INTRODUCTION:

Liquorice, the roots of Glycyrrhizia glabra has been used as a medicinal herb for over 4000 years. The active components of this plant have extensive therapeutic usage throughout the world and are subjected to enormous works in recent years¹. Licorice root has been used in both Eastern and Western medicine to treat a variety of illnesses ranging from the common cold to liver disease. Roots and rhizomes of Glycyrrhiza glabra are active pharmaceutical parts of plant and are used in estrogenic, demulcent, pectoral, antioxidant, antispasmodic, anti-inflammatory, antibacterial and expectorant drugs². Recently, the concept of marker-based standardization of herbal drugs is in demand. Identification of major and unique compounds in herbs as markers and development of analytical methodologies for monitoring them are the key steps involved in marker-based standardization^3-4.

The major active principle in Glycyrrhiza glabra is glycyrrhizic acid which mainly contributes to the pharmacological activity of the herb is recognized as analytical marker compounds for the quality control of herbal formulations containing Glycyrrhiza glabra. Glycyrrhizic acid is a triterpenoid saponin which is approximately 50 times sweeter than sugar⁵. Chemical structure of Glycyrrhizic acid is shown in Figure 1.

Figure 1. Molecular structure of Glycyrrhizic acid

There are reports on the application of various analytical methods for isolation and quantitation of glycyrrhizic acid present in Glycyrrhiza glabra^6-8. But no reported method deals with quantitative determination of glycyrrhizic acid in herbal formulations. In recent times, high performance liquid chromatography has emerged as an efficient tool for the phytochemical evaluation of herbal drugs because of its simplicity, sensitivity, accuracy, suitability for high throughput screening, etc. Hence it was thought worthwhile to develop a simple and rapid chromatographic method for determination of glycyrrhizic acid in crude drug and its herbal formulation. The method was validated and found to be sensitive and reproducible.

EXPERIMENTAL:

Reagents and materials:

Glycyrrhizic acid reference standard was purchased from Sigma Aldrich, USA. Methanol, distilled water, acetonitrile, (HPLC Grade), potassium dihydrogen phosphate, dipotassium hydrogen phosphate were obtained from Merck, Mumbai.

Plant Material:

The rhizome of Glycyrrhiza glabra plant is used for the quantitative analysis of glycyrrhizic acid and was purchased from local market. Rhizomes were pulverized to a fine powder and passed through 14 mesh. The fine powder was utilized for further experimental purpose.

Ayurvedic Formulation:

Herbolax Capsules manufactured by Himalaya Health Care were procured from the local market.

Instruments and analytical conditions:

Ultraviolet spectrophotometric analyses were carried out on a Elico –SL 164 spectrophotometer. The solubility of Glycyrrhizic acid was determined. From the solubility data, distilled water was selected as a solvent for the method because of its optimum solubility and easy availability. The spectrum of glycyrrhizic acid in distilled water was recorded. From the spectrum of glycyrrhizic acid the wavelength maxima was found to be 256 nm (Figure.2). Hence this wavelength was selected as detection wavelength for further analysis by HPLC.

Figure 2. Maximum wavelength of Glycyrrhizic acid in Distilled water (256nm)

The HPLC analysis was carried out on a Shimazdu, Prominence. The column used was BDS (250mm×4.6mm; 5µ particle size) maintained at room temperature. The detection was performed at 256nm by UV detector. The optimization was done by changing different compositions of the mobile phase and different ratios of mobile phase and chromatograms were recorded for glycyrrhizic acid. The most suitable mobile phase composition selected for the quantitative analysis of glycyrrhizic acid was Acetonitrile: phosphate buffer in the ratio 45:55 v/v at a flow rate of 1.0ml/min. The injection volume was 20µL and chromatogram was recorded (Figure 3)⁹.

Figure 3. Optimized chromatogram of Glycyrrhizic acid

Calibration curve for HPLC:

A stock solution of 100 μg/mL glycyrrhizic acid was prepared by weighing 50 mg of standard drug, initially dissolved in minimum quantity of mobile phase and later the volume was made up to 50 ml with mobile phase.

With the optimized chromatographic conditions, stock solutions of glycyrrhizic acid were prepared by using mobile phase, and various concentrations were prepared in the range of 20 –120 μg/mL of glycyrrhizic acid. 20 μL of each solution were injected individually. The chromatograms were recorded at 256 nm. The calibration curve was plotted using concentration against peak area.

Method Validation

The method was validated for linearity, accuracy, precision, robustness, sensitivity, system suitability and specificity by the following procedures:

Linearity

The standard stock solution was diluted further to get a concentration in the range of 20-120µg/mL of Glycyrrhizic acid. Each concentration was injected. A calibration curve was plotted using peak area Vs concentration. The correlation coefficient was found to be 0.999 for glycyrrhizic acid^9-11.

Precision

The precision was examined by performing the intraday and inter-day assays of the standard solution of glycyrrhizic acid. The intra-day assay precision was performed with the interval of 2 h in 1 day, while the inter-day assay precision was performed over 6 days for the median concentration of glycyrrhizic acid from the concentrations prepared for the calibration curve^9-11.

Limit of detection and limit of quantification

LOD and LOQ were determined by kSD/s where k is a constant (3 for LOD and 10 for LOQ), SD is the standard deviation of the analytical signal, and s is the slope of the concentration/response graph^9-11.

Specificity

The demonstration of specificity requires that the procedure is unaffected by the presence of impurities or excipients. In practice this can be done by spiking the drug substance or product with appropriate levels of impurities or excipients and demonstrating that the assay result is unaffected by the presence of these extraneous materials. The analyte is treated with different interferences like in the presence of acid, in presence of alkali, etc and checked for any degradation occurred^9-11.

Robustness:

Robustness of the proposed method was evaluated by changing the column [BDS (250mm×4.6mm; 5_micron particle size)] temperature.The effect of change in temperature was studied^9-11.

System suitability:

Standard solution was injected 6 times for each change. System suitability parameters and RSD were calculated for each peak. % RSDs were calculated for each component during each change^9-11.

Accuracy:

The accuracy of the method was determined by calculating the recoveries of Glycyrrhizic acid by the method of standard addition. Known amounts of the standards (80%, 100% and 120 %) were added to the pre-analyzed sample solution, and the amounts of these standards were estimated by measuring the peak areas and by fitting these values to the straight-line equation of calibration curve^9-11.

Solution stability and mobile phase stability:

Solution stability in the assay method was evaluated by leaving test solutions of sample and reference standard in tightly capped volumetric flasks at room temperature in the dark for 24 h. The same sample solutions were assayed every 6 h interval in the study period. Mobile phase stability was studied by assaying the freshly prepared sample solutions against freshly prepared reference standard solutions at 6 h intervals up to 24 h. Mobile phase was prepared and kept constant during the study period. The relative standard deviation (RSD) of the assay of glycyrrhizic acid was calculated for the study period during mobile phase and solution stability experiments^9-11.

Statistical Analysis:

The statistical analysis was performed using Microsoft Excel 2007.

Quantification:

Preparation of sample solutions:

Plant Material:

About 2gms of the Glycyrrhiza glabra rhizome powder was weighed accurately. To this 16 ml of methanol was added and was sonicated for one hour. After extraction, the solution was filtered through Whatman filter paper no. 41. Then it was evaporated to obtain residue^12-13. The dried residue obtained was weighed and 2 consecutive 10ml dilutions of extract with mobile phase were done for quantification which was injected and the chromatogram was recorded. The chromatograms recorded for the quantification of crude drug is given in Fig. 4. The amount of glycyrrhizic acid in crude drug was found to be15.193 mg/gm.

Ayurvedic Formulation:

About 20 capsules were weighed and average weight was calculated (389.54mg). From the powdered sample, approximately 0.38 g was weighed and extracted with 16 ml of methanol by sonication for one hour. After extraction, the solution was filtered through Whatman filter paper no. 41. Then it was evaporated to obtain residue ^[12-13]. The dried residue obtained was weighed and 2 consecutive 10ml dilutions of extract with mobile phase were done for quantification which was injected and the chromatogram was recorded. The chromatograms recorded for the quantification of herbal formulation is given in Fig.5. The amount of glycyrrhizic acid in herbolax capsule was found to be 8.58mg/capsule

Fig.4.Chromatogram of Glycyrrhiza glabra Plant Extract

Fig.5.Chromatogram of Herbal Formulation

RESULTS AND DISCUSSION:

An effort has been made to develop a simple, precise, specific and accurate HPLC method for the estimation of glycyrrhizic acid in crude drug and formulation. The mobile phase selected for the quantitative analysis of glycyrrhizic acid was Acetonitrile: phosphate buffer in the ratio 45:55 v/v. The calibration was done by using external standard calibration method. With the optimized chromatographic conditions, Standard stock solutions of glycyrrhizic acid were prepared by using mobile phase, and from the standard stock solution, various concentrations were prepared in the range of 20 –120 μg/mL of glycyrrhizic acid. The chromatograms were recorded at 256 nm. The correlation co-efficient value was found to be 0.999 indicates that the concentrations of glycyrrhizic acid had good linearity. The limit of detection and the limit of quantification were determined from the linearity studies. The limit of detection was found to be 0.704 μg/mL and the limit of quantification was found to be 2.348μg/mL. The precision of the method was confirmed by repeatable injection for five times. The percentage RSD value was found to be 0.542. The RSD values obtained for intraday and interday precision were within the acceptance criteria according to ICH guidelines. This indicates that the method has good precision. Accuracy of the developed method was satisfactory. The average percentage recovery obtained was 99.606% for glycyrrhizic acid. The robustness study indicated that the selected factors remained unaffected by small variations of the parameters. The RSD values of assay of glycyrrhizic acid during solution stability and mobile phase stability experiments were within 2.0%. The data obtained in both experiments proves that the sample solutions and mobile phase used during assay were stable up to 24 h. The amount of glycyrrhizic acid found to be present in crude drug and herbal formulation was found to be 15.193mg/gm and 8.58mg/capsule.

Table.1Summary of Validation parameters

S. No.	Parameters	Results
1	Wavelength	256nm
2	Linearity Concentration range Regression equation Correlation coefficient (r²)	R² = 0.999 20-120(µg/ml) 6.3247x+11.785 0.9993
3	LOD	0.704µg/ml
4	LOQ	2.348µg/ml
5	Precision Repeatability Intraday precision Interday precision	% RSD = 0.542 % RSD = 0.96, 0.17 % RSD = 0.431, 0.076
6	Accuracy	% Recovery = 99.67%
7	Specificity	No degradation
8	Robustness	Robust
9	Ruggedness	% RSD 0.825

Table.2. Quantification results

Category	Amount of Glycyrrhizic acid
Plant Extract	15.193 mg/gm
Herbolax Capsules	8.58 mg/capsule

CONCLUSION:

A simple, rapid and accurate HPLC method was developed for the determination of glycyrrhizic acid in crude drug and herbal formulation. The method showed excellent sensitivity, reproducibility, accuracy and repeatability, which is evidenced by low percentage relative standard deviation. The results obtained in recovery studies were indicating that there is no interference from the excipients used. Hence it is suggested that the proposed RP-HPLC method can be effectively applied for the routine analysis of glycyrrhizic acid in crude drug and herbal formulation in quality control analysis.

REFERENCES:

1. The Text book of Pharmacognosy by C.K.Kokate, A.P.Purohit, S.B.Gokhale

2. Indian Herbal Pharmacopoeia, Indian Drug Manufacturers Association, Mumbai, 2002, Revised New Edition, 206-213.

3. Wagner H, Bladt S. Plant drug analysis, A Thin Layer Chromatography Atlas. Springer, Berlin, Germany, 2002.

4. Mukherjee PK. Quality control of herbal drugs. Business Horizons, New Delhi, India, 2002; pp.147, 327.

5. http://www.ayurveda-recipes.com/yashtimadhu.html

6. SUN Chen, XIE Yuchun, LIU Huizhou Microwave-assisted Micellar Extraction and Determination of Glycyrrhizic Acid and Liquiritin in Licorice Root by HPLC Chin. J. Chem. Eng., (2007) 15(4) 474—477.

7. J.R. Hennell, S. Lee, C.S. Khoo, M.J. Gray, A. Bensoussan, The determination of glycyrrhizic acid in Glycyrrhiza uralensis Fisch. Ex DC. (Zhi Gan Cao) root and the dried aqueous extract by LC–DAD. Journal of Pharmaceutical and Biomedical analysis (2008) 494–500.

8. L. A. Baltina, n. G. Serdyuk, l. V. Krasnova, Preparation of glycyrrhizic acid from licorice extracts, Pharmaceutical chemistry journal, (1994) vol. 28, no. 9.

9. Synder K.L, Krikland J.J and Glajch J.L: Practical HPLC Method Development 2^nd Edn, Wiley-Interscience Publication, USA, 1983, 653-661.

10. ICH Harmonized Tripartite Guidelines, Validation of Analytical Procedures: Text and Methodology, Q2R(1).

11. ICH haromonizies tripati guide lines, Validation analytical procedures / methodology, 1996, 1-8.

12. Sethi, P.D. HPLC Quantitative Analysis of Pharmaceutical Formulations. 1^stedition., CBS publisher and Distributors, New Delhi, 2001, 5-10.

13. GU Hui-yan, GONG Li-dong, YU Jing-hua I, Measurement and comparison of glycyrrhizic acid contents in root of licorice (Glycyrrhiza uralensis Fisch.) from different cultivating areas, Journal of Forestry Research- 2002

Received on 18.03.2010 Modified on 02.04.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 703-706