Pharmacognostical evaluation of Drosera burmannii ... - NOPR - niscair

Indian Journal of Traditional Knowledge Vol. 8 (3), July 2009, pp. 326-333

Pharmacognostical evaluation of Drosera burmannii Vahl (Droseraceae) V Madhavan1, Hema Basnett1, MR Gurudeva2 & SN Yoganarasimhan1* Department of Pharmacognosy1, MS Ramaiah College of Pharmacy, Bangalore 560 054, Karnataka 2 Department of Botany, VV Pura College of Science, Bangalore 560 004, Karnataka E-mail: [email protected] Received 13 September 2008; revised 20 January 2009 Drosera burmannii Vahl, a member of Sundews group is an important medicinal and carnivorous plant. Species of Drosera are used in medications for ailments like asthma, cough, ulcers besides being useful in homoeopathy. Many medically active phytoconsitutents including naphthoquinones, plumbagin and hydroplumbagin are reported in species of Drosera. The pharmacognostical evaluation and HPTLC profile help in drug identification and to establish marker compound. The occurrence of plumbagin, an antifertility agent in the taxon is significant. Keywords: Drosera burmannii, Sundew, Pharmacognostical characters, HPTLC IPC Int. Cl.8: A61K36/00, A61P11/00, A61P11/06, A61P11/10, A61P29/00, A61P31/02

Drosera species (Droseraceae), popularly known as the Sundews is one of the largest genera of carnivorous plants with over 105 species; the members of Droseraceae lure, capture, and digest insects using stalked mucilaginous glands covering their leaf surface1,2. Botanical name (Greek Drosos = dew, dewdrops) as well as English name (sundew) refer to glistening drops of mucilage at the tip of each tentacle that resemble drops of morning dew. They are characterized by glandular tentacles, topped with sticky secretions that cover their laminae. Species like D. anglica Huds, D. intermedia Hayne, D. madagascariensis DC., D. ramentacea Burch. ex Harv. & Sond. and D. rotundifolia L. are used as medicinal herbs in cough preparations in Germany and other countries in Europe even today in some 200-300 registered medications, in ailments like asthma, coughs, lung infections, stomach ulcers, as an aphrodisiac, to strengthen heart, to treat sunburn and to prevent freckles2. D. rotundifolia is used in homeopathy for whooping cough, fever, mental disorders, stomach disorders, skin diseases while D. burmannii possesses rubefacient property1,3. Species of Drosera Vahl. contain several medicinally active compounds including quinones- plumbagin, hydroplumbagin glucoside, flavonoids (kaempferol, myricetin, quercetin and hyperoside) and rossoliside (7-methyl-hydrojuglone-4-glucoside), carotenoids, ____________ *Corresponding author

plant acids (butyric, citric, formic, gallic, malic and propionic acids), resins, tannins and ascorbic acid (vitamin C)4-6. D. burmannii contains naphthoquinones, flavonoids like quercetin and hyperoside4,7. Three species of Drosera L. viz. D. burmannii Vahl, D. indica L., and D. peltata J.E.Sm. are found in India8. D. burmannii and D. indica are distributed throughout the country, while D. peltata occur on hills of India9. D. burmannii is also distributed in Sri Lanka, Australia, China and West Africa, up to 2,700 m; it is known as Hulahidakana gida (Kannada), Krimibandha, rimibhaksha (Sanskrit), Azhukanni (Tamil) and Burada buchi (Telugu)1,19. Some pharmacognostical work is carried out on D. peltata and D. rotundifolia while no work has been carried out on D. burmannii3,10-12. Hence, the present investigation has been taken. Methodology Plant material collected from forests of Savanadurga, Bangalore, Karnataka was processed into herbarium specimens; voucher herbarium specimen (Hema Basnett 005) and crude drug sample are preserved at the herbarium and crude drug museum of Department of Pharmacognosy, MS Ramaiah College of Pharmacy, Bangalore13. The plant material was identified following local floras and authenticated at herbarium of the Regional Research Institute, Bangalore (RRCBI)9,14. For

MADHAVAN et al.: PHARMACOGNOSTICAL EVALUATION OF DROSERA BURMANNII VAHL

pharmacognostical studies, a small quantity of plant was collected and preserved in 70% alcohol; macroand microscopical observations were carried out15,16. Photomicrographs obtained by observing free hand sections of the drug under compound binocular microscope with built in analogue camera. Computer Images were captured using AVDigitaliser having Grand VCD 2000 – Capture Guard. Measurements of cells and tissues were carried out using Micro Image Lite Image Analysis Software. Physicochemical studies and Quinone test were carried out17,18. HPTLC was carried out using Camag HPTLC system with Linomat V sample applicator, Camag TLC Scanner 3 and CATS 4 software for interpretation of data. An aluminum plate (20 x 10 cm) precoated with silica gel 60F254 was used as adsorbent. The plates were developed using toluene: glacial acetic acid (55:1) and toluene: chloroform: glacial acetic acid (1:1:0.1) as mobile phase for alcohol and aqueous extracts, respectively in a Camag twin trough chamber to a distance of 8 cm each. A fingerprint profile was photodocumented using Camag Reproster-3 under 254, 366 uv and visible light. Reference standard plumbagin was procured. Results and discussion Drosera burmannii Vahl, Symb. Bot. 3:50.1794; Wt., Ic.t.944.1845; Gamble, Fl. Pres. Madras 1: 320. 1957 (repr ed), is an acaulescent, insectivorous herb, to 15 cm tall. Leaves radical, in a rosette, up to 1 x 1 cm, obovate or orbicular, greenish-red, clothed with numerous gland-tipped hairs, mucilaginous when fresh. Flowers white or pinkishwhite, few flowered, in terminal 5-9 cm long scape, arising from cluster of leaves. Sepals glandular. Petals reddish, eglandular. Capsule subglobose (Figs.1,2) Drug consists of dried whole plant which includes roots, scape or peduncle and leaves. Roots arise from indistinct condensed stem, weakly developed, run vertically downwards, slender, slightly curved, to 1.5 cm long, brownish black, without any characteristic odour or taste, lateral roots few, filiform. Leaves 20-30, dorsiventral, in 2-3 rosettes at the junction of root and highly condensed stem, overlapping, spathulate, upper surface depressed, narrow basally, attached directly to condensed stem, reddish, beset with glandular hairs all along margins, slightly bitter, without characteristic odour. Scape or

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peduncle 1-3, slender, smooth, glabrous, unbranched, 5-9 cm long, Flowers in unilateral raceme (Fig.3) Root in TS (Figs.4-9) shows cork, cortex, stele and central pith. Epidermis made up of rectangular cells. Cork 2-3-layered, cells rectangular, 10-21-43µ. Cortex 5-6 layered, cells 15-24-30µ, parenchymatous, tangentially elongated, contain simple starch grains. Endodermis consists of rectangular parenchymatous cells, 14-24-30µ. Ground tissue large, parenchymatous. Stele consists of vascular bundles arranged in multiple rings and central pith. Xylem radially arranged. Pith parenchymatous, cells 19-26-36µ. Root macerate (Figs.10-20) exhibits parenchymatous cells of variable size, 72-98-135µ; fibers slender, 262-243-275µ, with pointed ends and narrow lumen, septate or aseptate, sometimes with bordered pits; vessels of variable size, cylindrical and pitted, 47-123-197µ; tracheids with both simple and pitted thickenings, 10-11-14µ; xylem parenchyma with pitted thickenings. TS of leaf (Figs.21-31) exhibit undifferentiated mesophyll with lower and upper epidermis. Mesophyll cells 10-27-44µ, filled with chlorenchyma. Sessile or stalked glandular trichomes found along upper epidermis, 22-44-66µ. Stomata present on upper and lower epidermis, anomocytic or ranunculaceous, stomatal index 15.7. Veins reticulate; vein islet 5. Trichomes glandular or eglandular, abundant all along margins of both surfaces. Trichomes of three types: sessile glands, globular or dome shaped, few in number, found along upper epidermis, contain dense reddish content; uni or biseriate trichomes, sparsely distributed, without conducting tissue; and multiseriate glandular trichomes or digestive glands, abundant, consists of a stalk of variable length with a large globose head, vascular tissue traversing along the stalk, terminating in globose head. Globose head of glandular trichome divided into outer and inner secretory cell layers, outer part composed of two layers of glandular epidermal layers surrounding a suberized or cutinized layer, 262-356-494µ. Vascular bundles are found embedded in mesophyll. Leaf macerate (Figs. 32-37) exhibits unicellular trichome with bulbous base and tapering apex; parenchyma cells of variable shape (wedge shaped, balloon shaped and rectangular), 12-19-25µ; vessels with spiral thickenings, 15-20-26µ

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TS of scape or peduncle (Figs.38-42) exhibits outermost epidermis covered by thin cuticle. Epidermal cells rectangular, 1-layered. Hypodermis 2-3 layered, cells rounded, contain blackish to reddish contents. Sclerenchyma 2 - 4-layered, 12-21-34µ. Ground tissue parenchymatous, cells 5-9-15µ, contain simple starch grains, intercellular spaces found here and there. Vascular bundles arranged concentrically. Xylem consists of vessels, 5-8-11µ, xylem fibres and xylem parenchyma. Phloem consists of companion cells, sieve tubes and phloem fibres. Macerate (Figs.43-50) exhibits parenchyma cells of variable size and shape, 29-42-59µ, drum shaped or rectangular or pitted (xylem parenchyma); fibers with narrow lumen and pointed ends, 12-17-22µ; vessels with annular or spiral thickenings, 13-18-23µ. Root, leaf and peduncle sections when treated with reagents gave colour reactions showing presence or absence of cell contents (Table 1). Powder analysis under microscope (Figs. 51-59) revealed fragments of drug material consisting of Table 1 Histochemical tests of sections of D. burmannii Parts

Reagent

Iodine solution Phloroglucinol + HCl Root Con. H2SO4 Ferric chloride Millon’s reagent Dragendroff reagent Iodine solution Phloroglucinol + HCl Leaf Con. H2SO4 Dragendroff reagent Millon’s reagent Ferric chloride Iodine solution Phloroglucinol + HCl Peduncle Con. H2SO4 Dragendroff reagent Millon’s reagent Ferric chloride += Present; - = Absent

Test for

Colour change

Result

Starch Lignin Cellulose Tannin Proteins Alkaloids Starch Lignin Cellulose Alkaloids Proteins Tannin Starch Lignin Cellulose Alkaloids Proteins Tannin

Blue Pink Green Black Blue Pink Green Black Blue Pink Green Black

++ ++ + + + ++ + + ++ ++ + +

epidermal peel, digestive glands, trichomes, trichome base and fragments of lamina showing venation pattern. Physicochemical parameters of whole plant, viz. percentage of moisture content, total ash, and acid insoluble ash were found to be 2.5, 30.89, 22.98, and 26.77, respectively. Per cent alcohol extractive was 29.4 and water extractive 25.3. Successive Soxhlet extractive values, colour and consistency of extracts were found to be: petroleum ether (21.85, dark green, sticky mass), benzene (5.97, black, semi solid), chloroform (1.53, dark brown, sticky mass), acetone (6.13, imperial crimson, semi solid), ethanol (15.99, reddish brown, sticky mass) and water (3.38, light brown, semi solid) respectively. The petroleum ether, benzene, chloroform, acetone, ethanol and water extracts were subjected to preliminary phytochemical analysis. It was found that alkaloids, fixed oils, phenolic compounds, tannins, proteins, amino acids and volatile oils were absent in all extracts, phytosterols present in petroleum ether, benzene, chloroform and ethanol extracts, saponins in acetone and ethanol extracts, flavonoids in petroleum ether, chloroform, acetone and water extracts, gums and mucilage in petroleum ether, benzene, chloroform, acetone, ethanol and water extracts. Observations on the powder when treated with different reagents and under ultra-violet light are provided in Table 2. Alcohol and aqueous extract of TLC plates were dipped with aqueous 10% KOH solution. The alcohol extract exhibited majenta colour indicating presence of quinones (Fig.60). HPTLC studies of alcohol and aqueous extracts revealed different chromatographic profiles. Alcohol extract revealed seven peaks at Rf 0.12, 0.18, 0.21, 0.24, 0.29, 0.56, 0.81. Out of these, the most pronounced peak of maximum area was at Rf 0.56, corresponding to that of marker compound plumbagin (Fig.61). The other peaks at Rf 0.12, 0.18 and 0.21 were also significantly prominent. The specificity was confirmed by overlaying the spectra of plumbagin in reference standard (λmax425m) (Fig.62). The aqueous extract revealed two peaks at Rf 0.15 and 0.24 with no

Table 2 Ultra violet analysis of powder of Drosera burmannii Ultra-violet light

Treatment

Visible light

Powder as such Powder + 50% H2SO4 Powder + 50% HNO3 Powder + 5% KOH Powder + methanol Powder + 1N HCl Powder + 1N methanolic NaOH

Dark brown Mid buff Golden brown Golden brown Sandstone Sandstone Walnut brown

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short wave (254 nm)

long wave(365 nm)

No fluorescence Water green Cascade green Water green Cascade green Opaline green Dark green

No fluorescence No fluorescence No fluorescence No fluorescence No fluorescence No fluorescence No fluorescence

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Acknowledgement Authors are thankful to Gokula Education Foundation, Bangalore and VV Pura College of Science for providing facilities and necessary encouragement. References

Fig.61 HPTLC of alcoholic extract showing plumbagin

Fig. 62 Overlay spectra of plumbagin

peak corresponding to that of plumbagin reference standard (Rf 0.66). This is due to the hydrophobic nature of plumbagin. Drosera burmannii may be characterized by insectivorous habit, rosette of bright red coloured leaves, glandular trichomes, vascular bundle in multiple ring in root, vessels with annular and spiral thickenings in digestive glands. Conclusion Pharmacognostical evaluation of Drosera burmannii, an insectivorous, medicinal plant was carried out. HPTLC profile helps in establishing marker component. One marker component with Rf 0.56 was identified as plumbagin by comparing with standard plumbagin. The occurrence of plumbagin, an antifertility agent, is significant20.

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