INTRODUCTION

Glycyrrhiza glabra Linn. of the family Leguminosae, is a genus of perennial herbs and under shrubs is distributed in the subtropical and warm temperate regions of the world, chiefly in the Mediterranean and certain areas of

ARTICLE INFORMATION:

*Corresponding Author

Received 14th May, 2014

Accepted after revision 26th June, 2014 BBRC Print ISSN: 0974-6455 Online ISSN: 2321-4007

©A Society of Science and Nature Publication, 2014. All rights reserved.

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Asia. Licorice, the dried root of G. glabra, is widely used as flavoring and sweetening agent, but has also been proposed for various clinical applications, (Fiore et al., 2005). Traditions from ancient Assyrian, Egyptian, Chi- nese and Indian cultures have documented its extensive medicinal use as demulcent, expectorant and in ulcer

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Nargis Khan and Sharique A. Ali

healing (Armanini et al., 2002). Pharmacological effects of licorice including inhibition of gastric acid secretion, anti-inflammatory, antiviral and anti-atherogenic prop- erties have been well verified, (Fiore et al., 2008; Tanaka et al., 2008).

Pharmacological investigations have concluded that, flavonoides are the main bioactive components of lico- rice and have antioxidant and antibacterial bioactivi- ties, (Vaya et al., 1997; Sharma et al., 2013). Among them, the remarkable one such as Isoliquiritigenin (ISL) has been reported to carry strong biological activity. ISL is a flavonoid contained in licorice with a simple chal- cone structure (4,2’,4’-trihydroxychalcone) (Fig. 1). ISL has been shown to exert various actions including vaso- relaxant, antioxidant, neuroprotective, anti-platelet, anti-tumor, angiogenic, anti-allergic, and antispasmodic (on intestine) effects, and has estrogenic properties (Ii et al., 2004; Chin et al., 2007; Ye et al., 2009; Kang et al., 2010; Lee et al., 2012).

In addition, it has been reported that ISL can inhibit the activity of tyrosinase, a key enzyme of melanin bio- synthesis (Nerya et al. 2003). However, there has been no comprehensive study on its melanolytic activity on mammalian melanocytes .With the growing of its poten- tial pharmaceutical role, there is an increasing demand for analyzing ISL in plant extract and evaluating its melanolytic behaviour for a better understanding of the mechanism of its action and facilitating further research.

The separation and purification of ISL from licorice by conventional methods such as column chromatography and high-performance liquid chromatography (HPLC) is tedious, time consuming and usually requires mul- tiple chromatography steps (Cheng et al., 2005). Over the past few years, the applications of liquid chroma- tography coupled, with mass spectrometry (LC–MS) in natural product analysis have been dramatically grow- ing because of the increasingly improved separation and detection capabilities of HRLC–MS instruments.

Compared to other conventional detection methods, HRLC-MS can not only allow accurate determination of chemical structure of natural compounds with known and unknown structures, but also offers excellent sen- sitivity and attain high-quality data within minimum acquisition time. In particular, these analytical tech- niques greatly aid unequivocal identification and highly sensitive quantification of natural products at trace con- centrations in complex matrices (Ho et al., 2003; Sau- vage et al., 2006).

The present report deals with the identification, quan- tification and structure analysis of ISL from the root extract of G. glabra for the first time, which is respon- sible for anti browning activities and also to seek new tyrosinase inhibitors for application as whitening agents in cosmetic industry.

MATERIAL AND METHODS

Analytical grade methanol and ethanol were obtained from Qualigens Fine Chemicals, Mumbai, India. Ace- tonitrile (HPLC grade) was purchased from Sigma Aldrich (St. 21 Louis, MO, USA). ISL standard was procured from Alfa Aesar (USA). Licorice, the root of G. glabra, was pur- chased from local market of Bhopal. The plant materials were identified and authenticated by Dr. S.S. Khan of Botany Department, Saifia College, Bhopal. A voucher specimen (287/Bot/Saifia/11) is deposited at herbarium of Department of Botany, Saifia College, Bhopal.

EXTRACTION PROCEDURE AND SAMPLE PREPARATION FOR LC-MS ANALYSES

Extract was prepared according to the method of Cheng et al., (2005) with slight modification, where the roots of G. glabra were dried at 60°C and then pulverized. Five hundred grams of sample were put into a 3000 ml flask, to which 1200 ml 90% ethanol was added. After soaking extraction at room temperature for 24 h, the extraction procedure was repeated twice (1200 ml of 95% etha- nol each time). All the filtrates were combined, filtered and evaporated to brown syrup ointment under reduced pressure. The ointment was then dissolved in 500 ml hot water, and extracted with ether (3×300 ml). The ether extracts were combined and evaporated to dryness under reduced pressure, which yielded 15 g of crude fla- vonoid extracts. Obtained dry residue was resuspended in 1 ml methanol. 2 μL were used for LC–MS analysis.

Standard stock solution of ISL was prepared in meth- anol. Appropriate amounts of standard were dissolved in volumetric flasks to obtain a stock solution with a con- centration of 1 mg mL-1. Working solution was prepared by diluting the stock solution with methanol to different concentration.

HRLC-MS INSTRUMENT AND CONDITIONS

Chromatographic separations were performed on an Acquity UPLC system (Waters) equipped with a HSS T3 column18 (100 × 1.0 mm, particle size 1.8 μm; Waters); applying the following gradient at a flow rate of 0.5 mL min-1; 0-0.5 min, 10% A (acetinitrile) and 90% B (water); 1-3 min linear from 10 to 90% A; 3.5-5.5 min, isocratic 10% A. The injection volume was 3.1 μL (full loop injec- tion). The thermo stated auto sampler was kept at 4ºC.

Eluted compound was detected from m/z 50 to 3000 using a Micro-TOF-Q hybrid quadrupole time-of-flight mass spectrometer (Bruker micrOTOF-Q-II Daltonics) equipped with an Apollo II electrospray ion source in positive ion modes using the following instrument set- tings: nebulizer gas: nitrogen; drying gas flow: 7.0 L/min;

nebulizer pressure: 1.2 bar; capillary voltage: 4500 V; end

plate offset: -500 V; collision cell RF: 200 Vpp. drying gas

temperature: 200 ºC. System control and data acquisition were controlled by Bruker Compass Data Analysis 4.0

RESULTS AND DISCUSSION

High performance liquid chromatography (HPLC), High- speed counter-current chromatography (HSCCC), liquid chromatography-mass spectrometry (LC–MS) techniques were reported for qualitative and quantitative analyses of

Nargis Khan and Sharique A. Ali

ISL in licorice and rat plasma (Zhang and Ye, 2009; Tan- aka et al., 2010; Montoro et al., 2011). However there has been no report on accurate quantification of ISL from lico- rice extract using high resolution liquid chromatography - mass spectrometry (HRLC-MS). Licorice saponins and fla- vonoids are relatively polar compounds with carboxyl or phenol groups in the molecules, and thus could be readily ionized in the electrospray ionization (ESI) source.

The present method used slightly different mobile phases and a steeper gradient of water and acetonitrile. This allowed for the elution of all analytes within 6 min.

simultaneously acquired LC–MS total ion chromato- grams, UV characteristics and extracted ion chromato- gram of G. glabra extract and standard ISL are presented in Fig. 2. The UPLC analyses of the root extract from G. glabra shows several compounds where the purity ISL was 42% based on UPLC peak area percentage. ased on the UPLC analysis, a total amount of 106.25 μg mg-1 of ISL was quantified.

The average recovery of ISL at two different levels was found to be 99.49%.This method was found superior in linearity, recovery, and sensitivity compared to other chromatographic methods (Zhang et al., 2013; Wang and Yang, 2007) for quantitative estimation of ISL in the herbal extraction of G. glabra.

The identities, retention times, and observed molecu- lar and protonated ions for individual components are presented in fig. 3. Structure and molecular formula were confirmed by comparing retention time, UV/Vis spectra and MS data (accurate mass, isotopic distribu- tion in positive ion mode) of the compound detected. In the present study we found mass accuracy of our plant extract compared to standard ISL, matching with theoretical composition of C15H12O4. The total ion chro- matogram and computer-reconstructed selected ion chromatograms for the positive ion electrospray LC-MS was analysed. The exact mass of our plant extract ([M- H]+ m/z 257.0810) was identical to that of ISL ([M-H]+ m/z 257.0806), matching the theoretical composition of C15H12O4 (([M-H]+ m/z 257.0808). The positive ion elec-

trospray product ion mass spectra of sample and ISL are shown in Fig. 3 and are also indistinguishable. The UV absorbance spectra of sample and ISL were recorded during LC-UV-MS and showed absorbance maxima of 272 nm. The overall results of the present study indicate that H LC-MS is a powerful technique in separating and purifying ISL from herbal extracts of G. glabra, a possi- ble candidate for treatment of skin hyper pigmentation.

CONCLUSION

Despite the widespread medicinal and culinary uses of G. glabra, the levels of most phytoconstituents in com- mercial extracts are not all standardized and several specific compounds responsible for its pharmacological properties are not yet fully elucidated. The present study provides an unbiased multiplex approach, combining UPLC and high resolution MS techniques to reveal the characterization and quantification with unbiased mass accuracy of ISL in root extract of G. glabra coupled with multivariate data analyses.

ACKNOWLEDGEMENTS

The authors are grateful to the Secretary and Principal of Saifia College, Bhopal for rendering their support and help for the completion of this work. NK also wishes

to thank UGC New Delhi for the award of Maulana Azad National Fellowship (Award Letter Number: F1-17.1/2012-13/MANF-MUS-MAD-14855).

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