Sayani Majumder et al. IRJP 2013, 4 (1)

Page 212

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

PHARMACOGNOSTICAL AND PHYTOCHEMICAL INVESTIGATION OF

IPOMOEA SEPIARIA KOENIG EX. ROXB. LEAF Sayani Majumder*, Nishteshwar K

Department of Dravyaguna, Institute for Post Graduate Teaching & Research in Ayurveda , Gujarat Ayurved University, Jamnagar, India

Article Received on: 04/11/12 Revised on: 12/12/12 Approved for publication: 31/12/12

*E-mail- sayani1988sayani@gmail.com ABSTRACT Ipomoea sepiaria Koenig ex. Roxb. of the family Convolvulaceae is a perennial climber growing on the bank of streams, rivers, specially over hedges. Traditionally the whole herb is to possess number of therapeutic properties like, aphrodisiac, a good antidote to arsenic, rejuvenating, diuretic, aphrodisiac, laxative and also used in sterility in women and in diabetes. As the plant is not explored scientifically as yet, it was thought worth to evaluate each and every part of the plant thoroughly and the leaves being first selected for this. Present study deals with detailed macroscopy, qualitative & quantitative microscopy also includes its powder microscopy and its Chromatographic evaluations of leaf. Important diagnostic characters like, cluster crystals, starch grains, pitted vessels, laticiferous cells etc were observed. Physicochemical studies revealed loss on drying, total ash, etc. Preliminary analysis revealed the presence of various functional groups like alkaloids, saponins, phenols and resin. TLC & HPTLC study of the alcoholic, pet. ether and chloroform extract showed the presence of spots in short & long UV and after spraying developing reagent, respectively. The information generated by this particular study will provide relevant pharmacognostical and physicochemical data needed for proper identification and authentication of leaves of this particular species. KEYWORDS: Ipomoea sepiaria Koenig ex. Roxb., Lakhmana, Resin, Laticiferous cells, physicochemical study. INTRODUCTION Folk medicine often coexists with formalized, education-based, and institutionalized systems of healing such as Western medicine or Great traditional medicine systems like Ayurvedic, Unani medicine, and Chinese medicine, but is distinguishable from formalized or institutionalized healing systems1. I. sepiaria known as Lakshmana in Sanskrit2 is a herb known to possess good antidote to arsenic, uterine tonic, aphrodisiac, cures ‘tridosa’ and ulcers3, diuretic, aphrodisiac & tonic. It is also used in case of burning sensation, hyperdipsia, general debility & sterility in women4, laxative5 and diabetes6. The root powder in the dose of 1 teaspoon is administered with rice water for leucorrhoea (Nalgonda, Mehaboobnagar District of A.P.)7. In traditional practice the juice of the leaf is installed in the right nostril during 2nd and 3rd months of ante- natal period to the pregnant women for begetting male progeny known as “Pumsavana Karma” mentioned in Ayurvedic Classics. It is a glabrous or occasionally pubescent or hirsute, slender twinning with a slightly thickened or tuberous perennial root and very short stem producing annually or seasonally a number of terete villous, grayish purple branches bearing simple, cordate or ovate, variable median sized leaves, very often blotches with dull purplish patches in the centre and pink to purplish flowers in clusters on fairy long thickened clavate peduncles2,

8. As all the parts of the plant are medicinally important including leaf, though the present investigation is carried out to establish certain botanical and proximate chemical standards of leaf of I. sepiaria, which would help in crude drug identification as well as in checking adulteration, if any9. In the present study the pharmacognostical study,

HPTLC and phytochemical investigation of the leaf of I. sepiaria was carried out. MATERIAL & METHOD The plant was collected from the campus of GAU, Jamnagar, Gujarat in the month of December and was washed thoroughly with running water to remove adherent soil, dirt etc and dried in shade. Leaves were cut into 1to 2cm long pieces, few were preserved in FAA (Formalin9: Aceti acid7: Alcohol 3)10 for microscopic investigation. Free hand sections were taken from the preserved materials of the leaf and observed as such under the microscope for the presence of ergastic cell contents and other contents10. The section was cleared with chloral hydrate and then was stained with phloroglucinol and hydrochloric acid to observe the lignifications of the cell wall if any10. The histochemical tests were also performed to detect the location of various cell contents by using various reagents11. Photographs of sections and diagnostic characters of the powders were taken by using Swift Ives Camera Lucida. The methanolic, petroleum ether & chloroform extract was also subjected for Chromatography for separation and confirmation of chemical compounds by using various solvents and spraying reagents under visible and UV light of long and short wavelengths by following standard procedures12. Physicochemical constants13,14,15 and phytochemical analysis16,11 also carried out from shade-dried powder. The phytochemical screening was done with the methanolic and water extract of the leaf. Botanical identification was carried out by the Botanical Survey of India, Office of the Scientist-‘F’, Central National Herbarium, Botanic Garden, Howrah, West Bengal specimen No.- CNH/104/2011/Tech.II/581.

Table-1: Organoleptic characteristics of I. sepiaria Koenig ex. Roxb. leaf

Organoleptic characters Descriptions Colour Upper surface dark green and lower is pale green Odour Characteristic Taste Sharp mucilaginous

Texture Thin Touch Smooth Surface Glabrous, Shiny

Sayani Majumder et al. IRJP 2013, 4 (1)

Page 213

Table-2: Physico-chemical constant of of I. sepiaria Koenig ex. Roxb. leaf powder Tests Value

Loss on drying 10.07% w/w Ash value 14.38% w/w

Acid insoluble ash 1.45% w/w Water insoluble ash 4.35% w/w

Water soluble extractive 4.60% w/w Methanol soluble extractive 5.36% w/w

pH value 5.5

Table-3: Preliminary quantitative analysis of Ipomoea sepiariaKoenig ex. Roxb. leaf powder for the presence of various functional group Material Reagent/ Test Functional group Observation Result

Methanolic of leaf

Kellarkiliani Carbohydrates Reddish brown ring Present Fehling’s solution Carbohydrates Red ppt Present

Dragendroff’s reagent Alkaloids Orange brown ppt Present Legal’s test Glycosides Pink Present

Brontrager’s test Glycosides Pink Present Alkaline reagent test Flavonoids Colourless Present

FeCl3 Tannin Green Present Lead acetate test Tannin Presemt

Ammonia test Phenolic compounds Yellow Present Heat test Proteins No coagulation Absent

Biuret test Proteins No violet colour Absent Ninhydrine test Amino acid Present Salkowski test Steroids &terpenoids Red colour Present

Water extract of leaf

Resin test Resin White ppt Present Froth formation test Saponin Forth formation Present

Table-4: TLC analysis of Ipomoea sepiaria Koenig ex. Roxb. leaf powder Solvent system- Toluene: Ethyl acetate: Glacial acetic acid= 6.5:3.5:0.2

Condition Extract No. of spots Rf values 254 nm Methanol 2 0.33, 0.88

Pet. Ether 2 0.88, 0.94 Chloroform 1 0.90

366 nm Methanol 7 0.31, 0.42, 0.65, 0.69, 0.72, 0.79, 0.86 Pet. Ether 2 0.73, 0.79

Chloroform 3 0.74, 0.79, 0.88 Spraying with Anisaldehyde

sulfuric acid Methanol 7 0.15, 0.56, 0.60, 0.66, 0.72, 0.79, 0.95 Pet. Ether 6 0.63, 0.68, 0.72, 0.79, 0.87, 0.96

Chloroform 5 0.60, 0.68, 0.72, 0.79, 0.97

Fig 1 - Natural habitat

Fig2- Morphology of leaf

Fig 3 – Petiole of leaf

Fig 4 – Diagrammatic T.S of midrib

Fig 5– Diagrammatic T.S of lamina

Fig 6 – Diagrammatic T.S of petiole

Sayani Majumder et al. IRJP 2013, 4 (1)

Page 214

Fig 7– T.S of midrib showing lower epidermis and cortex cells

Fig 8– T.S of petiole showing centrally arc shaped vascular bundle.

Fig 9 – Lower epidermis of leaf

Fig 10 – Upper epidermis of leaf

Fig 11– Leaf epidermis showing sessile glandular trichome

Fig 12– Leaf epidermis showing stomata with striated cuticle

Fig 13– T.S of leaf showing sessile glandular trichome

Fig 14– T.S of leaf showing lactiferous cells

Fig 15– Leaf epidermis showing colouring matter

Fig 16– T.S of leaf showing enlarge view of xylem and phloem stained with

phloroglucinol+HCl

Fig 17– Epidrermal cell in surface view

Fig 18– Epidermal cells with striated cuticle and stomata

Fig 19– Sessile Glandular trichomes

Fig 20– Spiral vessel stained with phloroglucinol + HCl

Fig 21– Cluster crystalls embeded in parenchyma cells

Sayani Majumder et al. IRJP 2013, 4 (1)

Page 215

Fig 22– Starch grains stained with iodine

HPTLC PROFILE: Densitograms (visible spectrum comparison)

Track-1HPTLC for Methanolic extract of leaf of I. sepiaria Track-2 HPTLC for Chloroform extract of leaf of I. sepiaria

Track-3 HPTLC for Petrolium ether extract of leaf of I. sepiaria

Track 3(254 nm)

Track 1(254 nm)

Track 2(254 nm)

Track 1(366 nm)

Track 2(366 nm)

Track 3(366 nm)

Track 1 (After

spraying with Vanillin

HCl)

Track 2 (After

spraying with Vanillin

HCl)

Track 3 (After

spraying with Vanillin

HCl)

Fig 23- 254nm peak display Tract-1

Fig 24- 254nm peak display Tract-2

Fig 25- 254nm peak display Tract-3

Fig 26- 366nm peak display Tract-1

Fig 27- 366nm peak display Tract-2

Fig 28- 366nm peak display Tract-3

Sayani Majumder et al. IRJP 2013, 4 (1)

Page 216

Fig 29-Multiple Tracks (254 nm)

Fig 30- Multiple Tracks (366 nm)

Fig 31- U.V. Spectral comparison Rf 0. 76 of T-1, 2, 3

Fig 32- U.V. Spectral comparison Rf 0.91 of T-1, 2, 3

RESULT & DISCUSSION: Morphology: I. sepiaria is a climber. The leaves of the plant are thin and 4 to 6 cm long with 5 to 8 cm width. The leaves contain 2 to 5 cm long and 0.1 cm thick petiole. The leaf is alternate with acute, cordate & rounded basal lobes. The margin is repand or undulate with cordate which is sometimes hastate lamina. It has reticulate unicostate venation. It contains blotches with brownish or blackish patches towards the centre. Fresh leaf while cutting, latex is coming out. Occasionally the plant is having brownish leaf margin with various shape and size (Fig-1, 2, 3). The organoleptic characteristics of leaf are shown in Table-1. Microscopy: Leaf Diagrammatic T.S of the leaf passing through the midrib is broadly convex on the lower side and showing a notch at its upper side, showing an arc of centrally located meristele and with laterally extended dorsiventral lamina on its either sides. Detailed T.S shows, single layered epidermii, at both the sides, covered with thick striated cuticle and traversed with stomata and sessile glandular trichomes which are seen more in lower epidermis at places. In midrib portion underneath the lower epidermis lies , 5-6 rows of thick walled collenchymatous band without intercellular spaces; centrally located bicollateral vascular bundle composed of radial arrangement of xylem covered with narrow phloem at both the sides; 1-2 rows of palisade cells lies underneath the upper epidermis of mesophyll, followed by 4-5 layered, spongy parenchyma, with obliquely cutted vascular bundles cluster crystals of calcium oxalate observed especially underneath

both the epidermii and in the phloem region. The stomata are generally paracytic rarely anisocytic. The simple and compound starch grains and laticiferous cells are also seen throughout the section (Fig-4, 5). Petiole Diagrammatic T.S. of the petiole is oval in outline with two lateral winged margin on its upper side, shows cuticularised epidermis, collenchymatous hypodermis and centrally located arc shaped meristele. Detailed T.S of the petiole showed, single layered thick striated cuticularised epidermis at places being papillosed and transverse with stomata and trichomes following with 3-4 layers of thick walled, which being filled occasionally with purplish colouring matter and also embedded with cluster crystals of calcium oxalate; centrally shows an arc shaped bicollateral types of vascular bundle. Two lateral vascular bundles are also observed in parenchymatous ground tissue. Cluster crystals mostly found surrounded to vascular bundle and in the region of hypodermis (Fig-6-16). Powder microscopy: The powder microscopy of the leaf shows spiral vessel, sessile glandular trichomes, cluster crystals and starch. The fragments of stomata with straighted cuticle and surface of epidermal cells are found (Fig-17-22). Physicochemical study of all the ingredients was carried out by using various physiochemical parameters as mentioned in Ayurvedic Pharmacopoeia of India. The results are shown in Table-2. Phytochemical analysis for the presence of various functional groups was carried out on the methanol & water soluble extractive. The results are shown in Table-3.

Sayani Majumder et al. IRJP 2013, 4 (1)

Page 217

In HPTLC analysis, at short UV 254 nm, different spots was found in all three extracts i.e. methanol extract, chloroform extract and petroleum ether extract [Table-4, Fig-23, 24, 25 & 29] which indicates the presence of different phyto-components. At long UV 366 nm, methanol extract, chloroform extract and petroleum ether extract showed 7, 9 and 5 spots respectively [Table-3, Fig- 26, 27, 28 & 30]. After spraying with Vanillin sulfuric acid different spots were also seen. Presence of common Rf values 0.91, 0. 76 in all three samples, indicate the presence of common components in all three extracts [Fig-31, 32]. CONCLUSION: The presence of Pharmacognostical characters such as spiral vessel, sessile glandular trichomes, cluster crystals, simple & compound starch grains and fragments of stomata with straighted cuticle can serve as important diagnostic characters of the drug I. sepiaria leaf. The results of preliminary phytochemical and chromatographic study of the leaf showed the presence of saponins, glycosides, resins, flavonoids, alkaloids, tannins and other phenolic compounds. ACKNOWLEDGEMENTS: We acknowledged to the J.N. Pandey, Principal of Bengal Institute of Pharmaceutical Sciences (BIPS), a joint Venture Institute with government of West Bengal & to the Director, IPGT&RA, Gujarat Ayurved University, India for giving the necessary permission to carry out the research work in the Institute laboratories.

REFERENCES: 1. Barnerjee BG. Folk Illness and Ethnomedicine. New Delhi: Northern

Book Center; 1988. p.19. 2. AiyerK N, Namboodiri AN, Kolammal M. Pharmacognosy of

Ayurvedic Drugs. Series-I. Number-3. The Central Research Institute: University of Travancore; 1957. P. 57-63.

3. Kirtikar KR, Basu BD. Indian medicinal plant. vol.3.1960.p.1702-1712 4. Prajapati ND, Purohit SS, Sharma AK, Kumar T. A hannbook of Indian

Medicinal Plants, A complete Source Book. India: Agrobios; 2003 (Reprinted 2010). p. 291-292.

5. Venkataswamy R, Mubarack HM et al. Ethnobotanical Study of Medicinal plants used by Malasartribals in Coimbatore District of Tamil Nadu (South India). Vol 1 (2 ), ASIAN J. EXP. BIOL. SCI.Coimbatore, Tamilnadu 2010; 387 – 392.

6. Jain SK. Dictionary of Indian Folk Medicine and Ethnobotany. Deep Publication; 1991. p. 108.

7. NishteswarK. Herbs in Vasavarajeeyam. Delhi: Chaukhamba Sanskrit Pratishthan; 2003. p. 86.

8. Duthie JF. Flora of Upper Gangetic Plant and of the Adjacent Slwalik & Sub-Himalayan Tracts. Vol-II. Dehradun: Bishen Singh Mahendra Pal Singa; 1994. p. 114.

9. Rastogi PR, Melhotra BN. Compendium of Indian Medicinal Plant. Vol 3. Lucknow: Central drug research institute; 1999. p. 312.

10. Donald AJ. Plant Microtechnique. New York and London: McGraw-Hill Book Company; 1940. p.41.

11. Khandelwal KR. Practical Pharmacognosy - Techniques and Experiments. 9th ed. Pune: NiraliPrakashan; 2002. p.24-29 & 149-153.

12. Anonymous. Planner Chromatography, Modern Thin Layer Chromatography.Switzerland: 1999. p.2-16

13. Ayurvedic Pharmacopoeia of India. Part 1, Vol. 5. New Delhi: Government of India Publication; 2008.

14. Anonymous. The Ayurvedic Pharmacopeia of India. 1st ed. Part-1, Vol.-5. Delhi: Ministry of Health & Family welfare; 2004.

15. Anonymous. The Ayurvedic Pharmacopeia of India. 1st ed. Part-1, Vol.-1. Delhi: Ministry of Health & Family welfare; 2001.

16. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. United States: CBS Publishers and Distributors; 2005. p.169.

17. Gurdeep R, Chatwal, Sham K. Industrial Method of Chemical Analysis. 5th revised and Enlarged ed. Mumbai: Himalaya Publishing House; 2005. p. 2. 272-2.503, 2.599- 2.616, 2.673-2.700.

Source of support: Nil, Conflict of interest: None Declared IRJP is an official publication of Moksha Publishing House. Website: www.mokshaph.com. All rights reserved.