Vijay Thirugnanasambandan, IJMPR, 2016, 4(3) ): 134-138 CODEN (USA): IJMPMW ISSN: 2321-2624 International Journal of Medicine and Pharmaceutical Research Journal Home Page: www.pharmaresearchlibrary.com/ijmpr Research Article Open Access Phytochemical Studies of Grewia umbelliferrea in Different Solvent Fractions Vijay Thirugnanasambandan* Department of Biochemistry, Faculty of medicine, Ibn Sina National college for Medical studies, Gulail, Jeddah 21418, KSA A B S T R A C T The present study attempt to evaluate the phytochemical studies of Grewia umbelliferrea stem crude drug. The phytochemical screening was carried out and the berberine alkaloid was quantified in different fractions of extract by HPLC. The result shows that the methanol extracts have higher concentration of berberine when compared to other solvent fractions. The present study reveals the standardization profile and characterization of berberine compound from Grewia umbelliferrea, which would be of immense value in botanical identification and authentication of plant drug and may help us in preventing its adulteration. Keywords: Grewia umbelliferrea, berberine, phytochemical, standardization, characterization, HPLC. A R T I C L E I N F O CONTENTS 1. Introduction................................................................................ 134 2. Materials and Methods........................................................................135 3. Results and discussion........................................................................135 4. Acknowledgement........................................................................... 137 5. Conclusion..................................................................................137 6. References.................................................................................137 Article History: Received 28 February 2016, Accepted 31 April 2016, Available Online 10 June 2016 *Corresponding Author Vijay Thirugnanasambandan Department of Biochemistry, Faculty of medicine, Ibn Sina National College for Medical studies, Gulail, Jeddah 21418, KSA Manuscript ID: IJMPR2989 PAPER-QR CODE Citation: Vijay Thirugnanasambandan, et al. Phytochemical Studies of Grewia umbelliferrea in Different Solvent Fractions. Int. J. Med. Pharm. Res., 2016, 4(3): 134-138. Copyright 2016 Vijay Thirugnanasambandan, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. 1. Introduction The use of plant as medicine is an civilization. Herbal medicines have being old as human used by billions of people around the world for thousands of years. Herbal medicine is very popular in different system of medicines like Indian system of medicine, Chinese system of International Journal of Medicine and Pharmaceutical Research medicine, the most known traditional system such as Ayurveda, Siddha, Unani and Homeopathy were being practiced all over the world and are going demand there years [1]. In recent years, interest on plant-based drugs has increased considerably. According to an estimate, around 134
80% of the world population still depends on herbal products from about 20,000 plants species, for their basic medicinal and health care requirement. This is emphasized with the toxicity and side effects of the prescribed allopathic medicines and lack of drugs for many chronic ailments [2]. It demands for an urgent need to investigate new herbals leads to overcome the needs. Annual growth rate between 5 15% for trade of plant based drugs and raw materials is indicative of growing demand for herbal drugs. But the quality control and quality assurance still remains a challenge because of the high variability of chemical compounds involved. The major problem of quality assurance of herbal medicine has been solved to a great extent with the help of chromatographic and spectral finger prints analysis [3]. Grewia umbelliferrea is one such plant, which is widely used in indigenous system of medicine [4]. It is a large, glabrous, succulent, deciduous climbing shrub belonging to the family menispermaceae[5]. It is distributed throughout tropical India subcontinent, Sri Lanka and china, ascending to an altitude of 1200m. The stem of Grewia umbelliferrea is rather succulent with long filiform fleshy aerial roots from the branches. The bark is creamy white to grey, deeply left rosette like lenticels. The leaves are membranous and cordate. The flowers are small and yellow or greenish yellow [6]. Grewia umbelliferea (Tiliaceae) (GU) is herbaceous medicinal plant that has been distributed in Kanniya kumari district, Tamilnadu, India. The aerial plant part extract is much more useful in treatment like spleen damage, liver complications and cardio disorders. [7]. The large numbers of compounds have been isolated from the aerial parts and roots of Grewia umbelliferrea. GU is widely used in Ayurvedic system of medicine Rasayanas to the immune system and the body resistance against infections. In modern medicine Grewia umbelliferrea used for the treatment of general weakness, fever, dyspepsia, dysentery, gonorrhoea, urinary diseases, viral hepatitis and anaemia more recently, the immunomodulatory properties, antioxidant activity, anti-neoplastic, hypoglycemic activities have been reported [8,9]. The aim of the present research work was standardization of physical and chemical analysis and characterization of berberine from Grewia umbelliferrea stem extract in different solvent fractions. 2. Materials and Methods Plant materials: The stem part of Grewia umbelliferrea was collected fresh from Tirunelveli District area in Tamilnadu. The plant stem was authentified by the Herbarium of Botany Directorate in National Institute of Herbal Science, Plant Anatomy Research Center, Chennai. A voucher specimen (No: TC08) was deposited in the center. Preparation of plant extract: The dried powdered stem of Grewia umbelliferrea was allowed to pass through SS sieve (20 mesh). It was defatted by treating with petroleum ether (60 80oC) and then extracted to exhaustion (soxhlet) with various solvents like methanol, water, chloroform and the excess solvent was removed under vacuum to get the solid mass. Chemical materials: Acetonitrile, methanol, chloroform, petroleum ether, and other chemicals were used of AR grade (Merck, Darmstadt, Germany) and standard berberine from Sigma- Aldrich, Steinheim, Germany. Standardization: Physicochemical parameters were determined as per guidelines of WHO. Total ash, acid insoluble ash, watersoluble extractive value, alcohol soluble extractive value, heavy metals were determined by using standard procedures [10-12]. Microbial contamination screening: For the safe use of the drug, microbial count, total yeast, and mould count are within the prescribed WHO limits by analysis through standard procedure [13]. Phytochemical studies: Phytochemical screening of Grewia umbelliferrea stem different solvent extract fractions was done for the presence of various phyto constituents by using standard procedure [14-17]. HPLC- conditions [18-20]: Injection volume- 20µl; Flow rate 0.5ml/ min; Mobile phase Acetonitril: water (60:40); Detection wave length 265 nm; Mode isocratic; Retention time (Rt) berberine 5.15 min. 3. Results and Discussion The parameters of powdered Grewia umbelliferrea stem like Total ash, acid insoluble ash, water soluble extractive value, alcohol soluble extractive value, heavy metals and microbial contamination was carried out and there result are shown in Table 1. The deterioration time of plant material depends upon the amount of water present in plant materials. If the water content is high, the plant can be easily deteriorated due to fungus. The loss on drying at 105 C in stem was found to be 3.31%. Total ash value of plant material indicated the amount of minerals and earthy materials attached to the plant material. Analytical results showed total ash value content was 17.6%. The negligible amount of acid insoluble siliceous matter present in the plant was 1.16%. The water soluble extractive value indicates the presence of sugar, acid and inorganic compounds. The alcohol soluble extractive value indicates the presence of polar constituents like phenol, alkaloids, steroids, glycosides, and flavonoids and other compounds the result given in Table 2. The various different extract fractions of Grewia umbelliferrea stem phytochemical testes result indicate the presence of wide range of phytochemical contents. Alkaloids are heterocyclic indole compounds, which have proved to be having pharmacological properties [21]. The presence of flavonoids and tannins in all the plants likely to be responsible for the free radical scavenging effects observed. Flavonoids and tannins are phenolic compounds and plant phenolics are a major group of compounds that are as primary antioxidant or free radical scavengers [22]. Flavonoids show anti allergic, anti-inflammatory, anticancer activity [23]. The present HPLC method was conducted to identifying and quantifying the berberine from Grewia umbelliferrea medicinal plants stem various different extract. Berberine peaks from solutions of various extract like pet- ether; methanol, aqueous and chloroform were identified by comparing their Rt values with these International Journal of Medicine and Pharmaceutical Research 135
obtained by chromatography of the standard under the same conditions. The peaks of Rt 5.15min was observed in the chromatograms obtained from fractions like pet- ether, methanol, aqueous and chloroform, the chromatograms of standards and test samples are shown in Fig. 1 to 5 respectively. The berberine content of fractions of sample I (pet- ether), sample II (methanol), sample III (aqueous), sample IV (chlo roform) was 0.002% (W/W), 0.223% (W/W), 0.150% (W/W), 0.020% (W/W) respectively. The more amount of berberine was present in methanol extract when compared to other fractions. Table 1: Physico-chemical parameters of Grewia umbelliferrea stem Parameters Results Description Light brown to dark brown powder Loss on drying at 105 C (%W/W) 3.31 Total ash content (%W/W) 17.6 Acid insoluble ash (%W/W) 1.06 Water soluble extractive values (%W/W) 27.84 Alcohol soluble extractive values (%W/W) 35.76 Heavy metals Mercury 0.0153 ppm Lead 0.3186 ppm Cadmium 0.0347 ppm Arsenic 0.0180 ppm Microbiological analysis Test for E.coli/g Test for Salmonella/g Test for Shigella.sp/g Test for Entero bacterial sp. 102 org g-1 Total Heterotrophic bacteria 112 x 104 Test for Yeast and Mould 19x101 Table 2: Phytochemical Screening for different solvent extract of Grewia umbelliferrea stem Phytoconstituents Pet ether Methanol Aqueous Chloroform Alkaloids + ++ ++ + Carbohydrates - + + - Glycosides + ++ ++ - Protein & Amino acids - + ++ - Fixed oils & Fats - + - + Tannins - ++ - + Saponins - + + - Steroids (Phyto sterols) + ++ - - Flavonoids - + + - Phenols + ++ + + Key: ++ means abundant; + Indicates presence; - indicates absence; Figure 1: HPLC chromatogram of standard berberine. Figure 2: HPLC chromatogram of petroleum-ether extract Berberine peak at the Rt 5.15 min. detected at a wavelength of Grewia umbelliferrea peak at the Rt 5.15min. correspond of 265 nm. to berberine detected at a wavelength of 265 nm. International Journal of Medicine and Pharmaceutical Research 136
Figure 3: HPLC chromatogram of methanol extract of Grewia umbelliferrea peak at the Rt 5.15min. correspond to berberine detected at a wavelength of 265 nm Figure 4: HPLC chromatogram of aqueous extract of Grewia umbelliferrea peak at the Rt 5.15 min. correspond to berberine detected at a wavelength of 265nm. products of traditional medicine containing Grewia umbelliferrea as an ingredient can be explored. 5. Acknowledgements We would like to express our great thanks and sincere appreciation to Dr. Rashad Kashgari, the Dean of the Ibn sina National College, Jeddah, KSA for his valuable support to the Research Center. 6. References [1] V. Venkatesh, Medicinal plant Scenario in Indian foundation for Revitalization of Local Health Traditions, Bangalore, India, 2002. [2] N.K. Dubey, R. Kumar & P. Tripathi., Current science. 2004, 86, 37. [3] Y.Z. Liang, P. Xie & K.Chan., J. chromatography B., 2004, 812, 53. [4] K.R. Kiritikar & B.D. Basu. Indian medicinal plants. Volume I. Lalit Mohan Publishers, India., 1933, 77. [5] Anonymous. Wealth of India, Raw materials, Volume X, New Delhi, CSIR., 1976. [6] K.M. Nadkarni & A.K. Nadkarni, Indian materia medica, Vol- 1, 3rd ed., M/S. popular prakasan Pvt. Ltd, Mumbai., 1976. [7] V.V. Sivarajan & I. Balachandean, Ayurvedic Drugs and their plants sources, Oxford and IBH publishing Co. Pvt. Ltd, Delhi., 1999. [8] S.S. Singh, S.C. Pandey, S. Srivastava, K.S. Gupta, B. Patro & A.C. Ghosh, Indian.J. Pharmacol,2003, 35, 83. [9] T. Vijay, M.S. Dhana Rajan, K. Sarumathy, S.Palani, K. Sakthivel. Analysis of Phytoconstituents by GC-MS and Therapeutic efficacy of Grewia umbelliferea (GU) on Doxorubicin induced cardiotoxicity in albino rats. IJPI S Journal of Pharmacology and Toxicology. 2011; 1:3: 61-74 [10] Indian Pharmacopoeia, Physical and Determination, The Controller of publications, Govt. of India, New Delhi., 1996, A85. [11] J.B. Harborne, Phytochemical methods: A Guide to Modern Techniques of Plant Analysis, 2nd ed., 1984. Figure 5: HPLC chromatogram of chloroform extract [12] AOAC Official methods of analysis of AOAC of Grewia umbelliferrea peak at the Rt 5.15 min. International, 16th edition, AOAC International, correspond to berberine detected at a wavelength of 265nm. Suite 400, 2200 Wilson Boulevard, Arlington, 4. Conclusion Virginia, USA, 1995. [13] World Health Organization: Quality control It has been concluded from the present study that physicochemical methods for medicinal plant materials, Published parameters, toxic heavy metals, microbial by WHO, Geneva. 1998. contamination should be within the limits as per WHO [14] J.B. Harborne, Phytochemical Methods: A guide guidelines and is highly essential for raw drugs or plant to modern techniques of plant analysis, Chapman parts used for the preparation of compound formulation and Hall, New York, 1973, 279. drugs. The periodical assessment is essential for quality [15] C. K. Kolkate, Pharmacohnosy, 16th edition, assurance and safer use of herbal drugs. Phytochemical Nirali prakasham, Mumbai, India. 2001. screening tests useful in the detection of the bioactive [16] G.E. Trease & W.C. Evans, Pharmacognosy, principles and subsequently may lead to the drug discovery 13th ed., ELBS Oxford University Press, London, and development. The HPLC estimation of berberine is U.K., 1989, 245. used for standardization and quality control of herbal International Journal of Medicine and Pharmaceutical Research 137
[17] Indian Pharmacopoeia, Chemical tests and Assays, The Controller of publications, Govt. of India, New Delhi., 1996, A34. [18] U. Samyal, S. Bhattacharya, A. Patra & B. Hazra., J.Chromatogr. A., 1017, 225. [19] J.C. Touzhstone., Practice of Thin Layer Chromatography, USA, John Wiley and Sons., 1992. [20] J. Sherma & B. Fried, Handbook of Thin Layer Chromatography, Chromatography Science Series, New York: Marcel Dekker., 1996. [21] P B. Mallikharjuna, L N. Rajanna, Y N. Seetharam & Sharanabasappa G., E-Journal of Chemistry, 2007, 4, 510. [22] T. Vijay, M.S. Dhana Rajan, K. Sarumathy, A. Sudha. In-vitro cytotoxicity of Grewia umbellifera International Journal of Pharmacy and Life Sciences 2(12); 2011: 1293-1298 [23] Yamato and Gayor., Journal of clinical investigation, 2002, 493-503. International Journal of Medicine and Pharmaceutical Research 138