International Journal of Pharma and Bio Sciences PHARMACOGNOSTICAL AND PHYTOCHEMICAL INVESTIGATIONS OF COCCULUS PENDULUS (J.R. & G. FORST.

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1 H. RABARI *1, S. PANDYA 2, G. VIDYASAGAR 1, B. GAJRA 1 1 Veerayatan Institute of Pharmacy, Jakhania, Mandvi, Kachchh, Gujarat, India. 2 Pharmacy College, Rampura-Kakanpur, Panchmahal (District), Gujarat, India * Corresponding Author harirabari@yahoo.co.in ABSTRACT The leaves of Cocculus pendulus (J.R. & G. Forst.) Diels (Menispermaceae) is reported to have good medicinal values in traditional system of medicines. The present study deals with pharmacognostical examination of morphological and microscopical characters and phytochemical investigations of Cocculus pendulus leaves including determination of loss on drying, ash values and extractive values. The preliminary phytochemical screening of powdered drug was also carried out. The qualitative chemical examinations revealed the presence of various phytoconstituents like alkaloids, carbohydrates, phytosterols, proteins and mucilages in the leaf extracts. KEYWORDS Cocculus pendulus, Orap, Leaf microscopy, Phytochemical screening INTRODUCTION The genus Cocculus belongs to the family Menispermaceae. It comprises about 35 species, distributed throughout the tropical and subtropical countries of the world. The plants of this genus are shrubs or woody climbers. The members of this genus are used in the traditional system of medicine for various ailments 1-3. Only two species of Cocculus are found in Kachchh region of Gujarat, i.e. Cocculus pendulus and Cocculus hirsutus. The plant Cocculus pendulus (synonym: Cocculus leaeba) is a scandent shrub, growing particularly in cultivated areas, along rocks and in the dry mountainous areas of Nakhatrana, Mandvi and Bhachau region of Kachchh district. The roots of the plant are used in the indigenous system of medicine for the treatment of intermittent fevers and as a tonic. Hypotensive and anticancer activity has been attributed to the alkaloid fractions from the leaves and stem 4. The juice of the leaves contains mucilage which when mixed with water forms a jelly. This is taken as a cooling medicine for gonorrhoea 1-2. The roots and leaves are used in rheumatic pain 1,5. The present investigation was planned to study the detailed pharmacognostical and phytochemical aspects of Cocculus pendulus (J.R. & G. Forst.) Diels. 1

2 Figure 1 Twig of Cocculus pendulus MATERIALS AND METHODS Collection and authentication of the plant material Fresh leaves of Cocculus pendulus were collected from the outfield area of Jakhaniya village of Kachchh region of Gujarat (India) in the month of June The plant material was identified and authenticated from the Botanical Survey of India, Pune. The voucher specimen CPHAR-1 was also preserved for future reference. The collected leaves were shade dried for 15 days and size reduced by laboratory grinder in to coarse powder. It was stored in a well closed container free from environmental climatic changes till usage. Morphological investigation The healthy plants and their normal parts were selected for the morphological investigation. 2 The macroscopical characters like shape, size, colour and odour of the leaves were observed. Microscopical investigation Preparation of specimen: The healthy leaves were selected carefully for the study of microscopical characters. The specimen leaves of Cocculus pendulus were fixed in FAA (Formalin- 5ml + Acetic acid-5ml + 70% Ethyl alcohol-90ml). After 24 hours of fixing, the specimens were dehydrated with graded series of TBA (tertiary- Butyl alcohol). Infiltration of the specimens was carried out by gradual addition of paraffin wax (melting point ºC) until TBA solution attained super saturation. The specimens were cast into paraffin blocks.

3 PHARMACOGNOSTICAL AND PHYTOCHEMICAL INVESTIGATIONS OF Sectioning of specimen: The paraffin embedded specimens were sectioned with the help of Rotary Microtome. The thickness of the sections was µm. De-waxing of sections was done by customary procedure 6. The sections were stained with toluidine blue as per the specified method 7. Since toluidine blue is a polychromatic stain, the staining results were remarkably good and some cytochemical reactions were also observed. The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage and blue to the protein bodies. Wherever necessary, sections were also stained with safranin, fast-green and N/10 iodine to identify the presence of lignified cells and starch grains. To study the histology of stomata, venation pattern and trichome distribution, paradermal sections (parallel to the surface of leaf) were taken. The clearing of leaf was done with 5% sodium hydroxide or epidermal peeling by partial maceration, employing Jeffrey s maceration fluid 8. The mounting of macerated/cleared materials was performed with glycerine. Powdered materials of different parts were cleared with sodium hydroxide solution and mounted in glycerine medium after staining. Different cell components were studied and measured. Photomicrographs: Microscopic descriptions of tissues can be described with micrographs wherever necessary. Photographs of different magnifications were taken with Nikon labphoto-2 microscopic unit. For normal observations bright field was used. For the study of crystals, starch grains and lignified cells, polarized light was employed. Since, these structures have birefringent property under polarized light; they appear bright against dark background. Magnifications of the 3 figures are indicated by the scale-bars. Descriptive terms of the anatomical features are as given in the standard anatomy books Physical evaluation Physical constants of crude drugs like loss on drying, total ash, acid insoluble ash, water soluble ash, alcohol soluble extractive and water soluble extractive values were determined by using official methods Method of extraction Dried leaves of Cocculus pendulus (1 kg) were soaked in alcohol (4 liters) and kept covered for 4-5 days and filtered. The filtrate (alcohol extract) was concentrated under vacuum. The concentrate was acidified with 5% hydrochloric acid (200 ml) solution and the fatty material was removed by extraction with petroleum ether. The aqueous layer was extracted with chloroform. The chloroform extract was dried with anhydrous sodium sulfate and concentrated (CECp-1). The remaining solution (aqueous layer) was basified with ammonium hydroxide (up to ph 10) and extracted with chloroform. The chloroform extract was dried with anhydrous sodium sulfate, filtered and evaporated to dryness in vacuum (CECp-2). The remaining solution (aqueous layer) was extracted with ethyl acetate. The ethyl acetate extract was dried with sodium sulfate and evaporated to dryness in vacuum (EAECp). All the extracts were concentrated in vacuum and were preserved. The colour, consistency and percentage yield of the extracts were also noted. Preliminary phytochemical investigation The extracts obtained during the extraction process were subjected to preliminary phytochemical screening to determine the presence

4 PHARMACOGNOSTICAL AND PHYTOCHEMICAL INVESTIGATIONS OF of various phytoconstituents like alkaloids, carbohydrates and glycosides, phytosterols, fixed oils and fats, saponins, phenolic compounds, triterpenes, flavonoids, proteins and aminoacids, gums and mucilage, tannins and volatile oils by using reported methods Thin layer chromatography (TLC) of various extracts After concentration and drying of each extract in vacuum desiccator, identification of phytoconstituents was carried out by thin layer chromatography using different detecting reagents 17. The test extract was dissolved by using appropriate solvent in a concentration of 1 mg/ml and subjected for spotting. Silica gel G (mesh size 60) was used as a stationary phase and two solvent systems were used as mobile phase: Ethyl acetate-methanol-water (75:13.5:10) and Toluene-Ethyl acetate (93:7). Spots were detected by using KOH (for glycosides), Dragendorff s reagent (for alkaloids), UV light (for flavonoids) and vanillin-sulphuric acid (for saponins and volatile oils) as detecting reagents. The R f values were also noted. RESULTS Morphological investigation Morphological examination of Cocculus pendulus reveals the following interpretations: Leaves cm long, cm broad; oblonglanceolate, ovate or trapezoid; base truncate, cuneate, rounded or trilobed-hastate; apex obtuse, mucronate or emarginate; generally glabrous or slightly puberulous on both sides; basal nerves 3-5. Stem up to 15 cm in diameter; branches 5-6 m long. Flowers minute. Drupe reddish when fresh but turning black when dried; reniform, compressed, 4-7 mm long, 4-5 mm broad; endocarp ribbed on the lateral faces and without a prominent crest, not perforated in the centre. Microscopical investigation Microscopical examinations revealed that the leaf was bilateral, mesomorphic and stomatiferous. The surface of the leaf was even, smooth on the adaxial side and roughly echinate on the lower side due to heavy cuticular layer (Fig 2). 4

5 Figure 2 Anatomy of the midrib in Cocculus pendulus [AbE - Abaxial Epidermis; AdE - Adaxial Epidermis; BS - Bundle Sheath; Cu - Cuticle; Ph - Phloem; X - Xylem] Midrib: The midrib was fairly prominent and planoconvex in sectional view. It was flat on the adaxial side and hemispherical on the abaxial part (Fig 2). The midrib was 280 µm vertically and 320 µm thick horizontally. The ground tissue of the midrib consists of a wide circular zone of angular, compact, thin walled parenchyma cells forming a thick bundle sheath. The epidermis on the midrib region was squarish, even, smooth on the adaxial side and roughly echinate on the lower side. Adaxial epidermal cells were µm thick and abaxial epidermal cells were µm thick. Vascular bundles: The vascular bundle was small, single; top shaped and has collateral system. The xylem elements occur in a few multiples and a broad arc of phloem was seen on the abaxial part. The vascular strand was 120 µm vertically and 150 µm horizontally (Fig 2). 5 Lamina: Middle portion of the lamina was 160 µm thick and the marginal part was 110 µm thick. Adaxial epidermal cells were wide, cylindrical, thick walled, smooth, heavily deposited with cuticles; the epidermal cells were µm thick. The abaxial epidermis was narrow, cylindrical, uneven, roughly echinated, heavily cuticular; the epidermal cells were µm thick. Mesophyll: The mesophyll tissue consists of two layers of adaxial palisade and again two layers of abaxial palisade zones (Fig 3 and 4). Palisade cells: The palisade cells were short, cylindrical and loosely arranged; the median part of the lamina occur three or four layers of spongy mesophyll tissue with wide air spaces. Small and circular vascular bundles of the lateral vein were also seen in the spongy mesophyll tissue (Fig 3).

6 Figure 3 T.S. Cocculus pendulus showing lamina through lateral vein [AdE - Adaxial Epidrmis; BS - Bundle Sheath; PEp - Papillate Epidermal cells; Ph - Phloem; PM - Palisade Mesophyll; SM - Spongy Mesophyll; X - Xylem] Figure 4 T.S. Cocculus pendulus showing leaf margin [AdE - Adaxial Epidrmis; Cu - Cuticle; LM- Leaf Margin; MT - Mesophyll Tissue; Ve - Vein] Leaf margin: Leaf margin has similar structure as the middle part of the lamina. The epidermal cells were comparatively smaller but the cuticle was heavily thick and papillate (Fig 4). Epidermis: In surface view, the abaxial epidermal cells were angular and polyhedral. Anticlinal walls were thick and straight with the presence of papillate outgrowths. In the centre of the epidermal cells, the papillate outgrowths of the cuticle appear as circular 6 dark spots (Fig 3 and 4). Stoma were surrounded by a limited number of cells which were indistinguishable in size, shape or form, from those of the remainders of the epidermis, it was called as anomocytic type of stomata (Fig 5). Adaxial epidermal layer also has anomocytic type of stomata (Fig 5). The guard cells of the stoma were 20 µm in length and 10 µm in breadth. Some of these stomata were actinocytic type; with stoma surrounded by a

7 PHARMACOGNOSTICAL AND PHYTOCHEMICAL INVESTIGATIONS OF ring of cells, elongated radially to the guard cells. Venation pattern: The lateral veins and veinlets vary in thickness. They form dense network (Fig 6). In the middle part of the lamina, some of the vein-islets were distinct, wide and polygonal in outline. There was a distinct vein termination, which was branched, long, wavy and curved. The vein terminations were mostly branched one or more forming a dendroid outline (Fig 6). The cleared lamina shows dense reticulate pattern of venation. The lateral vein and veinlets were straight and become gradually thin. The vein-islets were polygonal in outline and vary in size and shape (Fig 7). The vein terminations were well defined and branched. The ends of the vein terminations often possess short, cylindrical tracheids. The terminal tracheids have spiral thickenings (Fig 8). Figure 5 Abaxial epidermis with stomata in Cocculus pendulus Leaf [EC - Epidermal Cells; GC - Guard Cells; SC - Subsidiary Cells] 7

8 International Journal of Pharma and Bio Sciences Figure 6 Paradermal section of Cocculus pendulus showing One vein-islet with vein-termination [VI - Vein-islets; VT - Vein-termination] Figure 7 Leaf section of Cocculus pendulus showing venation system [LV - Lateral Vein; Ve - Vein; VI - Vein-Islets; VT - Vein-terminations] 8

9 Figure 8 Leaf section of Cocculus pendulus showing vein-termination and tracheids (iii) Powder microscopy: The powder microscopy of the plant reveals the following microscopical characters: Epidermal cells: Fragment of abaxial epidermal cells showing stomatal histology. There are two types of stomata: one was anomocytic and another one was actinocytic. Both types were observed in powder. Epidermal cells were angular and polyhedral. The cell walls were thick and not clear in the powder (Fig 9). Veins and terminal tracheids: The vein-islets and vein-terminations were separated in the leaf [Tr - Tracheids] powder. Separated veins were shows spiral thickening (Fig 10). Vein-terminations have short and cylindrical tracheids at the ends (Fig 10). (iv) Physical evaluation: Physical evaluation revealed that loss on drying 3.73%, total ash 6.52%, acid insoluble ash 2.9%, water soluble ash 2.17%, alcohol soluble extractives 2.3%, water soluble extractives 3.4% w/w values were observed in leaves of Cocculus pendulus. The results were shown in Table 1. 9

10 Table 1 Physical parameters of leaves of Cocculus pendulus Sr. No. Parameters Determined value* (% w/w) 1 Loss on drying 3.73±0.9 Ash values 2 Total ash 6.52±0.2 Acid insoluble ash 2.90±0.5 Water soluble ash 2.17±0.3 3 Extractive values Alcohol soluble extractives 2.3±0.7 Water soluble extractives 3.4±1.2 * Mean value of three counts (v) Average extractive values: During successive solvent extraction of leaves of Cocculus pendulus, the percentage yield were determined as chloroform extract-1(cecp-1) 3.78 %, chloroform extract-2 (CECp-2) 0.98 % and ethyl acetate extract (EAECp) 2.72% w/w. The colour and consistency of the each extract were also noted during the extraction process as shown in Table 2. (vi) TLC profile of extracts: The preliminary phytochemical studies with the help of thin layer chromatography revealed that presence of alkaloid in all the three extracts prominently (R f values 0.23, 0.37 and 0.47 respectively). The results were shown in Table 3 and 4. (vii) Preliminary phytochemical investigation: The preliminary phytochemical screening with the various qualitative chemical tests revealed the presence of alkaloids in all the three extracts, and it was the main constituents present in the leaves of Cocculus pendulus. Carbohydrates, phytosterols, proteins and mucilage are the other phytoconstituents were present in the leaf extracts. The results were shown in Table 5. Sr. No. Table 2 Percentage yield of leaf extracts of Cocculus pendulus Extracts Colour and Consistency Average percentage yield (% w/w on dry weight basis) 1 CECp-1 Greenish black sticky mass CECp-2 Black sticky mass EAECp Brown sticky mass

11 Table 3 Thin layer chromatography of leaf extracts of Cocculus pendulus Solvent system used Detecting reagent Observation Inference C1 C2 E Ethyl acetate: KOH Red Anthraquinone Methanol: glycoside Water Dragendorff s Orange-red Alkaloid (75:13.5:10) reagent UV light Yellow/Green Flavonoid /Orange/Blue Vanillin Blue Saponin sulphuric acid Toluene: Vanillin Red/Yellow/ Essential oil Ethyl acetate (93:7) sulphuric acid Brown/Bluegreen [C1 Chloroform extract-1 (CECp-1); C2 Chloroform extract-2 (CECp-2); E Ethyl acetate extract (EAECp)] Table 4 R f values of leaf extracts of Cocculus pendulus Solvent system used Detecting reagent Observation R f values Ethyl acetate: Methanol: Water (75:13.5:10) Dragendorff s reagent Orange-red C1 C2 E Table 5 Preliminary phytochemical screening of leaf extracts of Cocculus pendulus Plant constituents Extracts Test/reagent used CECp-1 CECp-2 EAECp 1 Alkaloids Dragendorff s reagent Mayer s reagent Hager s reagent Wagner s reagent Carbohydrates and glycosides Molisch s test

12 Fehling test Benedict s test Legal s test Borntrager s test Phytosterols Salkowski test Liebermann-Burchard s test Fixed oils and fats Spot test Saponification test Saponins Foam test Phenolic compounds and tannins Ferric chloride test Bromine water Lead acetate test Tannins Triterpenes Flavonoids Proteins and aminoacids Gums and mucilages Volatile oils DISCUSSION This study on micro-morphological features of Cocculus pendulus, proposed a set of anatomical parameters may enable those who handle this plant to maintain its quality control. Morphological as well as microscopical studies of plants are the primary steps to establish its botanical standards before going to other studies. As per WHO norms, botanical standards are to be proposed as a protocol for the diagnosis of the herbal drug. The pharmacognostic parameters are helpful for the future identification and authentification of the plant in the herbal industry. The physical standards, such as loss on drying, ash values, extractive values will be useful to identify the authenticity of the drug even from the crushed or powdered plant materials. It will serve as a standard data for the quality control of the preparations containing this plant in the future. The information obtained from the ash values and extractive values are useful during the time of collection and also during extraction process. Using these standards, the plant can be differentiated from other related species. The leaf constants can be included as microscopical standards in Indian Herbal Pharmacopoeia. The curative properties of medicinal plants are perhaps due to the presence of various secondary metabolites such as alkaloids, flavonoids, glycosides, saponins, sterols etc. Thus the 12

13 PHARMACOGNOSTICAL AND PHYTOCHEMICAL INVESTIGATIONS OF preliminary screening tests may be useful in the detection of the bioactive principles and subsequently may lead to the drug discovery and development. Further, these tests facilitate their quantitative estimation and qualitative separation of pharmacologically active chemical compounds. Phytochemical study was also useful to isolate the pharmacologically active principles present in the drug. ACKNOWLEDGEMENTS The authors are thankful to the Director and Administrator, Veerayatan Institute of Pharmacy, Jakhania-Mandvi, Kachchh, Gujarat-India for providing necessary facilities to carry out the research work. Authors are also thankful to Joint Director, Botanical Survey of India, Pune, for authentification of plant sample. REFERENCES 1. Nandakarni KM and Nadakarni AK, Indian Materia Medica, 3 rd Edn, Popular book depot, Doota Paperhwar Prakashan, Bombay, (1954). 2. Chopra RN, Chopra IC, Handa KL and Kapoor LD, Indigenous Drugs of India, UN Dhar and Sons Pvt. Ltd., Calcutta, (1958). 3. Kirtikar KR and Basu BD, The Indian Medicinal Plnats: I, 2 nd Edn, Delhi, (1993). 4. Bhakuni DS and Joshi PP, Alkaloids of Cocculus pendulus (forsk) Diels. Tetrahedron, 31: , (1975). 5. Thakar JI, Plants of Kachchh and their utility, Pravin Publications, Rajkot, (1926). 6. Johansen DA, Plant Microtechnique, Mc Graw Hill Book Co., New York, (1940). 7. O Brien TP, Feder N and Mc Cull ME, Polychromatic Staining of Plant Cell Wall by Toluidine Blue: (1964). 8. Sass JE, Elements of Botanical Microtechnique, Mc Graw Hill Book Co., New York, (1940). 9. Easu K, Plant Anatomy, John Wiley and Sons, New York, (1964a). 10. Easu K, Anatomy of Seeds, John Wiley and Sons, New York, (1964b). 11. Anonymous, Indian Pharmacopoeia, Ministry of Health and Family Welfare, Government of India, Controller of publication, New Delhi, (1996). 12. The Ayurvedic Pharmacopoeia of India, Government of India, (2001). 13. Kapoor LD, Singh A, Kapoort SL and Strivastava SN, Survey of Indian Medicinal Plants for Saponins, (1989). 14. Kokate CK, Practical, 3 rd Edn, Vallabh Prakashan, New Delhi, (1991). 15. Harborne JB, Phytochemical methods, A guide to modern technique of plant analysis, Chapman and Hill, London, (1992). 16. Evans WC, Trease and Evan s. 14 th Edn. WB Sounders Company Limited, London, (1996). 17. Wagner H, Plants Drug Analysis. Verlas Publication, Berlin, (1989). 13