GJRMI - Volume 5, Issue 12, December 2016 by Dr. Hari Venkatesh.K.Rajaraman

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

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INDEX – GJRMI - Volume 5, Issue 12, December 2016 MEDICINAL PLANT RESEARCH Biology COMPARISION OF ANTIMICROBIAL ACTIVITY OF DIFFERENT EXTRACTS OF SILENE CORONARIA (Desr.) Clairv. ex Rchb. AND SILENE DENIZLIENSIS Aytaç. GROWN IN TURKEY Nur Ceyhan Güvensen*, Dilek Keskin, Kemal Yildiz

286–291

Pharmacognosy DIAGNOSTIC PARAMETERS ASSISTED WITH SUITABLE PHOTOMICROGRAPHS, PHARMACOGNOSTICAL AND PHYTOCHEMICAL STUDY OF ECLIPTA PROSTRATA (L.) L. Patnaik Sunita*, Mishra D N

292–299

INDIGENOUS MEDICINE Rasa Shastra & Bhaishajya Kalpana - Ayurveda PHARMACOGNOSTICAL VARIATIONS IN MARKET SAMPLES OF TINOSPORA SPECIES AND THEIR EXTRACTS Seetha Chandran*, Harisha CR, BJ Pagiri

300–312

Cover Page Photography: Dr. Hari Venkatesh K.R. Plant ID: Inflorescence of Antidesma montanum Blume* of the family Phyllanthaceae; Place: Koppa, Chikkamagalur District, Karnataka, India *Botanical Name validated from www.theplantlist.org as on 29/12/2016

Research article COMPARISION OF ANTIMICROBIAL ACTIVITY OF DIFFERENT EXTRACTS OF SILENE CORONARIA (Desr.) Clairv. ex Rchb. AND SILENE DENIZLIENSIS Aytaç. GROWN IN TURKEY Nur Ceyhan Güvensen1*, Dilek Keskin2, Kemal Yildiz3 Biology Department, Faculty of Sciences, Muğla Sıtkı Koçman University, Muğla, Turkey Çine Vocational High School, Adnan Menderes University, Aydın, Turkey 3 Biology Department, Faculty of Science and Letters, Celal Bayar University, Manisa, Turkey *Corresponding Author: E-mail: nurceyhan@msn.com 1 2

Received: 03/11/2016; Revised: 20/12/2016; Accepted: 23/12/2016

ABSTRACT Plants have been used for thousands of years to flavor and conserve food, to treat health disorders and to prevent diseases including epidemics. The antimicrobial activities of the extracts of ethanol, methanol, ethyl acetate and propanol made from Silene coronaria and Silene denizliensis were studied by disc diffusion method. These extracts were tested against five bacteria and one fungus, which revealed various levels of antimicrobial activity. The ethyl acetate extracts of S. coronaria leaves and branches showed the best antibacterial activity against S. aureus (11 mm). The propanol extracts of S. coronaria leaves and branches showed the best antibacterial activity against S.aureus (11 mm) and A. hydrophila (16 mm). The ethanol extracts of S. denizliensis leaves and branches showed the best antibacterial activity against E. coli (14 mm) and A. hydrophila (15 mm). The methanol extracts of S. denizliensis leaves and branches displayed the best antibacterial activity against E. coli and A. hydrophila (12 mm). When we compared theresults to that of antibiotic, the MIC value of erythromycin was higher than the propanol extracts of S. coronaria against B. cereus. Similarly the MIC value of erythromycin was higher than the methanol extracts of S. denizliensis against B. cereus. Therefore, S. coronaria and S. denizliensis leaves and branches extracts can be used as a natural antimicrobial agent in food additives or pharmaceutical products against infections. KEYWORDS: Silene coronaria, Silene denizliensis, Antimicrobial activity, Chemical content

Cite this article: Nur Ceyhan Güvensen, Dilek Keskin, Kemal Yıldız (2016), COMPARISION OF ANTIMICROBIAL ACTIVITY OF DIFFERENT EXTRACTS OF SILENE CORONARIA (Desr.) Clairv. ex Rchb. AND SILENE DENIZLIENSIS Aytaç. GROWN IN TURKEY, Global J Res. Med. Plants & Indigen. Med., Volume 5 (12): 286–291

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Global J Res. Med. Plants & Indigen. Med. | Volume 5, Issue 12 | December 2016 | 286–291

INTRODUCTION The use of plants for treating diseases is as old as the human species. Popular investigations on the use and efficacy of medicinal plants major contribute to the disclosure of their therapeutic properties, so that they are frequently prescribed, even if their chemical constituents are not always entirely known (Silva and Fernandes Júnior, 2010). The predicted number of medicinal species used is 80% of the world population in traditional medicines, and in the pharmaceutical sector between 53,000 and 72,000 species are being used (Boubakr et al., 2015a). Medicinal plants are considered essential raw material source for the finding of new molecules necessary for the development of future drugs (Boubakr et al., 2015b). Active compounds produced during secondary vegetal metabolism are usually liable for the biological characteristic of some plant species used throughout the globe for different aims, including treatment of infectious diseases (Silva and Fernandes Júnior, 2010). Food poisoning is usually assumed as an acute disease caused by contaminated food or beverages, with the bacteria as its major cause (Salek-Moghadam et al., 2000; García and Heredia, 2009). Other factors which can cause such disease are parasites, toxic plants, fungi and etc. (García and Heredia, 2009; Zendehdel and Babapour, 2010). Annually about 76 million persons are affected worldwide and most of the cases occur in summer. Generally children and old people are at a higher risk (García and Heredia, 2009). World Health Organization has estimated that in 2005, 1.8 million people have died due to diarrheal diseases and a considerable amount of these cases were after consumption of contaminated food and water (Wesley, 2009). Silene is represented by about 170 taxa in the Flora of Turkey. Silene is a genus of the family Caryophyllaceae, having the greatest number of species both in the Turkish floras (Davis, 1967; 1988; Güner et al., 2001). The antimicrobial effect of some species of Silene has been studied in previous researches

(Ertürk et al, 2006; Mahesh and Satish, 2008; Borchardt et al., 2008, Bajpai et al., 2008). As far as we know, this is the first study, an antimicrobial activity of four different extracts of Silene coronaria (Desr.) Clairv. ex Rchb. and Silene denizliensis Aytaç against five bacteria and one fungi were reported in this study from Turkey. MATERIALS AND METHODS Plant collection and preparation of extracts: Plant samples of the two species were collected from natural populations in Turkey. The plant specimens were identified taxonomically in accordance with Davis (1967, 1988) and Aytaç (1998). Voucher specimens of the plants were kept at the herbarium of Manisa Celal Bayar University, Faculty of Science and Letters, Department of Biology, in Turkey. Silene coronaria was collected from Turkey, Thrace (Marmara region), Kırklareli, Pınarhisar, above Yenice village, on forest road, at 625 m altitude in 25 May 2014, collector; Güngör Ay, Mehmet Kuh, Kemal Yıldız-0503 and Silene denizliensis was collected from Turkey, Aegean region, Denizli, Tavas, Kızılcabölük village, Çakıroluk district, 1550–1650 m altitude, in 04.July.2014, collector, Kemal Yıldız-0528. The leaves & stems of both the species used were dried and broken into small pieces under sterile conditions and 20 g of each plant was extracted with 150 mL of ethanol, methanol, etyl acetate and propanol (Merck) separately for 24 h by Soxhlet appartaus (Khan et al., 1988). Prepared extracts were dried at 30°C using a rotary evaporator until amount of each extracts was 1 mL. Microorganisms and media: Five bacteria (Staphylococcus aureus ATCC 6538/P, Bacillus cereus CCM 99, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Aeromonas hydrophila ATCC 19570) were obtained from the Biology Department of Mugla University, Science and Art Faculty. Cultures of these

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Global J Res. Med. Plants & Indigen. Med. | Volume 5, Issue 12 | December 2016 | 286–291

bacteria were grown in Nutrient Broth (NB) (Difco) at 37 ± 0.1oC for 24 h. One fungus (Candida albicans). Cultures of these fungi were grown in Sabouraud Dextrose Broth (SDB) (Difco) at 25 ± 0.1oC for 24 h. Antibacterial activity: The disc assay described by Bauer et al. (1966) was used for the antimicrobial activity. All of the extracts individually were injected into empty sterlized antibiotic discs having a diameter of 6 mm (Schleicher & Schül No:2668, Germany) in the amount of 25 μL. Discs injected with pure ethanol, methanol, etyl aceetate and propanol served as negative controls. The bacteria were incubated in Nutrient Broth (NB)(Difco) at 37±0.1°C for 24h, and then inoculated [106 mL-1 (NCCLS, 2000)] into petri dishes containing homogenously distributed 15 mL of sterilized Muller-Hinton agar (MHA, Oxoid) (Collins et al., 1989). Disc injected with extracts were applied on the solid agar medium by pressing slightly. The treated petri dishes were placed at 4oC for 1–2 h and then the injected plates with bacteria were incubated at 37 ±0.1oC for 18–24 h, (Collins et al., 1989; Bradshaw, 1992; Toroglu, 2007; 2011). Ampicillin (10 μg/disc) and erythromycin (10 μg/disc) discs were used as standard antibiotics (as positive control). After incubation, all plates were observed for zones of growth inhibition, and the diameters of these zones were measured in millimeters. The experiments were conducted in triplicates to ensure uniformity in the results. Antifungal activity: Antifungal assay was performed using disc diffusion method (Bauer et al., 1966). The respective fungal cultures were inoculated (105 mL-1) (NCCLS, 2000) into petri dishes containing homogenously distributed sterilized Saboraud Dextrose Agar (SDA) (Collins et al.,1989). Discs injected with extracts were applied on the solid agar medium by pressing slightly. The treated petri dishes were placed at 4oC for 1–2 h and then the injected plates with fungi were incubated at 25±0.1oC for 48 h. Nystatin 100 Units (10 μg/disc) discs were used

as positive control. Different plant extracts were used to saturate the disc and placed on the seeded plates. Respective solvents acted as negative controls. After incubation period, the antifungal activity was evaluated by measuring the zone of inhibition against test organisms. The experiments were conducted in triplicates to ensure uniformity in the results. GC/MS analysis: The steam-distilled components were analysed by GC/MS. A HP 6890 gas chromatograph equipped with a HP-PTV and a 0.32 m × 0.60 m HP-Innowax capillary column (0.5 µm coating) was employed for the GC analysis. GC/MS analysis was performed on a HP-5973 mass selective detector coupled with a 6890 gas chromatograph, equipped with a HP 6890 gas chromatograph, equipped with HP1capillary column. The column temperature was programmed from an initial temperature of 60oC to a final temperature of 250oC at 15oC/min. The carrier gas was helium (14,1 mL/min). Identification of the individual components was performed by comparison of mass spectra with literature data and by a comparison of their retention time (Rt) relative to a C8-C32 n-alkanes mixture (Adams., 1995). A computerized search was carried out using the Wiley 7n.l GC/MS library and ARGEFAR GC/MS library created with authentic samples. RESULTS AND DISCUSSION Antimicrobial activity results: The ethanol extracts of S. coronaria leaves and branches showed the best antibacterial activity against Pseudomonas aeruginosa and S. aureus (9 mm) (Table 1). The methanol extracts of S.coronaria leaves and branches displayed the best antibacterial activity against S.aureus (8 mm). The ethyl acetate extracts of S. coronaria leaves and branches showed the best antibacterial activity against S. aureus (11 mm). The propanol extracts of S. coronaria leaves and branches showed the best antibacterial activity against S.aureus (11 mm) and A.hydrophila (16 mm). The ethanol extracts of S. denizliensis leaves and branches

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Global J Res. Med. Plants & Indigen. Med. | Volume 5, Issue 12 | December 2016 | 286–291

showed the best antibacterial activity against E.coli (14 mm) and A. hydrophila (15 mm). The methanol extracts of S. denizliensis leaves and branches displayed the best antibacterial activity against E.coli and A.hydrophila (12 mm). The ethyl acetate extracts of S.

denizliensis leaves and branches showed the best antibacterial activity against A. hydrophila (10 mm). The propanol extracts of S. denizliensis leaves and branches showed the best antibacterial activity against E. coli (9 mm).

Table 1: Antimicrobial activity of Silene coronaria and Silene denizliensis leaf and branches extracts against test microorganisms by disc diffusion method. Microorganisms

Plants

Propanol

Ethanol

Methanol

Ethyl acetate

Erythromycin

Ampicillin

B.cereus

−

E.coli

P.aeruginosa

−

A.hydrophila

−

C.albicans

−

Nystatin

Ethyl acetate

S.aureus

Propanol

Methanol

Silene denizliensis (values in mm)

Ethanol

Silene coronaria (values in mm)

Antibiotics

(−): No inhibition, NT:Not tested

Table 2. MIC values of Silene coronaria and Silene denizliensis leaf and branch extracts, erythromycin, ampicillin, and nystatin against test microorganisms

1.6

B. cereus E.coli P. aeruginosa A. hydrophila C. albicans

0.8 12.8 25.6 6.4 12.8

Nystatin

1.6

Ampicillin

S.aureus

Antibiotics Erythromycin

Plants Microorganisms Silene coronaria Silene Propanol denizliensis Methanol

1.6 3.2 6.4 1.8 N.T

0.8 3.2 3.2 3.2 N.T

N.T N.T N.T N.T 3.2

NT:Not tested GC-MS results:

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Global J Res. Med. Plants & Indigen. Med. | Volume 5, Issue 12 | December 2016 | 286–291

MIC values: When it comes to MIC results, the propanol extracts of S.coronaria and methanol extracts of S.denizliensis showed same results against all of the tested microorganisms (Table 2). When we compared the results against the antibiotic the MIC value of erythromycin was higher than the propanol extracts of S.coronaria against B.cereus. Similarly the MIC value of erythromycin was higher than the methanol extracts of S. denizliensis against B.cereus. According to GC-MS results, 2-propanol was detected in the propanol extracts of S.coronaria, but no marked quantity of

essential oil was detected or the amount of the essential oil was very little. CONCLUSION: All the extracts showed varying degrees of antimicrobial activity on the microorganisms tested. Some of these plants were more effective than traditional antibiotics to combat the pathogenic microorganisms studied. On the basis of the present finding, S.coronaria and S.denizliensis leaves and branches possess the capabilities of being a good candidate in the search for a natural antimicrobial agent against infections and/or diseases caused by B. cereus and S. aureus.

REFERENCES Adams, R.P., (1995). Identification of Essential Oil Components by Gas Chromatography/Masss Spectrometry. Allured Publishing: Carol Stream, IL. 15–26. Aytaç Z, (1998). A new species of Silene L. (Caryophyllaceae) from Anatolia (Turkey). Th aiszia Journal of Botan, 8: 7–11. Bajpai, V.K., S. Shukla and S.C. Kang, (2008). Chemical Composition and Antifungal Activity of Essential Oil and Various Extract of Silene armeria L. Bior Tech, 99: 8903–8908. Bauer, A.W., W.M.M. Kirby, J.C. Sherris and M. Turck, (1966). Antibiotic Susceptibility Testing by a Standardized Single Disc Method. Am. J. Clin. Pathol., 45: 493–496. Bradshaw, L.J., (1992). Laboratory Microbiology, 4th edition. Saunders College Publishing, Fort worth, Philadelphia, USA.

Borchardt, J.R., D.L. Wyse, C.C. Sheaffer, K.L. Kauppi, R.G. Fulcher, N.J. Ehlke, D.D. Biesboer and R.F. Bey, (2008). Antimicrobial Activity of Native and Naturalized Plants of Minnesota and Wisconsin. Journal of Medicinal Plants Research, 2(5) 98–110. Boubakr, S., Z. Hakemi, A. Belaskri, H. Benhassaini, (2015a). Phytochemical study and antioxidant activity of methanolic extracts and alkaloids of the leaves of pistachio Atlas (Pistacia atlantica). Global Journal of Medicinal Plant Research, 3(3) 11–21. Boubakr, S., A. Latrech, Z. Mehdadi, Z. Hakemi, M. Dadache, B. Ammar, (2015b). Floristic, Ethnobotanical and Phytotherapy Studies of Medicinal Plants Spontaneous in the Area of Mountains Tessala, Western Algeria. Global Journal of Medicinal Plant Research, 3(5) 1–16.

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Collins, C.H., P.M. Lyne and J.M. Grange, (1989. Microbiological Methods. 6thEdn., Butterworhs, London, p 410. Davis, P.H. (ed.), (1967). Flora of Turkey and East Aegean Islands, Vol. 2. pp. 192, 193, 231. Edinburgh: Edinburgh University Press. Davis, P.H., Mill, R.R. and Tan, K. (eds.) (1988). Flora of Turkey and East Aegean Islands (Supplement), Vol. 10. pp. 76–81. Edinburgh: Edinburgh University Press. Ertürk, O., H. Kat, N. Yayl and Z. Demirbag, (2006). Antimicrobial Properties of Silene multifida (Adams) Rohrb. Plant extracts. Turk J Biol, 30: 17–21 García, S. and N. Heredia, (2009). Foodborne Pathogens and Toxins: An Overview. In: Microbiologically Safe Foods. John Wiley & Sons Inc., New York, p32. Güner, A., N. Özhatay, T. Ekim and K.H.C. Bayer, (eds.) (2001). Flora of Turkey and the East Aegean Islands, (Supplement 2). Edinburgh: Edinburgh University Press. Kremp GOW (1968.) Morphologic Encyclopedia of Palynology. Tucson: The University of Arizona Press, p170. Khan, N.H., M.S.A. Nur-E, Kamal and M. Rahman, 1988. Antibacterial activity of Euphorbia thymifolia Linn. Ind. J. Med. Res., 87: 395–397. Mahesh, B. and S. Satish, (2008). Antimicrobial Activity of Some Important Medicinal Plant Against Plant and Human Pathogens. World Journal of Agricultural Sciences, 4: 839–843.

Source of Support: NIL

NCCLS, (2000). National commitee for clinical laboratory standarts.Performance Standarts for Antimicrobial Disc Suspectibility Tests, 7th edition. Approved Standart M2-A7 NCCLS, Pennsyvania, USA. Salek-Moghadam, A., H. Forouhesh Tehrani, N. Mozafari and H. Ansari, 2000. Prevalence of Virulence Factors among E.coli Isolated from Food Materials from Iran University of Medical Sciences food microbial laboratory. Kaums. J. FEYZ, 4: 32–40. Silva, N.C.C. and A. Fernandes Júnior, 2010. Biological Properties of Medicinal Plants: A Review of Their Antimicrobial Activity. The Journal of Venomous Animals and Toxins including Tropical Diseases, 16(3) 402– 413. Toroglu, S., (2007). In-vitro Antimicrobial Activity and Antagonistic Effect of Essential Oils from Plant Species. J. Environ. Biol., 289: 551–559. Toroglu, S., (2011). In-vitro Antimicrobial Activity and Synergistic/Antagonistic Effect of Interactions Between Antibiotics and Some Spice Essential Oils. J. Environ. Biol., 32: 23–29. Wesley, I., (2009). Public Health Impact of Foodborne Illness: Impetus for the International Food Safety Effort.In: Garcia JS. Microbiologically Safe Foods. John Wiley & Sons Inc., New York, p83. Zendehdel, M. and V. Babapour, (2010). Study of Antinociceptive Effects of Ziziphora tenuior and Its Interference on Opioidergic and Serotoninergic Systems. J. Vet. Res. 65: 57–60. Conflict of Interest: None Declared

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Research article DIAGNOSTIC PARAMETERS ASSISTED WITH SUITABLE PHOTOMICROGRAPHS, PHARMACOGNOSTICAL AND PHYTOCHEMICAL STUDY OF ECLIPTA PROSTRATA (L.) L. Patnaik Sunita1*, Mishra D N2 1

Pratibha College of Commerce and Computer Studies, Pune Dist. (M.S.) 411019, India Sub-Centre (Swami Ramanand Teerth Marathwada University, Nanded), Ausa Road, Peth, Latur (M.S.) 413531, India *Corresponding Author: Email: suneeta.pattnaik@gmail.com 2

Received: 02/12/2016; Revised: 15/12/2016; Accepted: 31/12/2016

ABSTRACT Eclipta prostrata (L.) L. (Syn. Eclipta alba (Linn.) Hassk.) Known as Bhringaraj in Sanskrit is an important plant with diverse medicinal values. Traditional medicinal systems like Āyurveda as well as tribal practitioners of the countries in Indian subcontinent use this herb commonly for the treatment of gastrointestinal disorders, respiratory tract disorders (including asthma), fever, hair loss, graying of hair, liver disorders (including jaundice), skin disorders, spleen enlargement, cuts and wounds. But the expected therapeutic effect of it is attributed to genuine raw material for its quality parameters. Therefore, in the present study, a detailed pharmacognostical profile of Eclipta prostrata (L.) L. was attempted with a view to quantify the diagnostic parameters assisted with suitable photomicrographs. The pharmacognostical parameters studied, may be used as a tool for the correct identification of the plant and also to test the adulterants if any. KEYWORDS: Āyurveda

rin ara a , Eclipta prostrata (L.) L., pharmacognostical.

Cite this article: Patnaik Sunita*, Mishra D N (2016), DIAGNOSTIC PARAMETERS ASSISTED WITH SUITABLE PHOTOMICROGRAPHS, PHARMACOGNOSTICAL AND PHYTOCHEMICAL STUDY OF ECLIPTA PROSTRATA (L.) L., Global J Res. Med. Plants & Indigen. Med., Volume 5 (12): 292–299

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Global J Res. Med. Plants & Indigen. Med. | Volume 5, Issue 12 | December 2016 | 292–299

INTRODUCTION Plants produce wide ranges of secondary metabolites, making them a great source of various types of medicines. The WHO estimated that 65% of the world population still depends mainly on herbal and traditional remedies. India is one of the richest countries in the world blessed with huge diversity of medicinal plants, commonly used as herbal drugs (Rani J et al., 2015). Proper identification of the herbal drug depending on the descriptions of ancient literature and classical texts becomes difficult owing to different synonyms and vernacular names of the given to the plant (Payyappallimana U., 2008). There are no standardized descriptions of the crude drugs available in the classical Sanskrit medical literature. T e Āyurvedic system of medicine is also facing another major problem that is quality control of crude drugs due to adulterants (Gokarn Supriya R., Gokarn Rohit, 2014). For this, proper identification of the plant parts microscopically and morphologically is necessary. This can be achieved by Pharmacognostical study. A gross study of the plants with microscopical, external and internal morphology is now possible through pharmacognostical tools (Yadav P, Harisha CR & Prajapati PK, 2011). Eclipta prostrata (L.) L. is a herbaceous annual, 30–50cm high, erect or prostrate, much branched. It is a common weed of moist places found throughout India. It has high medicinal value and is well-known for its hepatoprotective activity (Kshirsagar AD et al., 2011; Patnaik S, Mishra DN, 2016). Significant and safe hepato-protective agents are unavailable in contemporary therapeutics. Therefore, due importance has been given globally to develop plant-based hepatoprotective drugs effective against a variety of liver disorders (Sapna Narasanagi, Sujnana VS, Jyoti Devangamath, Shreevathsa, 2015). This significant plant is also mentioned in important Pharmacopoeias (API, 2008; AFI, 2000). The major chemical composition of whole plant Eclipta prostrata (L.) L. contains Coumestan derivatives such as wedelolactone and dimethyl

wedelolactone (Jadhav et al., 2009) that makes the drug so valuable. ut present Āyurvedic Pharmacopoeia lacks photomicrographs of the descriptions provided to the plant or plant parts (API, 2008; AFI, 2000). Therefore, an attempt has been made to study and validate the findings with appropriate images along with preliminary phytochemical data for Eclipta prostrata (L.) L. MATERIALS AND METHODS Collection and Identification of Plant material The plant Eclipta prostrata (L.) L., was collected from Sunrise Agro Services, Wakad, Pune during the month of August, 2016. The Plant’s identification and aut entication was confirmed by Botanical Survey of India, Pune with Voucher No. 455. The specimen was kept for future reference at BSI, Pune. Pharmacognostic Studies The whole plant material was shade dried for more than 15 days and the dried material was coarsely powdered mechanically with the help of a grinder, passed through sieve no.44 and stored in an air tight container for further use. The anatomical features of fresh stem, root, and leaf material were studied by taking transverse hand sections and staining procedure (Evans, 2002). Microscopic characters, physicochemical parameters and phytochemical analysis were carried out using the powder extracts (Evans, 2002; Yadav P, Harisha CR & Prajapati PK, 2011) Organoleptic Analysis Various parameters such as colour, odour, taste, size, shape, texture of the powdered parts were studied with the help of various sensory organs like eye, nose, skin (Evans, 2002; Yadav, Harisha, Prajapati, 2011). Microscopic Analysis Microscopic characters were studied by taking hand sections from the fresh plant material and stained according to the standard

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Global J Res. Med. Plants & Indigen. Med. | Volume 5, Issue 12 | December 2016 | 292–299

procedures. (Evans, 2002; Yadav, Harisha, Prajapati, 2011). The Photomicrographs are taken with the help of scientific camera. Physicochemical Analysis Percentage of total ash value, acid insoluble ash, water soluble ash, and total percentage of sulphated ash were investigated (Evans, 2002; Yadav, Harisha, Prajapati, 2011). Fluorescence Analysis The powder was subjected to freshly prepared chemical reagents and tested after 24 hours to study the fluorescence behavior by exposing to visible light and UV light (short and long wavelength) (Evans, 2002; Yadav, Harisha, Prajapati, 2011). Extraction and Phytochemical Analysis Powdered material was subjected to Cold Extraction procedure. 10 g of powdered whole plant material was taken successively with 100 ml of different solvents (Petroleum ether, Fig. 1(a) Aerial part of plant E. prostrata (L.) L

Chloroform, Acetone, Ethanol, Methanol, and water) extracted for 48–72 hours. Preliminary phytochemical analysis was carried out according to the standard procedure (Evans, 2002; Yadav, Harisha, Prajapati, 2011). RESULTS Macroscopic Characters The morphological studies revealed the information about size and shape of the aerial parts of Eclipta prostrata (L.) L. Leaves are sessile, lanceolate, entire and were 3–5 cm in length and 2–3 cm wide. (Fig 1.b) Stem is cylindrical with longitudinal ridges, brownish in color and shows thickness of 0.2–0.3 cm. (Fig 1.a). Root is well developed, a number of secondary branches arise from main root up to 7 mm in diameter, cylindrical, grayish. Flower is solitary or 2, together on unequal axillary peduncles, involucral bracts, ovate, herbaceous, strigose with oppressed hairs, ray flowers ligulate as in (Fig 1.c). Fig. 1(b) Shows small sessile leaves of E. prostrata (L.) L

Fig. 1(c) shows flower with involucral bracts

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Organoleptic Analysis Organoleptic analysis is one of the parameters to identify crude drug macroscopically by its colour, odour, taste and texture by the sensory organs as mentioned below (Table 1). The organoleptic evaluation of the herb powder revealed that herb powder was dark green in color, with characteristic odour and bitter taste. Microscopic Characters Powder is dark green, shows vessels in large groups or broken pieces with pitted walls, trichomes present. Root cells of outer

secondary cortex are rounded while inner ones are elongated to irregular. Phloem rays broader towards the periphery, cells rounded and Xylem rays are distinct, run straight in tangential section, cells pitted as in Fig. 2 (a). Fig. 2 (c) shows stem with few epidermal cells elongated to form trichomes, secondary cortex composed of large cells having wide air spaces and collateral, endarch vascular bundle seen. Fig. 2(b) and (d) shows anomocytic and anisocytic stomata and non-glandular hairs are present on both surfaces, more abundant on lower side. Stomatal index 16.0–20.0 on upper and 21.5–25.0 on lower surface of the leaf.

Table 1: Organoleptic Analysis Colour Odour Taste Texture

Dark Green Characteristic Bitter Smooth

Fig 2 (a) T.S of Root Under 10x eyepiece and 10x objective lens Fig 2 (c) T.S of Stem under 5x eyepiece and 10x objective lens

Fig 2 (b) and Fig 2 (d) T.S of Leaf showing stomata under 10x eyepiece and 40x objective lens

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Table 2: Physicochemical Analysis Sr no.

Parameters

Values (w/w%)

1 2 3 4 5

Moisture Content Total Ash Acid Insoluble Ash Water Souble Ash Sulphated Ash

2.48% 14.1% 1.7% 34% 3.9%

Table 3: Fluorescence Analysis Sr no. Test with reagent

Sunlight

U.V. at 254 nm

U. V. at 365 nm Green

Powder

Dark Green

Light Green

Powder + 1 N NaOH

Dark Brown

Yellowish Brown Dark Green

Powder +50% HCL

White

Green

Light Green

Powder + 50% H2SO4

Black

Brown

Black

Powder + Petroleum ether Light Green

Dark Green

Green

6 7 8 9 10

Powder + Acetic Acid Powder + Methanol Powder + Chloroform Powder + Iodine Powder + 5% FeCl3

Yellowish Brown Yellowish Brown Green Yellowish Black Brown

Light Green Light Green Dark Green Black Dark Green

Light Brown Greenish Yellow Light Green Greenish Black Green

Physicochemical Analysis Moisture content, ash values, extractive values and determination of Sulphated ash are as mentioned below (Table 2). Fluorescence Analysis Powdered material of dried whole plant was tested with different chemical reagents and its fluorescence behavior with different wavelength of light is mentioned below (Table 3). This study helps in identifying some particular colored compounds through its fluorescence nature. Preliminary Phytochemical Analysis The dried powder was successfully extracted with various solvents by Cold

Extraction method and tested for various phytochemicals as presented in the Table 4. DISCUSSION The present study reveals and confirms that Eclipta prostrata (L.) L. has the presence of phytochemical constituents like alkaloids, flavonoids, carbohydrates, proteins, saponins, tannins, steroids as the phytochemical constituents (Table 4). Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms. They often have pharmacological effects and are used as medications. Most phytochemicals have antioxidant activity and protect our cells against oxidative damage and reduce the risk of developing certain types of cancer (Mishra, 2014). Bioassay-based fractionations of the

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extracts and purification of anti-Hepatitis–C Virus phytochemicals present in the active fractions have identified three compounds, wedelolactone, luteolin, and apigenin (Manvar et al, 2012). The standardized Eclipta alba

extract or its isolates can be used as an effective alternative and complementary treatment against Hepatitis–C Virus (Manvar et al, 2012).

Table 4: Preliminary Phytochemical Analysis, where + sign means presence and – sign means absence of corresponding components. Sr no. Phytochemical Petroleum ether Benzene Chloroform Methanol Ethanol Water Alkaloids − − − + + + 1 2 3 4 5 6 7 8 9 10 11

Tannins Flavonoids Proteins Carbohydrates Steroids Glycosides Phenols Saponins Terpenoids Anthroquinone

+ − + + + − + + + −

− − + + + − + + + −

− + − + − − − − + −

+ + + + + + + + + −

− + + + + − + + + −

Fig. 3 Structure of Phytoconstituents of Eclipta prostrata (L.) L.

Source Reference: (Jahan et al., 2014:1-22)

The plant has been reported to exhibit hepatoprotective action on subcellular levels of functional markers, in inflammation and liver injury (Jadhav V M, Thorat R M, Kadam and V J, Salaskar K P , 2009) . There has been clinical studies conducted that prove the effectiveness of E.alba therapy in jaundice in children (Jadhav V M, Thorat R M, Kadam and V J, Salaskar K P , 2009). Table 4 shows, for ethanol extract, all tests were positive except anthraquinones, which was absent in E. alba. In chloroform extract, the following tests were positive, fehling (solution B), terpenoids,

flavonoids and glycosides. All other tests were negative. In benzene extract, test for reducing sugars, terpenoids and saponins were positive and all other tests were negative. In petroleum ether extract, tests for anthraquinones, alkaloids, flavonoids and glycosides were negative and the rest were found positive. In water extract, test for anthraquinones, tannins and glycosides were found negative and the rest were positive. Our results showed in correlation with Nivedita and Vijay Priyanka, 2013 but in their result, chloroform extract shows only presence of Flavonoids and Terpenoids

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whereas in our result, Carbohydrates is also present. Different phytochemicals have been found to possess a wide range of activities, which may help in protection against chronic diseases. For example, alkaloids protect against chronic diseases. Saponins protect against hypercholesterolemia and antibiotic properties. Steroids and triterpenoids show the analgesic properties whereas steroids and saponins were responsible for central nervous system activities (Nivedita and Vijay Priyanka, 2013). CONCLUSION The present study places this indigenous herbal drug Eclipta prostrata (L.) L., a novel candidate for bioprospection and drug development for the treatment of liver disorders, where satisfactory cure managements are still not available. Correct identification of

this plant and its parts can be easily done by using the present results of pharmacognostic parameters and photomicrographs, which are not otherwise available in literature and adulterants if any in the marketed plant material can be traced out. Efforts are in progress to characterize the efficacy of this valuable medicinal plant. ACKNOWLEDGEMENTS Authors are very thankful to COCSIT Management for allowing to work in their lab at Latur and also thankful to the Principal and Management of Pratibha College of Commerce and Computer studies, Chinchwad, Pune for giving administrative support. We also thank BSI, Pune for authenticating and identifying the plant.

REFERENCES Anonymous (2008), The Ayurvedic Pharmacopoeia of India (API), Part I, Volume I to VI, Ministry of Health & Family Welfare, Government of India, New Delhi. Anonymous (2000), The Ayurvedic Formulary of India (AFI), Part B - Formulary Of Single Drugs, Ministry of Health & Family Welfare, Government Of India New Delhi. Evans W C (2002),. Trease and Evans Pharmacognosy, 15th ed, Saunders Ltd., London. Gokarn Supriya R., Gokarn Rohit (2014), “Problems faced in Ayurvedic Drug Research” Journal od Indian system of Medicine, 2(2), pp 100–103. Jadhav V M, Thorat R M, Kadam and V J, Salaskar K P (2009) “C emical composition, pharmacological activities of Eclipta alba” Journal of P armacy Research, 2(8), pp 1229–1231.

Jahan

Rownak, Al-Nahain Abdullah, Majumder Snehali, Rahmatullah Mohammed (2014), “Et nop armacolo ical si nificance of Eclipta alba (L.) Hassk. (Asteraceae)” International Scholarly Research Notices, Article ID 385969, pp 1–22.

Kadam Prasad V., Yadav Kavita N., Navasare Vidya S., Bhilwade Sumeet K., Patil Manohar J (2012) “Eclipta alba: A P ytop armaco nostic Study” International Journal of Pharmaceutical and Phytopharmacological Research, 1(6), pp 350–353. Kshirsagar A D, Mohite R, Aggrawal A S, Suralakar U R (2011), “Hepatoprotective medicinal plants of ayurveda” Asian Journal of Pharmaceutical and Clinical Research, 4(3), pp 1–8.

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Manvar Dinesh, Mishra Mahesh, Kumar Suriender, Pandey Virendra N. (2012), “Identification and evaluation of antihepatitis C virus phytochemicals from Eclipta alba” Journal of Ethnopharmacol, 144(3), pp 545–554.

Patnaik

Mishra Priyanka, Preya Jamdar, Sharav Desai, Dhara Patel and Dhananjay Meshram (2014) “P ytoc emical analysis and assessment of in vitro antibacterial activity of Tinospora cordifolia” International Journal of Current Microbiology and Applied Sciences, 3(3), pp 224–234.

Rani Jyoti, Singh Lalita, Singh Hosihar, Kapoor Manish, and Singh Gagandeep (2015) “Preliminary p ytoc emical analysis of different solvent extracts from leaf and stem of Tinospora cordifolia” International Journal of Phytotherapy, 5(3), pp 124–128.

Nivedita and Vijay Priyanka (2013), “P ysioc emical and p ytoc emical analysis of Eclipta alba” International Journal of Pharma and Bio Sciences, 4(3), pp 882-889. Payyappallimana U. (2008), Ayurvedic pharmacopoeia databases in the context of the revitalization of traditional medicine. In: Wujastyk D, Smith FM, editors. Modern and Global Ayurveda, Pluralism and Paradigms. Albany: SUNY (State University of New York) Press.

Source of Support: NIL

Sapna

Sunita, Mishra D N (2016), “Importance of botanical identities of plants quoted for the treatment of liver cirrhosis in , International Journal of Ayurvedic and Herbal Medicine, 6(3), pp 2233–2247.

Narasanagi, Sujnana VS, Jyoti Devangamath, Shreevathsa (2015), “Hepatoprotective property of varnya mahakashaya-An Innovative Insight”, Global Journal of Research on Medicinal Plants & Indigenous Medicine, 4(8), pp 172–181.

Yadav Pramod, Harisha C R, Prajapati P K (2011) “Validation of p armacopoeial characters of Bhringaraja (Eclipta alba Linn. Hassk.)” Journal of Current Pharmaceutical Research, 8(1), pp 17– 24.

Conflict of Interest: None Declared

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Research article PHARMACOGNOSTICAL VARIATIONS IN MARKET SAMPLES OF TINOSPORA SPECIES AND THEIR EXTRACTS Seetha Chandran1*, Harisha CR2, BJ Pagiri3 1

Phd Scholar, Dept. of Rasasastra and Bhaishajya Kalpana, I.P.G.T and R.A., GAU, Jamnagar, Gujarat, India 2 Head, Pharmacognosy, I.P.G.T and R.A., GAU, Jamnagar, Gujarat, India 3 Professor and HOD, Dept. of Rasasastra and Bhaishajya Kalpana, I.P.G.T and R.A., GAU, Jamnagar, Gujarat, India *Corresponding author: Email: seethaac@gmail.com; Mobile: +91 9496830565

Received: 09/11/2016; Revised: 27/12/2016; Accepted: 31/12/2016

ABSTRACT The Genus Tinospora belongs to Menispermaceae family, are usually climbing shrubs. There are mainly three species of Tinospora which are commonly available and used for medicinal purpose in India. They are commonly known as “Giloe” or “Guduchi” with significant medicinal importance in Ayurveda. Pharmacognosy, phytochemistry and medicinal property of Tinospora species depends on various factors like geographical location, season, samskara (processing techniques) etc. The safety and efficacy of herbal medicines are closely correlated with the quality of the source materials and the Samskara. In the current study, the market samples of two species of Guduchi collected from western and southern parts of India were evaluated pharmacognostically in three form of samples i.e., fresh stem, dried powder, and Ghana to study the action of Desha and Samskara. The study revealed that transverse section and powder of T. malabarica have abundant mucilage and brown content and less starch grains when compared to T. cordifolia. Simultaneously ghana sample of Tinospora showed entirely different characters from its powder form. KEY WORDS: Guduchi, Ghana, Menispermaceae, Pharmacognosy, Tinospora

Cite this article: Seetha Chandran*, Harisha CR, BJ Pagiri (2016), PHARMACOGNOSTICAL VARIATIONS IN MARKET SAMPLES OF TINOSPORA SPECIES AND THEIR EXTRACTS, Global J Res. Med. Plants & Indigen. Med., Volume 5 (12): 300–312

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INTRODUCTION Tinospora species of plants are climbing shrubs, which belongs to Menispermaceae family. About 60 species are distributed in tropical and subtropical regions of the world. Of these five species are indigenous to India (Nidhi et al., 2013). There are mainly three species which are commonly available and used for medicinal purpose in India: Tinospora cordifolia (Willd.) Miers, Tinospora malabarica (Lam.) Hook.f. & Thomson. / Tinospora sinensis (Lour) and Tinospora crispa (L.) Hook.f. & Thomson. (Nidhi et al., 2013). These three species are closely related within their morphology & chemical properties of stem, bark, leaves, flowers, etc. (Nidhi et al.,2013). This drug commonly known as Guduchi, (Syn: Chakralakshanika or Amruta) is an important drug of Indian medicine (Badani et al., 2015). T. malabarica is commonly seen in southern parts of India. The plant is used almost in the same way as T. cordifolia (Chadha et al., 2003). In northern and western part both T. cordifolia and T. crispa are widely seen. Meaning of word Guduchi in Sanskrit is ‘one which protects the body against diseases’. It is designated as Rasayana drug in Ayurveda and recommended for a number of diseases and also as adaptogen and immunomodulator (Choudhry et al., 2014). Many Ayurvedic pharmacies of south India are not uniformly using T. cordifolia for medicine preparations. T. malabarica though considered as an adulterant, it is used injudiciously in some industries of south India, considering its therapeutic efficacy. Till date no studies have been reported on the influence of geographical location and processing techniques on Tinospora species. Also the T. malabarica variety should be explored at all levels especially it’s clinical efficacy. So this study was aimed at exploring more about both the species. The drug is used in the form of Swarasa (fresh juice), kwatha (decoction), Vati (tablet), Choorna (powder), Sattva (extract) etc after certain samskara (processing techniques) in many diseases (Badani et al., 2015). Area of cultivation, stem thickness, season of collection, maturity of plant, etc affects the

morphological, microscopical and medicinal properties of Tinospora varieties. Keeping above into consideration, in the present study, the macro-microscopic characters of market samples of Guduchi collected from southern and western parts of India, were analysed pharmacognostically. Along with this the yield and macro-microscopical characters of ghana (extract) from these two varieties were also analysed and compared. These two methods, helped to evaluate the influence of region and samskara on Tinospora species. MATERIALS AND METHODS Material collection: Moderately thick stem of fresh Guduchi with uniform maturity were collected from kozhinjampara market of Palakkad district, Kerala (South India) and Darbar ghat (local market) of Jamnagar district, Gujarat (west India) in winter season (January) of 2016. Authentication: Pharmacognostical evaluation Anatomy of collected market samples was identified and authenticated by the pharamacognosist in the Institute of Postgraduate Teaching and Research in Ayurveda (IPGT & RA), Gujarat Ayurveda University (GAU), Jamnagar, Gujarat, India (Joshi. M. C, 2007). Samples of T. cordifolia and T. malabarica were assigned voucher no. PHM6206/15–16 and, PhM 6207/15–16, respectively. Among this market sample collected from Kerala was of T. malabarica variety and that collected from Gujarat was of T. cordifolia variety. The initial purpose of study was to confirm the authenticity and analyse the pharmacognostical difference between both samples. Another aim was to evaluate the pharmacognostical changes after preparing extract of the drug. So at first, section of fresh Guduchi stem was studied and compared, then the dried powder and ghana (extract) of both samples were subjected to organoleptic and microscopic evaluation separately at

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Pharmacognosy laboratory of IPGT & RA, GAU, Gujarat, India. Preparation of Guduchi Ghana Ghana is a semisolid extract of decoction. In the current study, Ghana (extract) was prepared from T. malabarica and T. cordifolia varieties of Guduchi. Fresh Guduchi stem was made into small pieces and kept overnight, soaked in water. Next morning it was subjected to heating. Boiling was continued till the quantity was reduced to 1/4th part. Then it was filtered through a single fold cotton cloth. This filtrate was collected as Guduchi Kwatha. It was further kept on mild flame. Continuous stirring was done till the Kwatha was converted into semi solid form (Acharya, Y. T., 1988). The product was collected in a glass tray and kept in oven at 50Â°C for three days, then it was dried under sunlight. When the moisture content of the samples got reduced, it was scraped and preserved in airtight container. Preparation of Powder: Stem of Guduchi was dried under sunlight. After that it was made in to small pieces. Then powdering was done with the help of a mixture and subjected to sieving. (Acharya, Sharngadhara., 2002).

OBSERVATION AND RESULTS Pharmacognostical evaluation: Morphology Tinospora cordifolia It has succulent stems, the bark was papery at first then corky with greyish brown colour, warty, deeply left spirally, the space in between was spotted with lenticels. Leaves were simple, alternate, estipulate, cordate, long petioles, roundish, and pulvinate. (Plate1:1) Tinospora malabarica Stem was, smooth, shining with light coloured, papery bark more or less worty. Leaves were membranous broadly ovate, cordate, acuminate, pubescent above, whitish tomentose beneath; petioles were Long, thickened & twisted at the base. (Plate1:2). Organoleptic Features: As per Ayurvedic Pharmacology, Guduchi has Katu, Tikta, Kashaya rasa, ushna veerya and Madura vipaka. The colour, odour, taste etc. of powder and Ghana of collected samples were analysed and are placed in table 1.

Plate 1: Morphology of T. Cordifolia & T. malabarica Western

Southern

Tinospora cordifolia (Willd.) Miers

Tinospora malabarica (Lam.) Hook.f.& Thomson.

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Table: 3.0 Organoleptic evaluation of Fresh stem & Dried powders & Ghana of both Tinospora species Features

Color Odour Taste Touch Sound

Fresh stem T.malabarica Creamy white Characteristic Slightly bitter

Dried powder T.cordifolia T.malabarica T.cordifolia Greyish brown Cream Ash brown

smooth

Characteristic Bitter

Ghana T.malabarica T.cordifolia Blackish Coffee Brown brown Characteristic Characteristic Characteristic Characteristic Slightly bitter Bitter Less bitter Bitter and strong Astringent astringent Fine Coarse Coarse Fine Coarse Slightly crispy Slightly crispy

Microscopic Evaluation Transverse section of Guduchi Stem Tinospora cordifolia: T.S. of fresh Guduchi of western region shows that outer cork is made up of 20–30 layered tangentially elongated cells with brown contents. Cortex was differentiated into outer 2–3 layered collenchymatous hypodermis and inner 3–4 layered parenchymatous cells, loaded with simple starch grains. Mucilage cavities were small and lesser in number. Cortex was followed by 8–10 layered lignified sclerenchymatous fibres forming arch like structure. Vascular bundle was open and collateral. Phloem situated above the xylem, was made up of phloem fibres and sieve elements. Xylem was wedge shaped, largely covered with xylem parenchyma and its fibres. Xylem bundles were separated by narrow and 7–8 layered multiseriate medullary rays, heavily loaded by the simple starch grains, and this gave spokes of wheel appearance. Central reduced pith was made up of parenchyma cells, loaded with simple starch grains (Plate 2.W1– W5). Tinospora malabarica: In the T.S. of fresh Guduchi stem of southern region outer cork was made up of 15– 20 layered tangentially elongated cells with brown contents. Cortex was differentiated into outer 1–2 layered collenchymatous cells and inner 5–6 layered parenchymatous cells, loaded with simple starch grains. Large numbers of mucilage cavities were distributed all over the

cortical zone. Cortex was followed by 6–7 layered lignified sclerenchymatous fibres, forming arch like structure. Vascular bundle was open and collateral. Phloem situated above the xylem, was made up of phloem fibres and sieve elements. Xylem was wedge shaped, covered with xylem parenchyma and its fibres. Xylem bundles were separated by broad and 6– 7 layered, multiseriate medullary rays, loaded with simple starch grains. Central reduced pith was made up of lignified parenchyma cells loaded with few starch grains (plate 2.S1–S5). Guduchi stem dried powder microscopy The mucilage content and brown content were more in T. malabarica compared to cordifolia variety where as less number of starch grains were found in T. malabarica. (Plate 3. W1–W9 & S1–S9). Guduchi Ghana microscopy The yield of Ghana from T. cordifolia variety was 2.3 % whereas yield from T. malabarica variety was 3.6%. In both the varieties, the channels of cell wall structure appear disturbed. Also the cellullar constituents were open and secondary metabolites were distributed more or less as such in raw form. Collenchyma structure was deformated and completely softened. The mucilage content were more in T. malabarica compared to T. cordifolia, but brown content were not observed in T. malabarica variety. Abundant simple starch grains were found in T. malabarica compared to T. cordifolia variety (Plate 4. W1–W5 & S1–S5).

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Plate 2. Fresh T.S of Guduchi stem T.Cordifolia

W1:Cork, cortex, medullary rays

T.Malabarica

S1:Cork, cortex, medullary rays

W2:Xylem, medullary rays

S2:Xylem, Pholem

W3:Medullary rays filled with ample starch grains

S3:Medullary rays filled with starch grains

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W4:Xylem medullar rays

S4:Xylem medullar rays

W5: Lignified xylem, xylem parenchyma and its fibre with pith

S5:Lignified xylem, xylem parenchyma and its fibre with pith

Plate 3. Guduchi powder microscopy T.cordifolia T.malabarica

Dried powder of stem of Guduchi

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W1.Fragment of lignified border pitted vessel

S1. Fragment of lignified border pitted vessel

W2.Iodine stained stained starch grains

S2. Iodine stained stained starch grains

W3. Oil globule with starch grains

S.3 Oil globule with starch grains

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W.4. Collenchyma cells

S.4 Collenchyma cells

W5. Brown content

S5. Brown content

W6. Lignified collenchyma cells

S6. Lignified collenchyma cells

W7. Lignified cork cells

S7. Lignified cork cells

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W8. Prismatic crystal of Calcium oxalate

S.8.Prismatic crystal of Calcium oxalate

W9. Simple fibre

S9. Simple fibre

Plate 4. Guduchi Ghana microscopy T. cordifolia

T.malabarica

W1: Powder of Ghana

W2:Clumped powder of Ghana

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W2:Soften and deformated collenchyma cells

S2:Soften and deformated collenchyma cells

W3:Brown content

W4: Fragment of fibre

S3:Fragment of fibre

W5:Simple strach grain

S4:Simple starch grains in ample amount

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DISCUSSION In Ayurveda, safety and quality of medicinal plants is based upon major factors like desha (habitat), kala (season) and Guna (properties) (Ranade et al., 2015). Period of accumulation, nature, and quality of metabolism in the plants also varies with such factors. Seasonal study indicates the best harvesting season which may be either winter or summer to obtain optimal yield of phytoconstituents of T. cordifolia for its best antioxidant potential and immunomodulator activity (Pradhan et al., 2013). In Ayurveda, the starch obtained from Guduchi stem is known as Guduchi Satva and is a well known drug (Pradhan et al., 2013). Moderately thick stem of Guduchi collected in a winter season (January) was proved to yield maximum starch. (Choudhry et al., 2014). So the drug was collected in winter season. Also properties of a medicinal plant vary depending upon the geographical location, dosage form etc. Samskara helps in preparing various dosage forms. Here the influence of samskara is assessed by analysing the extract prepared from both species. And effect of geographical location is assessed by analysing the herb collected from two different zones of India. Macroscopic features Stem of fresh T. malabarica (southern variety) was found to be more greenish compared to the T. cordifolia (western variety) which is having an ash coloured cork. The leaves of the T. malabarica were smooth, pubescent and small when compared to T. cordifolia. Lenticels, nodes and internodes were more in T. cordifolia but mucilage content was more in T. malabarica. Due to the environmental factors and species variations the characters differed in two species. Transverse section of stem Mucilage cavities and brown content were comparatively poor in T. cordifolia. Starch content was comparatively greater in T. cordifolia where the cells of medullary rays and those of cortex are abundantly packed with

starch grains. Reduced central pith and unlignified parenchyma cells loaded with simple starch grains were found in cordifolia whereas reduced central pith with lignified parenchyma containing less starch grains were seen in T. malabarica. The cork cells, mucilage and starch grains showed a major variation in two species mainly due to the difference in species and ecological factors. Powder The mucilage and brown content were considerably greater in T. malabarica. And the brown colour of powder of T. malabarica variety can be attributed to the presence of more brown content in it. Number of starch grains was noticeably less in T. malabarica. Ghana Yield of ghana from T. malabarica variety was comparatively more. And it’s colour was blackish brown due to saturation of brown content and mucilage in ghana. Because of the same reason brown content was not visible under microscope in T. malabarica variety. Also another major finding in Ghana is T. malabarica variety showed abundant starch grains compared to T. cordifolia, different from that of the T.S and powder microscopy findings. This may be because, the starch grains which were compacted in excessive mucilage content of T. malabarica variety might have released from the cellular constituents during heating process of Ghana preparation. As Guḍūci Sattva was observed to have hygroscopic nature, it readily gains moisture if kept exposed to environment, which in turn may promote microbial growth in it (Sharma et al., 2013). So it was stored in dry, sterile and airtight containers. Still ghana of T. malabarica variety captures environmental moisture and turned semisolid due to the presence of profuse starch and mucilage cells. Because, presence of starch and mucilage imparts cohesiveness (Sijo et al., 2013). In microscopy of Ghana form, the channels of cell wall structure appeared disturbed. Secondary

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metabolites were dissolved and uniformly distributed. This will increase the potency and efficacy of the finished product. During preparation of Ghana, Guduchi was subjected to the procedure of making decoction and further boiling till it became semisolid. These procedures are called as Toya sannikarsha (mixing with water) and Agni sannikarsha (preparing with fire) Samskara. Agni (fire) is said to be Ushna (hot), Tikshna (penetrating) and Sukshma (subtle). Due to Sukshma guna it enters minute channels and with Ushna and Tikshna it changes the shape which reflects as disturbed structure. Cell wall were seemed about to burst due to heavy fluid content. This is due to Toya Sannikarsha. According to the Ayurvedic principle, water produces looseness. Loosened tissue seems to be disturbed structurally. So the observed changes in structure can be understood with the help of principle of Gunantaradhan (change in attributes) (Badani, P et al., 2016). From this it is crystal clear that samskara (processing technique) will definitely cause changes in the property of a herb. CONCLUSION The study has revealed pharmacognostical variations between T. cordifolia and T. malabarica species collected from western and southern parts of India respectively. Also pharmacognostical difference was found in different forms (fresh stem, dried powder, and extract) of both species. Above two

observations reveals the impact of desha and samskara, respectively. Though more amount of mucilage and brown content were seen in T.S and powder form of T. malabarica variety, it was not visible in the microscopy of ghana. At the same time, there was abundant starch grains in its ghana compared to its T.S and powder microscopy findings and also to the ghana prepared from T. cordifolia sample. The process of speciation occurs due to various factors like geographical variation and seasonal variation. But it doesnâ&#x20AC;&#x2122;t means that only a particular species is having therapeutic efficacy and all others are adulterants. Very few studies have been conducted on the pharmacognosy of different Tinospora species. Also studies based on the geographical origin of various species and their comparative pharmacognosy should be done. Besides, therapeutic efficacy of these varieties has to be explored by conducting clinical trial. Through this we can also reduce the scarcity of herbal drugs in the field of Ayurveda to an extent and make it more beneficial to public health. ACKNOWLEDGEMENT: Authors acknowledge Dr. Vidya Unnikrishnan, Dr. Rahul shinagdiya, Dr.Harmeet kaur, Dr. Ankesh, Dr.K.P.Sajithkumar, Dr.N.Shemna, Mr.Sivanandhan, Mr. Aswin babulal Joshi for their needful actions at every stage of this work.

REFERENCES Acharya Sharnghadhara. (2002) Churna kalpana adhyaya- 1/1, Sharnghadhara samhitha, Varanasi: Choukampa surabharati prakashan. p. 355 Acharya, Y. T. (1988). Jwaradhikar-1/6, (11th ed.), Siddha Yoga Sangraha,. Nagpur: Baidhanath Ayurved Bhavan Ltd. p.4

Badani, P., Vyas, H., Harisha, C. R; Shukla, V.J. (2015). Impact of Samskara on Guduchi and Guduchi Ghanavati , I J Ayu Pharm Chem, [online] .volume 4(3). Available at:http://www. ijapc.com/volume4-thirdissue.shtml[Accessed 10 Nov.2016]

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Global J Res. Med. Plants & Indigen. Med. | Volume 5, Issue 12 | December 2016 | 300–312

Choudhry, N., Singh,S., Siddique, M.B. and Khatoon S . (2014), Impact of Seasons and Dioecy on Therapeutic Phytoconstituents of Tinospora cordifolia, a Rasayana Drug, BioMed Research International, 2014 (2014) Article ID 902138, 11 pages, Retrieved from https://www.hindawi.com/journals/bmri /2014/902138/[Accessed 10 Oct.2016] Nidhi, P., Swathi, P., R. Krishnamurthy. (2013). Indian tinospora species: natural immunomodulators and therapeutic agents. International journal of pharmaceutical, biological and chemical science (IJPBCS) 2(2) p. 2. Retrieved from http://www. jpbcs.net/view.php?iid=261 [Accessed 10 Nov.2016] Pradhan. Deepa. and Pandey. A. K. (2013) Estimation of starch extracted from different diameter thickness stem of Giloe (Tinospora cordifolia Willd Miers) , Journal of Pharmacognosy and photochemistry (JPP), 2 (4) pp. 162163. Retrieved from http://www. www.phytojournal.com/vol2Issue4/Issu e_nov_2013/37.pdf/ [Accessed 12 Oct.2016] Ranade. Anagha. Vishwas., Acharya. (2015) Influence of time factor on phytoconstituents in certain Ayurvedic medicinal plants: A comprehensive review, Journal of pharmaceutical and

Source of Support: NIL

scientific innovation, 4 (5) Retrieved from http://www. Jpsionline.com [Accessed 10 Nov.2016] Sharma. Rohith., Amin Hethal., Galib., Prajapati, P.K (2013) Validation of standard manufacturing procedure of Guḍūcī sattva (aqueous extract of Tinospora cordifolia (Willd.) Miers) and its tablets, Anc Sci Life 33 (1): 2734 doi: 10.4103/0257-7941.134564, Retrieved from https://www.ncbi.nlm.nih.gov/pmc/artic les/PMC4140018/ [Accessed 29 Dec.2016] Sijo. Davis, K., Sivakumar, R., Sajeeth, C, I. and Hari. B, Y., (2013) Evaluation of New Binder Isolated from Tinospora cordifolia for the Preparation of Paracetamol Tablets, Research Journal of Pharmaceutical, Biological and Chemical Sciences (RJPBCS), Volume 4(2)p:1184 Available at:http://www. rjpbcs.com/pdf/2013_4(2)/[128].pdf, [Accessed 29 Dec.2016] Joshi.M. C, (2007), Hand book of Indian medicinal plants, Jodhpur: scientific publishers of India, pp:3,4 Y.R. Chadha (Ed.). (2003), The wealth of India- Raw material series, Vol.X: spW, New Delhi, National institute of science communication and information resource Council of scientific and industrial research (CSIR)

Conflict of Interest: None Declared

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