ISSN 2277-4289 www.gjrmi.com International, Peer reviewed, Open access, Monthly Online Journal Research article DIAGNOSTIC PARAMETERS ASSISTED WITH SUITABLE PHOTOMICROGRAPHS, PHARMACOGNOSTICAL AND PHYTOCHEMICAL STUDY OF ECLIPTA PROSTRATA (L.) L. Patnaik Sunita 1 *, Mishra D N 2 1 Pratibha College of Commerce and Computer Studies, Pune Dist. (M.S.) 411019, India 2 Sub-Centre (Swami Ramanand Teerth Marathwada University, Nanded), Ausa Road, Peth, Latur (M.S.) 413531, India *Corresponding Author: Email: suneeta.pattnaik@gmail.com ABSTRACT Received: 02/12/2016; Revised: 15/12/2016; Accepted: 31/12/2016 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 r in 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
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
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
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 Dark Green Odour Characteristic Taste Bitter Texture 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
Table 2: Physicochemical Analysis Sr no. Parameters Values (w/w%) 1 Moisture Content 2.48% 2 Total Ash 14.1% 3 Acid Insoluble Ash 1.7% 4 Water Souble Ash 34% 5 Sulphated Ash 3.9% Table 3: Fluorescence Analysis Sr no. Test with reagent Sunlight U.V. at 254 nm U. V. at 365 nm 1 Powder Dark Green Light Green Green 2 Powder + 1 N NaOH Dark Brown Yellowish Brown Dark Green 3 Powder +50% HCL White Green Light Green 4 Powder + 50% H 2 SO 4 Black Brown Black 5 Powder + Petroleum ether Light Green Dark Green Green 6 Powder + Acetic Acid Light Brown Yellowish Brown Light Green 7 Powder + Methanol Greenish Yellow Yellowish Brown Light Green 8 Powder + Chloroform Light Green Green Dark Green 9 Powder + Iodine Greenish Black Yellowish Black Black 10 Powder + 5% FeCl 3 Green Brown Dark 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
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 1 Alkaloids + + + 2 Tannins + + + 3 Flavonoids + + + + 4 Proteins + + + + + 5 Carbohydrates + + + + + + 6 Steroids + + + + + 7 Glycosides + + 8 Phenols + + + + + 9 Saponins + + + + + 10 Terpenoids + + + + + + 11 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
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, 15 th 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.
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. 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. 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. Patnaik 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. 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. Sapna 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. Source of Support: NIL Conflict of Interest: None Declared