PHARMACOGNOSTICAL AND PHYTOCHEMICAL EVALUATION OF VIGNA UNGUICULATA LINN. (KULATTHA) SEED

Abstract

Kulattha (Vigna unguiculata Linn, Papilionaceae), one of the seed drugs described under dietetic group, is being used as both drug and diet, in different classical texts of Ayurveda. It is one of the drugs of choice for the management of urinary calculus (Ashmari). Though used as a source of both drug and diet, it is reported as a major causative factor of acid peptic disorder (Amlapitta). Seed of V.unguiculata can be identified microscopically by the presence of rhomboidal crystals, simple starch grains with hilum. Purity test shows loss on drying (91.89% w/w), total ash (4.89% w/w), acid insoluble ash (1.22% w/w), alcohol soluble extractive (1.31% w/w) and Water-soluble extractive (1.94% w/w). Preliminary analysis revealed the presence of starch, tannin and amino acid. HPTLC study of its methanolic extract showed the presence of four and seven spots in short and long UV respectively. The information generated by this study provides relevant Pharmacognostical and Physico-chemical data needed for proper identification and authentication of the seeds of this particular species. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Kulattha (Vigna unguiculata Linn, Papilionaceae), one of the seed drugs described under dietetic group, is being used as both drug and diet, in different classical texts of Ayurveda. It is one of the drugs of choice for the management of urinary calculus (Ashmari). Though used as a source of both drug and diet, it is reported as a major causative factor of acid peptic disorder (Amlapitta). Seed of V.unguiculata can be identified microscopically by the presence of rhomboidal crystals, simple starch grains with hilum. Purity test shows loss on drying (91.89% w/w), total ash (4.89% w/w), acid insoluble ash (1.22% w/w), alcohol soluble extractive (1.31% w/w) and Water-soluble extractive (1.94% w/w). Preliminary analysis revealed the presence of starch, tannin and amino acid. HPTLC study of its methanolic extract showed the presence of four and seven spots in short and long UV respectively. The information generated by this study provides relevant Pharmacognostical and Physico-chemical data needed for proper identification and authentication of the seeds of this particular species.

Key words: Kulattha, Pharmacognosy, Phyto-chemistry, HPTLC

Abbreviations: 7m - micrometer

INTRODUCTION

Kulattha (Vigna unguiculata Linn. Papilionaceae) is found throughout India from Punjab and Himalaya to Sikkim in the North (where it ascends to over 1500 m), the upper Gangetic plain, Central and South India till Kanyakumari. (Lakshmi1996). It is known as Horse gram In English, Kulathi in Hindi, Mudiraa in Malayalam, Kalathi in Gujarati, Kulitha in Marathi. (Anonymus, Database on Medicinal Plants 2008). It is extensively cultivated in Dehradun, Bengal, Chotanagpur, Deccan, and Coromandel Coast of Kerala as a food crop especially for Horses and Cattles as well as for its seeds which are considered nutritious. (Lakshmi, 1996) Classical text of Ayurveda, Charaka Samhita described it as one of the drug/diet causing Acid Peptic Disorders (Amlapitta) (Charaka Sutrasthana 25/40). Sushruta Samhita highlights its Anti-lithiatic (Shukrashmari) activity. (Sushruta Sutrasthana 46/37) In recent study V.unguiculata shows to have better results than the use of conventional potassium citrate in recurrence of renal calculus and can be used to reduce the recurrence of calcium oxalate stone. (Singh et al. 2010) Seed's extract also shows Anti-oxidant and Anti-free radical activities. (Hazra B et al. 2009)

Plant morphology

It is a Sub-erect downy to rarely glabrescent annual herb growing upto 30-60cm or more in height with a short erect stem and several elongate, diffuse, Sub-erect or at times twining branches. The young shoot usually covered with epidermal hairs, bearing pinnately trifoliate alternate stipulate leaves having ovate oblong or ovate lanceolate entire membraneous stipellate leaflets 2.5 cm or more long, very small pedicelled pale yellowish racemes axillary, with 2-6 flowers clustered at top of rachis and compressed, linear, falcate to much curved, four to six seeded pods 3.85 cm long and 6-9 mm wide (J.S. Gamble 1997).

Fruit: 4-6 seeded, with trichomes or downy linear, broadly linear falcate or scimitar shaped to much recurved (very slightly so in wild variety) compressed pod. Measuring 3.8-5cm long and 6-9mm broad tipped with the persistent style. Seeds reniform, compressed with a shiny hard testa of various colours, mostly reddish brown, grey black as well as mottled. Cotyledon orbicular to cordate and persists for a long time on seedling.

Though used extensively as a drug and diet, the detailed pharmacognostical and preliminary phytochemical characters of the seeds are not reported anywhere. Hence, in this article an attempt has been made to study the morphological and microscopical characters of the seed and its powder along with preliminary phytochemical characters including HPTLC study.

MATERIAL AND METHODS

Kulattha seeds were purchased from the local market in the month of March, authenticated by Pharmacognosy laboratory of IPGT&RA Jamnagar. Voucher herbarium specimen along with crude drug sample was preserved in Pharmacognosy laboratory, vide Ref No. 6036 March 2011. Pharmacognostical evaluation of Kulattha seeds including Histo-chemical studies were carried out by taking free hand sections. Powder microscopy of seeds was carried out following standard procedures (Wallis 1985) and the slides for powder microscopy were separately prepared first with distilled water, then stained with Phloroglucinol and concentrated HCl. Photomicrographs were taken using Carl Zeiss Binocular Microscope attached with Camera. Histo-chemical tests were carried out by taking thick sections following the standard procedure methods (Krishnamurthy 1988). Physicochemical parameters and chemical screening (Anonymous, Planner Chromatography 1999) studies were carried out following standard procedures. (Anonymous, API 2004) The methanol extract obtained during Physicochemical parameters was used for HPTLC. Sample was prepared by 30min sonication of drugs with methanolic medium and filtrate was used for experimental task. 'Benzene' was selected as the mobile phase. Chromatographic conditions were as follows. (Anonymous, Planner Chromatography 1999)

Chromatographic conditions

Application mode : Camag Linomat V

Development Chamber : Camag Twin trough Chamber.

Plates : Precoated Silica Gel GF254 Plates.

Chamber Saturation : 30 min.

Development Time : 30 min.

Development distance : 7 cm.

Scanner : Camag Scanner III.

Detection : Deuterium lamp, Tungsten lamp

Data System : Win cats software.

RESULTS AND DISCUSION

Macroscopic Characters of seed

Seeds shape reniform, 5-6mm long, 3-4mm broad and 2-3 mm in thickness, compressed with a polished or shiny and hard brown coloured testa. The micropyle was situated near the hilum. The hilum was 1-1.5 mm in length. The seed were exalbuminous. The testa was tough but comparatively thin except at the region of the hilum. The embryo which was exposed after removing the testa, by softening it through emersion of the seed in water, consists of two fleshy cotyledons, 5-6mm long and 4-5mm wide and an incurved radical which was 4mm long. [Figure 1.1]

Organoleptic evaluation

Organoleptic evaluation of powder of seeds of V. unguiculata revealed astringent (kashaya) taste, buffcolour and characteristic odour while the texture was coarse. [Figure 1.2]

Microscopic Character: T.S. of Seed

Detailed Transverse section passing through the center of the seed shows the testa having three layers, of which first and second layer were single celled, while the third layer consisted of several rows of thin walled narrow cells containing rhomboidal calcium oxalate crystals. The outermost row namely the epidermis was composed of vertically elongated palisade like cells, each cell with slight constriction nearer its upper end, 457m or more in height, 12- 157m in width at their broader part, 3-67m width at their narrow constricted part and 9- 127m in width at the extreme tip. [Figure 1.3, 1.4] There was a thin covering of cuticle over the epidermis about 37m in thickness. The palisade like cells were further characterized by the presence of narrow transverse light line at about two-third of their length from the base. The second or Sub-epidermal row consisted of shorter and broader column like cells with their outer and inner ends broader and the middle portion constricted. Cells with inter-cellular spaces had characteristic "hour glass" like shapes. They varied from 18-307m in height and 12-157m in width at the base, 6-97m in width at the narrow middle constricted region and 15-187m near the top. [Figure 1.5]. Third layer or zone was composed of 8-10 rows of thin walled narrow cells, some of which contained rhomboidal crystals that measured 30-607m × 18-277m, these parenchymatous cells were tangentially elongated with more or less oblique radical walls. These cells were lacking the inter cellular spaces. At the region of the hilum, two rows of palisade like cells were present (instead of one row). Beneath, there was a group of sclerenchyma cells with narrow elongated pits on their walls that appeared as an elongate lanceolate patch in T.S. The Sub-epidermal cells were columnar, expanded beneath the hilum into a cushion in which these groups of sclerenchymatous cells appeared embedded, surrounded by two layers of narrow thin walled elongated parenchymatous cells. [Figure1. 6, 1.7]

Powder Microscopy

Diagnostic characters of dried powder of V. unguiculata under the Microscope were prismatic crystals of Calcium oxalate from epidermis, dark brown coloured content; which was confirmed to be Tannin by adding Ferric chloride solution to it, which turned black; from Sub-epidermal region, simple starch grains with Hilum, Iodine stained starch grains, Loosely arranged parenchymatous cells. Clumped masses may be protein content, with some Aleurone grains. [Figure 1.8 to1.12]

Histochemical Test:

The results of the various Histo-chemical tests carried out, to detect Lignin, Calcium, Starch and Tannin are depicted in table no. 1

Phyto-chemical constituents

While observing the physicochemical characters for purity test, the loss on drying was not more than 91.89% w/w, ash value was not more than 4.89 % w/w, acid insoluble ash was not more than 58.14% w/w, water soluble extractive was not more than 1.94 % w/w and the methanol soluble extractive was not more than 1.34 % w/w. [Table no.2] Qualitative analysis showed presence of tannin, starch, amino acid and absence of alkaloids, cyanogenic glycosides and flavonoids. [Table no.3]. The methanol soluble extract was examined for high performance thin layer chromatography profile at 254nm frequency, using the solvent system of Benzen, Under high performance thin layer chromatography profile, at 254nm frequency four peaks were observed and at 366nm frequency seven peaks were observed. [Table no.4] [Plate 2]

CONCLUSION

Pharmacognostical and Phytochemical evaluation of seed and powder of Kulattha (V.unguiculata) market sample were found to be authentic and meet the standard parameters of API. Further Pharmacognostical findings i.e. rhomboidal calcium oxalate crystals, tannin, lignified fibres were important characters found during the study apart from what was mentioned in API. Phytochemical screening and HPTLC results can be considered as standards for further research works.

References

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Source of Support: Nil Conflict of Interest: None Declared