Abstract
The oleo-gum resin of Commiphora wightii (Arn.) Bhandari, a pharmacologically important balsamiferous woody shrub, has been used in treating various ailments and disorders since ancient times (2000 B.C.) due to the presence of steroidal compound guggulsterone. Two bioactive isomers of guggulsterone, E and Z, are responsible for lipid- and cholesterol-lowering and anti-cancerous activities. Further, guggul has been approved as food supplement by US-FDA as well as Council of Europe. Indiscriminate harvest of C. wightii from wild with negligible conservation efforts has lead to its inclusion in IUCN assemblage of endangered plant species. For identification of high guggulsterone yielding ecotypes of C. wightii, using high-performance thin-layer chromatographic (HPTLC) analysis, stem samples were collected from 50 plants from eleven locations in arid tracts of Haryana, Gujarat and Rajasthan. Dried, powdered material was subjected to extraction with petroleum ether using soxhlet apparatus. Samples were spotted on precoated activated silica plates (60F-254) and were developed using toluene–acetone (9:1 v/v) as mobile phase. The analysis was carried out in the absorbance mode at 250 nm using HPTLC scanner. The regression analysis data for the calibration plots for E and Z guggulsterone showed good linear relationship with R2 = 1 and 0.9897, respectively. Highest concentration of guggulsterone E (284 μg/g dry wt) was found in the accession collected from Palana, Bikaner whereas highest guggulsterone Z concentration (89.5 μg/g dry wt) was found in the accession collected from CAZRI, Jodhpur.
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Introduction
Commiphora (Burseraceae) encompassing approximately 165 species is a very slow growing genus with small thorny, sturdy, highly branched small balsamiferous trees with a short trunk and thin papery bark (Barve and Mehta 1993). C. wightii (Arn.) Bhandari grows widely in rocky tracts of arid and semi-arid regions of India, Bangladesh, Pakistan, China, Ethiopia, and Arabia, Tropical and Northern Africa and many other countries (Kant et al. 2010). In India it is found in arid, rocky and sandy tracts of Rajasthan, Gujarat, Maharashtra and Karnataka states. It is commonly known as “Indian Bdellium” or “Guggul” due to the presence of aromatic steroidal ketonic compound guggulsterone in vertical rein ducts and canals of bark. It has immense potential in treating various ailments and disorders like rheumatism, arterosclerosis, sciatica, anti-oxdative, gasterointestinal discomfort along with applications in perfumery and incense industry (see Kulhari et al. 2012). This resin was a native source of incense in India and was traded in ancient times; it is known from Classical Roman sources that Bdellium was traded from Indian ports (Yule and Burnell 1886; Miller 1968; Asouti and Fuller 2008). Its immense multifarious medicinal and therapeutic values were known from Ayurvedic medical texts namely Atharva Veda (2000 B.C.), Charaka (1000 B.C.), Sushruta Samhita (600 B.C.) and Vagbhata (seventeenth century A.D.) which describes the usefulness of guggul gum resin in various afflictions (Siddiqui 2011; Ramawat et al. 2008; Shishodia et al. 2008). In fact the herb finds mention as early as 3,000–10,000 years ago in the Vedas, the holy scriptures of India, for treatment of various human ailments (Dev 1987, 1999). Also, guggul has been approved as a food supplement by United States Food and Drug Administration in 1994, while Council of Europe included myrrh in the list of plants and parts thereof that are acceptable for use in foods in 1981 (FAO 1995; Council of Europe 1981). Excessive tapping for oleo-gum resin has led to extensive depletion of the species from nature making the plant vulnerable. Mostly the tapping of oleo-gum resin is done by local and tribal people adopting unscientific methods which generally results in the death of plants. Though, C. wightii is assigned to the Data Deficient category ver. 2.3 (1994) of the Red Data Book of IUCN, the Government of India has included it under RET (Rare, Endangered, Threatened) category (Samantaray et al. 2011). Currently, only few wild populations exist in the states of Rajasthan and Gujarat. According to an estimate, the demand of gum guggul is 1000 MT but India produces only 100 MT against its requirement (Maheshwari 2010). In spite of having many excellent traits, this plant is still in an early phase of domestication. Natural regeneration is almost negligible as compared to its depletion in nature due to lack of cultivation. Attention is required to prevent its exploitation and for sound management and conservation. This will ensure survival of this endangered gum resin species and will also save the fragile arid and semi-arid lowlands from desertification. Identification of high guggul yielding lines can pave the way for multiplication and mass propagation of elite germplasm through tissue culture and vegetative propagation methods.
The exudate of C. wightii is a complex mixture of resin (61 %), gum (29.3 %) and other chemicals (6.1 %). More than 150 compounds have been reported and new compounds continue to be reported (Fatope et al. 2003). Guggul comprises of several plant sterols, resin, gum, diterpenes, steroids, alcohols and other chemicals, however its multifarious benefits reside in two main inter-convertible isomeric forms of guggulsterone (C21H28O2) viz: E and Z (Verma et al. 1998; Agrawal et al. 2004a; Ramawat et al. 2008) which are involved in anticholesterol activity (Satyavati et al. 1969). Two different arrangements of CH3 at C20 in three-dimensional space and the hindered rotation about the carbon–carbon double bond at C17 and C20 classifies the guggulsterone into Z-{4,17(20)-cis-pregnadiene-3,16-dione} and E-{4,17(20)-trans-pregnadiene-3,16-dione} (Fig. 1).
Significant variations are likely to occur in the component content of guggul oleo-gum resin depending upon the climatic conditions under which the plants are grown and the resin is harvested (Agrawal et al. 2004a). Therefore, there is a need for reliable and consistent quantitative determination of bioactive ingredients of C. wightii oleo-gum resin for pharmaceutical applications. No reports are available regarding quantitative estimation of guggulsterone isomers in stem samples of C. wightii using HPTLC or HPLC methods. Few reports are available for quantification of guggulsterone in pharmaceutical dosage, in biological fluids and assessment of impurities related to stability of drugs in commercial Ayurvedic extracts and capsules (see Kulhari et al. 2012). Gradient HPLC method using PDA detector for quantification of E and Z stereoisomers with highest recovery (99.5 %) and precision was validated by Mesorb et al. (1998) in oleo-gum resin exudates of C. mukul Engl. along with its products (capsules). Soni et al. (2009) estimated the content of guggulsterone isomers using HPLC in resin while Dass and Ramawat (2009) used reverse phase column HPLC to determine guggulsterone content in cell and callus cultures of C. wightii. Verma et al. (1998) quantified the two isomeric forms of guggulsterone, simultaneously by HPLC (a C18 column using methanol and acetonitrile), in rat serum after administration of a single dose (50 mg/kg). Agrawal et al. (2004a) determined the concentration of E (Rf 0.38) and Z (Rf 0.46) guggulsterone in pharmaceutical dosage forms while Agrawal et al. (2004b) carried out stress degradation studies on guggulsterone using HPTLC. Quantitative analysis of many OTA products in USA was shown to contain significantly low or no content of guggul resin extracts and guggulsterone which was attributed to lack of quantitative analysis method or application of incorrect methods of analysis (Agrawal et al. 2004a). UV spectrophotometric analysis of guggulsterone content at 327 nm is non-specific and can produce erroneous results due to absorbance of this wavelength by all non-polar components of the resin (Rajpal 2002).
HPTLC, having the advantages of low operating cost, high sample throughput and requirement for minimum sample clean up, is becoming an analytical technique of choice especially in plants where no previous data is available for biochemical estimation of components. Unlike HPLC which require thorough sample clean up, many samples can be run simultaneously in a small quantity of mobile phase in HPTLC. Therefore, the aim of the present work was to develop a precise, specific and repeatable HPTLC method for accurate quantification of two isomeric forms of guggulsterone in stem samples of C. wightii collected from various geographical locations of North western India.
Materials and methods
Standard preparation
Two isomeric forms of guggulsterone (E and Z) procured from Chromadex, USA were used as reference analytes for quantitative estimation of guggulsterone. Standard stock solutions of E and Z guggulsterones were prepared by dissolving 5 mg in 5 ml chloroform. The standard stock solutions were further diluted to obtain working standard solutions of different concentrations ranging from 20 to 100 μg/ml and stored at 4 °C.
Extract preparation from raw material for HPTLC analysis
Stem, leaf and root samples were collected from 50 C. wightii plants growing in 11 different geographical locations of Rajasthan, Haryana and Gujarat states. The samples were dried in shade prior to extraction and 3 g of dried, powdered sample was extracted with petroleum ether in Soxhlet apparatus for 8–10 h, continuously. Extracted samples were concentrated under vacuum using rotary evaporator to near dryness. These concentrated sticky samples were reconstituted quantitatively in acetone. The concentrated extract was then subjected to HPTLC analysis.
Instrumentation and chromatographic conditions
Chromatographic experiments were preformed on precoated silica gel HPTLC plates, 60F-254 (20 cm × 10 cm, 250 μm thickness) (Merck, Germany). The plates were washed with methanol and activated at 70 °C for 15 min before chromatographic spotting. A constant spotting of individual stocks of E and Z guggulsterone samples along with standard were applied on the gel plate as 8 mm wide bands. The monochromatic bandwidth was set at 20 nm, with baseline correction. For better separation of individual components as well as spiky and proportioned linear ascending development of peaks, the TLC layer was developed in a Camag twin trough glass tank, pre-saturated with mobile phase toluene–acetone (9.0: 1.0, v/v) solvent system. The optimized chamber saturation analysis was performed for 30 min at 25 ± 5 °C temperature and 50 ± 5 % relative humidity under laboratory conditions. After development, the layer was air dried and densitometric scanning of developed chromatogram was performed on Camag TLC scanner III in the reflectance–absorbance mode at 250 nm. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm. These critical parameters were maintained during complete analysis of entire samples. Concentrations of both the compounds chromatographed were determined from the peak purity tests by capturing the intensity of diffusely reflected light. Evaluation was performed by calculating peak height with linear regression. All the samples were analyzed in triplicate and data was subjected to standard error calculation using SAS 6.0 software.
Results and discussion
HPTLC is a highly efficient, robust and rapid analytical method for detection and quantification of desirable components with optimum resolution. This process can be applied as a routine analytical method, even in small scale laboratories housing a minimum set of equipments, for testing of purity of drugs in herbal formulations as well as in identification of guggulsterone from resinous extracts of related plant species like Mangifera indica L., Acacia nilotica (L.) Willd. ex Delile, Ficus religiosa L., etc.
The leaf and root samples of C. wightii were found to contain negligible amount of guggulsterone compared to stem samples wherein substantial amount of guggulsterone was found therefore, further experiments were limited to stem samples only. The oleo-gum resin is mainly found in the stem only therefore; selective distribution of resin and its components is expected. TLC profiles of samples collected from different geographical locations, prepared in petroleum ether and acetone could elucidate the difference in guggulsterone content at 250 nm wavelength (Fig. 2). Peaks of E and Z guggulsterone were identified at a retention time of 0.36 and 0.42 min., respectively (Fig. 3). The isomers, individual as well as mixture, depicted a clear peak during separation without interference of any other constituents. A calibration plot was obtained by plotting peak height against concentration of guggulsterone (Fig. 4). A linear straight line was observed for guggulsterone E and Z standards using the regression equation Y = 0.0002x + 0.7183 and Y = 0.0004x + 0.7993 respectively (Table 1). Various components of the stem extract separated with discrete peaks along with the separation of E and Z isomers of guggulsterone (Fig. 5). Concentration of guggulsterone E varied from 67.7 to 184.4 μg/g while that of guggulsterone Z varied from 40.4 to 89.6 μg/g (Table 2). Amount of guggulsterone was higher in almost all locations of Rajasthan as compared to Haryana and Gujarat due to water deficiency and adverse climatic conditions. Among different geographical locations of Rajasthan, highest concentration of E guggulsterone (184.4 ± 0.0577 μg/g) was found in the sample collected from Palana (Bikaner, Rajasthan) while concentration of Z guggulsterone was found to be highest (89.6 ± 0.0305 μg/g) in sample collected from CAZRI (Jodhpur, Rajasthan). Total guggulsterone was highest in sample collected from Palana (257.2 μg/g) followed by that collected from Kailana lake, Jodhpur (247.6 μg/g).
Both the isomeric forms of guggulsterone are inter-convertible as was reported in callus and cell cultures of guggul (Ramawat et al. 2008). Soni et al. (2009) reported that Z isomeric form was dominating over E-guggulsterone however, the same trend was not found in the present study.
The oleo-gum resin is produced by arid zone plants as a stress mitigating mechanism therefore, concentration of its constituents is likely to be influenced by environmental factors (geographical and seasonal), individual plant performance (genotypes and morphotypes), as well as cultivation practices. Agrawal et al. (2004a) also reported that component content of guggul resin is dependent on climatic conditions under which the plant is grown and harvested. The content of guggulsterone produced in winters was very limited due to dormant phase of the plant while its production enhanced in summers. Similarly, Soni et al. (2009), using HPLC, reported that the guggulsterone content was significantly higher during summer (May–July, highest in May) and gradually decreased in the rainy seasons (Aug–Oct) and was lowest in winter (Nov–March). Variation in the content of guggulsterone was attributed to environmental factors like temperature and rainfall. With reference to the geographical locations northern, western and central part of Rajasthan showed maximum amount whereas southern part of the state produced lower amount of guggulsterone (Soni et al. 2009). They also depicted a strong relationship between average rainfall of the area and guggulsterone content. Influence of various parameters (seasonal variation, geographical variation, average rainfall, planting strength, genotype of plant, time of sowing, harvesting period and extracting solvents) on the concentration of bioactive agents have also been observed in other medicinal plants like Lepidium sativum L. (Nayak et al. 2009, 2012), Plantago ovata Forsk. (Mann and Vyas 1999) and Andrographis paniculata (Burm. f.) Wall. ex Nees (Saxena et al. 2000). Nayak et al. (2009) identified and quantified the sinapic acid in methanolic seed extract of Lepidium sativum using HPTLC and quantitative determination showed a clear difference in concentration of sinapic acid due to difference in date of sowing and harvesting period. Concentration of bioactive components was also found to be dependent on extracting solvent. Andrographolide derivatives were found to be maximum in methanol as compared to chloroform, ethyl acetate and ethanol extracts of Andrographis paniculata (Saxena et al. 2000).
The developed HPTLC technique is a precise, specific and accurate method for estimation of guggulsterone content in stem samples of C. wightii. Environmental factors were found to influence the guggulsterone content in the plants. Molecular profiling of these plants is underway to estimate the extent of genetic variability existing among them. Identification of high guggulsterone producing lines will play an important role in role in designing mass propagation as well as conservation strategies. Further study would be standardized to optimize the extracting solvent and conditions for maximum extraction and harvesting of guggulsterone for harnessing maximum benefits from this nutraceutical plant.
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Acknowledgments
A.K. thankfully acknowledges the financial assistance provided by Department of Biotechnology, Government of India, New Delhi, under the project sanctioned vide order no. BT/PR10526/NDB/51/164/2008. A.K. is also thankful to Director, CPB and CIRB for providing the lab facilities for research work.
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Kulhari, A., Sheorayan, A., Saxena, N. et al. HPTLC analysis of guggulsterone isomers in Commiphora wightii (Arn.) Bhandari: an endangered oleo-gum resin species heading towards extinction. Genet Resour Crop Evol 60, 1173–1180 (2013). https://doi.org/10.1007/s10722-012-9947-y
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DOI: https://doi.org/10.1007/s10722-012-9947-y