preventive promotive health care services depending on humeral dominance. The system records the interactions and results along with the bio-data for future references. It helps generate the hard copy of these recordings (Malayalam Resource Centre, 2013).

As the name indicates, this particular software deals with entirety of piles. It is intended for the diagnosis, prognosis, complications and treatments of piles. It was designed and developed by Gujarat Ayurveda University, Jamnagar, Gujarat in 1990.

BODY TUNE: Medicare/CAM):

(Computerized

Ayurvedic

This software has been developed by Dr.M.A Shajahan in 1983 in Govt. College of Indian Medicine, Bangalore in collaboration with Indian Institute of Science, Bangalore which was proved clinically successful by Gujarat Ayurveda University. A newer version of the software was developed in collaboration with Cyberveda Technologies in 1988. This particular software neither replaces a doctor nor avoids the importance of doctor-patient relationship. It helps organize diagnostic methods in a classical way envisaged by Indian sages of Ayurveda. This user friendly interactive software promotes accurate

PRAKRTI: This innovative and expert software has been designed and developed by Chaitanya Consultancy, Pune in 1989. It renders services on different functionalities of Ayurveda such as Prakrati (Constitution), dietary advices, advices on daily regimens, likelihood of an illness and its precautionary measures.

RASEX: This innovative software was designed and developed by Government Ayurveda College, Trivandrum in collaboration with CIRA (Center for Information Research and Action), and C-DAC (Center for Development of Advanced Computing), Thiruvanantapuram in 1992. This package attempts to correlate the pharmacological properties with that of therapeutic properties with the help of computer. A database was created after collecting, organizing and storing all the pharmacological and therapeutic properties of single rasa drug using DBase III plus. A list of drugs, which conforms to the physician’s specifications is collected and displayed in this package (Sushant Sud, 2013). Ayurinformatics: Ayur-informatics is a science dealing with the application of bioinformatics to the field of Ayurveda. It is the application of modern drug designing technology and bioinformatics to the field of traditional Ayurvedic system of medicine. This area is growing by leaps and bounds with some impressible and noticeable

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developments. Some of the recent works in this domain are as follows: 



An in-silico work based on Homology Modeling for the proteins of RAS, MYC, SRC, BRAC1, P53, and EGFR using Modeller9v7 software for establishing an Ayurvedic remedy for bronchial carcinoma was done by Preenon Bagchi et al., It reflects the use of classical Ayurveda drugs having importance in the cure of lung tumors (Preenon Bagchi et al.,2011). Another in-silico work based on COMT (Catechol-O-methyltransferase) gene was done by Preenon Bagchi et al., The work highlights the use of anti-psychotic herbal drugs as agonist to COMT mutation gene which inhibits the excessive dopamine production and cures Schizophrenia. The study reflects the use of herbs like Chandana (Santalum album), Shankhpushpi (Canscora decussata) and Jatamansi (Nardostachys jatamansi) which have mental regenerative & antipsychotic property, their active compounds such as βsantalol, xanthone and nardal respectively are docked with COMT mutation protein (Preenon Bagchi et al., 2012). An in-silico work based on Homology Modeling for the proteins of APP, APOE, Presenilin 1 and Presenilin 2 using Modeller9v7 and other software for establishing an in-silico Ayurvedic medication for Alzheimer’s disease was carried out by Mohini Gore Active compounds of medicinal herbs like Sankhapuspi (Canscora decussata), Jatamansi (Nardostachys jatamansi) and Kapikachu (Mucuna pruriens) have been used for the purpose of study (Mohini Gore 2010).

Ample of work is happening in the field of Ayurvedic education, teaching and training. This has not been reflected in the case with practice. A list of 4 PubMed indexed journals, 38 non PubMed indexed journals, 4 Hindi Ayurveda Journals, 26 Journals of Complementary and Alternative Medicine and 11 magazines of Ayurveda have been

documented (Ayurbhishak 2013). Many of the classical Ayurveda treatises such as Charaka Samhita, Sushrut Samhita, Astanga Sangraha, Astanga Hridaya, Sharangadhara Samhita, Madhav Nidan, Harita Samhita, Bhela Samhita and Kasyapa Samhita have been digitalized and are available on line (Ayurbhishak 2013). This academic and practice gap can be minimized by working on these issues in collaboration with renowned ISM practitioners with that of IT professionals. CONCLUSION: This era is rightly called as the digital age. Computer has immensely influenced human life. Computer has to do with every walk of human life so also the domain of medicine and health care. The medical informatics has grown by leaps and bounds but the same is not the case with ISM informatics. Things are relatively better and initiatives have already been started elsewhere but more and more endeavors are needed to place it on a noticeable height. As a specialized field this particular domain requires an integrative approach from both the field of Ayurveda and Information Technology. This judicious blend will definitely be of great help in different facets of Ayurveda be it clinical medicine, biomedical research or information storage and retrieval. Wider acceptance of these systems owing to their safe and efficacious therapeutics on many of human diseases has again created an urgent need for the development of ISM informatics. It has been widely observed that websites are mushrooming imparting information, education and communication in matters related to Indian systems of medicine but the authenticity of the same is skeptical which needs to be controlled with governmental support. Again there are lots of virgin areas which could be explored and worked out for better access, operation and above all better utility of Indian Systems of medicine. This can lead to the development of some work in the following domains of Indian System of Medicine. These may include; 

Computer retrieval

based

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medical

information

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      

Computers in Clinical Laboratory Computer assisted medical decision making Hospital information system Clinical information system Nursing information system Dietetic information system Computerized patient records

    

Computerized prescriptions for patients Computer Assisted Patient Education and Health care information Telemedicine Computer Assisted Drug Discovery and Development Computer Assisted Instruction in Medicine

REFERENCES: Ayurbhisak, Ayurveda News Repository. http://ayurbhishak.wordpress.com/ayurv edank/ (Accessed on 25/05/2013) Ayurbhisak, Ayurveda News Repository. http://ayurbhishak.wordpress.com/treati ses/ (Accessed on 25/05/2013) Center

Center

for Development of Advanced Computing, Pune, India. http://integrate.cdac.in/index.aspx?id=hi _pr_ayusoft (Accessed on 22/05/2013) for Development of Advanced Computing, Pune, India. http://integrate.cdac.in/index.aspx?id=hi _pr_vaidyasanmitra (Accessed on 22/05/2013) for Development of Advanced Computing, Pune, India. http://integrate.cdac.in/index.aspx?id=hi _pr_prakriti_vichaya (Accessed on 22/05/2013)

Center

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

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 7 | July 2013 | 546–553

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