Variation in the marker content of five different Dendrobium species : Comparative evaluation using validated HPTLC technique

Article history: Received on: 11/07/2015 Revised on: 27/07/2015 Accepted on: 26/08/2015 Available online: 28/10/2015 Northeast India represents a rich floristic wealth of India. Orchids form a very noticeable feature of this vegetation. Amongst orchids, the genus Dendrobium forms a very important group of plants which are used by the locals for the treatment of various ailments. A through literature survey revealed that there is a paucity of data on the phytochemical evaluation of Dendrobium species using analytical methods. The chromatographic technique such as HPTLC is reported to be useful for standardization of plant materials in terms of phytochemical markers and evaluation of their quality. Therefore, in this study, five different Dendrobium species have been evaluated and compared in terms of their marker content using validated HPTLC methods. All the samples analyzed were found to be a good source of β-sitosterol with maximum content in D. fimbriatum stem and minimum content in D. chrysanthum roots. D. nobile (roots and stem) and D. moschatum (stem) were also found to be a source of ursolic acid and lupeol, respectively. Thus, this work will contribute in the identification, quality evaluation and standardization of different Dendrobium species. On the basis of maximum bioactive marker content, specific part of Dendrobium species can be selected which may be supported by its efficacy.


INTRODUCTION
The genus Dendrobium is one of the largest groups of family Orchidaceae (Gutierrez, 2010).Different species of these orchids are found in Northeast India, China and Japan and are recorded in the Chinese Pharmacopoeia.These species are not only valued for their ornamental beauty but also have important therapeutic properties (Gutierrez, 2010;Vij et al., 1997).Various Dendrobium species have been reported to possess secondary metabolites such as phenols, alkaloids, coumarins, terpenes, flavonoids as the major therapeutic agents (Meng et al., 2013).Even primary metabolites such as polysaccharides from different Dendrobium species have been shown to promote proliferation in macrophages and increase their phagocytic activity (Lan Zhen et al., 2013).Dendrobium nobile Lindl., is a precious herbal plant in Chinese traditional medicine and it is one of the Dendrobium species specified in Chinese Pharmacopoeia, 2005(Luo et al., 2010).The stem of D. nobile has been used ethnomedicinally to nourish stomach, promote the production of body fluid and reducing fever.D. nobile has been a good source of compounds with anti-tumor activity (Singh et al., 2012).D. nobile, a highly prized orchid for its economic and medicinal importance is at the verge of extinction due to over-exploitation.Recently, the genetically stable regeneration protocol with increased phytochemical production and in vitro antioxidant activity of D. nobile has been reported (Bhattacharya et al., 2014).The efforts have also been made by researchers to propagate this endangered species through in vitro seed germination (Kumaria et al., 2005;Kumaria and Tandon, 2001).
Genetic characterization of this orchid, which is imperative for the prevention of elite germplasm erosion in breeding programs mainly during development of synthetic hybrids, has also been carried out using RAPD and SSR markers (Chattopadhyay et al., 2012;Zha et al., 2009).Phenanthrene and denbinobin isolated from aerial parts of D. nobile have been reported to possess cytotoxic activity against human lung and human ovary adenocarcinoma as well as antitumor activity (Gutierrez, 2010).The polysaccharides from different extracts of D. nobile possess antioxidant activity and activate macrophages (Lan Zhen et al., 2013;Luo et al., 2010).
Amongst the other Dendrobium species, Dendrobium fimbriatum Hook. is one of the native species to India (Singh et al., 2001).Infusion or decoction of its leaves is traditionally used to promote the production of body fluid and paste is applied on the fractured area to reset bones (Bi et al., 2003).D. fimbriatum is also used to treat liver disorders and nervous debility (Pant, 2013).
A number of phytochemical constituents like alkaloids, bibenzyl derivatives, flavonoids etc., have been reported from Dendrobium moschatum (Buch.-Ham.)Sw. (Singh et al., 2012), but compared to D. fimbriatum, detail investigation on phytochemical profile and therapeutic potential of D. moschatum has been overlooked.So far, two phenanthrenes viz.rotundatin and moscatin from D. moschatum have been reported to be useful in the inhibition of aggregation of platelets induced by arachidonic acid and collagen (Chattopadhyay et al., 2012).Also, in vitro propagation of this epiphytic orchid through thidiazuron induced high-frequency shoot proliferation has been studied (Nayak et al., 1997).
Dendrobium chrysanthum Wall.ex Lindl. is widely distributed in South China.Dried and ground leaves of D. chrysanthum are traditionally used as antipyretic, eye-benefiting, immunomodulatory agents and in the treatment of skin diseases (Li et al., 2001).A phenanthrene dendrochrysanene from this orchid has also been reported to possess anti-inflammatory activity (Yang et al., 2006).The studies carried out on the stem of D. chrysanthum revealed erianin isolated from it possesses anticancer activity (Gong et al., 2004;Li et al., 2001).
The extensive literature survey revealed that though, these orchids are extensively used for their ethnomedicinal importance; there is a dearth of scientific data in terms of their phytochemical evaluation.Till date, there is no work reported on other Dendrobium species in search of some substitute to the endangered species D. nobile in terms of their phytochemical profile.

Plant materials
Five different species of Dendrobium, namely; D. nobile, D. fimbriatum, D. moschatum, D. chrysanthum and D. chrysotoxum were collected from Northeast India.The representative sample of all five species of Dendrobium was authenticated from Dr. Suman Kumaria, Department of Botany, North East Hill University, Shillong.Different plant parts (roots and stem of D. nobile; and leaves, roots and stem of D. fimbriatum, D. moschatum, D. chrysanthum, D. chrysotoxum) were separated, thoroughly washed, cleaned and shade dried for a week followed by oven drying at 37°C for four days.The materials were powdered and sieved through 85 mesh (BSS) and stored in airtight containers.

Chromatographic characterization Extraction of phytochemical constituents from different samples of Dendrobium species
In order to extract terpenoids namely ursolic acid (Figure 1A), β-sitosterol (Figure 1B) and lupeol (Figure 1C) from the complex matrix of leaves, roots and stem of D. nobile, D. fimbriatum, D. moschatum, D. chrysanthum and D. chrysotoxum; each powdered sample (1.0 g) was extracted with 10.0 mL petroleum ether (Shailajan and Gurjar, 2015;Shailajan and Gurjar, 2014;Shailajan et al., 2012), vortex mixed for a minute and then sonicated for 20 min followed by filtration through Whatman filter paper no. 1.
The filtrate was evaporated to dryness under vacuum using rotary evaporator at 40 °C, reconstituted in an equal volume of methanol and subjected to HPTLC analysis for the separation of these terpenoids.In order to extract the phenolic compound gallic acid (Figure 1D), the powdered sample (1.0 g) was extracted with methanol (10.0 mL), vortex mixed for a minute and sonicated for 20 min followed by filtration through Whatman filter paper no. 1.The filtrate was subjected to HPTLC analysis for the separation of gallic acid.

Preparation of standard stock solutions
The standard stock solution of ursolic acid, β-sitosterol, lupeol and gallic acid (1000.0µg/mL each) was prepared in methanol.Serial dilution of the stock solution in methanol was carried out in order to prepare calibrant/ quality control samples.

Optimized chromatographic conditions for quantitation of markers High Performance Thin Layer Chromatography
The HPTLC system used consisted of CAMAG TLC Scanner 4 supported by winCATS software version 1.4.7 equipped with CAMAG Linomat 5 sample spotter and CAMAG Reprostar 3 system for photo-documentation.Chromatographic separation of the phytochemical constituents was achieved on TLC plates (E.Merck) pre-coated with silica gel 60 F 254 (0.2 mm thickness) on aluminium sheet support.
For simultaneous separation of ursolic acid, β-sitosterol and lupeol from leaves, roots and stem of D. nobile, D. fimbriatum, D. moschatum, D. chrysanthum and D. chrysotoxum, 10.0 µL of samples along with the standards-ursolic acid (10.0 µg/mL), β-sitosterol (50.0 µg/mL) and lupeol (10.0 µg/mL) of 10.0 µL each were spotted on TLC plates as bands of 8.0 mm and at a distance of 15.0 mm from the edges.Plates were developed up to a distance of 85.0 mm in CAMAG twin trough glass chamber pre-saturated with mobile phase toluene: methanol (8:1, v/v) for 15 min and derivatized using freshly prepared 10% methanolic sulphuric acid and dried in an oven preset at 110 °C for 10 min.For densitometric scanning, the source of radiation was a mercury lamp (366 nm).All measurements were performed at 22 ± 1 °C.Plates were photo-documented at 366 nm.To separate gallic acid from leaves, roots and stem of D. nobile, D. fimbriatum, D. moschatum, D. chrysanthum and D. chrysotoxum, the sample (10.0 μL) and gallic acid standard (100.0μg/mL, 10.0 μL) were .Gedsvfcrrdeswa spotted on TLC plate as bands of 8.0 mm and at a distance of 15.0 mm from the edges under mentioned instrumental conditions.The plate was developed up to a distance of 85.0 mm in CAMAG twin trough glass chamber pre-saturated with mobile phase toluene: ethyl acetate: formic acid (2:7:1, v/v/v) for 15 min.The plate was scanned and photo documented at 254 nm.

Method validation
The HPTLC methods were validated as per ICH guidelines for parameters like sensitivity, linearity, precision, selectivity, recovery and ruggedness (International Conference on Harmonization, 1997).

Statistical analysis
Microsoft Excel-2007 was used to determine mean, standard deviation (SD), relative standard deviation (RSD) and mean difference during the analysis.

RESULTS AND DISCUSSION
Quality evaluation of herbal medicine is an important factor and basic requirement for the herbal drug industry.The analysis and quality control of herbal medicines are moving a step ahead towards an integrative and comprehensive direction, in order to tackle the complex nature of herbal medicines.Highperformance thin layer chromatography (HPTLC) is one of the sophisticated instrumental techniques for standardization of the herbs and herbal drugs in terms of phytochemical constituents (Shivatare et al., 2013).Dendrobium species could be useful in the selection of some promising alternative to an endangered species like D. nobile.Therefore, in this study five Dendrobium species were comparatively evaluated for the presence of terpenoids (ursolic acid, β-sitosterol, and lupeol) and a phenolic compound (gallic acid).HPTLC analysis of different Dendrobium species was carried out using our recently published report on the estimation of ursolic acid, β-sitosterol, lupeol and gallic acid (Shailajan and Gurjar, 2015).Use of mobile phase composition toluene: methanol (8:1, v/v) showed a good resolution of terpenoids (ursolic acid at R f = 0.28 ± 0.02, β-sitosterol at R f = 0.48 ± 0.02 and lupeol at R f = 0.58 ± 0.02) during the HPTLC experiment.Separation of β-sitosterol was easily achieved from the phytochemical matrix of different Dendrobium species.Ursolic acid was detected in both the parts (roots and stem) of D. nobile, whereas lupeol was detected only in the stem of D. moschatum.The presence of terpenoids in the samples was confirmed by overlay and color of the characteristic band with that of the standard.The plate photo, overlay along with the chromatograms of standards and different parts of five Dendrobium species are represented in Figure 2   The HPTLC method was validated as per ICH guidelines and found rapid, specific, precise, accurate and rugged (Table 1).The regression analysis of calibrant samples for each marker (ursolic acid, β-sitosterol, and lupeol) resulted in the form of regression equation which was further used to estimate their content in samples.Along with the bands of selected markers, the same mobile phase also showed separation of few more bands as follows: 6 and 7 bands in D. nobile roots and stem, respectively; 7, 5 and 8 bands in D. fimbriatum leaves, roots and stem, respectively; 9, 6 and 8 bands in D. moschatum leaves, roots and stem, respectively; 7, 2 and 6 bands in D. chrysanthum leaves, roots and stem, respectively and 9, 6 and 8 bands in D. chrysotoxum leaves, roots and stem, respectively.The mobile phase toluene: ethyl acetate: formic acid (2:7:1, v/v/v) showed good resolution of a phenolic compound (gallic acid at R f = 0.53).But it was found absent in all the five Dendrobium species during HPTLC experiment (data not shown).This may be due to the presence of gallic acid in traces or presence of some other phenolic compounds in the plant samples.The content of phytochemical markers in different parts of five Dendrobium species is summarized in Table 2.
Amongst the five different species of Dendrobium, the maximum content of β-sitosterol was observed in stem of D. fimbriatum followed by leaves of D. chrysanthum and roots of D. nobile whereas ursolic acid was detected only in D. nobile and found maximum in its stem compared to the roots.Lupeol was detected only in the stem of D. moschatum (Table 2).This suggests the need of some other sensitive method to detect and quantitate markers such as ursolic acid and lupeol from these samples if present in traces.Although, ursolic acid, β-sitosterol, lupeol and gallic acid are not the plant specific markers, they were chosen for the quality evaluation of five different Dendrobium species because of their proven therapeutic efficacy against various ailments.As simultaneous marker-based quantitative methods would be complementary as well as additive approaches for the quality control and stability assessment of herbal preparations (Shailajan et al., 2013), the results of the present research work could be used for the chemical characterization of different Dendrobium species in order to check their uniformity.
The results of present research work suggest that, on the basis of maximum marker content, stem of D. fimbriatum could be the alternative source of D. nobile which should be equally supported by evaluation of safety and pharmacological profile.

Fig. 6 :
Fig. 6: TLC plate photo (A) and overlay of the chromatograms (B) showing the presence of a terpenoid -β-sitosterol at 366 nm in Dendrobium chrysotoxum leaves, roots and stem.Track details for plate A: a) Dendrobium chrysotoxum leaves, b) Dendrobium chrysotoxum roots, c) Dendrobium chrysotoxum stem, d) ursolic acid (10 µg/mL), e) β-sitosterol (50 µg/mL), f) lupeol (10 µg/mL), and g) mixture of ursolic acid, β-sitosterol and lupeol (10 µg/mL, 50 µg/mL and 10 µg/mL respectively).C represents the chromatogram of three terpenoids -ursolic acid, β-sitosterol and lupeol.D represents the chromatogram showing β-sitosterol in Dendrobium chrysotoxum leaves.E represents the chromatogram showing βsitosterol in Dendrobium chrysotoxum roots.F represents the chromatogram showing β-sitosterol in Dendrobium chrysotoxum stem.All the therapeutically active compounds (ursolic acid, βsitosterol, and lupeol) have been detected for the first time from the methanolic extract of D. nobile, D. fimbriatum, D. moschatum, D. chrysanthum and D. chrysotoxum following the reported extraction procedure and optimized HPTLC conditions.Although, ursolic acid, β-sitosterol, lupeol and gallic acid are not the plant specific markers, they were chosen for the quality evaluation of five different Dendrobium species because of their proven therapeutic efficacy against various ailments.As simultaneous marker-based quantitative methods would be complementary as well as additive approaches for the quality control and stability assessment of herbal preparations(Shailajan et al., 2013), the results of the present research work could be used for the chemical characterization of different Dendrobium species in order to check their uniformity.The results of present research work suggest that, on the basis of maximum marker content, stem of D. fimbriatum could be the alternative source of D. nobile which should be equally supported by evaluation of safety and pharmacological profile.

Table 1 :
Results of method validation experiment for simultaneous estimation of three terpenoids (ursolic acid, β-sitosterol and lupeol) and a phenolic compound (gallic acid).

Table 2 :
Content of terpenoids (ursolic acid, β-sitosterol and lupeol) and a phenolic compound (gallic acid) in different parts of five Dendrobium species.