|Year : 2016 | Volume
| Issue : 1 | Page : 59-62
Simultaneous determination of 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid in Glycyrrhiza glabra root by reversed phase high-performance liquid chromatography
Ambika Chamoli, Makhmur Ahmad, Mojeer Hasan, Bibhu Prasad Panda
Faculty of Pharmacy, Centre for Advanced Research in Pharmaceutical Sciences, Microbial and Pharmaceutical Biotechnology Laboratory, Jamia Hamdard, New Delhi, India
|Date of Web Publication||13-Apr-2016|
Bibhu Prasad Panda
Faculty of Pharmacy, Centre for Advanced Research in Pharmaceutical Sciences, Microbial and Pharmaceutical Biotechnology Laboratory, Jamia Hamdard, New Delhi - 110 062
Source of Support: None, Conflict of Interest: None
Background: The aim of the present research work is to develop a high-performance liquid chromatography (HPLC) method for simultaneous analysis of 18α-glycyrrhetinic acid (18α-GA) and 18β-GA (18β-GA) of Glycyrrhiza glabra. Materials and Methods: About 20 μL aliquots of each 18α-GA and 18β-GA were analyzed using reversed-phase C-18 column. The mobile phase was acetonitrile:tetrahydrofuran:water (10:80:10, v/v/v). The run time was 10 min at flow rate of 1 ml/min. Ultraviolet detection was carried out at 254 nm. Results: 18α-GA and 18β-GA were well resolved in reversed phase C-18 column using mobile phase acetonitrile: tetrahydrofuran: water (10:80:10, v/v/v, pH 7.9). The Rtof 18α-GA and 18β-GA was detected at 2.091 and 2.377 min, respectively. Conclusion: The developed chromatography method could be extended for potential quantification or simultaneous determination of these markers in plant as well as in herbal formulation.
Keywords: 18μ-glycyrrhetinic acid, 18β-glycyrrhetinic acid, Glycyrrhiza glabra, high-performance liquid chromatography
|How to cite this article:|
Chamoli A, Ahmad M, Hasan M, Panda BP. Simultaneous determination of 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid in Glycyrrhiza glabra root by reversed phase high-performance liquid chromatography. Drug Dev Ther 2016;7:59-62
|How to cite this URL:|
Chamoli A, Ahmad M, Hasan M, Panda BP. Simultaneous determination of 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid in Glycyrrhiza glabra root by reversed phase high-performance liquid chromatography. Drug Dev Ther [serial online] 2016 [cited 2017 Sep 19];7:59-62. Available from: http://www.ddtjournal.org/text.asp?2016/7/1/59/180168
| Introduction|| |
Glycyrrhiza glabra is very well-known medicinal plants of diverse pharmacological applications. The most important phytoconstituent present is glycone glycyrrhizin (GL). On acid, alkali or enzymatic hydrolysis, GL gives two molecules of glucuronic acid and one molecule of glycyrrhetinic acid (GA). The GL and GA are the most used liquorice saponins in pharmaceuticals and cosmeceuticals owing to their biological properties such as anti-inflammatory, anti-allergic, and anti-ulcerative activity. Its tumor protective activity has also been reported. Due to structural similarity to steroids, their mineralocorticoid-like effects and inhibition of metabolic enzymes for adrenocorticosteroids have been reported. Naturally occurring GL is β-isomer. The β isomers of GL (18β-GA, 18β-GL) and GA can be isomerized to their α-isomers (18α-GA, 18α-GL) under alkaline conditions. Since 18α-GA and 18β-GA showed different biological activities and physicochemical properties, their different stereochemistry is important for biological properties. Therefore, an accurate, simple, and efficient method is required to assess the optical purity of GA.
In the present paper, an accurate and precise reversed-phase high-performance liquid chromatography (RP-HPLC) method has been described for simultaneous determination of 18α-GA and 18β-GA present in biotransformed G. glabra extract.
| Materials and Methods|| |
Plant materials, microorganism, and chemicals
G. glabra root was procured from Global Herbs, New Delhi, and authenticated in NISCAIR, New Delhi, India. The bacterial strain Escherichia coli MTCC 1652 was obtained from the Institute of Microbial Technology, Chandigarh, India. It was grown in nutrient agar slants and maintained at 4°C. Standard 18α-GA (18α-GA) and 18β-GA (18β-GA) were received from Sigma Aldrich, Mumbai, India. HPLC grade acetonitrile, tetrahydrofuran, and water were procured from Merck, Mumbai, India.
Preparation of Glycyrrhiza glabra root extract
G. glabra root extract was prepared by using Soxhlet apparatus. Air-dried root of G. glabra (25 g) was coarsely powdered and extracted with water (100%) for 72 h. The extract was concentrated under reduced pressure in rotary evaporator at 40°C.
Biotransformation of glycone glycyrrhizin to 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid by Escherichia coli
To 50 mL of synthetic media, 5.0 mL of bacterial seed culture was inoculated and fermented at 37°C, 150 rpm for 24 h.
Extraction and analysis of glycone glycyrrhizin, 18α-glycyrrhetinic acid, and 18β-glycyrrhetinic acid
The fermented broth was subjected to sonication for 10 min. The disrupted cell was then centrifuged at 5000 rpm for 10 min. The supernatant was filtered, and the amount of 18α-GA and 18β-GA formed were analyzed.
High-performance liquid chromatography instrumentation and conditions
Chromatographic separation was achieved using analyticals technologies HPLC system (E67 and E68, Ravi Park, Vasna Road, Vadodara, Gujarat 390015) equipped with Model P3000A pump. The eluates were monitored by ultraviolet (UV) detector (Model UV3000) at 254 nm. Reversed phase C-18 column (LiChroCART ® 250) was purchased from Merck, Mumbai, India.
A volume of 20 µL aliquots of each 18α-GA and 18β-GA were prepared and analyzed. The analysis was carried out in mobile phase used acetonitrile:tetrahydrofuran: Water (10:80:10, v/v/v). The total run time was 10 min at flow rate of 1.0 ml/min and UV detection was carried out at 254 nm.
The method was validated for following parameters: Linearity, limit of quantitation (LOQ), limit of detection (LOD), and system suitability.
Following equations were used for calculating LOD and LOQ:
LOD = (3 × SD/slope) and
LOQ = (10 × SD/slope)
Linearity test solution for assay method was prepared from stock solution at different concentrations, and 25 µg/ml of each solution was analyzed and peak area of chromatograms was noted.
The interday precision of assay method was evaluated at four concentration (25, 50, 75, and 100 µg/ml) (n = 3). The interday precision was performed on three different, i.e., day 1st, day 2nd, and day 3rd at four different concentration (25, 50, 75, and 100 µg/ml) (n = 3). The relative standard deviation (%RSD) of the obtained assay values at four different concentrations was calculated.
The LOQ and LOD were based on standard deviation of response and slope of the constructed calibration curve (n = 3).
| Results|| |
Chromatographic separation of 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid
18α-GA and 18β-GA were well resolved in RP C-18 column. The mobile phase was different solvents with different concentrations to get sharp and well-separated peaks optimized. The different mobile phases were A (methanol: water 85:15, v/v), B (acetonitrile:methanol:water 25:15:60, v/v/v), C (acetonitrile:water 30:70, v/v), D (acetonitrile:tetrahydrofuran 10:90, v/v), and E (acetonitrile: tetrahydrofuran:water 10: 80: 10, v/v/v) were tested to get sharp chromatograms. When mobile phases A, B, C, D, and E at different flow rate (0.5 ml/min, 1.0 ml/min, and 1.5 ml/min) and different run time (10 min, 20 min, and 30 min) were run, the chromatograms obtained were overlapped at different Rt. However, sharp chromatograms were only obtained in mobile phase (E) containing acetonitrile: Tetrahydrofuran: water (10:80:10, v/v/v, pH 7.9) when the flow rate was maintained at 1.0 ml/min with total run time of 10 min. The resolution of both the molecules was strongly affected by pH of the mobile phase. At lower pH (4.5), the chromatograms were overlapped at 4.9 min of run time. When pH was increased to 7.2, decrease in retention time was observed and well-resolved chromatograms were detected. The Rt of 18α-GA and 18β-GA was detected at 2.091 and 2.377 min, respectively [Figure 1]. The biotransformation of GL to 18α-GA and 18β-GA by E. coli was studied and found that the sonicated E. coli has produced a maximum concentration of 18α-GA (705.985 µg/ml) and 18β-GA (133.036 µg/ml) since β-glucuronidase present in E. coli is an intracellular enzyme.
|Figure 1: High-performance liquid chromatography chromatogram of standard 18α-glycyrrhetinic acid (a), 18β-glycyrrhetinic acid (b), and their mixture (c)|
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The calibration curve was linear over the concentration range of 25–100 µg/ml [Table 1] and regression equation was found to be y = 2577.5x + 2,000,000 with correlation coefficient of 0.9923 for 18α-GA and y = 31779x + 605206 with correlation coefficient of 0.9944 for 18β-GA [Figure 2].
|Table 1: Linearity of 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid|
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|Figure 2: Regression plot of peak area against concentration of standard 18α-GA (a) and 18β-GA (b)|
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The RSD was found to be 0.001–0.447 (intraday) and 0.001–0.004 (interday) for 18α-GA [Table 2] and 0.001–0.006 (intraday) and 0.002–0.004 (interday) for 18β-GA [Table 3]. The LOQ and LOD for 18α-GA were found to be 0.052 and 0.015 µg/ml, respectively. The LOQ and LOD for 18β-GA were found to be 0.377 and 0.113 μg/ml, respectively. The proposed method was applied for the determination of 18α-GA and 18β-GA content in the fermented broth. RSD (%) of 18α-GA and 18β-GA was found to be 0.268 and 0.080 µg/ml, respectively. The LOQ and LOD for 18α-GA were calculated as 1.112 and 0.333 µg/ml, respectively. The LOQ and LOD for 18β-GA were 0.456 and 0.136 µg/ml, respectively.
|Table 2: Intra- and inter-day precision studies of 18α-glycyrrhetinic acid|
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|Table 3: Intra- and inter-day precision studies of 18β-glycyrrhetinic acid|
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| Discussion|| |
The present development of RP-HPLC method could be used as potential quantification method for the simultaneous determination of plant terpenoids isomers. These isomers are generally well resolved in the specific chiral column. Moreover, the present HPLC method was developed in RP C-18 column. Hence, this may reduce the cost and economy of the process.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]