Antioxidant and Antiinflammatory Activities of Licorice Root - American

Chapter 20

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 13, 2014 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch020

Antioxidant and Antiinflammatory Activities of Licorice Root (Glycyrrhiza uralensis): Aroma Extract Aki Tanaka and Takayuki Shibamoto Department of Environmental Toxicology, University of California, Davis, CA 95616

The volatile fraction obtained from a steam distillate of Licorice was examined for antioxidant activity by a hexanal/hexanoic acid assay. Antioxidant activity of the residual aqueous fraction was also tested by a malonadehyde/gas chromatography (MA/GC) assay. The volatile fraction inhibited hexanal oxidation by 99% over 40 days at a level of 50 μg/mL. The GC/MS analysis of the volatilefractionyielded hexanol (52.75 mg/mL) and hexanal (31.75 mg/mL) as major components. The volatile fraction exhibited strong anti-inflammatory activity when assayed by ELSA using lipoxygenase. The steam distillate also showed strong dose dependent anti-inflammatory activity. However, the aqueous solution with the volatiles removed did not show any anti-inflammatory activity.

© 2008 American Chemical Society

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Many traditional Chinese herbs, including licorice root, have been used to treat various diseases since the beginning of recorded history. Today, over seven thousand herbs have been employed by 80% of the world's population (/, 2). Licorice root, specifically the Glycyrrhiza species of the Fabaceae family (5), has been one of the most important medicinal herbs used in China, and one that has been in use there for over 6,000 years (4). Licorice root is one of the richest sources of biologically active compounds (5). For example, some flavonoids isolated from licorice root extract have been shown to exhibit strong antioxidant activity toward lard oxidation (6). Glycyrrhizin and glycyrrhetinic acid, which are the main components of licorice root, have been clinically used in the treatment of hyperlipemia, artherosclerosis, and allergic inflammation (2). Previous studies have focused on the non-volatile licorice root constituents, such as flavonoids with sugar moieties. However, plants also contain numerous aroma chemicals, which have been widely used in both folk medicine and aroma therapies (7), suggesting that they have some beneficial health effects in addition to their pleasant odor. Aroma chemicals are highly-volatile low-molecularweight compounds and have been isolated and identified in natural plants. Aroma chemicals have been studied mainly from the aspects of flavor and fragrance chemistry. However, medicinal activities of aroma chemicals have been discovered lately. For example, the antioxidant activities of aroma extracts obtained from spices and herbs have been reported (8), Essential oils, which are comprised mainly of aroma chemicals and which are isolated from medicinal plants such as chamomile, clove, and eucalyptus, have anti-microbial and antioxidant properties (9, 10). They have also been widely used for aromatherapy (//). Essential oils are prepared from natural plants, herbs, by steam distillation followed by solvent extraction. In the present study, aroma extract and residual aqueous solution isolated from licorice root by a water distillation were examined for antioxidant and anti-inflammatory activities to investigate the possible additional benefits of licorice consumption.

Materials and Methods Chemicals and Materials cc-Tocopherol was bought from Sigma Chemical Co. (St. Louis. MO). Glycyrrhizin was purchased from Wako Pure Chemical Industries (Osaka, Japan). Buffer solutions (pH 4.00, 7.00, and 10.00), dimethyl sulfoxide (DMSO), and 10% trichloroacetic acid solution (TCA) werefromFisher Co. (Pittsburgh, PA). Licorice root (Glycyrrhiza uralensis) was collected and dried naturally by Dr. John F. Louie (Sacramento, CA) in Heilongjiang Province, Northwestern


231 China. Lipoxygenase inhibitor screening assay (LISA) kit was purchased from Cayman Chemical Co. (Ann Arbor, MI).


Sample Preparation for Volatile Extract and Aqueous Solution Licorice root (50 g) was mixed with 1 L deionized water in a 2 L two necked-flask and allowed to stand for 1 h. The water in the flask was boiled using a mantle heater and 500 mL of water distillate was collected in a beaker. The distillate was extracted with 125 mL of dichloromethane for 6 h using a liquid-liquid continuous extractor. The extract was dried over anhydrous sodium sulfate for 12 h. After removal of the sodium sulfate, the extract was condensed to approximately 3 mL in volume using a rotary evaporator. The extract was further condensed to exactly 0.2 mL under a purified nitrogen stream (aroma extract sample) and then stored at - 20 °C until used for further experiments. Residual aqueous solution (aqueous solution sample) in the extractor was transferred into a flask and stored at 5 °C until used for further experiments.

Antioxidant Test of Aroma extract by Aldehyde/Carboxylic Acid Assay Antioxidant activity of the aroma extract was tested using its inhibitory effect on the oxidation of hexanal to hexanoic acid (12). Various amounts of the extract were added to a 2 mL dichlomethane solution of hexanal (3 mg/mL) containing 0.2 mg/mL of undecane as a gas chromatographic (GC) internal standard. The oxidation of the sample solution was initiated by heating at 60 °C for 10 min in a sealed vial and then storing at room temperature. A blank sample was prepared following the same procedure without a test sample. Butylated hydroxytoluene (BHT) and a-tocopherol were used as a positive control. The amount of hexanal was measured after 45 days.

Fractionation of Aqueous Solution Sample A residual aqueous solution from the dichloromethane extraction was condensed to a paste (dark brown color) using a rotary evaporator. The paste sample was placed in a glass column (40 cm x 4.5 cm i.d.) packed with Amberlite XAD-2 resin (Aldrich Chemical Co., Milwaukee, WI). The sample was eluted sequentially with a 1 L each water/methanol solution—100/0 (Fraction I); 95/5 (Fraction II); 80/20 (Fraction III); 50/50 (Fraction IV); 20/80 (Fraction V); and 0:100 (Fraction VI); and a final fraction of 1 L of acetone (Fraction VII). The seven fractions obtained were condensed to 0.3 mL using first a rotary evaporator and subsequently a purified nitrogen stream to 0.1 mL.


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Anti-inflammatory Test of Aroma Extract and Fractions from Aqueous Solution Anti-inflammatory tests were conducted using the lipoxygenase inhibitor screening assay (LISA) kit (75, 14). The solutions provided commercially in the LISA Kit were 0.1 M Tris-HCl assay buffer (pH 7.4), developing agents 1 and 2 (chromogen), soybean-enzyme 15-lipoxygenase (15-LOX) standard, arachidonic acid, and KOH. An assay buffer was diluted tenfold with HPLC-grade water before use. Chromogen, which was used within 1 h, was prepared by mixing equal amounts of developing agents 1 and 2. A blank well was prepared by adding an assay buffer solution (100 fiL) to a well plate supplied from the LISA Kit. A positive control well was made by mixing a 10 \\L of 15-LOX solution and a 990 \iL of assay buffer. A substrate solution was prepared by mixing 25 juL of arachidonic acid and 25 \iL of KOH in ethanol. After the substrate solution was vortexed, it was diluted with 950 \xh of HPLC-grade water. The substrate solution was used within 30 min to prevent degradation. The solution prepared was stored at 0 °C until used. A 15-LOX solution (10 |iL), a testing sample (10 \iL), and assay buffer (980 |iL) were placed in the testing well. The reaction was initiated by adding a 10 |LXL of substrate solution to a positive control well and a testing sample well. All testing wells, were covered and placed on a shaker (Bellco Biotechnology, Vineland, NJ) for 5 min. Chromogen (100 |uL) was added to the reaction wells to stop the enzyme catalysis and prevent further development of the reaction. The level of hydroperoxide in the samples was measured using a microplate reader (Molecular Devices Corporation, Sunnyvale, CA). The entire assay was performed in duplicates. The three concentrations (62, 125, 250 ng/mL) of glycyrrhizin (standard anti-inflammatory chemical), the volatile extract, and the fractions from the aqueous solution were tested. Concentration levels of 31, 62, and 125 |ig/mL were used for Fractions I and II because their original paste sample was highly viscous. The volatile extract and glycyrrhizin were diluted with DMSO. Fractions I, II, and III were diluted with HPLC-grade water. Fractions IV, V, and VI were diluted with methanol. Fraction VII was diluted with acetone. Analysis of Glycyrrhizin in Fractions from Aqueous Sample Glycyrrhizin in the aqueous Fractions was analyzed by an Agilent 1100 model HPLC system equipped with a 150 mm x 4.6 mm i.d.) Alltima C-18 5 JLX column (Alltech, Deerfield, IL) and a multiple wavelength detector. Mobile phase A was 10 mM citric acid and mobile phase B was methanol. The gradient


233 mode was initially set at A/B ratio of 85/15 from 0 to 5 min, then linearly increased to 60/40 at 40 to 85 min. The flow rate was 1.0 ml/min. The detector was set at 254; injection volume was 5 JLIL.


Results and Discussion Figure 1 shows the results of antioxidant tests on the volatile extract. The autoxidation of hexanal to hexanoic acid was monitored for 45 days. BHT (50 jug/mL), a synthetic standard antioxidant, inhibited hexanal oxidation by 100% throughout the 45 day period. a-Tocopherol, a natural antioxidant, inhibited hexanal oxidation by 95% during same period of time. A l l volatile extract samples, with the exception of 10 jig/mL volatile extract, inhibited hexanal oxidation by over 80% for 45 days.

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Antioxidant and Antiinflammatory Activities of Licorice Root - American

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