Chemometric Profile of Root Extracts of Rhodiola imbricata Edgew. with Hyphenated Gas Chromatography Mass Spectrometric Technique

Rhodiola imbricata Edgew. (Rose root or Arctic root or Golden root or Shrolo), belonging to the family Crassulaceae, is an important food crop and medicinal plant in the Indian trans-Himalayan cold desert. Chemometric profile of the n-hexane, chloroform, dichloroethane, ethyl acetate, methanol, and 60% ethanol root extracts of R. imbricata were performed by hyphenated gas chromatography mass spectrometry (GC/MS) technique. GC/MS analysis was carried out using Thermo Finnigan PolarisQ Ion Trap GC/MS MS system comprising of an AS2000 liquid autosampler. Interpretation on mass spectrum of GC/MS was done using the NIST/EPA/NIH Mass Spectral Database, with NIST MS search program v.2.0g. Chemometric profile of root extracts revealed the presence of 63 phyto-chemotypes, among them, 1-pentacosanol; stigmast-5-en-3-ol, (3β,24S); 1-teracosanol; 1-henteracontanol; 17-pentatriacontene; 13-tetradecen-1-ol acetate; methyl tri-butyl ammonium chloride; bis(2-ethylhexyl) phthalate; 7,8-dimethylbenzocyclooctene; ethyl linoleate; 3-methoxy-5-methylphenol; hexadecanoic acid; camphor; 1,3-dimethoxybenzene; thujone; 1,3-benzenediol, 5-pentadecyl; benzenemethanol, 3-hydroxy, 5-methoxy; cholest-4-ene-3,6-dione; dodecanoic acid, 3-hydroxy; octadecane, 1-chloro; ethanone, 1-(4-hydroxyphenyl); α-tocopherol; ascaridole; campesterol; 1-dotriacontane; heptadecane, 9-hexyl were found to be present in major amount. Eventually, in the present study we have found phytosterols, terpenoids, fatty acids, fatty acid esters, alkyl halides, phenols, alcohols, ethers, alkanes, and alkenes as the major group of phyto-chemotypes in the different root extracts of R. imbricata. All these compounds identified by GC/MS analysis were further investigated for their biological activities and it was found that they possess a diverse range of positive pharmacological actions. In future, isolation of individual phyto-chemotypes and subjecting them to biological activity will definitely prove fruitful results in designing a novel drug.


Introduction
To identify and evaluate the therapeutic potential of medicinal herbs, isolation of active components and structural elucidation of these compounds is very essential in medicinal chemistry and natural product research. In recent years a lot of attention has been given towards the study of organic compounds from medicinal herbs and to elucidate their pharmacological activities. Numerous extraction techniques and analytical systems like spectrophotometry, capillary electrophoresis, high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC), gas chromatography (GC) with flame ionization detection (FID), gas chromatography/mass spectrometry (GC/MS) have been developed for the analysis and characterization of active compounds from medicinal plants. GC/MS has become an ideal technique for qualitative and quantitative analysis of volatile and semi-volatile compounds of plant origin. It has the unique combination of a perfect separation system (GC) with the excellent identification and confirmation technique (MS) which has made it the best suited analytical system for plant compound characterization. Additionally, for rapid extraction and precise analysis of these active phyto-compounds, the experimental design should also be optimized to obtain enhanced recoveries, low solvent consumption, and reduced extraction time [1][2][3][4][5].
Rhodiola imbricata Edgew. (Rose root/Arctic root/Golden root/ Shrolo), belonging to the family Crassulaceae, is an important food crop and medicinal plant in the high altitude region of Indian trans-Himalayan cold desert. It is a popular medicinal plant in Pakistan, Nepal, India, Tibet, China, and many other countries and is widely used as food and traditional medicine around the world. A number of metabolites like phenylpropanoids, phenylethanol derivatives, flavanoids, terpenoids, and phenolic acids have been found in good quantity from these Rhodiola species and extracts of these plant species, particularly those from roots, have been shown to possess pharmacological activities. A survey of the literature showed that Rhodiola species influence a number of       Table 1. Phyto-chemotypes identified in the n-hexane root extract of R. imbricata by GC/MS. physiological functions including neurotransmitter levels, central nervous system activity, and cardiovascular function. It is being used to stimulate the nervous system, decrease depression, enhance work performance, eliminate fatigue, and prevents high-altitude sickness. Most of these effects have been ascribed to constituents such as salidrosides (rhodiolosides), rosavins, and ptyrosol. Many pharmacological studies on R. imbricata have demonstrated that this plant exhibits cardioprotective, antiinflammatory, antistress, dermal wound healing, and adaptogenic activities. It has also been found to possess antioxidant, antiaging, immuno-stimulant, radioprotective, and anticarcinogenic properties [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. All these reports validate its use in traditional system of medicine. However, the phytochemistry of the most important plant part having the medicinal and therapeutic potential, the root of R. imbricata has not been studied in considerable details. Hence, aim of the present investigation was to identify and quantify the chemotypes extracted successively in different solvents such as nhexane, chloroform, dichloroethane, ethyl acetate, methanol, and 60% ethanol, from roots of R. imbricata from trans-Himalayan cold desert of Ladakh, India, by hyphenated GC/MS technique.

Materials and Methods
Chemicals n-Hexane, chloroform, dichloroethane, ethyl acetate, methanol, ethanol, and water CHROMASOLV HPLC grade and all other chemicals used were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).

Ethics statement
All necessary permits were obtained for the described field studies. The permit was issued by Dr. B. Balaji (IFS), Divisional Forest Officer, Leh Forest Division, Jammu & Kashmir, India.

Plant materials and extraction
R. imbricata roots were collected from the trans-Himalayan region (Chang-La Top, altitude = 5330 m above mean sea level, Indus valley, Ladakh) of India in the month of October, 2011 after the period of senescence, with the prior permission from the local authorities. The plant roots were washed thoroughly and cut into small pieces and shade dried at room temperature for 15 days. Then they were finely powdered and used for extraction. The root powder (20 gm) was taken for the sequential extraction in six solvent systems with increasing polarity viz. n-hexane, chloroform, dichloroethane, ethyl acetate, methanol, and 60% ethanol by Soxhlet apparatus (Borosil GlassWorks Limited, Worli, Mumbai, India) at 40uC. The extracted fractions were concentrated under vacuum and reduced pressure (BUCHI Rotavapor R-205, BUCHI Labortechnik AG CH-9230, Flawil, Switzerland) at 40uC by circulation of cold water using thermostat maintained at 4uC in order to minimize the degradation of thermolabile compounds. The dry extracts were then stored in a 280uC freezer till further analysis.

Preparation of sample for GC/MS analysis
The 25 mg of concentrated n-hexane, chloroform, dichloroethane, ethyl acetate, methanol, and 60% ethanol root extracts were redissolved in the respective solvents, vortexed properly and filtered through 0.22 mm syringe filter (Millipore Corp., Bedford, MA, USA). One microlitre aliquot of the sample solution was injected into the GC/MS MS system for the requisite analysis.

Instrumentation and chromatographic conditions
GC/MS analysis was carried out on a Thermo Finnigan PolarisQ Ion Trap GC/MS MS system comprising of an AS2000 liquid autosampler (Thermo Finnigan, Thermo Electron Corporation, Austin, TX, USA). The gas chromatograph was interfaced to a mass spectrometer instrument employing the following conditions viz. Durabond DB-5 ms column (30 m60.25 mm60.25 mm),  operating in electron impact [electron ionisation positive (EI + )] mode at 70 eV, helium (99.999%) was used as carrier gas at a constant flow of 1 ml/min, an injection volume of 0.5 EI was employed (split ratio of 10:1), injector temperature 280uC, and transfer line temperature 300uC. The oven temperature was programmed from 50uC (isothermal for 2 min), with gradual increase in steps of 10uC/min, to 300uC. Mass spectra were taken at 70 eV, a scan interval of 0.5 s, and full mass scan range from 25 m/ z to 1000 m/z. The data acquisition was performed on Finnigan Xcalibur data acquisition and processing software version 2.0 (ThermoQuest, LC and LC/MS Division, San Jose, California, USA).   Results GC/MS chromatograms of n-hexane (Fig. 1), chloroform (Fig. 2), dichloroethane (Fig. 3), ethyl acetate (Fig. 4), methanol (Fig. 5), and 60% ethanol (Fig. 6) root extracts of R. imbricate as per the experimental procedure discussed above, showed various peaks indicating the presence of different chemotypes in the respective extracts.

Discussion
We have conducted the present investigation to identify the major volatile and semivolatile components in the root of R. imbricata. The presence of various bioactive compounds justifies the use of the plant by traditional practitioners of 'Amchi' system of medicine in trans-Himalayan Ladakh region. Also, extensive pharmacological studies were conducted by different researchers with the plant root extracts [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] and the results were very promising to justify the use of this plant as therapeutic agent.

Phyto-chemotypes
Root extracts n-Hexane Chloroform Dichloroethane Ethyl acetate Methanol 60% Ethanol in plant extracts permits the complete phenotyping of genetically or environmentally adapted plant systems and such investigations draw on simple extraction procedures that have been shown to be very robust and have permitted broad range of high-throughput applications in plant metabolomics. [25][26][27].
Most of the pharmacological studies were conducted with the aqueous, ethanol, and hydro-alcoholic root extracts of this plant and it was found to have numerous biological activities such as anti-stress, adaptogenic, anti-hypoxic, immune-stimulatory, anticancer, cytoprotective, radioprotective, anti-hemolytic, anti-inflammatory, and wound healing potential [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. Our investigations conclude that the compounds present in the ethanol and water extracts have the potential to perform these functions.   Though, the root extracts of the plant obtained by polar solvent extraction have been investigated for their pharmacological actions in considerable detail, non polar root extracts were not studied till date. Hence, our primary objective in the present work was to find the bioactive constituents present in the non polar extraction of root of this herb. These findings will definitely usher in new directions in pharmacological and therapeutic investigations with the root extracts obtained from non polar solvent extraction such as n-hexane, chloroform, dichloroethane, and ethyl acetate.

Conclusion
In the present study, sixty three phyto-chemotypes have been identified from n-hexane, chloroform, dichloroethane, ethyl acetate, methanol, and 60% ethanol root extracts of R. imbricata by GC/MS analysis. It showed the existence of various bioactive principles that confirm the application of R. imbricata for various ailments in traditional system of medicine. However, isolation of individual phyto-chemotypes and subjecting them to biological activity will definitely give fruitful results to find a novel drug. It could be concluded that R. imbricata contains various bioactive phyto-chemotypes having phyto-pharmaceutical importance. However, further studies will need to be undertaken to ascertain its bioactivity, toxicity profile, effect on the ecosystem, and agricultural products.