Estimation of Guggulsterone E and Z in the Guggul-based Commercial Formulations Using High-performance Thin-layer Chromatography.

BACKGROUND: Guggulsterone (GS) is a plant steroid and bioactive compound present in gum Guggul of Commiphora wightii. An Indian herbal medicine system "Ayurveda" has a long history of use of gum Guggul and plant extract of C. wightii as medicine for the treatment of various illnesses. Complex nature, low availability, and inconsistency of phytoconstituents make its analysis of difficult tasks. AIMS: In this work, six different Guggul-based herbal formulations were examined for estimation of GS and their isomers (E and Z) through high-performance thin-layer chromatography technique.
MATERIALS AND METHODS: For that various concentrations of standard E-GS and Z-GS (50 ng-250 ng/spot) with samples (20 μg/spot) were applied on silica gel coated aluminum plate and developed with the mobile phase of toluene: ethyl acetate: formic acid: methanol (6:2:1:0.5). The scanning was performed at 254 nm wavelength and the absorbance (scan) spectrum of E-GS and Z-GS peak was generated at 200 nm-400 nm wavelength range. RESULTS AND
CONCLUSIONS: Rf value and scan spectrum pattern of the samples reveal that they contain either one form of GS (E-GS, Z-GS) or both. The quantity of E-GS and Z-GS within the samples was ranged from 0.230 ± 0.0040-0.926 ± 0.0168% to 0.537 ± 0.0026-0.723 ± 0.0177%, respectively.

INTRODUCTION

For the past few decades, compounds from natural sources have been gaining importance because of their vast chemical diversity, and many of them are being used as a medicine. Herbal-based medicines have traditional value in the curing of various diseases, and now it has become more popular due to its scientifically known pharmacological activity with fewer side effects.[1] With the constant increase in the demand of herbal medicines/formulations worldwide, the safety and quality of herbal raw materials and their products have become a major concern for health authorities as well as pharmaceutical industries. The analysis of phytoconstituents is a difficult task because of their complex nature, usually low availability and inconsistency even within the same plant species.[2] Due to the complex nature of combined/mixed herbal products in comparison to the chemical drug, it needs more attention and various analytical methods can be used for its quality assessment.[3] The WHO has taken a keen interest in the quality control of herbal medicine and recommends the use of high-performance thin-layer chromatography (HPTLC) for the characterization and quality assurance of herbal medicines.[3]

HPTLC is preferred over other analytical method due to its several advantages such as automation, reproducible, fast, simple, many samples, as well as standards simultaneously, cost-effective, and hyphenation.[24] The manufacturing of herbal product after proper quality assurance through HPTLC can enhance the efficiency and value of the herbal product.[56]

Guggulsterone (GS) (4,17 (20)-pregnadiene-3,16-dione) is a plant polyphenol (steroid) and bioactive compound present in gum Guggul of Commiphora wightii, (family Burseraceae; synonyms: Balsamodendron mukul or Commiphora mukul).[78] GS occurs in two isomeric forms, namely Z-GS and E-GS (Patil et al., 2015). GS act as antagonist ligands for the bile acid receptor, farnesoid X receptor, and as active ingredients responsible for the hypolipidemic activity.[910] Both GS isomers were demonstrated to suppress lipopolysaccharide-induced inflammation by inhibiting IκB-α degradation and NF-κB activation.[1112] In addition, GS was reported to protect cardiac and neuronal damages in animal models.[13] GS has medicinal properties such as anti-inflammatory,[1415] hepatoprotective,[1617] muscle relaxing,[18] hypocholesterolemic and anti-obesity,[192021] antimycobacterial, antischistomal, larvicidal, and molluscicida.[22232425262728] There are several gum Guggul-based standardized formulations are being used as cholesterol-lowering agents and also in the curing of joint pains.[2930] The GS is the main active ingredient of gum Guggul which has various pharmacological importance; therefore, it is essential to ensure that the high quality and right quantity of GS in the Guggul-based commercial formulations. In the present study, six different Guggul-based herbal formulations were examined for estimation of GS and their isomers (E and Z) through HPTLC technique.

MATERIALS AND METHODS

Preparation of sample and standard

Gum Guggul-based six herbal formulations (Mf-A to Mf-F) of different manufacturers available in the Indian market were used. Tablets of these formulations were crushed and properly dissolved in methanol (10 μg/μl). The mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant was used as a sample. Standard E-GS and Z-GS (Chromadex, USA) were prepared separately in methanol with concentrations of 50 ng/μl and 100 ng/μl.

Development of high-performance thin-layer chromatography plate

The samples were applied using Linomet 5 (CAMAG) as a form of 7 mm long bands on prewashed (with methanol) 20 cm × 10 cm, 200 μm layered silica gel 60 F254 coated aluminum HPTLC plate (Merck) using the stream of neutral gas (Nitrogen) at a constant rate of 150 nl/s. Since the standardization of E-GS and Z-GS estimation, two different amount standards (200 and 400 ng/spot) and samples (20 and 40 μg/spot) were applied to the plate, while in the estimation process, standards were applied in the concentration varied from 50 ng to 250 ng/spot and 20 μg/spot samples were applied in two replicates.

After sample application, plates were developed in CAMAG Automatic Developing Chamber 2 with the mobile phase of toluene: ethyl acetate: formic acid: methanol (6:2:1:0.5) solution. The developing chamber was saturated with mobile phase (20 ml) for 20 min, and 10 ml mobile phase was allowed to migrate up to 70 mm distance on the plate at the room temperature under controlled humid condition (60–80% with NaCl solution).

Estimation of E-guggulsterone and Z-guggulsterone

The developed plate was scanned in CAMAG TLC scanner 3 at 254 nm wavelength in deuterium lamp (D2) with 6.00 mm × 0.45 mm slit dimension, 10 mm/s scanning speed, and 100 μm/step data resolution. The scan spectrum of E-GS and Z-GS peak was generated through scanning at a wavelength range of 200 nm–400 nm with slit dimension of 6.00 mm × 0.10 mm.

The presence of E-GS and Z-GS was assessed only in the samples of which scan spectrum was similar to standard E-GS and Z-GS. The quantity of E-GS and Z-GS was estimated through the calibration curve between standard E-GS and Z-GS concentration and their peak area using WinCATs Planner Chromatography manager software (CAMAG) Switzerland Excel (Microsoft Office 10) USA. The E-GS and Z-GS content (percentage) in the samples were calculated using following formula:

RESULTS

Presence of E-guggulsterone and Z-guggulsterone in the sample

Standard E-GS and Z-GS had 0.43 and 0.48 Rf value. According to these Rf values, E-GS was present in all the samples except sample Mf-E, while Z-GS was present in all samples. The Rf of E-GS on the sample Mf-A, Mf-B, Mf-C, Mf-D, and Mf-F were ranged from 0.43 to 0.44, and the Rf of the Z-GS in all samples were ranged from 0.48 to 0.52 [Table 1 and Figures 1 and 2]. The pattern of scan spectra and λmax of all the samples (λmax 249–250 nm) were almost similar to the standard E-GS (λmax of 250 nm) except sample A (λmax of 213 nm), similarly the scan spectra and λmax of all the samples (λmax of 251 and 257) and standard Z-GS (λmax of 250) had nearly same except sample Mf-F (λmax of 235) [Table 1 and Figures 3 and 4].

Table 1

Rf and λmax of E-guggulsterone and Z-guggulsterone in the standard and sample

Figure 1

Peak of guggulsterone E; (a) in the standard; (b) in the sample

Figure 2

Peak of guggulsterone Z; (a) in the standard; (b) in the sample

Figure 3

Absorbance spectra of guggulsterone E peaks

Figure 4

Absorbance spectra of guggulsterone Z peaks

Quantification of guggulsterone

In the estimation of E-GS and Z-GS, the respective linear calibration curves were plotted with five different concentrations of standard E-GS and Z-GS (50–200 ng) and their peak area. The correlation coefficient (r2) of E-GS and Z-GS calibration curve was 0.9979 and 0.9991, while the residual standard deviation of the standard point (sdv) was 3.142 and 2.1873, respectively. All the samples contain GS, either in the form of E, Z, or both. Sample Mf-A and sample Mf-E had only Z-GS with 0.590 ± 0.0205 and 0.723 ± 0.0177%, respectively, while sample F had only 0.926 ± 0.0168% GS in the form of E trans isomer. Both forms of GS were present in the sample Mf-B, Mf-C, and Mf-D with 0.382 ± 0.0025, 0.230 ± 0.0040, and 0.240 ± 0.0206% E-GS and 0.573 ± 0.0028, 0.537 ± 0.0026, and 0.245 ± 0.0034% Z-GS, respectively [Table 2 and Figures 5 and 6].

Table 2

Quantity of E-guggulsterone and Z-guggulsterone in the samples

Figure 5

Calibration curve for estimation of guggulsterone E

Figure 6

Calibration curve for estimation of guggulsterone Z

DISCUSSION

Traditional medicinal plants are being used in curing the diseases, particularly in remote parts of the developing countries having few health facilities. However, herbal-based medicine is now becoming popular in the developed countries[31] might because medicinal plants are the best source to obtain a variety of drugs due to its various advantages over other drugs. The use of herbal medicine in the form of formulations/extract would have certain drawbacks because the inconsistent amount of main constituents in the raw material, therefore, raw materials should be used in the drug formulation after proper investigation of their properties, safety, and efficacy.[32] Based on traditional knowledge of herbal medicines, its scientific validation using standardized techniques may be a safe path of reverse pharmacology approach.[33] This may play a vital role in drug discovery, development, and therapeutics.

Guggulipid extract has been used in Ayurveda medicine to treat various diseases such as dyslipidemia, inflammation, and arthritis and also have cholesterol-lowering properties and thyroid-stimulating activity. The cis and trans GS (E-GS and Z-GS) are the main ingredients in guggulipid and preliminary studies indicate that the medicinal value of guggulipid extract is due to these isomers of GS.[34353637] Looking the necessity of quantitative estimation of GS in herbal medicine, six Ayurveda formulations were undertaken using HPLTC because HPTLC was found to be specific, reproducible, and precise for the estimation of E-GS and Z-GS.[3839] This estimation process would reveal the presence of either both the isomers of GS or only one isomer of GS in the observed herbal formulations, which subsequently leads the authentication of proper pharmaceutical doses of GS.[3840]

For HPTLC method development, the suitable stationary and mobile phase is the primary requirement. Silica gel 60 F254-coated aluminum plate as a stationary phase used in more than 80% of HPTLC analysis for the quantification of compound. In context with mobile phase, various combinations of organic solvents were used for GS estimation like light petroleum: ethyl acetate (3:1),[41] n-hexane: chloroform: ethyl acetate: methanol in (10:3:3:1),[39] toluene: acetone with the ratio of 9:1[424344] and 9.3:0.7[45] and petroleum ether: ethyl acetate: methanol (6:2:0.5).[40] In addition to these combinations of solvent toluene: ethyl acetate: formic acid: methanol (6:2:1:0.5) solution is preferred for quantitative estimation of GS in the herbal formulations.[38]

Both the isomers of GS absorb the same wavelength of light,[42] which was 250 nm in the present study. Thus, for the quantitative estimation of GS isomers, it is necessary that they should be discriminated through their Rf. The used mobile phase properly separates these isomers with Rf value of 0.35 and 0.42 for E-GS and Z-GS, respectively. However, the presence of compound within the samples is also determined by the pattern of the absorbance spectrum (scan spectrum). The absorbance spectrum of the sample has to be similar to the standard of the compound analyzed and the purity of the absorbance spectrum band was checked by overlaying both the absorbance spectrum band at start, middle, and end position.[40] Using these criteria, GS was found in the all six samples, while the both forms of GS were present only in four samples.

The correlation coefficient (r2) near to 0.999 and sdv lesser than 5 is better for the quantification of the compound through the linear calibration curve.[46] The regression analysis data for the calibration plots for E-GS and Z-GS showed a linear relationship with a correlation coefficient (r2) of 0.9979 and 0.9991 and sdv of 3.142 and 2.187, respectively, showed that the quantity of GS within the samples would be precise and accurate. The content of E-GS and Z-GS was estimated in the ayurvedic medicine, which reveals that in the sample, the content of E-GS form was varied from 0.230 ± 0.0040 to 0.926 ± 0.0168%, while the content of Z-GS form varied from 0.245 ± 0.0034 to 0.723 ± 0.0177%, which was near about to earlier studies upon the herbal formulations such as Rakta Dosh Nashak vati[40] and Jivitprada vati.[38]

High-performance liquid chromatography (HPLC) was used by many researchers for estimation of E-GS and Z-GS in blood serums, commercial formulations, plant materials, and health supplements; reversed-phase liquid chromatographic method was used for the estimation of E-GS and Z-GS in rate blood plasma[47] and human blood serum.[484950] Reversed-phase HPLC and HPLC methods successfully used for the estimation of GSs E and Z in many commercial formulations, i.e., in nine commercial formulations available in the US market,[51] in Guggul formulations,[52] in nanoemulsion formulation and Habb-e-Khardal Unani tablet,[53] in a polyherbal formulation containing C. wightii extracts,[54] in polyherbal formulation, Yogaraja Guggul tablets,[55] and in Ariflex tablet formulation.[56] This method was also used for the estimation of E-GS and Z-GSs in plant materials,[5758] as well as in vitro generated plant callus.[5960]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.