Determination of Glabridin in Licorice Root (Glycyrrhiza glabral.) Using High Performance Liquid Chromatography Faridah a, Siti Umrah Noor a, Rahmawati T a. Abstract Public opinionwho define that beautiful is someone who has a white skin, hasled to abuse of dangerous skin bleach, such as mercury and hydroquinone without labelconcentration and most of them are sold in free market. A natural whiteningcan be obtained from plant, for example licorice root that contains glabridin as a whitening compound with high activity. The content of Glabridin in licorice root is very small, soit requires a specific and sensitive method in order to determine glabridin concentration in licorice root namely HPLC. The result showed that concentration of glabridin in licorice extract from powder with particle size of1 mm (sample A) and particle size of 4,7 mm (sample B) were 0.2062 mg/g ±0.4779% and 0.4578 mg/g± 0.5006% respectively. in addition,the detection limit of 0.0388 ppm and the limit of quantitation of 0.1176 ppm.the content of glabridin in licorice extract in sample B is higherthan thatin sample A using HPLC method. Keywords: glabridin, licorice root, Glycyrrhiza glabral, HPLC a Faculty of Pharmacy Pancasila University Corresponding author email address: idaffup@gmail.com Introduction Currently almost everybody wants their skin looks white, because in public there is an opinion that a beautiful people is someone who has a white, clean and bright skin. Not only women but also men want white and brighter skin. The bright white skin is influenced by several factors, including the location geography of residence, genetic parents, race of nation and personal content of melanin.melanin is a naturally pigment or dye in living organisms. Initially, a white skin is only owned by people who have a genetically white skin such like Chinese, Japanese, British, American but now white skin can be owned by everyone who is using a certain cosmetics. Hence, some cosmetics manufacturers only orient to produce bleach skin with hazardous materials such as mercury, hydroquinone and kojic acid which can harm the users. White skin can be produced by inhibiting the formation of melanin by blocking the work of the melaninforming enzyme tyrosinase enzyme (tyrosinase inhibitors) which is a compound that can inhibit melanin production. Tyrosinase inhibitors are currently used in cosmetic products as an inhibitor of melamine production in the epidermis and make the skin look brighter. Nowadays, it has been discovered a new compound from licorice being glabridin which has an ability to brighter the skin (4) This study aimed to obtain the highest content of glabridin in licorice extract using HPLC Methodology Preparation of extracts A total of 100 g of dry powder are macerated with 70 % ethanol under stirring and then filtered. Continue the maceration until all the compounds are perfectly extracted. Collect and concentrate the resulting extract by vacuum rotavapourator. Determination ofthe optimum conditions of HPLC a. Determination themaximumwavelengthofglabridin This experiment is done by making the absorption spectrum of 5 ppm glabridin standard at 200-600 nm. The wavelength of maximum absorbance obtained is used to detect glabridin on the HPLC. b. Selection of mobile phase and flow rate 1 Preparation of the solution Glabridin 5 mg are dissolved in ethanol to 10ml. 1.0 ml the solution is diluted up to 100 ml. 20 µl the diluted are injected into the chromatograph,. Green Chemistry Section 4: Organic Chemistry,Faridah, et al. P a g e 289
ISBN 978-602-285-049-6 a. The mobile phase tested are: Methanol Water = 71 : 29 and Methanol Water = 72 : 28 b. Tested flow rates were 1.0 ml/min The mobile phase flow rate chosen gave the best separation results with a defined retention time. Identification of glabridin in licoriceextract. 1. Extract Approximately 50 mg of extract was diluted with ethanol up to 10 - ml 2. Glabridin standard Approximately 5 mg glabridin was dissolved in ethanol to 10 ml and 1.0 ml of the solution was diluted with ethanol up to 100 ml. 7. Recovery Test Proceedings of The 9 th Joint Conference on Chemistry a. Preparation of standard solution Approximately 5 mg of glabridin were weighed carefully and dissolved in 10 ml of the mobile phasethen1.0 ml diluted up to 20 - ml. b. Test Solution The recovery test used two series, namely 50 mg of liquorice extract was added to 1 ml of standard solution of glabridin (25 ppm) for I series and 50 mg of liquorice extract which was added 2 ml of standard solution of glabridin 25 ppm for the series II. Calculate the recovery test. Results and Discussion Determination of Maximum absorption wavelength of Glabridin 3. Identification method Inject 20 µl of the solution into the chromatograph, and then compare the retention time glabridin in sample with glabridin standard. 4. Linearity test The test is performed to determine the relationship between the concentration with the peak area. to perform the test solution 1 series with 7 different concentrations (0.5 ; 1 ; 1.5 ; 2 ; 2.5 ; 3 and 3.5 ppm) was prepared. Then made a relationship curve between the concentration as the x-axis and the area as y-axis, find the regression line equation Y = a + bx. 5. System suitability test This test is performed to determine whether the tools, methods and conditions form a single analysis system. This test was done by measuring the standard glabridin 5 times at the same concentration, then the relative standard deviation was calculated Figure 1.Absorption spectra of glabridin (5 ppm) Based on the absorption spectra obtained glabridin maximum absorption wavelength is 281 nm, because the wavelength of 233.60 nm is likely absorption of the solvent. Mobile phase and Flow Rate Selection of mobile phase composition used are acetonitrile-water (70:30), methanol-water (71:29) and methanol-water (72:28) with a UV detector at a wavelength of 281 nm and a flow rate of 1.0 ml/min. 6. Determination of glabridin in licorice extract using HPLC Weighed approximately 5 mg of glabridin standarddissolved in ethanol until 10 - ml, then 1.0 mldiluted until 100ml. Weighed approximately 100 mg extract then dilute with ethanol up to 10 ml. 20 µl of each solution is injected into the chromatograph, and then measurethe peak and calculate the concentration of glabridin. Figure 2.chromatogram of the glabridin standard with a mobile phase of methanol-water (72:28) 290 P a g e Green Chemistry Section 4: Organic Chemistry, Faridah, et al.
System Suitability Test Figure 3.Chromatograms of glabridin with a mobile phase of methanol-water (71:29) Mobile phase composition used are acetonitrile-water (70:30) does not give good results From the selection of the wavelength, mobile phase, and flow rate obtained the best conditions for glabridin analysis are: Stationary phase: C18 Column Mobile phase: methanol-water (72:28) UV detector: 281 nm Flow rate: 1.0 ml/min Identification of Glabridin in Licorice Extract Identification was carried out to ensure glabridin in licorice extract by comparing the retention time of liquorice extract with the reference standard of glabridin. System suitability test was performed to ensure the suitability and effectiveness of the operating system among others, including tools, methods, analyte and other operational conditions that can form a single analytical system. Parameters assessed were recurrence and reinjection of standard solution expressed in relative standard deviation. The data of retention time, peak area and relative standard deviation glabridin were obtained by injecting 5 times. Relative standard deviation of glabridin is 0.588 % which meets the requirements of the relative standard deviation value ( 2 %). It can be concluded that the condition of high performance liquid chromatography can be used for the assay of glabridin. No. Table 1. System suitability test Retention Time (Rt) 1 13.360 142164 2 13.368 141454 SD RSD (%) 3 13.359 143378 835.99 0.588 4 13.044 142120 5 13.150 141237 Analysis of glabridin in licorice extract Table 2. Glabridin Analysis Results in sample A N o Weight s (mg) Concen -tration (ppm) Conten t (mg/g) Averag e (mg/g) RSD(% ) 1 100.2 10020 5813 2 0.2007 2 100.5 10050 6148 1 0.2117 3 100.4 10040 5923 3 0.2042 0.2062 0.4779 Figure 4.Chromatograms of glabridin standard (5 ppm) (Rt = 13,086 minutes) 4 100.7 10070 5 100.2 10030 6104 1 5926 8 0.2098 0.2047 Figure 5.Chromatograms of liquorice extracts (Rt = 13,062) Green Chemistry Section 4: Organic Chemistry,Faridah, et al. P a g e 291
ISBN 978-602-285-049-6 Proceedings of The 9 th Joint Conference on Chemistry Table 3. Glabridin Analysis Results in sample B No Weights (mg) Concentration (ppm) Content (mg/g) Average (mg/g) RSD(%) 1 100.4 10040 131173 0.4522 2 100.6 10060 134193 0.4617 3 100.7 10070 135002 0.4640 0.4578 0.5006 4 100.4 10040 132530 0.4569 5 100.3 10030 131569 0.4540 Table 4. Recovery test results No Weights(mg) Glabridin(mg/g) Concentration St Sample Sum St Sum Recovery(%) Average(%) RSD (%) 1 50.45 2.309 4.809 139622 90.144 2 51.03 2.5 2.336 4.836 76503 139307 88.6535 3 50.82 2.326 4.826 139852 89.7659 0.4578 4 50.63 2.318 7.318 199665 90.2453 5 50.15 5 2.396 7.296 146162 198382 89.8075 6 50.74 2.323 7.323 199845 90.2684 89.84 0.6084 Linearity Test Linearity test is used to see the relationship between the concentration of the standard solution of glabridin and peak areas. in this test glabridin standard solution was added to a certain amount of extract. If the correlation coefficient (r) approaches ± 1, it means that there is a linear relationship between the concentration of glabridin standard solution added to the extract and a broad peak. The results of the linearity test is a regression line with an equation as follow: y = 6083,7995+ 29229,4900 (x) withr = 0.9996 Limits of detection and quantitation Test The result of the limits detection and quantitation calculated using data obtained from the linearity test are 0,0388 and 0.1176, respectively. Recovery test Recovery test results are shown in Table 6.The average recovery obtained was 89.84 % with RSD of 0.6084 %. This data shows that the high performance liquid chromatography method used is accurate and a thorough method, in which this can be shown by the results of the recovery that meets the requirements of analytical method validation, i.e. 80-110 %. Large range of values is due to the amount of analyte in the sample was in a low concentration, so that the error factor will be greater because it takes too sensitive instrument to detect analytes. from the obtained results HPLC is an excellent method because it provides accuracy, precision and good linearity. Conclusion HPLC with 72 % methanol as mobile phase, C18 as stationary phase,flow rate of 1 ml/min using a UV-Vis detector at 281 nm is an acceptable method for analysis of glabridin in the ethanol extract of liquorice. The content of glabridin in licorice extract in sample B is higherthan thatin sample A using HPLC method. Acknowledgement This research was fund by Indonesian Government in Hibah Bersaing. References 1. Mingle, T, Youn,Y H, and Kyung, R. Simultaneous Extractionand Separation of Liquiritin, Glycyrrhizic Acid, and Glabridin from Licorice Root with Analytical and Preparative Chromatography. Journal of Biotechnology and Bioprocess Enginering; Korea, Depertment of Chemical Engineering Inha University; 2006. 2. The United States Pharmacopeia Commiion. The United Stated Pharmacopeia Vol.2. The United States Pharmacopeial Convention; 2009,p.1741-4 3. Republuk Indonesian Ministry of Health. Indonesian Pharmacopoeia. Edition IV. Jakarta: 292 P a g e Green Chemistry Section 4: Organic Chemistry, Faridah, et al.
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