Indian Journal of Experimental Biology Vol. 55, September 2017, pp. 622-627 Pharmacognostical evaluation of Indian folk-traditional plants Coelogyne cristata and Pholidota articulata used for healing fractures Chetan Sharma1, Saba Irshad2, Sayyada Khatoon2 & KR Arya1* 1Botany Division, CSIR-Central Drug Research Institute, Lucknow-226 031, India. 2Pharmacognosy & Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow-226 001, India. Received 07 May 2015; 14 September 2016 Coelogyne cristata Lindley (CC) and Pholidota articulata Lindley (PA) (Fam. Orchidaceae), locally known as ‘hadjojen’(bone jointer) are the most effective traditional remedies for healing fractures in Uttarakhand Himalaya, India. Recent pharmacological investigations of crude extracts and isolated compounds from these plant species revealed rapid fracture healing properties and osteogenic potential. This paper provides macro and microscopic characteristic features, physicochemical properties and HPTLC profiles of both the species. Microscopic studies of leaf, pseudobulb and powdered materials showed collateral vascular bundles containing large number of mucilage cells, parenchymatous cells with pitted banded lignified or beaded with mesh-like network, septate and aseptate fibres, rhomboidal crystals of calcium oxalate and pitted parenchyma. Comparative HPTLC profile showed blue and pink florescent band at different Rf values with the distinct characteristic bands at Rf 0.31, 0.47 and 0.62 corresponding to the analytical marker compounds: ursolic acid, -sitosterol and lupeol respectively in both CC and PA. Findings of this study can be used a standardized pharmacognostical markers of CC and PA for identification and authentication of their genuine herbal drug formulations as quality control markers. Keywords: Bone jointer, Hadjojen, Herbal, HPTLC, Osteogenic, Orchids, Pharmacognosy Coelogyne cristata Lindley (CC) and Pholidota and PA are known with the similar local trade articulata Lindley (PA) belongs to family name‘Hadjojen’(bone jointer). Poultice made from Orchidaceae and are distributed throughout mountane the whole parts of the plants were used as fracture to submountane zones from Uttarakhand Himalayas healing remedies in folk tradition of Kumaon and (Kumaon and Garhwal) to Arunanchal Pradesh and Garhwal region of Uttarakhand Himalaya, India4. Our Indo-China to Malaysia1. These ancient medicinal recent chemical and pharmacological investigations plants, has long been used as a remedy for acute or on crude extract and its isolated compounds revealed chronic bronchitis, toothache, duodenal ulcer and rapid fracture healing properties in ovariectomized fracture healing2-4 and proved to be a rich store-house estrogen deficient rats and focused as potential plants of phytochemicals including alkaloids, flavonoids, for the treatment of osteoporosis during menopausal glycosides, benzyl derivatives, phenanthrenes, terpenoids, disorders10,11. Standardization of pharmacognostical phenolic compounds, pyrone derivatives, etc.5-7 with markers for quality of products due to deliberate promising biological activities for antitumor, anti- adulteration is one of the major challenging tasks to inflammatory, anticancer, anticonvulsive, antidiabetic, authenticate and maintain the quality of herbal osteoprotective, etc.6,8,9. Nearly 200 species of products12. In view of its diverse medicinal Coelogyne and 46 species of Pholidota are distributed applications and in order to ensure the quality, all over the world1,3 and only 5-6 species have been authenticity and assay as well as lack of investigated in some details6. pharmacognostic studies, the present investigation During ethno botanical and local herbal drug was undertaken with an objective to evaluate both the market survey revealed that both the plant species CC plant species on various pharmacognostical parameters, such as macroscopic, microscopic, —————— physiochemical, fluorescence and phytochemical *Correspondence: studies. Taxonomically both the plants species are Phone: +91 522 2772467; Fax: +91 522 2771941 E-mail: [email protected] different in their morphological characters along with SHARMA et al.: PHARMACOGNOSTICAL EVALUATION OF INDIAN TRADITIONAL ORCHIDS 623 their chemical constituents and biological porcelain crucible at 550°C in a muffle furnace. activities10,11. No pharmacognostical investigations Physicochemical values were calculated as per are available so far on these two plant species. Standard Indian Pharmacopoeial methods13. The pharmaceutical application for fracture Powdered material (10 g) was extracted with healing properties of these plant species are now methanol on a water bath for 25 min, consecutively in public domain. Standardization of pharmacological thrice, concentrated and dried the extract. Known markers for identification and authentication of quantity of extract was dissolved in methanol for these important herbal drug formulations seem to HPTLC. One mg of -sitosterol, lupeol and ursolic be logical. This paper reports a detailed acid (Sigma) as reference markers were also dissolved pharmacognostical study of CC and PA pertaining separately in 10 mL methanol. with the macro and microscopical studies, physico- phytochemical parameters and HPTLC profiling as Chromatographic conditions standard pharmacognostical markers for identification Known quantity of methanolic extracts along with and authentication of their herbal products. chemical markers (lupeol, sitosterol and ursolic acid) were applied on to Higlachrosep nano silica UV Materials and Methods 254 HPTLC plates (10 ×10 cm) with 0.2 mm nano silica with fluorescent indicator (S.D. Fine-Chem. Plant materials and processing Ltd. India) using CAMAG Linomat 5 Applicator, Fresh plant material (leaves and pseudobulb) of CC band size 5mm, positioned 10 mm at X axis and and PA were collected from Bajun forest range of 10 mm at Y axis, 10.5 mm distance between two Nainital district, Kumaon, Uttarakhand, India and tracks. Plate was eluted to a distance of 8.5 cm at identified by one of the senior author (KRA) 1 room temperature (24±2ºC) in a solvent system- according to Flora of District Garhwal . Voucher toluene: ethyl acetate: formic acid (8: 2: 0.1) in specimens KRA-24462 and KRA-24460, respectively previously saturated twin trough chamber (CAMAG). have been deposited in the departmental herbarium of Photographs were taken under UV light 254 and CSIR-Central Drug Research Institute, Lucknow. 366 nm using CAMAG Reprostar 3 video Plant materials were chopped into small pieces and documentation unit (CAMAG). Plate was derivatized completely shed dried. All precautions were by dipping in anisaldehyde sulphuric acid reagent and undertaken to avoid any types of microbial after heating plate at 110ºC for 10 min documented contamination. The dried material was powdered under visible light. Plate was scanned at wavelength using grinder. For microscopic studies, transverse 580 nm using CAMAG TLC Scanner 3 with software sections (TS) were prepared and stained. Leaf winCATS 4.3.2. constant i.e. stomatal number and stomatal index was studied according to the standard methods13,14. Results and Discussion Samples were dried at 50±2ºC in a hot air oven, Macroscopy stored at 25±2ºC in air tight container. Dried plant Coelogyne cristata Lindley material of both the species was powdered and sieve Flowers are white, borne in 3-10 flowered clusters through the 85 mesh. A small quantity of powdered 15-20 cm long hanging clusters, 5-9 cm across, with a material was washed with water and then cleared by white lip with 4 yellow ridges at the base between the heating gently with saturated chloral hydrate solution, lateral lobes, 2 broad crenulate yellow plates on the cooled and mounted in glycerin for microscopic mid-lobe, Sepals and petals are 4-5 cm long, observation of powder. oblong blunt with wavy margins, Bracts are oblong and persistent, Spur is absent. Leaves are paired, Physicochemical studies linear-lance shaped 15-30 cm long, 2-3 cm broad. Physicochemical analysis such as the percentage of Pseudobulbs are oblong ovoid, 5-8 cm, arising from a ash values and extractive values were carried out stout rhizome (Fig. 1A). The flowering season is according to the official methods prescribed in Indian 13,15 March-April. Pharmacopoeias and the WHO guidelines on quality control methods for medicinal plant Pholidota articulata Lindley materials16. About 1 g of each dried powder was Flowers greenish white or white and slightly tinged weighed and subjected to dry-ashing in a well-cleaned with reddish pedicel. Dorsal sepal oblong or elliptic, 624 INDIAN J EXP BIOL, SEPTEMBER 2017 Fig. 1 — (A) Coelogyne cristata Lindley; and (B) Pholidota articulata Lindley. concave, 9-10 × 4-5 mm, dorsally carinate, 5-veined; lateral sepals ovate, oblique, slightly wider than dorsal sepal. Petals oblong-lanceolate or suboblanceolate, 7 × 2-2.5 mm, 5-veined; lip broadly oblong in outline, contracted at apical 1/4-1/3 into epichile and hypochile; hypochile cymbiform, slightly wider than Fig. 2 — T.S. of CC and PA leaf (i) T. S. of CC leaf (ii) T. S. of epichile, with 5 longitudinal lamellae near base; PA leaf (iii) T. S. through parallal vein of PA leaf(iv) T. S. epichile transversely elliptic, 3-4 mm wide, margin through laminar region of CC leaf (v) T. S. through mid-rib portion of CC leaf; Pholidota articulata [Cu-Cuticle, Uep- Upper crisped. Column 2.5-3 mm, 1 mm wide, stout, apex Epidermis, Hypo- hypodermis, Aer- Aerenchyma, Vb- Vascular winged;