Indian Journal of Natural Products and Resources Vol. 1(1), March 2010, pp. 38-43 Pharmacognostical studies on the leaves of Cocculus hirsutus (Linn.) Diels – Chilahinta , an Ayurvedic drug V Madhavan 1, Mohamed Sajid Ullah 1, M R Gurudeva 2 and S N Yoganarasimhan 1* 1Department of PG Studies and Research in Pharmacognsoy, M S Ramaiah College of Pharmacy, Bangalore-560 054, Karnataka, India 2Department of Botany, V V Pura College of Science, Bangalore-560 004 Received 22 September2008; Accepted 5 May 2009 Cocculus hirsutus (Linn.) Diels known as Chilahinta in Ayurveda and Kattu kodi in Siddha is an important medicinal plant belonging to the family Menispermaceae. The leaves are used to treat several diseases like polyuria, fevers, piles and is said to possess aphrodisiac property. The present study provides taxonomical, pharmacognostical and physico-chemical details helpful in laying down standardization and pharmacopoeial parameters. Some diagnostic characters are presence of unicellular ribbon shaped trichome both on lamina and petiole, presence of sunken stomata, excretory sacs in mesophyll. Physico-chemical studies revealed total moisture content (6.67%), total ash (5.07%), acid insoluble ash (0.57%), water soluble ash (0.65%), alcohol soluble extractive (32.63%) and water soluble extractive (26.85%). Ultraviolet analysis exhibited considerable variation and preliminary organic analysis revealed presence of alkaloids, flavonoids, fixed oils, fats, mucilage, glycosides and phytosterols. HPTLC profile of alcoholic extract of leaves gave 16 phytoconstituents. Keywords : Cocculus hirsutus , Menispermaceae, Leaves, Pharmacognosy, Physico-chemical analysis, HPTLC, Alcohol extract. IPC code: Int. cl. 8—A61K 36/59, A61K 127/00 Introduction leaf is not available 14-16 . Hence, the present Cocculus Linn. (Menispermaceae) consists of investigation which helps not only in the 20 species of mostly scandent or rarely suberect herbs identification of the drug but also provides requisite or shrubs, distributed in tropics and subtropics 1. material for establishing the biomarker/bioactive Cocculus hirsutus (Linn.) Diels is a scandent shrub, compound. known as Chilahinta in Ayurveda and Kattu kodi in Siddha system of medicine; the roots and leaves are Materials and Methods used to treat polyuria, eczema, dysuria, abdominal Fresh leaves were collected from Kudur village, disorders, rheumatoid arthritis, fevers, piles, syphilis, 2,3 Bangalore Rural district, Karnataka, during March, disorders of blood and as an aphrodisiac . Roots 2006, preserved in 70% ethyl alcohol for histological and leaves possess antimicrobial, cardiotonic, studies. Botanical identification was carried out using hyperglycaemic, diuretic, laxative and epileptic 17,18 4-6 local floras , identified by senior Plant Taxonomist activity . Several isoquinoline alkaloids are present and authenticated at the herbarium of the Regional in leaves and roots; the important ones are cohirsine, 7-11 Research Institute (Ay), Bangalore (RRCBI). Voucher cohirsinine, cohirsitinine, hirsutine and jamtine . herbarium specimen (Sajid Ullah 001) was prepared Some histological studies on the root, rhizome and 12,13 and is preserved along with crude drug sample at the leaf has been carried out but detailed herbarium of M S Ramaiah College of Pharmacy, pharmacognostical studies including macerate details, Bangalore 19 . Pharmacognostical evaluation including powder study exhibiting the various elements and histochemical, macerate and powder studies were fluorescence analysis besides HPTLC studies on the carried out by taking free hand sections following Johansen 20 , Wallis 21 and Evans 22 . Safranin (4%) was _________ *Correspondent author: used to stain transverse sections. Reagents like E-mail: [email protected] potassium iodide, ferric chloride, Sudan III, MADHAVAN et al : PHARMACOGNOSTICAL STUDIES ON THE LEAVES OF AN AYURVEDIC DRUG 39 concentrated HCl, ruthenium red and phloroglucinol tomentose beneath. Veination reticulate with 5 to 6 with dilute HCl were used for histochemical tests. pairs of alternating lateral veins; first two pairs of Concentrated nitric acid (50%) with pinch of lateral veins arise basally giving a multicostate potassium chlorate crystals was used as the appearance; secondary and tertiary veinlets macerating fluid. Photomicrographs were obtained by anastomose to form reticulation with free end observing free hand sections of drug under compound included in meshes. Petiole greyish-tomentose, with a binocular microscope (Olympus-CH20i model) with distinct swelling at proximal and distal ends. Leaves built in analogue camera (CMOF, 1.4 mega pixel). greenish, odourless and mucilaginous when fresh, Computer Images were captured using AV-Digitaliser brittle and powdery on drying, without characteristic having Grand VCD 2000-Capture Guard. taste, prolonged contact produces itching sensation Measurements of cells and tissues were carried out (Plate 1 Fig. B). using Micro Image Lite Image Analysis Software Transverse section of petiole is circular in outline (Cybernetics, Maryland, USA). Physico-chemical with trichomes emerging all over the surface, consists constants, organic analysis, ultra-violet analysis and of epidermis, cortex and stellar region with prominent chromatographic studies were carried out from shade- pith. Epidermis single layered, made up of thick 23-27 dried powder following prescribed methods . walled rectangular cells. Hairs or trichomes emerge HPTLC studies were carried out on alcohol extract from some epidermal cells. Hypodermis is made up of using Camag HPTLC system equipped with Linomat collenchymatous cells, measure 5-7-10µ. Cortex V sample applicator, Camag TLC scanner 3 and consists of hexagonal cells, demarcated into inner CATS 4 software for interpretation of data. An region of larger cells and outer region of smaller cells, aluminium plate (5×10 cm) precoated with silica cells contain abundant chloroplast and sparsely gel 60F254 (E Merck) was used as adsorbent. distributed starch grains. Endodermis is single The plates were developed using toluene:ethyl layered. Pericycle is multilayered, heterogeneous, acetate:chloroform:glacial acetic acid (6:2:3:0.5) sclerenchymatous over the vascular bundle, in a Camag twin trough chamber to a distance of parenchymatous in between vascular bundle; 8 cm each. conjunctive tissue is found in between vascular bundle (Plate 1 Fig. C-D). Stele consists of vascular Results bundle varying from 6 to 8, arranged in a ring; Botanical description vascular bundle conjoint; collateral, endarch (Plate 1 Cocculus hirsutus (Linn.) Diels in Engler, Fig. E). Xylem cells measure 5-7-8µ, consists of Pflanzenr. 46, 236.1910; Dunn in Gamble, Fl. Pres. vessels and fibres; phloem cells measure 5-7-8µ, Madras 1, 21. 1957 (repr. ed.). Menispermum consists of sieve tube and companion cells. hirsutum Linn., Sp. Pl. 341. 1753. Cocculus villosus Transverse section of leaf consists of lamina and (Lam.) DC., Syst. 1, 525. 1817; Hook.f. & Thoms. in midrib regions (Plate 1 Fig. F). Lamina exhibits upper Hook. f. Fl. Brit. India, 1 , 101. 1872. Menispermum and lower epidermis; lower epidermal cells smaller, villosum Lam., Encycl . 4, 97. 1797. measure 5-6-7µ, upper epidermal cells measuring Hirsute climbers or stragglers. Leaves 2.5-5×2-2.5 8-9-10µ; epidermal cells rectangular, filled with cm, simple, ovate, obtuse, mucronate, truncate at chloroplast. Stomata anomocytic, found on lower base, softly villous. Flowers small, greenish-yellow. epidermis, many, sunken, each surrounded by 4 to 6 Male flowers in 5 to 6 cm long axillary cymose epidermal cells. Mesophyll comprises of palisade and panicles. Calyx and corolla lobes 6, obovate. Stamens spongy parenchyma, cells filled with chloroplast and 6, enclosed by corolla lobes. Female flowers in starch grains. Palisade cells columnar, 1-layered, axillary fascicles, rarely in racemes. Fruits globose, except near midrib where it is 2 to 3-layered. Spongy green, shining (Plate 1 Fig. A). Distributed throughout parenchyma 2 to 3-layered, cells elongated, thin Tropical Africa and India in most districts. walled and enclose air spaces in between and Macro- and microscopical characters of leaves excretory sacs (Plate 1 Fig. G). Midrib exhibits Leaves dorsiventral, variable, simple, ovate to crescent shaped vascular bundle enclosed by ovate-oblong or slightly lanceolate with truncate to sclerenchymatous bundle sheath. Next to bundle cordate base, apex sometimes mucronate, margins sheath lies parenchymatous ground tissue; some entire or slightly wavy, lamina hairy, greyish-white peripheral cells are collenchymatous; vascular bundle INDIAN J NAT PROD RESOUR, MARCH 2010 40 Plate 1: Figs. A-U. Macro- and microscopical characters of the leaves of Cocculus hirsutus. A. Fresh twig. B. Fresh crude drug. C-E. T S of petiole (C. outline of tissues; D. regions enlarged; E. vascular bundle enlarged). F-I. T.S. of lamina and midri (F. outline of lamina and midrib; G. Mesophyll enlarged; H. Lamina enlarged; I. Vascular bundle in midrib region enlarged). J-U. Macerate elements (J. Epidermal peel showing epidermal cell and trichome base; K. Epidermal peel showing stomata; L. Stomata enlarged; M, N. Unicellular trichome; O. Parenchyma cell; P. Collenchyma cell; Q, R. Fibres; S, T. Vessel; U. Venation pattern) (Abbreviations: CJT-conjunctive tissue; COL-collenchyma; COR-cortex; END- endodermis; EP-epidermis; EPC-epidermal cell; EXS- excretory sac; GT-ground tissue; HYP-hypodermis; LAM-lamina; LOE-lower epidermis; MID-midrib;