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Ulmus Wallichiana Planchon: an Indian Traditional Plant for Osteogenic Activity

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Ulmus Wallichiana Planchon: an Indian Traditional Plant for Osteogenic Activity Indian Journal of Traditional Knowledge Vol. 12 (4), October 2013, pp. 664-669 Development of quality control markers for Ulmus wallichiana Planchon: An Indian traditional plant for osteogenic activity K R Arya1*, Sayyada Khatoon2 & Brijesh Kumar3 1Botany Division, 3Sophisticated Analytical Instrument Facility CSIR- Central Drug Research Institute, Lucknow- 226001, India, 2Pharmacognosy & Ethnopharmacology Division, CSIR- National Botanical Research Institute, Lucknow- 226 001, India E-mails: [email protected], [email protected]; [email protected] Received 17.01.12, revised 31.05.13 Ulmus wallichiana Planchon, is an Indian folk traditional plant used for the treatment of fractured bones in folklore tradition of Uttarakhand Himalaya, India. During chemical investigations, three major compounds, Ulmoside A & B, and Naringenin-6-C-β-D glucopyranosideis for osteogenic activity have been isolated from bark of this plant species. This paper presents macro and microscopic study, physicochemical parameters and chemo-profiling of stem bark of U. wallichiana through Q TOF HRMS for development of quality control markers and identification of crude samples. Transverse section (TS) stem bark and powder microscopy shows crushed rhytidoma consisting plenty of mucilaginous canals followed by crushed cork, cells of the cork filled with dark brown content. Whereas, authenticity and quality of raw materials can be determinate with exact calculated mass value of marker compounds for osteogenic activity at 467, 451 and 435 using Q TOF HRMS technique. Ursolic acid, β-sitosterol and lupeol at Rf value 0.25, 0.36 and 0.44 has also identified as HPTLC marker for identification and authentication of crude drug samples of stem bark of U. wallichiana. Keywords: Ulmus wallichiana, Bone healing, Q TOF HRMS, Quality control marker, HPTLC IPC Int. Cl.8: A61K 36/00, A01D 20/00, A01D 9/02, G07C 3/14, A62B Himalayan elm, Ulmus wallichiana Planchon, treatment of post menopausal osteoporosis9 and are family Ulmaceae, locally known as Chamourmou now in public domain for commercial exploration. is commonly used for healing fractured bones in Source of raw materials for pharmaceutical animal as well as human being in folk tradition production is only from wild. Unprecedented demand of Uttarakhand Himalaya, India1-2. Plant species is and scarcity of raw materials may lead to chances rare3-4 and endemic to Western Himalaya5. Habitat of adulteration and substitution of genuine drug. degradation, biotic interferences and unsustainable Chemical and biological evaluation, physico-chemical harvesting of bark shows decline in species richness parameters, HPTLC finger printing is useful for and total tree density in Western Himalaya, India. identification and authentication of genuine drugs and Globally, about 45 species of this genus is found their quality evaluation10-13. This study provides in North temperate region, while 3 species, i.e. macro and microscopic studies of stem bark of U wallichiana, U. lancefolia and U. parviflora U. wallichiana pertaining with HPTLC profiling of are reported from India6. Two novel nonestrogenic ethanolic extract, physico-chemical parameters and osteoprotective compounds Ulmoside A and chemical fingerprinting through Quadrupole-Time- Ulmoside B rich in C-glycosylated flavanoid of-Flight High Resolution Mass Spectrometer and third known compound Naringenin-6-C-β-D (Q-TOF HRMS) to insure quality and authenticity glucopyranosideis isolated from same extract7 of crude drugs. showed peak bone achievement and prevention of menopausal bone loss in ovariectomized rat Methodology and promotes osteoblast function and inhibited 8-9 Ulmus wallichiana Planchon, large deciduous tree adipogenesis . During pharmacological investigation with rough grey exfoliating bark, up to 30 m high; these compounds revealed therapeutic potential for young branches pubescent to tomentose. Leaves —————— elliptic-acuminate to obovate-cuspidate,7-15 cm long, * Corresponding author 4-6 cm broad, base obliquely cuneate to rounded, ARYA et al.: QUALITY CONTROL MARKERS OF OSTEOGENIC ULMUS WALLICHIANA 665 sharply biserrate, teeth arching with 2-4 secondary (4 × 25 mL, each time for 15 min) under reflux on a teeth, upper surface pubescent to scabridulous, water bath at 100°C. Extracts were filtered through lower surface densely pubescent to tomentose; Whatman No. 1 filter paper (separately for each petiole 6-10 mm long, pubescent. Inflorescence axis sample), concentrated under reduced pressure, and elongated, pedicels more than 5 mm long, articulated; lyophilized17-18. A solution (10 mg mL-1) of these 1/3rd lower portion of pedicel uniformly pilose; extracts and standard solution (0.1 mg mL-1) of flowers in clusters appearing before leaves on β-sitosterol, ursolic acid and lupeol were also branches of previous season, perianth tube narrowed prepared in methanol. Known quantity of methanolic into the pedicel, lobes 5-6, obtuse, pubescent to extracts along with chemical markers (β-sitosterol, sub glabrous; stamens 5-6, filaments longer than ursolic acid and lupeol) were applied on to the perianth, anthers red; ovary slightly pubescent Higlachrosep nano silica UV 254 HPTLC plates of all over; fruit samara, orbicular-obovate, 12-15 mm, 10x10 cm with 0.2 mm nano silica with fluorescent narrowed into a short stipe, 2-3 mm long, stipe indicator (S. D. Fine-Chem. Ltd. India) with the longer than the perianth, central seed hirsute to help of CAMAG Linomat Applicator 5, positioned sub glabrous, wing membranous, reticulate, margin 15 mm from side and 10 mm from bottom of the ciliolate (Fig. 1A). plate. Plate was eluted to a distance of 8.0 cm at Stem bark of Ulmus wallichiana was collected room temperature (24ºC) in a solvent system – from three different altitudinal locations toluene: ethyl acetate : formic acid (9 : 1 : 0.1) in of Uttarakhand Himalaya, viz. Nainital (1800 M), previously saturated twin trough chamber (CAMAG). Almora (2500 M) and Bageshwar (3000 M) Plate was derivatized by spraying with anisaldehyde during April, 2009. All the samples were properly sulphuric acid reagent. After heating at 110°C for identified by one of the senior author (KRA) 10 min plate was documented under visible light and compared with Flora of The District Garhwal, and scanned at wavelength 600 nm using CAMAG North West Himalaya6. Herbarium specimens TLC Scanner 3 with software Win Cats 3.2.1. were deposited in departmental herbarium, CDRI, Lucknow, vide voucher specimen numbers KRA Results and discussion 24432, KRA 24443, and KRA 24444, respectively. Analytical grade chemicals from SD Fine, Mumbai Macroscopic studies (India) and standards (Lupeol, β-sitosterol and Bark fibrous, rough grey exfoliating in diamond ursolic acid) were procured from Sigma, St. Louis, shaped flakes. Dried mature bark slightly curved, 1 to MO, USA. 1.5 cm in thickness, outer surface blackish brown Macro and microscopy of bark and powder were in colour, rough due to the presence of irregular studied13-15. Air dried plant material was used for cracks and rhytidoma which peeled off at some quantitative determination of ash and extractive places leaving rough, protuberated brown coloured values16. Swelling Index was calculated by modifying surface; internal surface brown in colour, smooth WHO method due to high percentage of mucilage. or with very fine, wavy, transverse and longitudinal 100 mg powder was kept in 25 ml of water in a striations; fracture hard and splintery; mucilaginous glass-Stoppard measuring cylinder; the material and slightly acrid in taste with pleasant odour was shaken repeatedly for 2 hrs and then kept (Figs.1 B & C). for 24 hrs to settle down. Volume of the mixture (ml) was measured as swelling Index. Marker compounds Microscopic studies Ulmoside A, B and Naringenin-6-β-D glucopyranoside TS bark shows crushed rhytidoma consisting were identified through Q-TOF HRMS fitted with of plenty mucilaginous canals followed by crushed an electro spray (ESI) interface. Analyses utilized cork, cells of the cork filled with dark brown the positive ion mode m/z (M+H)+ for detection of content. Cork cambium is not distinct. Phelloderm is compounds. very broad traversed with plenty schizogenous mucilage canals, some stone cells, fibres and Chromatographic analysis and HPTLC profile prismatic crystals of calcium oxalate. Parenchymatous Dried stem bark of U. wallichiana were powdered cells also filled with mucilage. Secondary phloem and sieved through 44 meshes. Powdered sample is very narrow as compared to phelloderm. Phloem (5 gm) was exhaustively extracted with methanol consists of sieve tubes, companion cells, phloem 666 INDIAN J TRADITIONAL KNOWLEDGE, VOL. 12, NO. 4, OCTOBER 2013 Fig. 1—A- Ulmus wallichiana tree; B- Outer surface of stem bark; C- Inner surface of stem bark. Table 1—Physico-chemical values of U. wallichiana collected from Nainital, Almora and Bageshwar districts in Uttarakhand S.No. Parameters Nainitala Almoraa Bageshwara 1. Total ash % 15.29 ± 0.315 16.04 ± 0.235 16.23 ± 0.156 2. Acid insoluble ash % 0.94 ± 0.054 0.96 ± 0.016 0.99 ± 0.031 3. Alcohol soluble extract % 22.00 ± 0.100 23.00 ± 0.300 24.30 ± 0.100 4. Swelling Index (mL) 12.50 ± 0.500 13.20 ± 0.300 12.80 ± 0.250 a Mean value of 5 readings ± SD parenchyma, abundant mucilage canals and tangential Physico-chemical analysis bands of crystalloid fibres interrupted by medullary Ash values (total, acid insoluble, water-soluble), rays. Mucilage canals are arranged in tangential alcohol soluble, and water-soluble extractives are and
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