Indian Journal of Natural Products and Resources Vol. 2(1), March 2011, pp. 74-80

Macroscopical, anatomical and physico-chemical studies on of Coccinia indica Wight & Arn., growing wildly in eastern Uttar Pradesh region of India

Arshad Hussain *, Shadma Wahab, Aleza Rizvi and Md. Sarfaraj Hussain Faculty of Pharmacy, Integral University, Dasauli, Kursi Road, Lucknow-226026, Uttar Pradesh, India.

Received 10 March 2010; Accepted 22 January 2011

Coccinia indica Wight & Arn. of family , called Ivy Gourd in English and Kundru or Bimb in Hindi, is a wildly growing climber or trailing weed in eastern Uttar Pradesh region of India. Its leaves are traditionally used for treatment of various diseases like wounds, ulcers, inflammation, skin diseases, fever, asthma, cough, diabetes and anemia. An attempt has been made to highlight this folk herbal medicine through present study which will assist in the identification of fresh as well as dried crude samples of leaves anatomically and physicochemically. TLC fingerprint profiling and fluorescence analysis of powdered leaves were also carried out and the salient qualitative and quantitative parameters are reported. These studies will provide referential information for correct identification and help in checking adulteration in market samples used in the preparation of various herbal medicines. The present observation will also be helpful in differentiating the leaves of this species from closely related species of same genus and family.

Keywords : Coccinia indica , Ivy Gourd, Kundru , Anatomy, Physico-chemical, Cucurbitaceae, Wild, Medicinal .

IPC code; Int. cl. (2011.01)—A61K 36/00

Introduction traditional medicine for their primary health care needs. Search for eternal health and longevity and to seek In almost all the traditional medicines, the medicinal remedy to relieve discomfort prompted man to develop play a major role and constitute the backbone of diverse ways and means of health care. The early man the traditional medicines. Standardization of natural explored his immediate natural surroundings, tried products is a complex task due to their heterogeneous many things like plants, animals and minerals and composition, which is in the form of whole plant, plant developed a variety of therapeutic agents 1. The parts or extracts obtained thereof 5,6. To ensure knowledge gathered by generations was either reproducible quality of herbal products, proper control documented or passed on to the posterity and this of starting material is utmost essential. The first step practice was generally termed as Traditional Medicine. towards ensuring quality of starting material is Plants are also appreciated in pharmaceutical research authentication . Despite the modern techniques, as the major resource for new medicine and a growing identification of plant drugs by pharmacognostic body of medical literature supports the clinical efficacy studies is more reliable. According to the World Health of herbal treatments 2. Today, about 40% doctors, Organization (WHO, 1998), the macroscopic and especially in India and in China (the Mystic Orient) microscopic description of a medicinal plant is the first have reverted to increasing use of indigenous drugs and step towards establishing the identity and the degree of natural medicines. Steadily, a sizeable section of purity of such materials and should be carried out scientists in biological, biochemical and biomedicinal before any tests are undertaken 7,8. discipline have embarked on research on medicinal Coccinia indica Wight & Arn., belonging to the plants, which are the staple sources of many indigenous family Cucurbitaceae grows wildly throughout India drugs 3,4. The World Health Organization (WHO) and to a limited extent also cultivated in various parts. estimates that about 80% of the population living in the It is commonly known as Kundru . The whole plant is developing countries relies almost exclusively on traditionally used for various medicinal purposes and leaves are used in Indian folk medicine for treatment ——————— of number of ailments including diabetes, wounds, *Correspondent author E-mail: [email protected] ulcers, inflammation, in eruptions of skin, fever, asthma Phone: +91(0522) 2890812, 2890730 and cough. Scientific investigations have shown that the HUSSAIN et al : STUDIES ON LEAVES OF COCCINIA INDICA 75

crude extract possesses hepatoprotective, antioxidant, per standard methods. Calibrated digital pH meter was anti-inflammatory, anti-nociceptive, anti-diabetic, hypo- used to measure the pH of 1 and 10% aqueous extracts lipidemic, antibacterial and antitussive activities. and also loss on drying was noted 27,29-30 . Though the plant has been reported for many biological activities, no scientific data is available to identify the Preliminary phytochemical screening genuine sample. The present investigation was therefore, Preliminary phytochemical screening for the taken up to establish identity of fresh and dried leaves detection of various chemical constituents was carried morphologically microscopically and physicochemically out by using standard procedures described by 31 32 for the standardization of the drug 9-24 . Harborne and Khandelwal .

Thin layer chromatography and high performance thin layer Materials and Methods chromatography (HPTLC)

Collection and authentification Thin layer chromatography of the ethanol and The fresh leaves of wildly growing plant C. indica chloroform extracts was carried out in various solvents at 30°C using silica gel G as adsorbent and the R f were collected from the field areas of eastern Uttar 34 Pradesh region during the month of February, 2009. values were determined . The same mobile phase was For identification and taxonomic authentication, used for the HPTLC profiles of these extracts Every 33-35 sample of plant material was given to National part of this plant is valuable in medicine . Botanical Research Institute (NBRI), Lucknow, India which confirmed the authenticity of plant specimen. Results

(voucher specimen no. NBRI-SOP-202 receipt no. Macroscopic/morphological characters and date 19/72, 24-02-09). The fresh leaves were used It is climbing perennial herbs with tuberous for the study of macroscopic and anatomical rootstock producing annual stems up to several meters characters. Collected plants were shade-dried and long, which is found spreading on ground and twilling coarsely powdered. This coarse powder was used for around the trees and supports around it (Plate 1). the determination of ash values, extractive values. Leaves are triangular or pentagonal in shape. Margin Preliminary phytochemical investigation was done as is dentate, upper surface glabrous and attachment of per standard methods. and major vein branching occurs. Apex Preparation of leaves extract obtuse, petioles 1-3 cm long and tendrils are Air dried leaves (100 g) coarse powder was packed unbranched. Flowers monoecious, solitary, rarely in in four pouches of muslin cloth and subjected to axillary clusters of 2-3, pedicels 15-50 mm long, Soxhlet extractor for continuous hot extraction with corolla lobes white, ovate, hypanthium 10-15 mm distilled water, ethanol, petroleum ether and long. Fruit is slimy in touch, pulpy and ovoid to chloroform for 8 h, separately. Each extracts were ellipsoid shaped. It is green in color when young filtered and filtrate was evaporated to dryness. The which turns to scarlet red when it ripes, 2.5-5 cm long percentage yield of the water, ethanol, petroleum and 1.3-2.5 cm in diam., glabrous, pulp red. The fruit ether and chloroform extracts was 23.8, 7.03, 2.45 and possesses numerous seeds which are oblong, 6-7 mm 4.15%, respectively. long, margins thickened 36,37 .

Macroscopic and microscopic studies Leaves are simple, subflashy, alternate, ovate, The macromorphology of the leaves were studied palmately 3 to 5 lobed with obtuse apex, ranging from according to standard methods 25-27 . For anatomical 5-8 cm long and 3-6.5 cm wide. It shows reticulate studies hand section of the was taken, stained and venation with glabrous surface, dentate margin and mounted following usual microtechniques 28 and cordate base. Petiole stout, cylindrical, smooth, 1.2- representative diagrams were taken with the help of 3.0 cm long, slightly flashy. The leaves have bright inverted microscope for photodocumentation (Leitz, green upper surface and pale-green underneath, with Japan). characteristic odour and astringent taste.

Physicochemical analysis Microscopic characters Physicochemical analysis i.e. alcohol (90% ethanol) and water soluble extractive values, fluorescent Midrib region analysis, total ash, acid-insoluble ash, water-soluble Microscopical study shown, transverse section ash, swelling indices and foreign matter was done as passing through midrib protrudes at the lower side 76 INDIAN J NAT PROD RESOUR, MARCH 2011

Plate 1 ―Coccinia indica - a. Whole plant; b. Leaves; c. Flower; d. Fruit and flat at the upper side. Single layered upper and layered collenchymatous and 2-6 layered circular, thin lower epidermis was observed with straight walls. walled, chlorenchymatous cells with intracellular Epidermis was covered with thick cuticle bearing few spaces were observed. Bicollateral vascular bundles short glandular trichomes. Below the upper epidermis were arranged in a single ring. Some bundles were single layered palisade cells were observed with capped by one or two layered, thick walled, lignified, spongy mesophyll cells and in between were the polygonal pericyclic sclerenchyma. In centre very xylem and phloem vessels. Xylem and phloem are wide pith was observed which was composed of large arranged in ring. Xylem ring present towards the parenchymatous cells (Plate 3). center and is surrounded by phloem ring. Small collenchymatous patch lied under the upper epidermis Physico-chemical parameters and 1-3 layers of well developed collenchymatous The physicochemical characters of powdered cells were on the lower side. Vascular bundles were leaves of C. indica such as total alcohol soluble semicircular, vessels arranged in radial rows, extractive, water soluble extractive, ash value, acid bicollateral, three, one centrally located was bigger in insoluble ash, water-soluble ash, loss on drying, size, and two lying under the upper epidermis were swelling index and foreign matter are presented in smaller in size (Plate 2). Table 1. The fluorescence analysis of the powdered drug of C. indica in various solvents and chemical Petiole reagents was performed under normal and Ultra Transverse section of petiole shows single layered Violet (UV) light (Table 2). The pH of 1% solution epidermis, consisting of flattened, elongated cells and 10% solution of powdered drugs was reported as with covering of cuticle. Under the epidermis 2-5 7.23 and 6.98, respectively. HUSSAIN et al : STUDIES ON LEAVES OF COCCINIA INDICA 77

Plate 2 ―T.S . of Coccinia indica leaf through midrib with lamina

Plate 3 ―T.S. of Coccinia indica leaf passing through petiole Table 1 ―Physico-chemical parameters of leaves of Ethyl acetate (8.5:1.5) as mobile phase, respectively Coccinia indica and the Rf values were recorded (Table 4). The

S. No. Quantitative parameter Values obtained (%) w/w visualizing reagent employed was anisaldehyde- 1 Alcohol soluble extractive 8.4 sulphuric acid reagent to effect visualization of the 2 Water soluble extractive 24.60 resolved spots (Figure 1). TLC and HPTLC finger 3 Total ash 15.33 printing studies on ethanol extract showed presence 4 Acid-insoluble ash 1.683 of various phytoconstituents with their respective Rf 5 Water-soluble ash 8.83 values. The ethanolic extract was developed on 6 Swelling index 2.0 chromatographic plates with many ratios of different 7 Loss on drying 10.27 8 Foreign matter content 1.2 solvents and the best eluent mixture was used further for HPTLC profile to minimize errors in TLC Preliminary phytochemical screening The preliminary phytochemical investigation of the pattern. The preliminary HPTLC studies revealed that the solvent system Toluene:Ethyl acetate aqueous, ethanol, petroleum ether and chloroform (8.5:1.5) was ideal and gave well resolved sample extracts of leaves showed the presence of phytosterols, peaks. The spots of the chromatogram were flavonoids, terpenoid saponins, carbohydrates, tannins, visualized at 366 nm (Figure 2). glycosides alkaloids proteins organic acids (Table 3).

Thin layer chromatography and high performance thin layer Discussion chromatography (HPTLC) As a part of standardization study, the Thin layer chromatography of the ethanol and macroscopical examination of leaf was studied. chloroform extracts was carried out using Toluene: Macroscopical evaluation is a technique of qualitative 78 INDIAN J NAT PROD RESOUR, MARCH 2011

Table 2 ―Fluorescence analysis of powdered leaf of Coccinia indica

S. Solvent used Observation under No. UV light (254 nm) UV light (366 nm) 1 NaOH in methanol Light green Yellowish brown 2 NaOH in water Light green Dark brown 3 Benzene Fluorescent green Reddish brown 4 Acetone Yellowish green Orange 5 Ethyl acetate Yellowish green Orange 6 Chloroform Light green Creemish green 7 Distilled water Light green Blackish brown 8 Dil. HNO 3 Light green Bluish green 9 Dil.H 2SO 4 Light green Dark green 10 Conc. HCL Yellowish green Yellowish brown Table 3 ―Qualitative analysis of phytochemicals in Coccinia indica leaf extracts

Extracts Pet. ether Chloroform Ethanolic Aqueous Sterols + + + - Tannins - + + - Flavonoids - - + + Fig. 1 ―TLC finger printing of ethanolic and chloroform extract Proteins and amino - + + + of leaf of Coccinia indica acid - + + + Glycosides - - + + Phenols - - - - Acidic compounds - - + + Carbohydrates - + + - Saponins - + + + Alkaloids + Present - Absent Table 4 ―Thin layer chromatography of leaf extracts of Coccinia indica

S. Test extract Solvent system Number of spots Rf value No. 1 Chloroform Toluene: 6 0.07, extract Ethylacetate 0.15, (8.5:1.5) 0.31, 0.69, 0.76, 0.99

2 Ethanol Toluene: 5 0.07, extract Ethylacetate 0.15, (8.5: 1.5) 0.72, Fig. 2 ―HPTLC fingerprinting of ethanolic extract of leaf of 0.79, Coccinia indica scanned at wavelength 366 nm 0.99 extractives in different solvents indicated the quantity evaluation based on the study of morphological and and nature of constituents in the extracts. The sensory profiles of drugs. The macroscopical extractive values are also helpful in estimation of characters of leaf of plant can serve as diagnostic specific constituents soluble in particular solvent. The parameters. The extractive value, ash value, loss on fluorescence analysis of the powdered drug from the drying and fluorescent analysis of the leaf extracts leaves of C. indica in various solvents was performed have been carried out. The results showed greater under normal and UV light. All the leaf extracts extractive values in hot extraction, indicating are examined in short UV (254 nm) and long UV the effect of elevated temperature on extraction. (366 nm) to detect the fluorescent compounds. Thin Percentages of the extractive values were layer chromatography (TLC) is particularly valuable calculated with reference to air-dried drug. The percent for the preliminary separation and determination of HUSSAIN et al : STUDIES ON LEAVES OF COCCINIA INDICA 79

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