Indian Journal of Experimental Biology Vol. 55, September 2017, pp. 622-627

Pharmacognostical evaluation of Indian folk-traditional 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, . 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. ), 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 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- 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 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, and are 4-5 cm long, observation of powder. oblong blunt with wavy margins, 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 (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 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. 2.5-3 mm, 1 mm wide, stout, apex Epidermis, Hypo- hypodermis, Aer- Aerenchyma, Vb- Vascular winged; rostellum broadly ovate, 1.4-1.8 mm. Capsul Bundle, Ep- Epidermis, Bs- Bundle Sheath, Ph- Phloem, e ellipsoid to obovoid-ellipsoid, 1.8-2 cm, slightly Xy- Xylem] 3-ridged; fruiting pedicel and ovary 6-7 mm. abaxial surfaces. Mesophyll region is 4-5 layered, Pseudobulbs connected to each other at both ends and chlorenchymatous, cells thin walled, without stem like, subcylindric, (2-)4-12 cm × 5-10(-25) mm, intercellular spaces. Raphides are present in sometimes slightly narrowed, branching or not, mesophyll region. Vascular bundle are in one row; sometimes with very short between them large in midrib region and comparatively very small and producing a few roots (Fig. 1B). Leaves 2, at apex fibrous bundles in laminar region surrounded by of new pseudobulb; leaf blade obovate-elliptic, sclerenchymatous bundle sheath (Fig. 2). Stomata are oblong, or narrowly elliptic, 7-17.5 × 2.7-6.2 cm, anomocytic type and present only on lower surface. veins plicate, subacute or obtuse; petiole 1-1.5 cm. Stomatal number 50 per cm2 and stomatal index at apex of new pseudobulb, 6-18 cm; ranges from 09.09 to12.25. rachis 10- or more flowered, flexuous; floral bracts deciduous during flowering, narrowly ovate-oblong, Psudobulb 1.5-2.5 × 0.5-0.7 cm. ca. 2.5 mm. Flowering Jun-Aug, The TS of pseudobulb is circular in outline fr. Oct-Dec. 2n = 38, 38 + 2B, 40, 40 + 2-6B. with number of vascular bundles distributed all

Microscopy over the ground tissue. Cuticle thick, epidermis Coelogyne cristata Lindley is well developed with sunken stomata at places. Leaf Hypodermis 4-5 layered with compactly arranged The TS leaf lamina showed prominent midrib sclerenchymatous cells. Ground tissue is made up of having collateral vascular bundle and lateral fibrous loosely arranged parenchymatous cells interrupted bundles. The lower and upper epidermal cells are thin with large mucilage containing cells and small walled, pitted, polygonal, some cells with spiral chlorophyllous cells. Parenchymatous cells are pitted, thickening and covered with thick cuticle. The banded, lignified or beaded with mesh like network. hypodermis consists of a single layer of polygonal Some cells contain starch grains and raphides of cells comparatively larger than epidermal cells and calcium oxalate needles. Vascular bundles are interrupted by 1-2 water storage cells on adaxial and abruptly distributed all over the ground tissue. SHARMA et al.: PHARMACOGNOSTICAL EVALUATION OF INDIAN TRADITIONAL ORCHIDS 625

Towards the periphery vascular bundles are smaller in conjoint, bicollateral, closed and surrounded by size; more in number where as towards the centre they schlerenchymatous bundle sheath. are bigger size and less in number. Each vascular bundle is oval, conjoint, bicollateral and closed Powder capped with sclerenchymatous bundle sheath at both Powder of both the species is greenish-brown in ends (Fig. 3) colour, aromatic with a characteristic taste. Under the microscope it shows groups epidermal cells, Pholidota articulata Lindley anomocytic stomata, fragments of septate and aseptate Leaf fibres raphides of calcium oxalate needles and The TS leaf lamina showed collateral vascular rhomboidal crystals of calcium oxalate, pitted bundles of almost uniform sized in a row. Cuticle is parenchyma cells etc (Fig. 4). moderately developed on both surfaces. Epidermis is single layered made up of sclerenchymatous, Physicochemical analysis isodiametric and squared cells interrupted with water Statistical analysis of all the physicochemical storage cells and sunken stomata on adaxial surface. parameters i.e. extractive value, total ash and acid Slight depressions on both the surfaces are present at insoluble ash value were done (Table 1). the position of vascular bundles which are collateral, closed and surrounded by scherenchymatous bundle HPTLC analysis sheath and arranged in a row at the median line of The results of HPTLC profiles of CC and PA lamina. Mesophyll is not differentiated, 4-5 layered, methanolic extract are given in Table 2 which showed chlorenchymatous, some cells with intercellular spaces and some having spiral thickening. Raphides are present in chlorenchymatous cells. Stomata are anomocytic type and present only on lower surface. Stomatal number varies from 25 to 50 per cm2 and stomatal index ranges from 3.70 to 6.66.

Psudobulb The TS of psuedobolb is circular in outline with number of vascular bundles distributed all over the ground tissue and more compactly arranged towards the centre. Outermost epidermis layer is made up of isodiametric cells covered with comparatively thin cuticle. Hypodermis 2-3 layer with large cells, ground tissue parenchymatous with spiral thickened mucilage containing cells and small chlororenchyma cells with starch grains, lacunae also present in ground tissue, vascular bundles scattered, bundle sheath

scleranchymatous present at both phloem and xylem Fig. 3 — T.S. of Pseudobulb and vascular bundle. (A and B) of poles (Fig. 3). Ground tissue is made up of CC; and(C and D) of PA, respectively. [Cu-Cuticle, parenchymatous cells which are loosely arranged Uep-Upper Epidermis, Aer- Aerenchyma, Vb- Vascular Bundle, towards the peripheral region interrupted with large Ep- Epidermis, Bs- Bundle Sheath, Ph- Phloem, Xy- Xylem]. mucilage containing cells and small chlorophyllous cells while compactly arranged, much smaller in size Table 1 — Physicochemical details of methanolic extract of CC and PA towards the centre. Parenchymatous cells are pitted, banded, lignified or beaded with mesh like network. Parameter Percent value (wt/wt) Some cells contain starch grains and raphides of CC PA calcium oxalate needles. Vascular bundles are smaller Alcoholic extractive 1.25±0.05 1.14±0.10 in size and scattered towards the periphery while Water extractive 2.27±0.04 2.69±0.08 these are closely placed at the centre and much Total ash 5.92±0.60 7.10±0.31 larger in size. Each vascular bundle is oval, Acid insoluble ash 3.90±0.25 4.50±0.25 626 INDIAN J EXP BIOL, SEPTEMBER 2017

Table 2 — Rf details of HPTLC profile of methanolic extract of CC and PA Sample Colour UV UV after Rf 254nm 366nm derivatization CC PA CC PA CC PA 0.10 Brownish + - - - + - 0.14 Light pink - + - + + + 0.17 Purple + + - - + + 0.20 Blue/black + + + + + + 0.23 Blue + - + - - - 0.26 Orange + + - - + + 0.31 Purple/black + + - - + + (Ursolic acid) 0.37 Dark pink/black + - - - + - 0.47 Light gray - - - - + + ( sitosterol) 0.40 Blue - + - - - + 0.51 Green/gray - - + + + + 0.62 Purple (lupeol) - - - - + + 0.67 Black/gray + + - - + + 0.75 Black/gray + + - + + 0.85 Black/gray + + - - + +

Table 3 — Quantity of reference compounds present in methanolic extract of CC and PA Reference Marker µg/g Extract (range) CC PA Beta Sitosterol 5.51-8.28 4.83-7.69 Lupeol 2.66-3.74 2.80-3.63 Ursolic acid 4.05-7.84 4.05-8.98

(dark pink) were observed under visible light after derivatization (Fig. 5). Three chemical markers viz. ursolic acid, -sitosterol and lupeol given in Fig. 5C showed best representation through densitometric scanning profile at 580 nm by simultaneous bands at Fig. 4 — Powder microscopic characters of (A) PA (i-v); and (B) Rf 0.31, 0.47, 0.62, respectively. Comparative CC (vi-x). [(i) Anomocytic stomata, (ii) tracheids with spiral HPTLC profile with marker component showed thickenings, (iii) raphides of calcium oxalate, (iv) septate fibres, (v) epidermal cells and ground tissue having some presence of aforesaid three markers in both CC and parenchymatous cells with raphides; and (vi) Anomocytic PA (Fig. 5 E & D). Slight variations were observed in stomata, (vii) Aseptate fibres, (viii) rhomboid crystals, (ix) the quantity of these marker compounds, the details of Vascular bundle surrounded by schlerenchymatous bundle sheath which has been given in the Table 3. and (x) a patch of pitted parenchyma cells] Both the plant species CC and PA are well similar and differentiating bands. Two common bands known for their bone healing properties in folk at Rf 0.20 (blue) and 0.51(green) under UV 366 nm tradition of Uttarakhand Himalaya, India3,4. It is and eight bands at Rf 0.17, 0.20, 0.26, 0.31, 0.40, notable that the ethanolic extract and bioactive 0.67, 0.75 and 0.85 under UV 254 nm and eleven compounds isolated from these plant species bands at Rf 0.14, 0.17, 0.20, 0.26, 0.31, 0.47, 0.57, were also proven their bone healing potential 0.62, 0.67, 0.75, 0.85 were observed under visible during post menopausal osteoporosis10,11. light after derivatization in both the species. The Pharmaceutical products, for long term use, require identifying blue fluorescent band at Rf 0.23 under UV authentication following established standardized 366 nm was observed only in CC and pink at Rf 0.14 quality control markers. Till date no reports are was present only in PA. However, blue band at Rf available on the pharmacognostical evaluation of 0.40 in PA while band at Rf 0.10 (brown) and 0.37 these plant species. The comparative macro SHARMA et al.: PHARMACOGNOSTICAL EVALUATION OF INDIAN TRADITIONAL ORCHIDS 627

Though the HPTLC profile of both the plants showed several common bands under visible and florescent light, presence of the blue florescent band at Rf value 0.23 under 366 nm and pink band at Rf value 0.14 and Rf values at 0.31, 0.47, 0.62 in both CC and PA corresponding to the marker compounds (ursolic acid, -sitosterol and lupeol) at 540 nm could be considered as standard identifying parameters for authentication of herbal drug samples or any other marketed products originated from these plant species.

References 1 Gaur RD, Flora of District Garhwal, North West Himalaya (Trans Media, Srinagar, Garhwal, India), 1999. 2 Zhong Hua Ben Cao: The Chinese Herbal. Vol. 8, (Shanghai Xin Hua Press, Shanghai), 1999, 7904. 3 Jalal JS, Tiwari LM & Pangtey YPS, Pholidota articulata Lindl., An Orchid used in bone jointing in Kumaon region, Western Himalaya. Ethnobot Leaflets, 13 (2009) 147. 4 Sharma C, Kumari T & Arya KR, Ethnopharmacological survey on bone healing plants with special references to Pholidota articulata and Coelogyne cristata (Orchidaceae) used in folk tradition of Kumaon, Uttarakhand, India. Int J Pharma Res Health Sci, 2 (2014) 185. 5 Majumder PL, Sen S & Majumder S, Phenanthrene derivatives from the orchid Coelogyne cristata. Phytochemistry, 58 (2001) 81. 6 Bandi AKR & Lee DU, Chemical constituents and bio-activities of plants from the Pholidota. Chem Biodivers, 57 (2011) 33. 7 Hossain MM, Therapeutic orchids: Traditional uses and recent advances- an overview. Fitoterapia, 82 (2011) 102. 8 Gutierrez RMP, Orchids: A review of uses in traditional medicine, its phytochemistry and pharmacology. J Med Pl Res, 4 (2010) 592. 9 Singh D, Sati SC & Sati MD, Evaluation of anti- inflammatory activity of Pholedota articulata extract. Eur J Pharm Med Res, 3 (2016) 362. 10 Sharma C, Mansoori MN, Dixit M, Shukla P, Kumari T, Bhandari SPS, Narender T, Singh D & Arya KR, Ethanolic extract of Fig. 5 — TLC profile of two orchid species, Pholidota articulata Coelogyne cristata Lindley (Orchidaceae) and its compound (PA) and Coelogyne cristata (CC) with three chemical markers: Beta Sitosterol (R1), Lupeol (R2) and Ursolic Acid (R3). coelogin promote osteoprotective activity in ovariectomized Visualization under (A) UV366 nm; (B) 254 nm; (C) after spray; estrogen deficient mice. Phytomedicine, 21 (2014) 1702. and (D and E) densitometry HPTLC profile at 580 nm of 11 Sharma C, Dixit M, Singh R, Agrawal M, Mansoori MN, Pholidota articulata and Coelogyne cristata Kureel J, Singh D, Narender T & Arya KR, Potential osteogenic activity of ethanolic extract and scopic characters of both the plant species discussed oxoflavidin isolated from Pholidota articulata Lindley. here and also described previously1 provide essential J Ethnopharmacol, 2015. DOI: 10.1016/j.jep.2015.04.045 information for taxonomical identification of these 12 Thatte U, Challenges in clinical research on herbs. important plant species. However, presence of Indian J Nat Prod, 19 (2003) 35. 13 Anonymous, Ayurvedic Pharmacopoeia of India (API). Part I, collateral vascular bundles and anomocytic type of Vol II, (Government of India, Ministry of Health and Family stomata on lower surface of leaf, large number of Welfare, New Delhi, India), 2000. mucilage cells, pitted banded lignified or beaded with 14 Evans WC, Trease and Evans Pharmacognosy. 15th ed. mesh like network of parenchymatous cells, septate or (London: Saunders), 2003, 545. 15 Anonymous, Indian Pharmacopoeia. Vol II, 4th ed. aseptate fibers, raphides of calcium oxalate needle (Government of India, Ministry of Health and Family and rhomboidal crystal of calcium oxalate pitted Welfare, New Delhi, India), 1996, A53. parenchyma can serve as a diagnostic features during 16 WHO, Quality Control Methods for Medicinal Plant microscopic studies. Materials. (World Health Organization, Geneva) 1992, 9.