Phytochemical studies of Hybanthus enneaspermus Linn., F Muell.,

World Journal of Pharmaceutical Sciences ISSN (Print): 2321-3310; ISSN (Online): 2321-3086 Published by Atom and Cell Publishers All Rights Reserved Available online at: http://www.wjpsonline.org/ Original Article Phytochemical studies of Hybanthus enneaspermus Linn., F Muell., Baviya Sampath*, R. Radha, R. Vadivu Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai -600003, Tamilnadu, India ABSTRACT Received: 06-02-2016 / Revised: 22-02-2016 / Accepted: 25-02-2016 / Published: 28-02-2016 Hybanthus enneaspermus is a shrub with pink violet flowers belonging to family Violaceae. Plants belonging to Violaceae family contain volatile oils, anthocyanins, flavonoids importantly Rutin, carotenoid pigments and cyclotoxic cyclotides. Used as diuretic, to balance kapha and pitta, urinary calculi, dysentery, vomiting, wandering of the mind, blood troubles, asthma, The desire of the study is to understand the phytochemical nature of the entire plant. It was grind to coarse and successively extracted with solvents of increasing polarity in a soxhlet extractor, extracts were subjected to qualitative analysis, fluorescence analysis, quantitative analysis and chromatographic analysis to know the noteworthy of the plant and make it effectual for human kind in treatment of illness. Keywords: Hybanthus enneaspermus, Qualitative analysis, Quantitative analysis, Fluorescence analysis, Chromatographic analysis INTRODUCTION Though technology has taken up the world, people still rely on nature which has blessed human kind with lashings of medicinal plants for prevention and cure of disease. Hybanthus enneaspermus is a one such suffrutescent herb with pink purple flowers belonging to family violaceae. The entire plant is of medicinal value used in Ayurveda, Siddha and other traditional systems of medicine for curing various ailments. It consists of steroids, triterpenes, sugars, alkaloids, phenols, flavones, catechins, tannins, anthraquinones and amino acids. [1] Hybanthus enneaspermus is of great importance in traditional system of medicine. The plant is bitter and acrid; easily digested, removes kapha and pitta, urinary calculi, stangury, pain, dysentery, vomiting, burning, wandering of the mind, urethral discharge, blood troubles, asthma, epileptic fits, cures cough, gives tone to the breast, alexetric (Ayurveda). In south west Nigeria, Hybanthus enneaspermus locally known as Abiwere, is used by traditional birth attendants to make delivery less laborious. In Tamil it is called orilaithamarai or orithalthamarai, in Hindi it is called ratanpurus, in Sanskrit as amburuha, atichara, avyatha, charati, Padma, padmacharini, padmavati, padmavah, ramya, sthalapadmini, supushkara. In Ayurveda it is called [2] Sthalakamala. The study deals with the phytochemical screening of the entire plant of Hybanthus enneaspermus for phytoconstituents, fluorescent compounds, estimation of active constituents and chromatographic studies. MATERIALS AND METHODS Plant Collection: The fresh entire plants of Hybanthus enneaspermus Linn., (F) Muell. was collected from Irula tribal farm Thirukalzukundram. It was authenticated by Dr. Aravind M.D.(s), M.Sc., Medicinal plants, Assistant professor National Institute of Siddha (Government of India) Tambaram. A voucher specimen was deposited in the Department of Pharmacognosy, Madras Medical College for future reference. The plants were shade dried and coarsely powdered Preparation of extracts: The shade dried plant material was extracted in soxhlet extractor successively with solvents of increasing polarity like n-hexane, chloroform, ethyl acetate and ethanol. Each time before extracting with next solvent, the marc is dried. All the extracts were concentrated using rotary vacuum evaporator. The percentage yield was calculated for every extract in *Corresponding Author Address: Baviya Sampath, Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai -600003,Tamilnadu,India; E-mail: baviya235@gmail.com

terms of dried weight of plant material. The colour and consistency of the concentrated extract was also noted. Qualitative chemical tests [,3,4,5] : Plant powder and all the extracts were subjected to qualitative phytochemical analysis to identify the various phytoconstituents present in it, as per the standard procedures. Baviya et al., World J Pharm Sci 2016; 4(3): 440-447 using CAMAG Linomat 5 injector. The derivatizing agent is 10% anisaldehyde sulphuric acid. Plates were scanned at 400-800nm with tungsten lamp. The data obtained from scanning were brought in to integration through CAMAG software. Chromatographic finger print was developed for the detection of phytoconstituents present in the each extracts and R f values were tabulated. Fluorescence Analysis: Fluorescence analysis for the extracts was carried out and the powdered drug with various reagents was performed to identify the presence of chromophores. The importance of fluorescence analysis is that UV light shows the fluorescent nature of the compound where fluorescence cannot be observed in day light. Hence it is performed according to the standard procedures. Chromatographic Analysis [6] Thin layer chromatography: All the extracts were subjected to thin layer chromatographic studies using various solvent systems. Several mobile phases were tried for the separation of maximum components by trial and error method. The solvent system selected was ethyl acetate: formic acid:acetic acid:water(5:3:1:1), ethylacetate:nbutanol:ethanol:water (4:1:0.1:5) ethanol: Glacial acetic acid: formic acid:water(3:0.9:0.9:0.5). R f values were noted down for each selected extracts after elution by using different detecting agents such as Dragendroff s, Ninhydrin, Libberman Burchard, Con. Sulphuric acid & Ferric chloride. Estimation of phytoconstituents and flavonoids [7] : Quantitative estimation of phytoconstituents was done by HPLC method using validation of ambient extraction method. Different solvent systems were used for the separation of alkaloids, steroids, flavonoids and phenolic compounds. It is compared with the standard graph and quantified using standard formula. Estimation of heavy metal: Heavy metals in the sample was digested by wet digestion or dry digestion or high pressure microwave digestion and the amount of heavy metals were determined, i.e. arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg) by using graphite furnace atomic absorption spectrophotometer (GF-AAS) and flow injection analysis system -atomic absorption spectrophotometer (FIAS-AAS) and the values were compared with the WHO standards. HPTLC [8] : The extracts were dissolved in same solvent and 10µl quantity of sample applied on the HPTLC aluminium coated silica gel-merk F254 graded plate sized 6cm x 10cm as narrow bands RESULTS AND DISCUSSION The percentage of successive extractive values for the entire plant of Hybanthus enneaspermus Linn., (F) Muell. is tabulated in Table 1. Successive extractive values revealed the particulars regarding solubility and polarity of the metabolites in the plant powder. Percentage yield of various extracts were as follows: n-hexane (2.18%), chloroform (2.36%), ethyl acetate (1.4%) and ethanol (1.7%). Hexane and chloroform extract showed high extractive yield among other extracts. Qualitative preliminary phytochemical analysis was performed with different respective chemical detecting agent to detect the phytoconstituents and their presence in each extract and powder which is tabulated in table no.2. Hexane and chloroform extracts showed the presence of steroids, flavonoids, alkaloids. Ethyl acetate extract was found to contain flavonoids, alkaloids and carbohydrates. Ethanol and aqueous extracts showed the presence of carbohydrates, flavonoids, saponins and alkaloids. Fluorescence analysis of powdered drug and extracts were viewed in day light, short UV and long UV wavelengths. No fluorescence was observed for the powder as well as extracts revealed absence of chromophore in the plant which is tabulated in table no. 3 and 4. Estimation of phytoconstituents were done by HPLC equipped with 270nm detector with 4.6mm*25cm column and flow rate of 1.5ml per min., alkaloids, phenols, steroids and flavonoids were estimated and known to contain flavonoid Rutin in higher quantity. By comparing with standards and the values are tabulated in table no.5. Qualitative chromatographic analysis of these extracts was performed to separate and identify the phytoconstituents using thin layer chromatography. The hexane extract showed 2 spots R f values 0.78 0.76, chloroform extract showed one spot (0.64), whereas a spot found with ethyl acetate extract 0.60 and 4 spots were found in ethanol extract R f values (0.30, 0.61, 0.65, 0.73). TLC was performed for the identification of different components in the extracts qualitatively. HPTLC plate was scanned at 400-800nm with the best solvent to detect the maximum number of 441

components and peak abundance qualitatively and quantitatively at higher resolution which is presented in fig 1. HPTLC fingerprint is one of the versatile tools for qualitative and quantitative analysis of active constituents of multicomponent sample and also a diagnostic method to find out the adulterants to check purity. CONCLUSION Entire plant of Hybanthus enneaspermus was extracted with solvents of increasing polarity, Baviya et al., World J Pharm Sci 2016; 4(3): 440-447 subjected to fluorescence analysis with various reagents revealed absence of chromophore. The constituents were identified and estimated by qualitative and quantitative analysis by TLC, HPTLC and HPLC showed presence of active flavonoid constituents along with other phytoconstituents, complete phytochemical studies have been carried to bring out the importance of various phytochemical constituents present in the entire plant and their use in pharmacology and usefulness to human kind. S.NO Table no. 1 Successive solvent extraction and their yield METHOD OF PHYSICAL EXTRACT EXTRACTION COLOUR NATURE YIELD (%W/W) 1. Hexane Semisolid Dark green 2.18 Continuous Hot 2. Chloroform Semisolid Dark green percolation method using Soxhlet apparatus 3. Ethyl acetate Semisolid Dark green 2.36 1.46 4. Ethanol Semisolid Dark brown 1.7 5. Aqueous Cold maceration Semisolid Dark brown 1.5 Chemical constituents Powdered drug Table no. 2 Qualitative phytochemical analysis Hexane Extract Chloroform Extract Ethyl acetate Extract Ethanol Extract Steroids + + + + + + Glycosides - - - - - - Saponins + - - - + + Flavonoids + + + + + + Tannins + - - - - + Triterpenes + + + + + + Proteins - - - - - - Alkaloids + + + + + + Carbohydrates + + + + + + Terpenoids + - - - + + Fats and oils - - - - - - Water 442

Table no. 3 Fluorescence analysis substance Day light Short UV Long UV powder Pale green and brown black greenish brown Powder+ H2O Pale green and brown black greenish brown Powder +1NHCl Pale green and brown black Orange green Powder +1N HNO3 Brown black orange Powder + acetic acid Pale green Dark green Greenish yellow Powder +1N NaOH Pale green Black Pale green Powder +alc.naoh Pale green Black Pale green Powder +1N KOH Pale green black Pale green Powder +alc.koh Pale green black Pale green Powder+H2So4 black Black black Powder+NH4 Pale green Black green Powder+I2 Reddish brown Black brown Powder+FeCl3 Green Black Brown Powder+ethanol Pale green Black Greenish brown Table no. 4 Fluorescence analysis of extracts Extract Day light Short UV Long UV Hexane Dark green Black Dark green Chloroform Dark green Black Black Ethyl acetate Dark green Black Dark green Ethanol Dark brown Black brown Table no. 5 Estimation of phytoconstituents Constituents Hexane Chloroform Ethyl acetate Ethanol Alkaloids 0.36 0.56 1.45 0.98 Phenols 0.37 1.67 3.06 5.38 Steroids 0.43 0.39 0.39 0.44 Flavonoids Quercetin 0.101 0.021 0.046 0.145 Rutin 0.618 0.365 0.107 0.355 Gallic acid 0.201 0.314 0.462 0.355 Thymoquine Nil Nil 0.111 0.031 Gallangin Nil Nil 0.144 0.166 443

Fig no.1 HPLC of hexane extract of Hybanthus enneaspermus Fig no.2 HPLC of chloroform extract of Hybanthus enneaspermus Fig no.3 HPLC of ethyl acetate extract of Hybanthus enneaspermus Fig no.4 HPLC of ethanol extract of Hybanthus enneaspermus 444

Table no.6 Estimation of heavy metals Element Hexane Chloroform Ethyl acetate Ethanol Specification as per WHO Guidelines Mercury Arsenic Lead Cadmium Nil Nil Nil Nil Not more than 0.5ppm Nil Nil Nil Nil Not more than 5.0ppm 0.04 0.05 0.08 0.22 Not more than 10ppm Nil Nil Nil Nil Not more than 0.3ppm Fig.5 HPTLC analysis of all four extracts Fig.6 HPTLC of Hexane extract of Hybanthus enneaspermus 445

Fig.7 HPTLC of chloroform extract of Hybanthus enneaspermus Fig.8 HPTLC of ethyl acetate extract of Hybanthus enneaspermus 446

Fig.9 HPTLC of ethanol extract of Hybanthus enneaspermus Fig.10 HPTLC of all four extracts of Hybanthus enneaspermus REFERENCE [1] Kritikar K.R and Basu B.D, Indian Medicinal Plants. Vol, 1, 212-213. [2] Khare C, Indian Medicinal Plants an Illustrated Dictionary, Reprint 2007, 329. [3] J.B. Harborne. Phytochemical Methods. A guide to modern techniques of plant analysis, London, Chapman and Hall; 2nd ed, 1973, 4-34. [4] C.K. Kokate, Purohit, S.B. Gokahle. Pharmacognosy, Pune: Vallabh Prakashan, 24th ed, 2003,108-109. [5] V.E. Tyler, L.R. Brady, J.E. Robbers. Pharmacognosy, Philadelphia USA: Lea and Febrigorer, 9th ed,1998, 78. [6] Afsal unnisa, Suraj Kumar Sahoo, industrial Pharmacognosy, 2.40 2.43 [7] British pharmacopoeia-system simulation limited,7 th edition,2013:1828. [8] H. Wagner. Plant Drug Analysis. A thin Layer chromatography Atlas, Heidelberg: Springer-Verlag Belgium 2nd ed, 2002, 210. 447