| Abstract|| |
Jivitprada vati is an Ayurvedic formulation, which is consists of Guggul, Shilajit and Galodhan Guggulsterone E & Z and Tinosporaside are active constituents that are used for rheumatoid arthritis and erectile dysfunction. The purpose of this study was to develop an HPTLC method of quantitative estimation of marker compounds, Guggulsterone E & Z and Tinosporaside in laboratory prepared authentic formulation and compare with three different marketed formulation. The marker compounds were isolated from plant material and authenticated by comparing its UV spectrum, IR spectrum and GC-MS fragmentation pattern with standard marker and its literature studied. The four formulations were subjected to methanol extractions by Soxhlet apparatus. Guggulsterone E & Z and Tinosporaside were quantified in the above extracts by HPTLC method. The detection and quantification was performed at a wavelength of 230 nm. The laboratory formulation was found to contain 2.18% of Guggulsterone E, 1.898% of Guggulsterone Z and 0.954% of Tinosporaside while in case of marketed formulation MF-1, MF-2 and MF-3 was found to contain, respectively, 1.06%, 0.527%, 0.318% of Guggulsterone E and 0.914%, 0.487%, 0.24% of Guggulsterone Z in the methanolic extracts of formulations, whereas in MF-1 and MF-2 was found very less amount of Tinosporaside (respectively, 0.347% & 0.14%) except in MF-3 which was devoid of Tinosporaside. The method was found to be linear, precise and accurate for quantitative estimation of E & Z Guggulsterone and Tinosporaside in different
Keywords: Guggulsterone E and Z, HPTLC, Jivitprada vati, laboratory formulation, Tinosporaside
|How to cite this article:|
Jariwala JK, Saluja AK, Anajwala CC, Dakhara SL. Simultaneous estimation of Guggulsterone E & Z and Tinosporaside in Jivitprada vati by HPTLC method. Int J Green Pharm 2011;5:113-7
|How to cite this URL:|
Jariwala JK, Saluja AK, Anajwala CC, Dakhara SL. Simultaneous estimation of Guggulsterone E & Z and Tinosporaside in Jivitprada vati by HPTLC method. Int J Green Pharm [serial online] 2011 [cited 2013 Jun 19];5:113-7. Available from: http://www.greenpharmacy.info/text.asp?2011/5/2/113/85168
| Introduction|| |
The recognized Indian Systems of Medicine are Ayurveda, Siddha and Unani, which use herbs and minerals in the formulations. Indian traditional systems of medicine in Ayurveda, Siddha and Unani backed by a strong manufacturing base are appropriately positioned to provide holistic health care not just within the country but internationally too1  Jivitprada vati consists of Guggul (Commiphora wightii),  Shilajit (Asphaltum)  and Galodhan (Tinospora cordifolia).  Guggulsterone E & Z , and Tinosporaside, , are active constituents of guggul and galodhan, respectively. Nowadays, Ayurvedic or Siddha formulations must require standardization.
| Materials and Methods|| |
HPTLC system equipped with a sample applicator Camag Linomat 5, Camag TLC scanner 3, Silica gel coated on aluminium sheets, Twin through glass chamber, Deuterium lamp (Switzerland).
Toluene, ethyl acetate, formic acid and methanol were obtained from ACS Laboratory, Gujarat, India. Guggulsterone E & Z and Tinosporaside were isolated from C. wightii and T. cordifolia. C. wightii, T. Cordifolia and Shilajit were procured from Mahavir Enterprise and Hakim Chichi, Gujarat, India. These were identified and authenticated on the basis of their various morphological as well as microscopical characters.
The market formulation of "Jivitprada Vati (Tablet)" of different manufacturers (Aasfa, Surat, Shree Bhuvaneshwari Aushadashram, Gondal and Shree Mahanarayan Ayurvedic Pharmacy, Ahmadabad) was procured from the local market. These market formulations were named as MF-1 (Aasfa), MF-2 (Bhuvaneshwari), MF-3 (Mahanarayan) and fourth formulation was prepared in the laboratory and marked as LF (Lab or In-House formulation).
HPTLC Method for Estimation of Guggulsterone E & Z and Tinosporaside
Preparation of mobile phase
A mixture of 6 ml toluene, 2 ml ethyl acetate, 1 ml formic acid and 0.5 ml methanol previously filtered through filter paper in a flask was used as a mobile phase.
Preparation of standard solution
Stock solution of isolated E and Z - Guggulsterones and Tinosporaside (1 mg/ml) was prepared by dissolving 10 mg of reference compound E and Z - Guggulsterones and Tinosporaside in 10 ml of methanol individually. From this stock solution 100 μl/ml was prepared by transferring 1 ml stock solution to 10 ml volumetric flask and adjusted the volume with methanol.
Pre-coated silica gel G60 - F 254 aluminium sheet, (E. Merck, Germany) (100 Χ 50 mm, thickness layer 0.2 mm, pre-washed with methanol and dried at room temperature)
Mobile Phase - Toluene : Ethyl acetate : Formic acid : Methanol (6 : 2 : 1 : 0.5)
Chamber saturation time - 30 min
Distance run - 80 millimetre
Temperature - 27°C
Wavelength - 254 nm
HPTLC analysis (calibration curve)
Semi-automatic spotter was used containing a syringe having capacity of 50 μl. Approximately 10 μl standard solution was filled in syringe and under nitrogen stream it was applied in form of bands of desired concentration range (200-1000 ng/spot) of standard solution on pre-coated plates. For E- Guggulsterone, Z-Guggulsterones and Tinosporaside individual plates were prepared. Plates were developed using Toluene : Ethyl acetate : Formic acid : Methanol (6:2:1:0.5) at 27°C (±2°C) and dried in air. Developed plates were subjected to densitometric measurements in absorbance mode at wavelength 230 nm using Camag TLC scanner 3. Plots of peak area vs. concentration for isolated E- Guggulsterone, Z-Guggulsterone and Tinosporaside are shown in [Figure 1],[Figure 2],[Figure 3].
Preparation of Jivitprada Vati
Firstly galodhan was treated with the Guduchi kwath in slightly warm condition and mixed properly by stirring well continuously. Then add the previously accurately weighed and prepared Suddh guggul and Suddh shilajit in this galodhan and Guduchi kwath semisolid mixture and properly mix by stirring continuously. And at that same time add the mixture mentioned in [Table 1] and prepare mould Vati with hand. They are then dried under shade at sun temperature, packed in air tight container and labelled as Lab formulation Jivitprada vati [Table 1]. 
Preparation of extracts
Weigh accurately 1 g of each powdered formulation and extract with methanol for four times using 25 ml methanol each time (4΄25), combine the extract and concentrate to 25 ml. One millilitre of concentrated solution was taken in 100 ml volumetric flask and diluted up to 100 ml (sample solution). Sample solution was used for the quantification.
Injection : Camag Linomat 5, Switzerland
Scanner : Camag TLC scanner 3, Switzerland
Stationary phase : Silica gel coated on aluminium sheets
Development chamber : Twin through glass chamber
Detector : Deuterium lamp
Recovery was carried out by estimating the Guggulsterones E and Z and Tinosporaside content in the pre-analysed MF-1, MF-2, MF-3 and LF spiked with E and Z-Guggulsterones and Tinosporaside at different concentration levels.
| Results|| |
Ayurvedic physician uses many traditional formulations for the treatment of arthritis. Non-medicinal person also claim to treat the disorder with the so-called plants drugs. Preparation given by such practitioners is often kept confidential, but some preparations are also available in market, whose composition is well known and not yet standardized properly. We attempted to standardize some of the formulation, containing C. mukul, T. cordifolia and Shilajit by determining the percentage of marker compound of drug in different formulation by HPTLC method.
Validation of HPTLC Method
Linearity of an analytical method is its ability to elicit test results that are directly or by a well-defined mathematical transformation, proportional to the concentration of analyte in a sample within a given range. The range of the analyte method is the interval between the upper limit and lower limit of the analyte in the analytical method.
The linear range is 100-500 ng/spot for E-Guggulsterone, Z-Guggulsterone and Tinosporaside with correlation coefficient of marker compound by HPTLC was, respectively, 0.9901, 0.9947 and 0.9961.
Accuracy: (% recovery)
The accuracy of analytical method closer to the test results were obtained by the method of the value. The accuracy may often be expressed as % recovered by the assay by adding known amount of analyte. The recoveries of marker compound are shown in [Table 2],[Table 3],[Table 4].
The specificity of an analytical method is its ability to measure accurately and specifically the analyte in the presence of component that may be expected to be present in the formulation. Amount of added maker compound was estimated without interference of other component present in the formulation.
Limit of detection
It is the concentration of analyte in the sample that can be detected but not necessarily quantified under the stated condition. The limit of detection was found for E-Guggulsterone, Z-Guggulsterone and Tinosporaside.
Limit of quantification
It is the lowest concentration of analyte in the sample that can be determined with acceptable decision and accuracy under the stated experimental conditions. The limit of quantification for different marker compound was 70 ng/spot, 80 ng/spot and 10 ng/spot.
It is a measure of reproducibility of data within a series of results. Results within a series, which agree closely with one another, are said to be precise. Precision data of E-Guggulsterone, Z-Guggulsterone and Tinosporaside showed that the method was found to be precise [Table 5],[Table 6],[Table 7].
It is defined as precision under the same concentration over a short interval time. It can be affected by factor such as analyst, environment condition or instrument error. Summary of all validation parameters of E and Z-Guggulsterone and Tinosporaside are shown in [Table 8],[Table 9],[Table 10].
| Discussion|| |
Jivitprada Vati (tablet) was prepared according to the Nidan Chikitsha Hastamalka by Ranjitrai Desai and also by slightly modification in the method of preparation. Quality control parameters according to WHO guidelines were examined, such as extractive value, Ash value, volatile oil content, moisture content, tablet weight variation, disintegration study, friability etc. The disintegration time noted were slightly higher than recommended limit but it would not adversely affect the dose delivery to patients. Proximate analysis of formulation was carried out and higher water-soluble extractive value indicated the presence of more of polar constituents. Successive solvent extraction with different solvents like petroleum ether, chloroform, acetone, methanol and water were carried out and their extractive values were found.
Qualitative chemical examination of various extracts of formulation revealed the presence of different phytoconstituents like alkaloids, glycoside, steroids, essential oil, terpenes, fixed oils, saponins etc. Thin layer chromatography of formulation was carried out to confirm the presence of E and Z Guggulsterone and Tinosposide [Figure 4],[Figure 5],[Figure 6]. TLC fingerprints of formulations were carried out to confirm the presence of E and Z Guggulsterone and Tinosposide. HPTLC method was developed for quantification of E and Z Guggulsterone and Tinosposide and results show the moderate amount of E and Z Guggulsterone and Tinosposide in market formulation as compared with the laboratory formulation. The method was validated and method is found simple, accurate, precise and specific. It can be used for quantification of marker compound for market formulation.
|Figure 4: UV-366 photos with various formulations and compare with marker|
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|Figure 5: UV-254 photos with various formulations and compare with marker|
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|Figure 6: Derivation photos with various formulations and compare with marker (MF-1: Market formulation-1, MF-2: Market formulation-2 MF-3: Market formulation-3 LF: Lab or Inhouse formulation, G-1: E-guggulsterone, G-2: Z-guggulsterone, T-1: Tinosporaside)|
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| Conclusion|| |
The developed HPTLC method was found to be rapid, simple and accurate of quantitative estimation of E and Z Guggulsterone and Tinosporaside in different formulation. The recovery value of E and Z Guggulsterone and Tinosporaside shows the reliability and suitability of the method. The coefficient of variation for E and Z Guggulsterone and Tinosporaside proves the accuracy and precision of the analysis. The method was found to be useful in detecting the genuineness of the formulation and thus suitable to evaluate various formulation available in the market. The method also revealed the suitability of methanol solvent in extracting the phytomarkers present in the formulation. The proposed solvent system and the scanning wavelength are suitable to identify and estimate for E and Z Guggulsterone and Tinosporaside simultaneously.
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Jitesh K Jariwala
Department of Pharmacognosy, Babaria Institute of Pharmacy, Varnama, Vadodara, Gujarat
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]