Evolvulus Species
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Indian Journal of Traditional Knowledge Vol. 14(4), October 2015, pp. 564-570 Comparative pharmacognostical studies of two medicinally important Indian Evolvulus species Saba Irshad1, 2, Siddhartha Pragyadeep1, Ajay Kumar Singh Rawat1, PK Misra2 & Sayyada Khatoon1* 1Pharmacognosy and Ethnopharmacology Division, CSIR-National Botanical Research Institute, Post Box No. 436, Rana Pratap Marg, Lucknow -226001, India; 2Department of Botany, University of Lucknow, Lucknow- 226007, India E-mails: [email protected]; [email protected]; [email protected] Received 18 November 2014, revised 19 December 2014 The genus Evolvulus consists of small herbs or under shrubs distributed in the tropical and warm temperate regions of the world. Two species of this genus have been reported from India, viz. E. alsinoides L. and E. nummularius L. (Convolvulaceae). Both the species are traditionally being used as nerve tonic and for the treatment of skin diseases. The current study was carried out to provide comparative macro-microscopy, physicochemical parameters and TLC finger print profiles of aforesaid Indian Evolvulus species. The microscopy showed anisocytic stomata in E. alsinoides while diacytic and paracytic stomata in E. nummularius; presence of stellate trichomes, characteristic pith with spindle shaped deposition of calcium oxalate crystals and starch grains only in E. nummularius. Comparative TLC profile showed presence of some common as well as differentiating bands in methanolic extract of Evolvulus sp. However, the chemical markers, viz. ferulic acid, caffeic acid, β sitosterol and lupeol were present in both the species. The macro-microscopy and TLC profiles may play an important role for identification and quality evaluation of two medicinally important Evolvulus species. Keywords: Evolvulus alsinoides, Evolvulus nummularius, Macro-microscopy, Pharmacognosy, TLC IPC Int. Cl.8: A61K, A61K 36/00, A61J 3/00 Convolvulaceae, commonly known as morning glory Traditionally the whole plant is used as a medicine for family, represented by 60 genera and more than 1,650 hysteria, to cure burns, cuts, wounds and scorpion species and many of them have medicinal properties. stings7. Pharmacologically it is reported for wound Evolvulus, is a genus of this family, consists of small healing8, as anthelmintic9, poor sedative and herb or under shrubs distributed in the tropical and anticonvulsant10. On account of aforesaid medicinal warm temperate regions of the world. Although more properties, quality control markers of EA and EN are than 70 species are reported from all over the world required. Preliminary pharmacognostical data but only two species of this genus have been reported reported for EA11 only. from India, viz. E. alsinoides L. (EA) and 1 EA contains several alkanes, alkaloids, fatty acids, E. nummularius L. (EN) . Both the species are flavonols, flavonoids, organic acids, phenolics, reported for their therapeutic claims in the traditional phytosterol, saponins, and tannins12. Some of them are systems of medicines and in folklores. EA is known betain, shankhpushpine and evolvine13; scopoletin, by different vernacular names, viz. Shankhpushpi, scopolin, umbelliferon, ferulic acid easters, Shnkha valli, Vishnugandhi, Vishnukarandi, Vishnuka kampferol-7-O-β-glucopyrenoside and quecitin-3-O-β ranta, etc. Traditionally it is used all over India glucopyrenoside and caffeic acid14. EN reported to for the treatment of various ailments, viz. fever, contains β-sitosterol and its glucoside, stigmasterol, cough, cold and jaundice, as nerve tonic, hysteria, ursolic acid, oleanolic acid, palmitic acid and to cure burns, cuts, wounds and scorpion stings, nummularic acid15. etc.1-2. Pharmacologically it is reported to possess antiulcer and anticatatonic3; immunomodulator, Therefore, present study is plan out to antiinflammatory4 and nootropic5 activities. Another investigate comparative macro-microscopical species EN also called by various names, viz. characters, physicochemical parameters and TLC Aakhukarni, Muusaakarni and Chhinipatra6. fingerprinting for EA and EN with the aim to set the quality control parameters of these two important —————— *Corresponding author Indian medicinal plants. IRSHAD et al.: QUALITY CONTROL MARKERS OF INDIAN EVOLVULUS SPECIES 565 Materials and methods (S D Fine Chemicals, India). Samples (20 µL) Collection and processing of plant material and standards (10 µL) were applied to the plates The selected plant species of Evolvulus were by means of a Camag (Switzerland) Linomat collected from Chitrakoot and Lucknow in July 5 sample applicator. The plates were developed to and August 2013, their herbarium specimens a distance of 8 cm from the lower edge of the were prepared as per standard herbarium plate with 20 mL toluene–ethyl acetate–formic acid procedure16 and deposited in the herbarium of (8.5:1.5:0.1 v/v/v) as mobile phase, in a Camag CSIR-National Botanical Research Institute, twin-trough chamber, previously saturated with Lucknow wide voucher specimen number LWG-30 mobile phase vapor for 30 min at 25±2 °C. and LWG-33 for E. alsinoides and E. nummularius, After removal from the chamber, plates were respectively. completely dried in air at room temperature (25±2 °C) and documented under UV 254 nm and Macro-microscopical studies UV 366 nm. The plates were dipped in anisaldehyde The macroscopy of Evolvulus species was sulphuric acid reagent, dried and heated at 110±2 °C described with the help of Floras17. Qualitative and for 5 min and documented in visible light after quantitative microscopy, powder studies and derivatization. florescent analysis were done according to the 18-20 standard methods . Results and discussion Comparative macroscopy of two studied Physicochemical studies Evolvulus species (Fig. 1A & 2A) has been shown Total ash, acid insoluble ash, alcohol and that root is straight; cylindrical; 8 to 10 cm long, water soluble extractives were calculated as per 21 rooting at the nodes in both the species. Stem- Pharmacopoeial methods . Sugar and starch numerous; prostrate; often more than 30 cm long; were also estimated according to Montgomery 22 usually clothed with long silky hair in EA although method . in EN stem are not covered with silky hairs. In EA Preparation of sample solutions for TLC finger printing leaf-simple, alternate, sessile, lamina oblong, For TLC studies, plants were air-dried at lanceolate, epiculate usually acute at the base, room temperature in the shade and were used for shallowly, emarginate and mucronate at the apex solvent extraction. The whole plant of EA and while in EN, leaf- simple, alternate, petiolate, lamina EN ground to coarse powder and placed in circular or circular-obovate, obtuse, base truncate to appropriately sized volumetric flasks. 25mL subcordate, glabrous or sparingly pubescent beneath. methanol was added to 4 gm of powder of each In EA, flower-one to three with peduncle, axillary; plant, shaken on shaker for 2 hrs, kept at rest calyx-densely silky, 5 lobed, acute, lanceolate; overnight. The methanolic extracts were filtered corolla- bluish purple, five, shallow lobed, funnel through Whatmann No. 1 filter paper. The procedure shaped with small white eye inside but in EN, was repeated thrice with methanol (25 mL) at room flowers-solitary or rarely paired, pedunculate, temperature (25˚C ± 2˚C). The extracts were axillary; calyx-ovate to elliptic ovate, acute; corolla- concentrated under reduced pressure at a temperature white, subrotate, deeply 5 lobed, lobes obovate. of 45±2˚C. Accurately weighed 10 mg of the extract Capsule- globose, 4 valved, 3mm in diameter, seeds was dissolved in 1 mL methanol, and filtered brownish in EA and bivalve, 3–4 mm. in diameter, through a 0.45 µm filter membrane, the filtrate was seeds brown to black in EN. The characteristic used as sample solution. 1 mg each of ferulic acid, microscopic features are - anisocytic stomata in EA lupeol and β-sitosterol were dissolved in 10 mL while diacytic to paracytic stomata in EN; simple methanol to get 0.1 mg/ mL solution of standard and glandular trichomes in EA and stellate trichomes markers. in EN; tenniniferous cells in EA while schizogenous mucilaginous canals and calcium oxalate crystals in TLC finger printing EN. Further, pith is parenchymatous in the TLC was performed on 10x 20 cm silica transverse view of both stem but embedded with TLC aluminium sheet, coated with 0.2 mm layer of spindle shaped deposition of starch grains and silica gel containing UV 254 fluorescent indicator calcium oxalate crystals only in EN. Comparative 566 INDIAN J TRADIT KNOWLE, VOL 14, NO. 4, OCTOBER 2015 Table 1—Comparative microscopical details of E. alsinoides and E. nummularius S No. Part E. alsinoides E. nummularius 1. TS Root Outer most cork composed of 3-4 layers of tangentially Similar to EA. elongated, thick walled suberized cells. Cortex many layered parenchymatous, large Cortex many layered parenchymatous, traversed by yellowish intercellular spaces, more deposition of starch brown tenniniferous cells and starch grains. grains with calcium oxalate crystals. Phloem region about 1.5-1.9mm broad, composed of Phloem region 2.5-3.2 mm broad, rest of the sieve elements, phloem parenchyma. Xylem vessels about characters are similar to EA (Fig. 2B). 41.83-54.34µm broad and mostly solitary (Fig. 1B). 2. TS Stem Epidermal cells covered with cuticle, long unicellular thick Similar to EA; epidermal cells with high tannin walled simple and glandular trichomes and stomata; deposition; hypodermal cells 2-3 layered.