Indian Journal of Traditional Knowledge Vol. 9 (4), October 2010, pp. 629-634 Pharmacognostical studies on the root of Nothosaerva brachiata Wt. – A botanical source of the Ayurvedic drug, Pashanabheda V Madhavan1, Priyanka Goswami K1, Gurudeva MR2 & SN Yoganarasimhan1* 1 Department of Pharmacognosy, MS Ramaiah College of Pharmacy, Bangalore 560 054, Karnataka; 2 Department of Botany, VV Pura College of Science, Bangalore 560 004, Karnataka E-mail: dr_yogan@yahoo.co.in Received 23.01.2009; revised 26.05.2009 Pashanabheda is an important Ayurvedic drug. Several species belonging to different families are used as the botanical source of Pashanabheda while the accepted source is Bergenia ciliata (Haw.) Sternb. The roots of Nothosaerva brachiata Wight is used is South India as one of the sources of Pashanabheda. The study comprising taxonomy of the species, macroand microscopical characters, physicochemical and ultra-violet analysis besides chromatographic details of the root of N. brachiata, helps in the identification of the plant and the drug but also contribute towards establishing pharmacopoeial standards. HPTLC studies helps to identify the species in drug form and to establish the biomarker compound. Keywords: Nothosaerva brachiata, Pharmacognosy, Pashanabheda, Ayurvedic drug, Medicinal plant Int. Cl.⁸⁸ : A61K36/00, A61P1/16, A61P5/00, A61P5/10, A61P9/14, A61P13/00, A61P13/02, A61P15/00 Pashanabheda is an Ayurvedic drug used in the treatment of urinary calculi (ashmari), dysuria (mootrakrichchhra), polyuria (atimutra), fevers (jwara), piles (arsha), dysentery (pravahika) and uterine disorders (garbhashaya vikara)1. The main Ayurvedic formulations of the drug are ashmarihara kashaya, mootra virechaniya kashaya, pashanabhedadi kvatha, pashanabhedadya ghrita, sarasvata ghrita, varunadi kvatha and vidari ghrita, to mention a few2. The accepted botanical source of Pashanbheda is Bergenia ciliata (Haw.) Sternb. belonging to family Saxifragaceae3. Different botanical sources are used in the name of Pashanabheda in different parts of the country, viz. Aerva lanata (L.) Juss. ex Schult., A. persica (Burm.f.) Juss. ex Schult., (both of Amaranthaceeae), Ammania baccifera L. (Lythraceae), Bauhinia racemosa Lam. (Caesalpiniaceae), Bergenia stracheyi (Hook.f. et Th.) Engl. (Saxifragaceae), Bridelia crenulata Roxb., B. retusa (L.) Spreng., B. stipularis Blume, Homonoia riparia Lour. (all Euphorbiaceae), Didymocarpus pedicellata R.Br., (Gesneriaceae), Gentiana kurroo Royle, G. lutea L. (both Gentianaceae), Iris pseudacorus L. (Iridaceae), Kalanchoe pinnata (Lamk.) Pers., K. integra (Medic.) Kuntze, (both Crassulaceae), Lepidagathis trinervis Wall. ex Nees, (Acanthaceae), Nothosaerva brachiata Wight ___________ *Corresponding author (Amaranthaceae), Ocimum tenuiflorum L., Plectranthus amboinicus (Lour.) Spreng. (both Lamiaceae), Rotula aquatica Lour. (Boraginaceae) and Trianthema triquetra Rottl. ex Willd. (Aizoaceae)4-6. Of these, A. lanata, B. crenulata, B. stipularis, H. riparia, N. brachiata and R. aquatica are used as Pashanabedha in South India while B. ciliata, D. pedicellata, Iris pseudacorus, K. pinnata and O. tenuiflorum are used as Pashanabheda in North India7,8. The part used in Pashanabheda as per Ayurvedic literature is the root1. Pharmacognostical investigation with macerate details and powder analysis besides HPTLC studies on the roots of N. brachiata which help in the identification of crude drug is not available in the literature and hence the study was undertaken9,10. Methodology Fresh roots were collected from the vicinity of Tirunelveli, Tamil Nadu, during February, 2007, preserved in 70% ethyl alcohol for histological studies. Botanical identification of the plant was carried out11,12. Voucher herbarium specimen (Goswami Priyanka Kantivan) is preserved along with crude drug sample at the herbarium of MS Ramaiah College of Pharmacy, Bangalore13. Pharmacognostical evaluation including histochemical, macerate and powder studies were carried out14-16. The vernacular names are provided17. 630 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 9, No. 4, OCTOBER 2010 Photomicrographs were obtained by observing free hand sections of drug under compound binocular microscope with built in analogue and computer images were captured. Measurements of cells and tissues were carried out using Micro Image Lite Image Analysis Software. Physicochemical constants, organic analysis, ultra-violet analysis and chromatographic studies were carried out from shade dried powder following prescribed methods18-22. HPTLC studies were carried out on alcohol extract for interpretation of data. An aluminium plate (5 x 10 cm) precoated with silica gel 60F254 was used as adsorbent. The plates were developed using chloroform: acetone: formic acid (7:2.4:0.6) for alcohol extract and n-butanol: glacial acetic acid: water (7:1:2) for aqueous extract in a Camag twin trough chamber to a distance of 8 cm each. The time of saturation for alcohol and aqueous extracts were 3 hrs and 2 hrs, respectively. The development time for alcohol extract was 25 min whereas for aqueous extract, it was 1h 45 min. Results and discussion Nothosaerva brachiata (L.) Wight, Ic. t. 1776B. 1852. Achyranthes brachiata L., Mant. Pl. 50. 1767. Tamil: Seerupilai; Hindi: Silabheda; Sanskrit: Pashanabheda; Rajasthani: Dhaula phindawari. Annual erect herbs. Leaves opposite or alternate, entire. Inflorescence of axillary, solitary or clustered spikes. Utricle rupturing irregularly. Seeds black (Figs. 1&2). It is a monotypic genus distributed from Tropical Africa to Asia23,24. Roots are slender, brownish, measuring 7-10 cm long. Several lateral filiform roots arise from the tap root. Taste is not characteristic, smell is pleasant. The roots are sold in the market in small bundles, which often consists of portions of aerial stem (Fig. 3). Microscopical characters of the root Transverse section of the root is circular in outline. It shows cork, secondary cortex and a wood region showing anomalous secondary growth. The cork is 4-7 layered and is made up of thick walled cells, measuring 20-50-37 x 8-15-12µ. Next to the cork lies a single layer of cork cambium. Secondary cortex occupies a large region, made up of several layers of parenchyamatous cells measuring 23-46-70 x 24-2938µ. Some cells are tanniferous, some others contain druses types of calcium oxalate crystals. Endodermis and pericycle are indistinct. Next to the secondary cortex, is situated 3-4 layers of secondary phloem consisting of thin walled parenchymatous cells, measuring 10-23-23 x 4-8-12µ. Secondary phloem cell measure 11-18-26 x 6-9-14µ and is demarcated by secondary xylem by 3-4 layers of vascular cambium. The vascular bundles are arranged in 3 rings exhibiting anomalous feature. It can be differentiated into an outer ring of vascular bundle showing secondary growth, traversed by interfascicular sclerenchyma and patches of intra-xylary parenchyma, cells measuring 5-6-8 x 2-5-7µ; middle ring of vascular bundle is conjoint, collateral and is traversed by parenchymatous conjunctive tissue, cells measure 16-27-45 x 9-11-15µ; the inner ring of vascular bundle exhibit secondary growth and is traversed in between by sclerenchymatous conjunctive tissue similar to that found in outer ring, cells measure 12-16-19 x 7-13-18µ; xylem cells measure 4-7-17 x 2-4-9µ. Druses type of calcium oxalate crystals are found in cells of conjunctive tissue, measure 28-29-30x24-25-26µ. The central portion is occupied by sclerenchymatous pith (Figs. 4-10). Macerate Macerate of the root exhibit the following elements: cork cells which are thick walled, rectangular or polyhedral, measure 65-67-70 x 38-48-57µ (Figs.11&12); parenchyma cells of different size and shape, globose or elliptical, measure 140-160-186 x 6632-38µ (Fig. 13); xylem parenchyma with simple pits, measure 115-116-117 x 65-73-80µ (Fig. 14); fibres of different size and shape with broad lumen and narrow pointed ends, measure 220-250-300 x 11-15-17µ (Figs. 15); druses type of calcium oxalate crystals (Fig. 16); and vessels of different size and shape – narrow, cylindrical, barrel shaped or with pitted thickenings, measure 59-108-178 x 16-30-58µ (Fig. 17). Powder study Root powder is greenish, odourless, bitter, fibrous; when treated with chloral hydrate solution, stained in 1% safranin for 5 -10 minutes, mounted in 50% glycerine exhibits fragments of cortical parenchyma and druses type of calcium oxalate crystals. Histochemical tests The root sections of N. brachiata when treated with phloroglucinol and dilute HCl gave red colour indicating the presence of lignin; with Millons’s reagent turned red indicating presence of proteins, with ferric chloride turned black showing presence of MADHAVAN et al.: PHARMACOGNOSTICAL STUDIES OF PASHANABHEDA 631 632 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 9, No. 4, OCTOBER 2010 MADHAVAN et al.: PHARMACOGNOSTICAL STUDIES OF PASHANABHEDA tannins and with concentrate HCl effervescence was observed showing the presence of crystals; when treated with iodine no blue colour, with ruthenium red and Sudan III, no red colour, with Dragendorff’s reagent, no brown colour was observed indicating the absence of starch, mucilage, fixed oil and alkaloids, respectively. Physicochemical studies The percentage of moisture content was 8.5%, total ash 5.07%, acid insoluble ash 1.33%, water soluble ash 4.5%, alcohol soluble extractive 19.2% and water soluble extractive 28%; the colour, consistency and percentage of successive extractive values of extracts were: petroleum ether (60-80ºC) (tata mimosa, sticky mass, 1.4%), benzene (olive green, sticky mass, 1.16%), chloroform (sand stone, sticky mass, 4.8%), acetone (brown, sticky mass, 3.24%), ethanol (brown, semi solid, 4.5%) and water (brown, semi solid, 4.72%). Preliminary organic analysis A known quantity of dried powder was extracted in a Soxhlet with petroleum ether (60-80ºC), benzene, chloroform, acetone and ethanol (95%) and finally macerated with chloroform-water (2%) for 24 hrs successively and tested for different constituents. It revealed the presence of carbohydrates, glycosides, phenols, tannins, saponins, flavonoids, proteins and amino acids in ethanol and water extracts, whereas gums and mucilage were found in water extract. Chromatographic studies HPTLC profile of alcohol extract of roots revealed when scanned at 254 nm, 9 phytoconstituents at Rf 0.11, 0.14, 0.24, 0.28, 0.56, 0.78, 0.82, 0.86, 0.88 (Fig. 18), out of these, spots at Rf 0.56, 0.88 were pronounced, spots at Rf 0.24, 0.78, 0.86 were less pronounced, whereas spots at Rf 0.11, 0.14, 0.28, 0.82 were least pronounced; at 366 nm, 7 phytoconstituents at Rf 0.10, 0.13, 0.22, 0.26, 0.50, 0.80, 0.92 were observed (Fig. 19), out of these, spots at Rf 0.10, 0.13, 0.80, 0.92 were pronounced, spot at Rf 0.26 was less pronounced, whereas spots at Rf 0.22, 0.50 were least pronounced. All spots in alcohol extract quenched green fluorescence under 254 nm, showed blue fluorescence under 366 nm, while no spots were visible under 425 nm. Aqueous extract at 254 nm revealed 7 phytoconstituents at Rf 0.06, 0.16, 0.32, 0.40, 0.53, 0.60, 0.73 (Fig. 20), out of these, spots at Rf 0.53, 633 0.73 were pronounced, spot at Rf 0.40 was less pronounced, whereas spots at Rf 0.06, 0.16, 0.32, 0.60 were least pronounced; at 366 nm, 8 phytoconstituents at Rf 0.10, 0.18, 0.29, 0.36, 0.43, 0.50, 0.66, 0.88 were observed (Fig. 21), out of these, spots at Rf 0.29, 0.36, 0.43, 0.50 were pronounced, spot at Rf 0.88 was less pronounced, whereas spots at the Rf 0.10, 0.18, 0.66 were least pronounced and at 425 nm 6 phytoconstituents at Rf 0.30, 0.38, 0.43, 0.51, 0.72, 0.88 were found (Fig. 22), out of these, spots at Rf 0.38, 0.43 were pronounced, spots at Rf 0.30, 0.88 were less pronounced, whereas spots at Rf 0.51, 0.72 were least pronounced. All spots in aqueous extract quenched fluorescence under 254 nm, showed blue fluorescence under 366 nm whereas under 425 nm all spots showed yellow fluorescence. Ultra-violet analysis Powdered drug under ultra-violet and ordinary light when treated with different reagents emitted various colour radiations (Table 1) which help in identifying the drug in powder form. Table 1 Ultra-violet analysis of the roots of N. brachiata Treatment Powder as such In methanol In 1N methanolic NaOH In ethanol (70%) In 1N ethanolic NaOH In 1N HCl 50% H2SO4 50% HNO3 5% KOH Visible light Ultra-violet light Short wave Long wave (254 nm) (365 nm) Pista Opaline green No fluorescence Tata mimosa Opaline green No fluorescence Tata mimosa Mint green No fluorescence Mid buff Water green No fluorescence Tata mimosa Mint green No fluorescence Mid buff Pale cream Mid buff Mid buff Water green Water green Mint green Water green No fluorescence No fluorescence No fluorescence No fluorescence Discussion Pashanabedha is considered as one of the controversial drugs in Ayurveda4. Different botanical sources are used in various parts of the country as Pashanabedha5,8. Nothosaerva brachiata is distinguished from other botanical sources by the following diagnostic characters. The plant is identified by the presence of flowers in sessile spikes; the root drug is characterised by the presence of anamolous secondary growth, secondary xylem arranged in three rings, presence of druses, intraxylary parenchyma and sclerenchymatous pith. 634 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 9, No. 4, OCTOBER 2010 Conclusion The pharmacognostical and phytochemical studies of the root of N. brachiata were carried out. Preliminary phytochemical analysis revealed presence of carbohydrates, glycosides, phenols, tannins, saponins, flavonoids, proteins and amino acids in ethanol and water extracts, whereas gums and mucilage were present in water extract. HPTLC studies help in identification of the drug and provide leads for establishing the biomarker compound. Acknowledgement Authors are thankful to the Gokula Education Foundation and to the Principal, VV Pura College of Science, Bangalore for evincing interest in the work. Authors are also thankful to Sri V Chelladurai of Tirunelveli for providing authentic plant material. 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