148 | P a g e International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 5(1): January-February 2016 INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093*** ICV 5.13*** Pharmaceutical Sciences RESEARCH ARTICLE …………!!!

MICROMORPHOLOGICAL, MICROMETRIC INCLUDING HISTOCHEMICAL EVALUATION OF LEAF MADHAVILATA ( BENGHALENSIS) LINN. (KURZ) Madhavi Patel1, Harisha C.R2. 1Lecturer, Dravyaguna Department, Indian institute of Ayurvedic pharmaceutical sciences, Gujarat Ayurved University, Jamnagar, Gujarat, . 2Head, of Pharmacognosy, I.P.G.T. & R.A., Gujarat Ayurved University, Jamnagar, Gujarat, India. ABSTRACT KEYWORDS: Linn. (Kurz). belonging to family Hiptage benghalensis . Commonly known as Madhavilata is a used by Linn. (Kurz.), Leaf, the indigenous people for the treatment of wound and obesity in Madhavilata, Malpighiaceae. India. Till date there is no pharmacognostical scientific work has For Correspondence: been done properly on its leaves. In the present study detailed Madhavi Patel * pharmacognostical evaluation has been done. Results showed that Address: Lecturer, Dravyaguna presence of rosette, cluster crystals and starch grains in , Department, Indian single layer of palisade parenchyma, annular, pitted, spiral vessels in institute of Ayurvedic leaf, stomatal index i.e. 39 in lower epidermis. Powder microscopy pharmaceutical sciences, Gujarat Ayurved of sun dried powder showed cluster and rosette crystals, tannin University, Jamnagar, content and oil globules. Gujarat, India.

Full Text Available On www.ijupbs.com

149 | P a g e International Standard Serial Number (ISSN): 2319-8141

INTRODUCTION: The exploration of traditional knowledge for cures to common diseases is attractive, but also overwhelming. Hiptage benghalensis Linn. (Kurz). commonly known as ‘Madhavilata’ is an important medicinal plant distributed throughout India, predominantly in Western ghats, Konkan, Deccan, Kumaon, Assam, Nepal and Andaman island, chiefly in damp places up to an altitude of 2000m[1,2]. It is a stout, high-climbing or large , with white or yellowish hairs on the stem. Its leaves are lanceolate to ovate-lanceolate and approximately 20 cm (8 in) long, and 9 cm (4 in) broad; petioles are up to 1 cm long. It has scandent branches up to 5 m (16 ft) high. Flowers produce fragrant borne in compact ten-to-thirty- flowered axillary . The flowers are pink to white, with yellow marks. Fruits are samaras with three spreading, papery oblanceolate to elliptic wings, 2–5 cm long, and propagate via wind or by cuttings[3,4]. The leaves of H. benghalensis Linn. (Kurz) are used in treating skin diseases in Burma. In India, it is widely used to treat cough, asthma, leprosy and also to quench thirst[5,6]. Although there are some pharmacognostical studies carried out by previous scholars regarding different parts of the plant. But those studies were mainly focused on morphological and microscopical aspect of the plant in general terms. In the present study of micrometric evaluation was very much encouraging and rationale in its own term. MATERIAL AND METHODS: Collection & authentication of raw drug: The fresh leaf of. H. benghalensis Linn. (Kurz).collected from natural habitat at Indian Institute of Ayurvedic Pharmaceutical Sciences, Gujarat Ayurved University, Jamnagar, Gujarat. Plant identification The correct identity of the and its family were confirmed by studying its morphological characters and authenticated by comparing them with the characters mentioned in various floras and text book of botany and by subject expert. Collected samples were made into herbarium wide no. PHM. 6144/15 preserved in Pharmacognosy department, I.P.G.T. & R.A., Gujarat Ayurved University. Processing and preservation Genuine and good quality materials, required for the present study, free from any filth and worm infection were cut and separated, washed, dried in sun light, shade and stored in air

Full Text Available On www.ijupbs.com

150 | P a g e International Standard Serial Number (ISSN): 2319-8141 tight dried container. Coarse powder of sufficient quantity was prepared and stored in air tight container. The powder was prepared for powder microscopy. Pharmacognostical analysis: Morphological : Under the Morphological study, the basic structure; the types of formation of a particular organ & their arrangement in different parts of H. benghalensis Linn. (Kurz). leaf was studied in detail. These observations were noted properly and then matched with the data available in taxonomic book & with the flora[2,7 ]. Macroscopic: The collected sample was identified and authentified by studying their characters were studied systematically as per the methods described in the textbooks of Pharmacognosy. The specimen was observed as such with necked eyes. The samples were cleaned properly and macroscopic study of the different leaf parts was carried out. The individual character of the leaf like petiole and their macroscopic characters like size, shape, and colour were noted in detail [8-9]. (plate 1b) Organoleptic: Evaluation of the raw sample was done by their various characters like, colour, texture, odour, taste etc.[7] Microscopic: Free hand sections of the plant material observed under distilled water under the microscope for the presence of primary and secondary metabolites, like starch grains which were confirmed by staining them with iodine. The sections cleared with chloral hydrate to observe the various ergastic cell contents like, crystals of calcium oxalate, calcium carbonate, and silica if present any. The natures of these crystals were also confirmed by performing some tests like, solubility of them in acids (HCl). The sections then stained with Phloroglucinol and HCl for detecting lignified elements like fibres, sclereids, xylem vessels, tracheids etc Repeat the same method for the powder samples [9]. Histochemical evaluation: Sample thick sections subjected to Histochemical tests to find starch grains, tannin, calcium etc. by treating various reagents[8]. Leaf surface preparation To make thin leaf transparent, cut a pieces of leaf into two portions, turn one piece upside down, add a few drops of chloral hydrate, and heat to boil directly on a slide. As soon as

Full Text Available On www.ijupbs.com

151 | P a g e International Standard Serial Number (ISSN): 2319-8141 bubbles start to escape, remove the slide from the flame. When bubbles stop to appear, heat until the fragments are transparent. Add 1 or 2 drops of chloral hydrate solution, apply cover glass and observe under microscope. For thicker leaves that do not become transparent easily when prepared by this method clear by boiling with strong chloral hydrate solution in test tube, transfer a fragment onto a slide, cut into two portions, scrape the surface of the fragment by scalpel. Wash the epidermis by chloral hydrate solution. add 1 or 2 drops of chloral hydrate solution, cover it with a cover glass and observe under microscope[9]. Powder characters study method: For powder microscopy, slides were prepared by using water, chloral hydrate as a clearing agent, stained with phloroglucinol and conc. HCl for lignified tissues, iodine for starch grains and glycerin as mountant[8]. Micrometric evaluation: Under the micrometric study, the measurement of a particular character or a particular band/zone which was made up of similar kind of tissues/cells was taken into consideration, which was otherwise not possible to make them accountable aided with our naked eyes. These characters are microscopic in nature & when they are labelled with their measurement, those characters can be very effective tools to standardize a particular drug due to their specificity & preciseness[9]. RESULTS Macroscopic study Under the macroscopic study shape, size & color of the different parts of the leaf was measured & noted appropriately. Leaf is simple and the shape of the leaf was lanceolate to ovate. Margin entire, slightly wavy, apex acuminate, upper surface of leaf is dark green and glossy while lower surface is light in colour, venation alternate, veinlet is net like structure. Then the observations were documented in table no.1. Macroscopic study has been demonstrated in the fig (plate 1a-c). Microscopic study Different parts of the leaf like petiol, leaf, midrib were separated then transverse section of each individual part was studied thoroughly. The results obtained from transverse sections of various parts of the leaf were described below.

Full Text Available On www.ijupbs.com

152 | P a g e International Standard Serial Number (ISSN): 2319-8141

Transverse section of Petiol : Petiol had multicellular, warty, covering trichome measuring 4.5mm in length & 0.5mm in width. Barrel shaped unicellular epidermis with thin cuticle. Epidemis was interrupted by both warty trichome, rarely interrupted by stomata. The total area of petiol was of 7X8mm in 40X. Rossate and cluster crystals of calcium oxalate embedded in cortical region and in epidermal layer. Prismatic crystal of calcium oxalate rarely seen in epidermal layer of the petiol. Below the epidermal layer single endodermal layer rich in starch grains. Cortex is embedded beneath the endodermis 2X2 mm in 40X. Two meristele with one major vascular bundle. 6X4mm in 40X Vascular bundle consists of radially arranged pericyclic fibre, parenchyma of xylem, uniserriate medullary rays. (plate. 2a,b) Localization: Tannin- epidermis, pericyclic fibre and some of the cortical cells. Starch grain- endodermis – simple starch grains. Transverse section of Leaf : Midrib Tangentially elongated, single layered, barrel shaped, upper epidermis with thick cuticle, measuring of 0.9X0.4mm in 40X, some of the lower epidermal cells interrupted by stomata. Size of the lower epidermis was of 0.7X0.4mm in 40X, covering with thick cuticle. The lower epidermal cells were smaller than the upper one. Mesophyll cells diffrernciated in to upper single layer celled parenchyma along with some of the cluster and rosette crystal. At the end of parenchymatous layers, a distinctive band of vascular bundles were present. This band of vascular system was arranged in a semi lunar form. (plate 3 a,b) Lower and Upper surface Thin upper and lower surface prepared by simple peeling method the lower epidermis shows the numerous stomata, where as upper epidermis devote of stomata, wavy, had beaded parenchyma stomata mainly of paracytic, epidermal cells, prismatic and rosette crystals of calcium oxalate. (plate 4 a-c) Powder microscopy Orgenoleptic charecters: Green colour, characteristic odour, sweet, sour and astringent in taste with fine texture. Diagnostic characters of the leaf are stomata in surface view, cut fragments of trichome, oil globules, fibres, cut fragments of epidermis, rosette and cluster crystals of calcium oxalate, annular, pitted and spiral vessels, tannin containing cells. (plate 5 a-h)

Full Text Available On www.ijupbs.com

153 | P a g e International Standard Serial Number (ISSN): 2319-8141

Micrometric analysis: Stomatal length, breadth, stomatal index and other constituents were scientifically measured (3 successive readings) the mean value taken into consideration and results depleted in table no. 2. Histochemical tests: Various Histochemical tests were conducted on sections of the leaf. The results are depleted in table no. 3. CONCLUSION: Pharmacognostical work reveals that petiole shows large number of cluster crystals. Leaf T.S showed 10-12 layers of palisade parenchyma cells, cluster crystals. Surface study shows paracytic stomata. The values extracted from micrometric studies were scientific and constant. Leaf section and its powder shows the presence of calcium oxalate crystals, large amount of tannin and oil helpful in the treatment of cuts and wounds may be considered for further research works. REFFRENCES: 1. The Ayurvedic pharmacopoeia of India, (2008), Ministary of Health and Family welfare, Department of Ayurved, Yoga & Naturopathy, Unani, Siddha and Homeopathy, New Delhi, part I, vol. VI, 106-107. 2. J.S Gamble, Flora of the presidency of Madras; Secretary of state for India in council. Londan, vol. 1, 127- 128. 3. Bailey, L. H. and E. Z. Bailey. 1976. Hortus. 3rd ed. Hortus third: A concise dictionary of cultivated in the United States and Canada. Macmillan, New York, pp. 1290. 4. Whistler, W.A, 2000, Tropical Ornamentals. Timber Press, Portland. 5. P.C Sharma, M.B. Yelne & T.J.Dennis, Database on Medicinal plants used in Ayurveda, CCRAS, Vol. 3, 398-403. 6. Meenaa Venkataramani and Sasikumar Chinnagounder (2012) Preliminary phytochemical screening and GC-MS profiling of Hiptage benghalensis (L.) kurz, Journal of Pharmacy Research,5(5),2895-2899. nd 7. Edmund N Gathercoal, Elmer H Wirth. Pharmacognosy. 2 ed. Philadelphia: Lea & Febiger; 1949. p. 34. 8. Khandelwal KR. Practical Pharmacognosy. Pune: Nirali Prakashan; 2008. p. 161. 9. Trease and Evans, Pharmacognosy, 15th Ed., W.B. Sunders Company Ltd. 1996 p.569,570.

Full Text Available On www.ijupbs.com

154 | P a g e International Standard Serial Number (ISSN): 2319-8141

TABLE 1: MORPHOLOGICAL STUDY OF LEAF Sr. Characters Measurement no. 1 Leaf Length: 22X18 cm Breadth: 7.5X9 cm 2 Petiol 0.7 cm 3 Main nerves 7 pairs TABLE 2: MICROMETERIC ANALYSIS OF LEAF Sr. Characters Lower epidermis Upper epidermis No.

1. Stomata length 5μm --

2. Stomata breadth 3 μm --

3. Stomata radius 68.1μm --

4. Stomatal Index 39 --

5. Pallisade ratio -- 1-4

6. Epidermal cells 0.7X0.4mm 0.9X0.6mm

TABLE 3: HISTOCHEMICAL TESTS OF LEAF Sr. no Reagent Observation Characteristics Result 1. Phloroglucinol+Conc. Red Lignified cells + HCl 2. Iodine Blue Starch grains + 3. Phloroglucinol+Conc. Dissolved Ca Ox - crystals + HCl

4. Fecl3 solution Dark blue Tannin cells + 5. Ruthenium red Red Mucilage - 6. Sudan III Red Oil globule +

7. Hcl Effervesces Ca Co3 +

8. H2SO4 Dissolved Silica - 9. Iodine Reflecting Aluerone grains -

Full Text Available On www.ijupbs.com

155 | P a g e International Standard Serial Number (ISSN): 2319-8141

Plate 1: Natural Habitat, leaf and its powder- Hiptage benghalensis Linn. (Kurz).

a. Natural habitat b. Leaf c. Powder of leaf Plate 2 T.S. of Petiole- Hiptage benghalensis Linn. (Kurz).

a. Diagrammatic T.S. of Petiole b. Stained T.S. Of Petiole

Full Text Available On www.ijupbs.com

156 | P a g e International Standard Serial Number (ISSN): 2319-8141

Plate 3: T.S. of Leaf through midrib - Hiptage benghalensis Linn. (Kurz).

a. Diagrammatic T.S. of Midrib b. Stained T.S. of Midrib

Plate 4 : Surface study - Hiptage benghalensis Linn. (Kurz).

a. Upper epidermis in b. Lower epidermis c. Upper epidermis surface view

Full Text Available On www.ijupbs.com

157 | P a g e International Standard Serial Number (ISSN): 2319-8141

Plate 5: Powder characters - Hiptage benghalensis Linn. (Kurz).

a. Rosette crystal b. Cluster crystal c. Cut fragment of fibre

d. Fragment of lower e. Pitted vessels f. Sclerid epidermis

g. Trichome h. Paracytic stomata

Full Text Available On www.ijupbs.com