Serco ISSN : 0974-0376 Volume - IV She Gcoscan An International Quarterly Journal of Environmental Sciences Pt SUMO c econ NECN EIB OUCH weCecleCo Sey ; p> @ SOs Cen nate een YY ‘, FE 3 | Ey BA 8 croreoa renin 2 fle Genes _ i a] PHYTOCHEMICAL AND CHROMATOGRAPHIC STUDIES IN THE STEM BARK EXTRACT OF FICUS RECEMOSA (L) V. Rajendra Chary and Estari Mamidala KEYWORDS Ficus racemosa Phytochemicals ne Plant extracts R, Retention factor, Proceedings of International Conference on Harmony with Nature in Context of Ecotechnological Intervention and Climate Changees BP ShaGeses {B \V. RAJENDRA CHARY AND ESTAR! MAMIDALA* Department of Zoology. Kakativa University, Warangal - 506 009,Andhra Pradesh, INDIA E-mail: estarimamidala@gmail.com, RCSL Tes ‘To separate the bioactive compounds from ‘crude hexane extract, chloroform extract, ethyl ‘acetate extract, acetone extract and methanol ‘extracts of Ficus recemosa stem bark. Prelimi- rary phytochemical screening so as to detect the major class of bio active compounds present in Ficus recemosa by Phytochemical analysis was performed by various qualitative ‘methods and TLC profiling was carried out for those plant extracts using hexane : ethyl acetate (1:1, 2:1, 3:1, 9:1 and 2-3 drops of CH,COOH was added to each solvent sys- tem) as mobile phase. The qualitative phy- tochemical analysis shows the presence of al= INTRODUCTION Ficus racemosa Linn is commonly known as cluster fig and udumbara (Sanskrit) belongs to the family Moraceae, is an evergreen, moderate sized, deciduous tree and used as herbal medicine from ancient times. Its leaves are dark green, ovate and having traditional medicinal importance (Abu Hasant et al., 2011). This is native to Australia, South East Asia and the Indian subcontinent. Itis unusual in this plant that its figs grow on or close tothe tree trunk Joseph et al, 2010). Itis one of the herbs mentioned in all ancient scriptures of Ayurveda. It has various synonyms like yajnanga, yajniya, yainayoga, yajnyasara etc. suggesting its use in ritual sactifice. The plant grows all over India in many forests and hills. It is frequently found ‘around the water streams and is also cultivated (Joseph et a., 2010). In the traditional system of medicine, the plant is used for various health problems and diseases laloids, carbohydrates, glycosides, saponins, tannins, phenols, flavonoids, quinones, ste- roids, amino acids and terpenoids in various extracts. In TLC profiling maximum eight bands were observed in methanolic extract of Ficus recemosa stem bark with Rf value be- ‘tween 0.14-0.87 and 10 bands in acetone ex- ‘tract with RF value between 0.08 -0.96. Ficus recemosa showed presence of so many phytochemicals play an important role when used as herbal medicine. The application of (Paarakh et al., 2009), (Chopra et al,, 1992). Ficus recemosa bark is acrid, cooling, galactagogue and good for gynaecological disorders. The stem bark is used to treat menorthagia, leucorthoea, gonorthoea, urinary diseases, hemorthage and skin diseases (Patil etal, 2009). Ficus recemosa., a widely distributed genus in North East India and abundant in Warangal district, Andhra Pradesh having used by warangal peoples to reduce diarthea and stop bleeding along with some other ethno medicinal practices (Rajendra Chary and Mamidala, 2012) So, Itwas the need of time to explore some of the species ofthis genus specially Ficus recemosa which is available in Southern part of Andhra Pradesh, India, for better upgradation of knowledge regarding the investigated plant species in various drugs was ‘based on their phytochemical content.1LC profiling of all extracts gives an idea about the presence of various phytochemicals. Differ- tent Rf (Retention factor) value of various phytochemicals provide valuable clue regard- ing their polarity and selection of solvents for separation of phytochemicals, values phytochemicals of this genus. Therefore, the aim of this work is to carry out a phytochemical screening of stem bark extracts of Ficus recemosa L. in order to know the composition of secondary metabolites. MATERIALS AND METHODS Plant Material Based on ethno botanical investigation and literature surveys, the Ficus recemosa plant was selected for study. Ficus recemosa stem bark was collected from rural areas of Venkatapuram forest, Warangal Dist, AP, India. The taxonomic identities Of these plants were confirmed by Department of Botany, Kakatiya University and Warangal, India. the collected fresh stem bark of plant free from diseases was bought to laboratory in sterile polyethylene bags and washed thoroughly 2-3 times with running tap water and then shade dried for three weeks, subsequently ground into fine powder using mechanical grinder and motor driven grinding mill, The powder was used for extraction of crude extracts. Preparation of plant extracts ‘Around 2kg of stem bark of Ficus recemosa was powdered by using of electric, grinder to obtain coarse powdered which is best sulted for extraction. The powder was weighed and plant powder material was extracted successively with maceration method. In this method using solvents ranging from nonpolar to polar i.e. hexane, enechloroform, ethyl acetate, acetone and methanol for 24h.The ‘extract was concentrated under vacuum using rotary vacuum evaporator. The obtained extracts were stored at 4°C till to se for phytochemical investigation. Preliminary Phytochemical analysis Preliminary phytochemical screening of the extracts for alkaloids, carbohydrates, glycosides, saponins, tannins, phenols, flavonoids, quinones, steroids, amino acids and terpenoids using standard phytochemical screening methods (Harborne et al., 2009, Siddiqui et al., 1997 and Earnsworth et al, 1974). Thin Layer Chromatography TLC was carried out to identify the components that were present in most effective extracts of plant. TLC studies were carried out for different extracts on Silica gel plates which are commercially available. The different solvent systems of diferent polarities were prepared and TLC studies were caried ‘out to select the solvent system capable of showing better resolution. The four different solvent systems Hexame (H): Ethyl Acetate (EA) = 1:1, H: EA=2:1 and H: EA=3:1 H:EA=9:1 ‘were used. The above prepared plant extracts were applied on pre-coated TLC plates by using capillary tubes and developed in a TLC chamber using above mobile phase. The developed TLC plates were air dried at room temperature. In each case, the spots were visualized by exposure of the plates, to iodine vapour. Different bands were observed and corresponding R, values were determined. R, value of each spot was calculated as:- R,= Distance Travelled by the Solute / Distance Travelled by the Solvent PHTOCHEMICAL AND CHROMATOGRAPHIC STUDIES RSS TT Yield of Extracts of Ficus recemosa stem bark The yield of sequential extracts (g) is shown in [Table 1]. The 500 grms of the plant material used for extraction with each solvent and the amount of the hexane extract obtained from the extraction was (7.3) (1.46 % w/w yield), chloroform extract, was 6.6g(1.32% w/w yield, ethyl acetate extract 5.38 (1,06 % wiw yield), acetone extract 2.9 g (0.58 % w/w yield) and methanol extract was 47.6g (9.52 % w/w yield). Phytochemical Screening of Ficus recemosa stems bark Phytochemical screening of the sequential extract of Ficus recemosa stem bark revealed the presence of various bioactive ‘components of which phenolics, saponins, steroids, alkaloids, flavonols, , terpenoids, tannin, and cardiac glycosides are the most prominent components and the result of phytochemical tests presented in [Table 2}. Among these phytochemical tests, methanol extract was found to contain maximum saponin content along with plant phenolics such as alkaloids, glycosides and tannins. Chromatographic profile of Ficus recemosa stem bark extractions This has been presented in Fig. 1, Fig, 2, Fig. 3 and Fig. 4. MUN aT There is an abundant medicinal plants throughout the world but only small amounts areinvestigated for its biological activity (Awadh Ali et al,, 2001). Ficus recemosa plant has medicinal value like treatment of skin problems, bronchitis, dysentery, Table 1: Barack yield Hexane Chloroform Ethylacetate ‘Acetone Methanol Powder (@ 500 500 500 500 500 Yield (@) 73 66 53 29 476 Yield hw) (%) 1.46 132 1.06 058 9.52 Table 2: Qualitative phytochemical screening of Sequential Extraction of Ficus recemosa stem bark Phytochemicals Test conducted Extracts Hexane Chloroform __Ethylacetate Acetone ___Methanol Alkaloids Mayer's test a : = ss - Hager’ test tte +ee aa ee +44 Phenols Lead acetate test ate : : oe +t Ferric chloride test ++ ++ +44 Terpenoids Test for Terpenoids-1 c + i + + Test for Terpenoids-2 + + + + s Carhohydeates Fehling'stest tet tet tee ++ Sis Benedict’s test tte +ee +e hide aes Starch lodine test tte tte tHe a ate Glycosides Borntrager’s test ra +t tte 44+ 44+ Brown fing test 4te en tee +ee +44 Flavonoids Alkaline test +44 tet + + + Quinones, Test for Quinones +e +e ++ ++ + Tannins Test for Tannins P : : + + Saponins Saponification test ++ 5 + + ate Steroids Test for Steroids + e + + + Proteins and Amino acids Miillon’s test : : : Biuret test : : a Nin hydrin test : zs\V RAJENDRA CHARY AND ESTARI MAMIDALA Table 3: R, values of sequential extracts of Ficus recemosa stem bark S\No. Solventsystem Extract No. of | Ryaluel Ryvaluell___-RValuelll__RValuelV_R,valueVRvalueVI_R, valueVil Hexane:éthyl spots acetate detected Ton W 2 O53 042 - 5 = | c 2 0.92 ort ; ‘ : : | cy 3 0.89 073 0.60 - . . | A 2 089 014 : 2 : : M 4 0.86 0.80 0.64 0.08 : d 2 ana H 3 089 031 ot : : : © 3 0.83 0.50 0.26 : : : cy 4 0.89 0.82 071 01 : E A G os2 0.89 oz 0.62 0.50 0.23 - M 4 0.61 0.50 0.30 og : : : 3 aaa H 1 0.17 - : : : : a 3 071 032 0116 : : : : A 3 0.85 0.78 0.64 x s fs : A 7 0.96 089 0.80 or 044 0.33 os M 4 0.87 0.70 058 08 a ’ 5 4 aoa H a 0.93 0.50, : : : : : c 1 04s : 5 ss : : : A 2 0.69 0.26 s : . m A 5 0.93 0.86 0.58 043 032 : : M 3 075 0.62 0.22 i 3 5 Scarcetor Raia mouth wash, dysentery, menorthagia, effective remedy in glandular swelling, abcess, chronic wounds, cervical adenitis, haemorthoids, boils alleviates the edema in adenitis, parotitis, orchitis, traumatic swelling, toothache, vaginal disorders, diarrhoea particular in childrens and also aphrodisiac. Latex is applied externally on chronic infected wounds to alleviate edema, pain and to promote the healing, treating piles, root sap is used for treating diabetes, treatment of skeletal fracture, treat constipation, anaemia and haemoptysis in spongy gum(Shiksharthi and Mittal, 2011) and easily available. 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. The steroids and saponins were responsible for central nervous system activities. (Kubmarawa et al., 2007). It has been known that plant extracts which contain phenolic and flavonoid compounds have antioxidant Figure 1, figure 2, figure 3 and figure 4 are showing that mobile phase of all solvent extract ifferent solvent systems and antibacterial effects (Da Silva et al, 2006 and Pereira ct al,, 2007). Previous study of the phytochemical analysis of ficus recemosa report as found that most of the biologically active phytochemicals were prsent in the ethanolic extract of Ficus racemosa bark (Poongothai et al., 2011). The results confirm the presence of constituents which are known to exhibit medicinal as well as physiological activities (Ismaila et al,, 2011). The results confirm the presence of constituents which are known to exhibit medicinal as well as physiological activities (\smaila et al, 2011). In this study TLC profiling ofall 5 extracts gives an impressive result that directing towards the presence of number of phytochemicals. Various phytochemicals gives different R , values in different solvent system (Das talukdar et al., 2010). This variation in R , values of the phytochemicals provides a very important clue in understanding of their polarity and also helps in selection of appropriate solvent system for separation of pure compounds by column chromatography. Mixture of solvents with variable polarity in diferent ratio can be used for separation of pure 30‘compound from plant extract. The results obtained in this study thus suggest that the identified phytochemical ‘compounds may be the bioactive constituents responsible for the efficacy of the stem bark of the plants studied (Ogu et al, 2012). Qualitative tests performed on the stem bark extracts of Ficus recemosa indicate the presence of alkaloids, carbohydrate, sglycosides, saponins, tanins, phenolicompounds, proteis, flavonoids, terpenoids, quinines, steroids indifferent extracts. TLC profiling also suggests the presence of diverse kinds of phytochemicals in all those stem bark extracts which could bbe useful substrate for the synthesis of new drug to treat the physiological disorders. Further purification, identification and charectorization of the active compounds would be our priority in the future studies. ACKNOWLEDGEMENT = ‘The authors wish to acknowledge the Department of Botany, Kakatiya University, Warangal for identification and preservation of the plant and also thank to traditional healers of my research area, REFERENCES LI Abu Hasant, Md., Zulfiker, M. S., Shammy, S. and laizuman, N, 2011. Hypoglycemic and Invitro antioxidant activity of Ethanolic leaf fextract of Ficus racemosa linn, Am. . Scientific and Industrial Res 2(3): 391-400, ‘Awadh Ali, N. A. Juelich, W. D., Kusnick, C. and Lindequist, U. 2001. Screening of Yemeni medicinal plants for antibacterial and ‘ytotoxic activities. J. Ethnopharmacol. 74: 173-179, ‘Chopra, R. N., Chopra, I. C. and Varma, B. S. 1992. Supplement to Glossary of indian Medicinal Plants, (reprinted edn), CSIR: New Delhi. pp, 29:30. ‘Das Talukdar, Dutta, C. M., Chakraborty, M. and Dutta, B. K. 2010. Phytochemical screening and TLC profiling of plant extracts of Cyathea gigantea (Wall. Ex. Hook.) Halt. and Cyathea brunoniana, Wall. ex. Hook. (Cl. and Bak), Assam University J. Science and Technology: Biological and Environmental Sciences. (0: 70-74, DasSilva, |. F. My DeSouza, M. C,, Matta, §. R., De-Andrade, M. R. and Vidal, F. V. N. 2006. Correlation analysis between phenolic FHAYTOCHENICAL AND CHROMATOGRAPHICSTUDIES levels of brazilian propolis extracts and their antimicrobial and antioxidant activities. Food Chemistry. 99: 431-435, Harborne, J.B. 2009. Phytochemical methods. A Guide 0 Modem Techniques of plant analysis, Chapmanand Hall, J, Sci. Res. 12) 393. 390, Ismaila, V.S., Denban, M. K.and Emmanuel, K.M. 2011, Nutritional and phytochemical screening of Senna obtusifolia indigenous to Mubi Nigeria. Advances in Applied Science Research. (3) 432-437. Joseph, B. and Raj, S. J. 2010. Phytopharmacological and phytochemical properties of three Ficus species - an overview Int. |. Pharma. Bio Sci. 1: 246-253, Kubmarawa. D.. Ajoku. G. A.. Enworem, N. M. and Okorie, D. A. 12007. Roles of agricultural biotechnology in ensuring adequate food security in developing societies. A. |. iotechnol.6: 1690-1696. Eamsworth, N.R., Berderka, J.P,, Moses, M. 1974, |, Pharma, Sci. 63: 457-459, Ogu, G. |, Tanimowo, W. O., Nwachukwu, P. U. and igere, BE 2012. Antimicrobial and phytochemical evaluation ofthe lea, stem bark and root extracts of Cyathula prostrata(L) Blume against some human pathogens. J. Intercult. Ethnopharmacol. 1): 35-43. Paarakh, P.M. 2009. Ficus racemosa Linn.-An overview. Nat. rod. Radiance. 8: 84-90, Patil, V. V., Pimprikar, R. B. and Sutar, N. G. 2009, Anti Hyperslycemic activity of Ficus racemosa Linn leaves. . Pharm. Res 2:54.57, Pereira, J. A. Olivera, I, Sousa, A., Valentao, P. and Andrade, P. 2007. Walnut uglans regia (.) leaves: Phenolic compounds. antibacterial activity and antioxidant potential of different cultivars. Food and Chemical Toxicology. 45: 2287-2295, Poongothai, A., Sreena, K. P., Sreejth, K., Uthiralingam, M. and ‘Annapoorani,S. 2011. Preliminary phytochemicals screening of Ficus racemosa linn. Bark, Int J. Pharma. Bio Sci. 2): 35-46, Rajendra Chary, V. and Mamidala, £. 2012. Ethnobotanical investigations among traditional healers in Warangal district of Andhra Pradesh, India, Phcog J. 4-34: 13-17. Shiksharthi, A. and Mittal, 8.2011. Ficus Racemosa: Phytochemistry, Traditional Uses and Pharmacological Properties: A Review. International J, Recent Advances in Pharmaceutical Research, 4: 6. \. A. and Ali, M. 1997, Practical Pharmaceutical Chemisty Ist Edition. 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