|Year : 2016 | Volume
| Issue : 1 | Page : 69-72
Screening and bioconversion of glycyrrhizin of Glycyrrhiza glabra root extract to 18β-glycyrrhetinic acid by different microbial strains
Makhmur Ahmad1, Jalaluddin2, Mohammad Ali2, Bibhu Prasad Panda2
1 Microbial and Pharmaceutical Biotechnology Laboratory, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India; Department of Pharmaceutics, Buraydah College of Pharmacy and Dentistry, Buraydah, Al-Qassim, Saudi Arabia
2 Microbial and Pharmaceutical Biotechnology Laboratory, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India
|Date of Web Publication||13-Apr-2016|
Bibhu Prasad Panda
Faculty of Pharmacy, Microbial and Pharmaceutical Biotechnology Laboratory, Jamia Hamdard, New Delhi - 110 062
Source of Support: None, Conflict of Interest: None
Objective: The objective of the present study is to perform screening of different microorganisms (7 bacteria and 14 fungi) for conversion of glycyrrhizin (GL) to 18β-glycyrrhetinic acid (GA). Penicillium chrysogenum produced the highest concentration of β-glucuronidase enzyme (61 U/mL) and produced GA of 52 μg/mL while E. coli produced the highest β-glucuronidase of 376 U/mL with GA concentration of 2.1 μg/mL. Materials and Methods: Submerged and solid state biotransformation of GL was carried out. To 9.0 mL of bacterial supernatant, 1.0 mL 0.2% w/v of aqueous Glycyrrhiza glabra root extract was added and incubated at 37°C for 24 h. β-glucuronidase activity was measured and high-performance liquid chromatography analysis was carried out. Results and Discussion: Induced-Escherichia coli produces 2.1 μg/mL of GA with an enzyme activity of 376 U/mL which shows that the enzyme has a potential biotransformation capability. Rhizopus oryzae and P. chrysogenum have the potential ability to biotransform GL to GA with 2.6 μg/mL and 61 μg/mL of GA with enzyme activity of 569 U/mL and 61 U/mL, respectively. Conclusions: G. glabra roots containing GL can be hydrolyzed by microbial β-glucuronidase enzyme under sub-merged fermentation (SmF). β-glucuronidase, an enzyme of E. coli, was found to be the best microbial source of enzyme which biocatalyzed the reaction than fungal strain under SmF.
Keywords: 18β-glycyrrhetinic acid, β-glucuronidase, biotransformation, screening
|How to cite this article:|
Ahmad M, Jalaluddin, Ali M, Panda BP. Screening and bioconversion of glycyrrhizin of Glycyrrhiza glabra root extract to 18β-glycyrrhetinic acid by different microbial strains. Drug Dev Ther 2016;7:69-72
|How to cite this URL:|
Ahmad M, Jalaluddin, Ali M, Panda BP. Screening and bioconversion of glycyrrhizin of Glycyrrhiza glabra root extract to 18β-glycyrrhetinic acid by different microbial strains. Drug Dev Ther [serial online] 2016 [cited 2017 May 27];7:69-72. Available from: http://www.ddtjournal.org/text.asp?2016/7/1/69/180170
| Introduction|| |
Glycyrrhiza glabra root, commonly known as sweet root, is widely used in the Indian traditional system of medicine. The active component of G. glabra root is glycyrrhizin (GL), and it is composed of one molecule of 18β-glycyrrhetinic acid (GA) and two molecules of glucuronic acid. It inhibits cyclooxygenase activity and prostaglandin formation, all factors in the inflammatory process by inhibiting phospholipase A2 activity., GL is potentially valuable for HIV therapy., GA is absorbed into the systemic circulation and produces pharmacological action. Further, it is metabolized to 3 β-D-(monoglucuronyl) 18β-GA in the liver and excreted out from the body in the urine. The biological activity of GA is 20 times more than GL. High concentration of GL or GA in serum resulted severe adverse reaction. Therefore, scientists are now trying to produce GA from GL by biocatalysis and by bioprocess under in vitro means.
The objective of this study was to investigate the concentration of GL and GA present in fermented G. glabra root extract by high-performance liquid chromatography method.
| Materials and Methods|| |
Plant material, microbes, and chemicals
The roots of G. glabra were collected from the Global Herbs, New Delhi, and authenticated. Different bacterial strains and fungal strains were isolated and identified. All the chemicals, reagents, and microbiological medium were obtained from Hi-media, Mumbai, India.
Biotransformation of glycyrrhizin to glycyrrhetinic acid
Submerged biotransformation of GL was carried out in 250 mL Erlenmeyer flasks. Nutrient broth was prepared, and pH was adjusted to 7.2. A loop full of different bacterial cultures was inoculated and incubated for 2 days at 37°C at 170 rpm. Bacterial broth was filtered, pH was adjusted to seven, and centrifuged at 3000 rpm. To 9.0 mL of supernatant, 1.0 mL 0.2% w/v of aqueous G. glabra extract was added and incubated at 37°C for 24 h. The β-glucuronidase activity was analyzed at 0 h. The reaction mixture was analyzed for bio-converted product (GA) during and after biotransformation reaction.
Beta-glucuronidase enzyme assay
The β-glucuronidase activity was measured at 37°C by mixing cultured broth with 3 mM phenolphthalein-β-d-glucuronic acid buffered with 75 mM potassium phosphate buffer with 1.0% bovine serum albumin at a pH of 6.8.
Extraction of glycyrrhetinic acid and glycyrrhizin
The extraction of GL and GA from fermented broth was carried out under biphasic condition. Methanol was used for extraction of GL and GA from the fermented mass.
Chromatographic condition and analysis of glycyrrhetinic acid and glycyrrhizin
The chromatography was carried out by RP C18 column, the mobile phase consists of methanol:water (85:15, v/v) at a flow rate of 1.0 mL/min with run time of 10 min. Ultraviolet detection was carried out at 254 nm.
| Results and Discussion|| |
Isolation of bacterial and fungal strain
Different bacterial stains were isolated and identified as Bacillus subtilis, Bacillus megaterium, Lactobacillus acidophilus, and Escherichia coli. All the bacterial strains were tested for bioconversion of GL to GA under submerged fermentation (SmF). Among fungal strains, Penicillium chrysogenum MPBL1 and Rhizopus oryzae MPBL2 were identified. The chromatograms of standard GL and GA are shown in [Figure 1],[Figure 2],[Figure 3].
|Figure 1: High-performance liquid chromatography chromatograms of standard glycyrrhizin (75 μg/mL) and standard glycyrrhetinic acid (75 μg/mL) and their mixture (75 μg/mL)|
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|Figure 2: High-performance liquid chromatography chromatograms of produced glycyrrhetinic acid in different bacterial (Bacillus subtilis, unidentified B2, unidentified B3, unidentified B4, Lactobacillus acidophilus, Bacillus megaterium, and Escherichia coli) fermented broth|
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|Figure 3: High-performance liquid chromatography chromatograms of produced glycyrrhetinic acid in different fungal (unidentified F1, unidentified F2, unidentified F3, Rhizopus oryzae, unidentified F5, unidentified F6, unidentified F7, unidentified F8, unidentified F9, unidentified F10, unidentified F11, unidentified F13, Penicillium Chrysogenum, and unidentified F14) fermented broth|
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Bioconversion of glycyrrhizin to glycyrrhetinic acid with enzyme activity under submerged fermentation
E. coli produces 2.1 μg/mL of GA with an enzyme activity of 376 U/mL [Table 1]. The chromatograms of all the bacterial fermented samples were shown in [Figure 1]. Higher enzyme activity was observed without the presence of inducer for bacteria as well as fungi. This may be due to the presence of water extract of drug as it also has antimicrobial action, which inhibits the growth of microorganism and inhibits the production of enzyme. Enzyme activity of bacteria was much more than fungal strain under SmF due to higher water activity. Some bacterial strains were found to have good enzyme activity but were not capable of biotransforming GL to GA, which may be due to bioconversion of GL into unknown molecules or lack of selectivity of bond.
|Table 1: Concentration of glycyrrhizin, glycyrrhetinic acid, and enzyme activity under submerged biotransformation process by different bacterial strains|
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R. oryzae and P. chrysogenum have higher capability to biotransform GL to GA. The concentration of 2.6 μg/mL and 61 μg/mL of GA with enzyme activity of 569 U/mL and 61 U/mL was produced by R. oryzae and P. chrysogenum, respectively [Table 2]. The chromatograms of all the bacterial fermented samples were shown in [Figure 2]. Some fungal strains were found to have good enzyme activity, but they do not biotransform GL to GA. Moreover, biotransformation under submerged condition showed that bacterial strains were having higher capability to biotransform GL to GA than fungal strains, since bacterial cells grow faster in the presence of large volume of water, but fungal cells require solid support to grow.
|Table 2: Concentration of glycyrrhizin, glycyrrhetinic acid, and enzyme activity under submerged biotransformation by different fungal strains|
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| Conclusion|| |
Twenty-one microbial strains (bacterial and fungal) were screened for biotransforming GL to GA in G. glabra root. E. coli was found to be the best source of enzyme (β-glucuronidase) for biotransformation among all bacterial strains. It has been found that bacterial strains produce higher amount of β-glucuronidase enzyme than fungal strain during SmF.
The authors are highly thankful to Bioactive Natural Product Laboratory, Jamia Hamdard, New Delhi, for their kind support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Farag MA, Porzel A, Wessjohann LA. Comparative metabolite profiling and fingerprinting of medicinal licorice roots using a multiplex approach of GC-MS, LC-MS and 1D NMR techniques. Phytochemistry 2012;76:60-72.
Hennell JR, Lee S, Khoo CS, Gray MJ, Bensoussan A. The determination of glycyrrhizic acid in Glycyrrhiza uralensis
Fisch. ex DC. (Zhi Gan Cao) root and the dried aqueous extract by LC-DAD. J Pharm Biomed Anal 2008;47:494-500.
Ohuchi K, Tsurufuji A. A study of the anti-inflammatory mechanism of glycyrrhizin. Mino Med Rev 1982;27:188-93.
Okimasu E, Moromizato Y, Watanabe S, Sasaki J, Shiraishi N, Morimoto YM, et al.
Inhibition of phospholipase A2 and platelet aggregation by glycyrrhizin, an antiinflammation drug. Acta Med Okayama 1983;37:385-91.
Numazaki K, Umetsu M, Chiba S. Effect of glycyrrhizin in children with liver dysfunction associated with cytomegalovirus infection. Tohoku J Exp Med 1994;172:147-53.
Shabani L, Ehsanpour AA, Asghari G, Emami J. Glycyrrhizin production by in-vitro
cultured Glycyrrhiza glabra
elicited by methyl jasmonate and salicylic acid. Russ J Plant Physiol 2009;56:621-6.
Kerstens MN, Guillaume CP, Wolthers BG, Dullaart RP. Gas chromatographic-mass spectrometric analysis of urinary glycyrrhetinic acid: An aid in diagnosing liquorice abuse. J Intern Med 1999;246:539-47.
Lu DQ, Hui L, Yan D, Ping-Kai O. Biocatalytic properties of a novel crude glycyrrhizin hydrolase from the liver of the domestic duck. J Mol Catal B Enzym 2006;43:148-52.
Morana A, Lazzaro AD, Lerina ID, Ponzone C, Rosa MD. Enzymatic conversion of 18-β-glycyrrhetinic acid from Glycyrrhiza glabra
L. Biotechnol Lett 2002;24:1907-11.
Combie J, Blake JW, Nugent TE, Tobin T. Morphine glucuronide hydrolysis: Superiority of beta-glucuronidase from patella vulgata. Clin Chem 1982;28:83-6.
Siracusa L, Saija A, Cristani M, Cimino F, D'Arrigo M, Trombetta D, et al.
Phytocomplexes from liquorice (Glycyrrhiza glabra
L.) leaves – chemical characterization and evaluation of their antioxidant, anti-genotoxic and anti-inflammatory activity. Fitoterapia 2011;82:546-56.
Wang J, Sun Q, Gao P, Wang JF, Xu C. Bioconversion of glycyrrhizinic acid in liquorice into 18β-glycyrrhetinic acid by Aspergillus parasiticus
speare BGB. Appl Biochem Microbiol 2010;46:421-66.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]