Indian Journal of Traditional Knowledge Vol. 14(4), October 2015, pp. 564-570

Comparative pharmacognostical studies of two medicinally important Indian 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 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. (). 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 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 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 . 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 , 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. hypodermis 3-4 layered. Cortical region is 2-3 layered; endodermis and Cortical region 4-5 layered; endodermis distinct; pericycle pericycle not well distinguish; phloem region characterized by discontinuous layer of fibres; phloem 2.7-3.5mm broad; medullary rays uni or biseriate; 1.5-2.3mm thick; medullary rays uniseriate; pith pith parenchymatous with spindle shaped parenchymatous with intercellular spaces (Fig. 1C). deposition of starch grains and calcium oxalate crystals (Fig. 2C). 3. TS Leaf Dorsiventral with meristele, 2 layered palisade cells and Same as in EA, 2-3 layered palisade cells 3-4 layered spongy mesophyll, without intercellular spaces and 4-5 layered spongy mesophyll (Fig. 1D & E). interrupted by schizogenous mucilaginous canals (Fig. 2D & E). 4. Quantitative Stomatal number ranges 250-300; stomatal index 25.25-27.35 Stomatal number ranges 150-200; stomatal index microscopy on upper epidermis while on lower epidermis stomatal number 14.28-20.50 on upper epidermis while on the lower of leaf ranges 300-450 and stomatal index ranges 27.50-2935. epidermis stomatal number ranges 200-250 and Palisade ratio ranges from 2.0-3.0. stomatal index ranges 25.0-25.25. Palisade ratio ranges from 5.0-6.0. 5. Powder Thick walled simple and glandular trichomes of different Simple and stellate trichomes, epidermal cells sizes, epidermal cells with wavy margins and anisocytic with diacytic to paracytic stomata, reticulate stomata, xylem vessels with spiral thickening, fibres and and pitted xylem vessels, fibres prismatic crystals paranchymatous cells (Fig. 1F). of calcium oxalate and parenchymatous cells (Fig. 2F). microscopical details of EA and EN are given in colour in the visible light, however, ultra violet light Table 1 and shown in Figs. 1 & 2. produces fluorescence in many natural products Air dried material was used for quantitative (e.g. alkaloids like berberine). If the substances determination of physicochemical values. themselves are not fluorescent, they may often be Extractive values (alcohol and water soluble), ash converted into fluorescent derivatives by applying values (total and acid insoluble ash), total sugar different reagents hence some crude drugs are often and starch content were repeated for six times assessed qualitatively in this way and it is an and mean values ±SD was presented in Fig. 3. important parameter of pharmacognostical Higher water extractive value of EN indicating evaluation24-25. The results of fluorescence study of the possibility of considerable amount of polar whole plant powder using different reagents are given compounds and also high starch content in EN in Table 2. showed the high rate of photosynthesis and low TLC fingerprint profile showed similar and 23 growth rate . Total ash value of EA was relatively differentiating bands in Figs. 4A, B & C. low, which may be due to low content of carbonates, Common bands at Rf 0.12, 0.23, 0.30, 0.59 phosphates, silicates and silica. Ash value is useful (under UV 366 nm) at Rf 0.19, 0.30, 0.62, in determining authenticity and purity of drug and 0.75 (under UV 254 nm) and at Rf 0.30, 0.48, 0.62 also these values are important quantitative and 0.75 (under visible light after derivatization) 21 standards . were observed in both the species. However, the Fluorescence is an important phenomenon identifying blue fluorescent band at Rf 0.88 under exhibited by various chemical constituents present in UV 366 nm and greyish blue band at Rf 0.71 after plant material. Some constituents develops different derivatization were observed only in EN. Similarly, IRSHAD et al.: QUALITY CONTROL MARKERS OF INDIAN EVOLVULUS SPECIES 567

Fig. 1—Microscopy of the whole plant of E. alsinoides. A: Whole plant, B: T.S. Root, C: T.S. Stem, D; E: T.S. Leaf and F: Powder - a: prismatic crystals, b: parenchymatous cells, c: xylem vessel, d: epidermal cells with stomata, e: trichome, f: bundle of xylem fibres and vessels, g: fibre, h: glandular trichome. Abbreviations: ck, cork; ct, cortex; glt, glandular trichome; hyp, hypodermis; lepi, lower epidermis; p, pith; pal, palisade layer; ph, phloem; sg, starch grains; sm, spongy mesophyll; uepi, upper epidermis; v, vessels; xy, xylem. the characteristic blackish grey band at Rf 0.68 Quality of herbal drug in term of active principal under UV 254 nm and after derivatization while and their efficacy necessitates the need of quality another puplish grey band at Rf 0.79 after control studies of raw drug materials. Global derivatization in EA were observed (Fig. 4, Table 3). marketing has also created awareness towards Three chemical markers viz. ferulic acid, β-sitosterol validation of plant based drug to maintain the quality, and lupeol showed simultaneous bands at Rf safety and efficacy. As these two medicinally 0.30, 0.48, 0.62, respectively (Fig. 4) in both the important Evolvulus species being a nerve tonic in species. traditional bsystems of medicine, have a potential to 568 INDIAN J TRADIT KNOWLE, VOL 14, NO. 4, OCTOBER 2015

Fig. 2—Microscopy of the whole plant of E. nummularius. A: whole plant, B: T.S. Root, C: T.S. Stem, D: T.S. leaf passing through lamina; E: T.S. Leaf passing through midrib; F: Powder - a: prismatic crystals, b-d: xylem vessels, e: stellate trichomes, f: simple trichome, g: epidermal cells with stomata. Abbreviations: ck, cork; ct, cortex; gt, glandular trichome; hyp, hypodermis; lepi, lower epidermis; sc, schizogenous mucilagenous canal; p, pith; pal, palisade layer; ph, phloem; sg, starch grains; sm, spongy mesophyll; uepi, upper epidermis; v, vessels; xy, xylem.

Fig. 3—Physicochemical parameters of E. alsinoides and E. nummularius as the mean value ± standard deviation (n≥ 6) IRSHAD et al.: QUALITY CONTROL MARKERS OF INDIAN EVOLVULUS SPECIES 569

Table 2—Florescence analysis of the powder of whole plant of E. alsinoides and E.nummularius S.No Reagent Under UV 366 Under UV 254 Visible light EA EN EA EN EA EN 1 Powder+1M NaOH DB DG DB DG YG DG

2 Powder+CH3COOH LB DB LB DB LG RB 3 Powder+5% I2 LG BG BG DB LG DB 4 Powder+5% FeCl3 DG RB DG BG DG DG 5 Powder+Conc. HNO3 DB LB DB DG RB BG 6 Powder+Conc. NH3 LG DG LG DG YG DG Abbriviations: Dark Brown (DB), Dark Green (DG), Yellowish Green (YG), Light Brown (LB), Light Green (LG), Raddish Brown (RB), Brownish Green (BG).

Table 3—Comparative Rf details of E. alsinoides Conclusion and E. nummularius From the present studies, it can be concluded that Sample→ Colour Under UV Under UV After the characteristic macro-microscopical features, Rf ↓ 366 254 derivetization physicochemical parameters and distinguishing bands EA EN EA EN EA EN in the TLC profiles are very important as quality 0.12 Blue + + - - - - control markers of medicinally important Indian 0.19 Black - - + + - - Evolvulus species. 0.23 Blue + + - - - - 0.30 Dark + + + + + + Acknowledgement Blue/Black 0.48 Greyish blue - - - - + + The Authors are thankful to Director CSIR- 0.59 green + + + - - - National Botanical Research Institute, Lucknow for 0.62 purple - - + + + + providing lab facility for this research work and first 0.68 black - - + - + - author is also thankful to Indian Council of Medical 0.71 Greyish blue - - - - - + Research, New Delhi for financial support. 0.75 Blackish green - - + + + + 0.79 Purplish grey - - - - + - References 0.88 Blue - + - - - - 1 The Wealth of India, Raw material, Vol 3, D-E, (Council of Scientific and Industrial Research, New Delhi), 1952, 233-234. 2 Chopra RN, Nayar SL & Chopra IC, Glossary of Indian Medicinal plants, (Council of Scientific and Industrial research, New Delhi), 1956, 116. 3 Purohit MG, Shanthaveerappa GK & Badami S, Antiulcer and anticatatonic activity of alcoholic extract of Evolvulus alsinoides (Convolvulaceae), Indian J Pharm Sci, 58 (1996) 110–112. 4 Ganju L, Karan D, Chanda S, Srivastava KK, Sawhney RC & Selvamurthy W, Immunomodulatory effects of agents of plant origin, Biomed Pharmacother, 57 (2003) 296–300. 5 Nahata A, Patil UK & Dixit VK, Anxiolytic activity of

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