Asian Journal of Biochemical and Pharmaceutical Research

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1 ISSN: CODEN (USA): AJBPAD Asian Journal of Biochemical and Pharmaceutical Research Research Article Pharmacognostical Studies on Stem & Fruit of Syzygium alternifolium (Wight) Walp., - An Endemic to South Eastern Ghats, India A. Sudhakar 1 *, C. Ramesh 1, N. Nagaraju 1, S. Vedavathy 1 & K. Sri Rama Murthy 2 1. Herbal Folklore Research Centre, Department of Botany, Sri Venkateswara Arts College, Tirupati - Andhra Pradesh, India 2. School of Conservation Biology and Plant Biotechnology, Department of Biotechnology, Montessori Mahila Kalasala, Vijayawada, A. P., India Received: 26 December 2011; Revised: 09 January 2012; Accepted: 19 January 2012 Abstract: Syzygium alternifolium (Wight) Walp. known in vernacular as Mogi is a moderate-sized deciduous endemic tree growing abundantly in Seshachalam Hill ranges of Southern Eastern Ghats, especially in Tirumala Hills. The stem and fruits are known to be used in the treatment of diabetes since long in the traditional system of medicine. The fruits are also used for duodenal and gastric ulcers. Since the plant is endemic and abundantly used in native medicine, the authors carried out pharmacognostic studies on its parts in order to find out adulterations, if any, which is presented here. Keywords: Syzygium alternifolium, stem, fruit, Pharmacognostical, Phytochemical studies INTRODUCTION: Herbal medicine is a triumph of popular therapeutic diversity. Plants above all other agents have been used for medicine from time immemorial because they have fitted the immediate personal need are easily accessible and inexpensive. Human population in countries around the world has been using plants from thousands of years for treating various ailments of humans and animals 1. Syzygium alternifolium (Wight) Walp. (Myrtaceae) is one of the dominant species on the hill plateau of Tirumala, is a medium sized tree up to 12 m tall, with slightly fissured, grayish bark. The leaves are dark green, shining above and measured x 7-9 cm. Berries are dark purple, globose and show considerable variation in size, shape and taste.the tree species is commonly found in Tirumala hills up to an altitude of 930 M. The stem and fruits have been used by various local tribes (Yanadi, Yerukula, Sugali) and rural folk for a long time in Rayalaseema districts (Chittoor, Kadapa, Kurnool and Anantapur). Fruits with seed powder along with water 3 times a day given after food as a remedy in controlling diabetes. Stem decoction is given to regulate blood sugar level. Fruit pulp is used in local tribes to treat bacillary dysentery. Fruit decoction (20 ml) is given orally once a day for a month to cure ulcers in the stomach. Except for some chemical and pharmacological investigations on the species, no references are available in the literature on its Pharmacognosy [2-7]. This traditional knowledge about the plants can be transferred to several generations only by proper documentation of their botanical, physicochemical, phytochemical characters and along with their medicinal uses in the form of monographs. The monograph of these plants are prepared according 127

2 to the WHO guidelines and presented as herbal pharmacopoeia. These guidelines enable to identify, authenticate, detect adulterants and standardize the plant material[1] Since the stem and fruit powder are sold in the various local drug markets, these were collected, identified and compared with genuine material for detecting adulteration if any and in view of the importance of the stem bark and the fruit in the indigenous system of medicine since long, a detailed Pharmacognostical studies on them were undertaken. MATERIALS AND METHODS: The fresh stem and fruits were collected from a healthy and well developed tree of Syzygium alternifolium (Wight) Walp, from Seshachalam hill range i.e. Tirumala, India in June 2002, when the flowering was in bloom. They were identified as stem and fruits of Syzygium alternifolium by comparing the morphological characters described in the literature[8]. voucher specimen number was given as AS : 159 and deposited at Herbal Folklore Research Centre, Department of Botany, Sri Venkateswara Arts College, Tirupati India for future reference. The stem and fruits were made free from aerial parts and thoroughly washed under running tap water to remove adherent dirt materials, then peeled and shade dried. Few samples of stem and fruit were stored in formalin aceto-alcohol solution and out of remaining; few kept as such i.e. entire form and others are powdered to #40 and stored in airtight glass containers. Transverse sections of fresh stem and fruit of Syzygium alternifolium were taken by microtome and free hand sectioning. Numerous temporary mounts of transverse sections were prepared using lactophenol as a mounting agent and examined microscopically. Histochemical reactions were applied with hydrochloric acidphloroglucinol to reveal lignified elements, iodine-iodide for starch, Sudan IV for lipophilic substances, Dragendorff s reagent for alkaloidal substances, ruthenium red for mucilage and ferric chloride for phenolic compounds[9]. Photomicrographs of the microscopical sections were taken with the help of photomicroscope[10]. The measurements of different cells and cell contents were done with the help of calibrated ocular micrometer. The powdered stem and fruit was subjected to preliminary phytochemical screening for qualitative detection of phytoconstituents. The dried and coarsely powder (50 g) was extracted in (300ml) methanol by Soxhlet method (Hot methanolic extraction) and for cold maceration (cold methanolic extraction) 25g powder in 150 ml methanol. The concentrated extracts were evaporated to dryness and the extracts were then weighed. Their percentages were calculated in terms of initial air dried plant material. The colors of the extracts were observed. The extracts as mentioned above, were subjected to various qualitative phytochemical tests for the identification of chemical constituents present in the plant material as per standard procedure[10-14]. Physicochemical properties such as the percentage of total ash, Acid insoluble ash, Water soluble ash, Alcohol soluble extractive & water soluble extractive values were determined as per the standard procedure[10]. Percentage of ash value is indicative of the purity of the drug and extractive values represent the presence of polar and non polar compounds in the extract. 128

3 Fluorescence study is an essential parameter for first line standardization of crude drug. The crude powders were subjected to these studies & their fluorescence patterns were noted. The powder material were treated separately with different reagents & exposed to visible, ultraviolet light to study their fluorescence behavior[15-16] The colors obtained by application of different reagents in different radiations were recorded All standard procedure were followed as per the references for studies of characteristics like presence of Total Ash, Acid insoluble Ash, Water soluble Ash, Alcohol soluble extractive value, water soluble extractive value, and qualitative chemical test for presence of secondary metabolites[17-19]. RESULTS AND DISCUSSION: Macroscopic Characters: Stem: The stem pieces are hard, measure about 1-2 cm across, brownish in color, slightly zigzag, with remnants of branch lets at the internodes. The surface is smooth, covered with numerous longitudinal lenticels. A transverse section shows a thick woody region and a thin bark. The fracture is hard and not easy to break. It has no characteristic odor and is tasteless. Fruit: The fruit shows considerable variation in size, shape, color and taste. Ovoid or globose; black, blue or violet; brownish when dry with longitudinal faint striations and a large terminal copular attachments of the calyx showing the inferior condition. Fruit contains a seed which is free from the pericarp. It has no characteristic odor and sweetish taste when mature. Microscopic Characters: Stem: Transverse section of the stem shows the following characters. The cork region is made up of layers of rectangular cells. The cells are brownish in color. A few rod shaped crystals are found in some of the cork cells. Next to the cork is the cork cambium which is one layered, made up of thin walled tangentially elongated cells. This is followed by the several layered secondary cortex region. The cells of the secondary cortex are thin walled; some cells are also found here and there. N ext is the secondary phloem region which is multi layered, consisting of secondary phloem and uni to biseriate medullary rays. In this region groups of stone cells are found arranged concentrically in 2 to 3 layers. This followed by one layered thin walled vascular cambium. The secondary wood is diffuse, porous consisting of small and big vessels, xylem fibers, xylem parenchyma and uni to biseriate medullary rays. Transverse section of the root is circular in outline. The cork cells are thin walled, Macerate : Macerate consists of the following elements. 1. Cork cells which are rectangular and containing calcium oxalate crystals. 2. Thin walled parenchymatous cells. 3. Sclereids of different size and shape with striations, pitted thickenings and a broad lumen. 4. Xylem parenchyma cells, elongated with pitted thickenings. 5. Fibers which are long, narrow, pointed at both ends and with a broad lumen. 6. Vessels with simple, pitted thickenings. 129

4 Diagnostic characters 1. Presence of rod shaped calcium oxalate crystals in some cells of the cork region. 2. Presence of oil cells in the secondary cortex region. 3. Presence of group of stone cells in concentric rings in secondary cortex. 4. Presence of uni to biseriate medullary rays in the secondary phloem. FRUIT: A transverse section of fruit shows single layered epidermis covered by cuticle. This is followed by many layered, thin walled, rounded, brown colored parenchymatous cells with or without intercellular spaces constituting the mesocarp region. Some cells of the mesocarp contain calcium oxalate crystals and some others contain oil globules while a few contain tannin. Next to this region is endocarp represented by groups of elongated stone cells with narrow lumen. Some cells of the endocarp contain tannin. Vascular region is represented by strands with spiral thickenings. In between elongated stone cells, rounded to polygonal stone cells are also present. Seed region is made up o many layered, rounded and thin walled parenchymatous cells. Starch grains are simple and abundant; some cells with oil globules are found here and there. Macerate: Macerate shows the parenchymatous cells which are thin walled.2. Epidermal cells which are thin walled, hexagonal in shape and closely packed.3. Vascular strands are narrow and with spiral thickenings.4. Stone cells or polymorphic sclereids with narrow or broad lumen and of various size and shape ie, hammer shaped, osteo sclereid, idiofibro sclereid and fusoid sclereid. Diagnostic characters: 1. Presence of abundant, elongated, polygonal to rounded stone cells with heavily thickened cell walls with narrow lumen and pits.2. Presence of abundant brown to chocolate colored tannin. 3. Cotyledonary region shows abundant and simple starch grains. 4. Presence of calcium oxalate crystal.the measurements of various cell and tissues are provided respectively (Table - I) Histochemistry: Histochemical tests were carried out on the genuine material for starch, tannins and alkaloids. For these tests free hand sections were taken and these were treated with different reagents. The results are given. (Table II). Phytochemistry: Phytochemical tests were carried out with different extracts for the presence of alkaloids, flavonoids, indoles, leucoanthocyanins, steroids, carbohydrates, phenols, steroidal nucleus, proteins, lignins tannins, saponins, terpinoids, methylenedioxy functional compounds and coumarins (Table -III ). Physico-Chemical Studies: The stem and fruit powder was studied for their physico-chemical constants which include ash values, successive extractive values and solubility. It shows higher ethanol and chloroform extractive values 23.04%w/w and 23.34% w/w respectively (Table -IV). Fluorescence Studies:Fluorescence analysis of drug powder and its various extract treated with acid, alkali was studied and tabulated). It shows yellowish for the powder drug and yellowish green fluorescence for ethyl acetate extract under white and visible light (Table-V). 130

5 CONCLUSION: The pharmacognostic study on the stem and fruit of Syzygium alternifolium (Wight) Walp is known in vernacular as, Mogi or Konda neredu is a well known local drug used in the treatment of diabetes and frequently used for ulcers. The fruit and seed of this plant has long been sold in local drug markets of Seshachalam catchments area, hence the detailed pharmacognostic studies have been carried out which includes macroscopic, microscopy, histochemistry, phytochemistry, physicochemical studies and fluorescence analysis, to detect adulteration if any. The original scientific data on the Pharmacognosy of the species help not only in the standardization of the drug but also in laying down pharmacopoeial standards. ACKNOWLEDGEMENTS: The first author is gratefully acknowledged to Dr. S. N. Yoganarasimhan and Dr. Bikshapathi, Pharmacognosy division, Regional Research Centre, Bangalore for providing laboratory facilities. Also extends his thanks to M.R. Guradeva, Department of Botany, Visveswarapura College of Science, Bangalore and Dr. Mrs. Sailaja, Department of Pharmacognosy, Institute of Pharmaceutical Sciences, S. P. Mahila University, Tirupati. Table I. Measurements of different cells and tissues of Syzygium alternifolium Tissue Measurement (µ) Stem and Fruit bark T.S Stem Fruit Cork x Epidermis x Parenchyma (cortex) x Parenchyma(mesocarp) x Xylem fibre x Parenchyma(seed) x Vessle x Stone cells x Stone cells x Starch grains (diameter) Macerate Cork x Parenchyma x Xylem parenchyma x Parenchyma(surface) x Vessel x Stone cells x Xylem fibre x Vascular strand (diameter) Stone cells x Oil cell x

6 Table II. Histochemcial tests for stem and fruit of Syzygium alternifolium Drug Reagents Test for Reaction Results Stem Fruit Stem Fruit Section Iodine solution Starch Blue color Blue color + ++ Section Ferric chloride solution Tannin Black color Black color + ++ Section Sudan III solution Oil globules Pink color Pink color + ++ Section Con. HCl Crystals effervescence effervescence + + Section Pinch of phloroglucinol + dil. HCl + alcohol Lignin Magenta color(xylem and phloem fibres) Magenta color ++ + Table III: Phytochemical screening of extracts of stem and fruit of Syzygium alternifolium Test Benzene Ethanol Hexane Chloroform Stem Fruit Stem Fruit Stem Fruit Stem Fruit Alkaloids Flavonoids Indoles Leucoanthocyanins Steroids Carbohydrates Phenols Steroidal nucleus Proteins Lignins Saponins Terpinoids Methylenedioxy functional compounds Table IV: Physico-chemical analysis of Syzygium alternifolium Parameter Values (% W/W) Stem Fruit Ash Values Total ash Water soluble ash(%w/w Acid insoluble ash(%w/w) Extractive values Water Ethanol Chloroform Pet. ether

7 Table- V: Fluorescence analysis of Syzygium alternifolium S.No Particulars Visible light UV light 254nm 1 Alcocholic extract Stem Yellowish Fruit yellow Stem Pale yellw Fruit Light green red 2 2 ml Alcoholic extract Pale yellow Green Aqueous Ag NO 3 (1 drop) 3 2 ml Alcoholic extract N aqueous NaOH ( 3 drops) Dark brown Brownish green 4 2 ml Alcoholic extract + 0.1N Purple Yellowish aqueous NaOH ( 2 drops) 5 Powder as such Light yellowish brown Light brown Purple green Purple 6 Powder + 1 N NaOH in Deep purple Dark brown methanol 7 Powder + 1 N NaOH in H 2 O Violet Greenish violet 8 Powder + 1 N H Cl -- Purple Deep purple 9 Powder + 50 % HNO Greenish violet Greenish violet 10 Powder + 50 % H 2 SO Greenish purple Purplish green Fig.I. Microscopical characters of the fruit of Syzygium alternifolium 133

A. T.S. of the fruit (semi diagrammatic) EC: Epicarp, PAR: Parenchyma, OG: Oil globule, MC: Mesocarp, SCR: Sclereids, VS: Vascular strand B. T.S showing details of cell structure CU: Cuticle, EP: Epicarp, CR: Crystal, OG: Oil globule, TC: Tannin cell, STCL: Stone cell layer, VS: Vascular strand, TC: Tannin cell, Fig.

8 A. T.S. of the fruit (semi diagrammatic) EC: Epicarp, PAR: Parenchyma, OG: Oil globule, MC: Mesocarp, SCR: Sclereids, VS: Vascular strand B. T.S showing details of cell structure CU: Cuticle, EP: Epicarp, CR: Crystal, OG: Oil globule, TC: Tannin cell, STCL: Stone cell layer, VS: Vascular strand, TC: Tannin cell, Fig. II Microscopical characters of the fruit macerate of Syzygium alternofolium A: T.S. of seed showing parenchymatous cells with abundant starch cells VC: Vscular cambium; OG: Oil globule; PAR: Parenchyma; SG: Sstarch grain B: Parenchyma cells, C: Stone cells, D: Vascular strands, E: Epidermal cells 134

9 Fig. III. Microscopical characters of the Stem macerate of Syzygium alternifolium A - Cork cells with crystal, B - Parenchyma, C - Sclereids, D - Xylem parenchyma, E - Xylem fibres, F - Vessels 135

10 Fig. IV. Microscopical characters of the Stem of Syzygium alternifolium STEM : showing cellular details; CR: Crystal ; CK: Cork; CAM: Cambium; SCOR: Secondary cortex; OC: Oil cell; STC: Stone cell; SPH: Secondary phloem; MR: Medullary ray; STC: Stone cell; VCAM: Vascular cambium; MR: Medullary ray; V: Vessel 136

11 REFERENCES: 1. Ashok Kumar, S. Ramachandra Setty & Laxmi Narsu., RJPBCS., 2010, 1, N. Nagaraju & K.N. Rao., Folk medicine for diabetes from Rayalaseema of Andhra Pradesh, Ancient Sci. Life., 1989, 9, S. Vedavathy, A. Sudhakar & V. Mrudula., Tribal Medicinal Plants of Chittoor District. Ancient Science of Life., 1997, XVI 4, A. Sudhakar., Pharmacognosy of some indigenous medicinal plants of Chittoor district, AP. India. Ph.D., thesis, S.V. University, Tirupati C. Venkataramaiah, K. Harikumar Reddy & K. N. Rao., Environ.&Ecol.,1988, 6, J. Rajasekhar Rao & R. Srinivasa Rao., Indian J. Chem., 1991, 30, S. Vedavathy.,Studies on medicinal plant of Titumala and Tirupathi, Chittoor district, Andhra Pradesh, Ph.D., Thesis, S.V. University, Tirupati T. Pullaiah & S. Sandhya Rani., Trees of Andhra Pradesh, Regency publications, New Delhi., D.A. Johansen., Plant Microtechnique, McGraw Hill Book Company, New York,1940, C.K. Kokate.,Practical Pharmacognosy, 4th Edition. New Delhi, Vallabh Prakashan, 2006, R.R. Mendonça-Filho.,in Modern phytomedicine: turning medicinal plants into drugs - Bioactive Phytocompounds: New Approaches in the Phytosciences, (Ahmad I, Aqil F and Owais, M., Eds.), WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.,2006, K.R. Khandelwal., Practical Pharmacognosy, Techniques and Experiments, 12th Edition. Nirali Prakashan. 2004,

12 13. The Ayurvedic Pharmacopoeia of Indian Ministry of Health and Family Welfare New Delhi, 1996; Part-1, J.B. Harborne., Method of extraction and isolation In. Phytochemical Methods. Chapman & Hall, London., 1998, C.J. Kokoski, R.J. Kokoski & F.J. Salma., J.Am.Pharm.Asson., 1958, 47, C.R. Chase & R. Pratt., Jour. Amer. Pharm. Asso. (Sci. ed)., 1949, C.K. Kokate, A.P. Purohit & S.B. Gokhale., Pharmacognosy., 29 th Edn., Nirali prakashan, Pune,2009, J.B. Harbone., Phytochemical Methods., Edition-III, London, Chapman and Hall., The Indian Pharmacopoea, 2 nd Edn., Govt. of India, publications, Delhi., 1966, 947. *Correspondence Author: A. Sudhakar *Herbal Folklore Research Centre, Department of Botany, Sri Venkateswara Arts College, Tirupati , Andhra Pradesh, India 138

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