Journal of Herbal Medicine and Toxicology 4 (1) 97-102 (2010) ISSN : 0973-4643 Original Article IN VITRO ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITY OF GLYCYRRHIZA GLABRA ROOT EXTRACTS Bhawya Dayananda, Anilakumar Kandangath Raghavan*, Farhath Khanum and Bawa Amarinder Sngh Biochemistry & Nutrition Discipline, Defence Food Research Laboratory, Mysore-570011, India. * E-mail: anilakumarkr @gmail.com Received- 21st Oct. 2009, Revised -12th Dec. 2009, Accepted- 30th Dec. 2009 Abstract: Glycyrrhiza glabra (Fabaceae) commonly called as licorice is one of the oldest and most widely used herbs in Eastern traditional medicine. The powdered extracts of Glycyrrhiza glabra were prepared using solvents based on polarity and their antioxidant properties were evaluated employing various established in vitro systems viz. 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion radicals scavenging, hydroxyl radicals scavenging, metal ion chelation, reducing power along with polyphenols and flavonoids. Proximate composition and mineral content analysis was carried out along with vitamins and tannins in the powdered root. The study revealed a notable inhibitory activity of ethanol extract, followed by methanol and water extracts. Key words: Glycyrrhiza glabra, free radicals, superoxide anion, metal ion chelation, reducing power. INTRODUCTION Many of the diseases are mainly linked to oxidative stress due to free radicals Antioxidants have been reported to prevent oxidative damage caused by free radicals and may prevent the occurrence of diseases. It can interfere with the oxidation process by reacting with free radicals, chelating, and catalytic metals and also by acting as oxygen scavengers . Currently available synthetic antioxidants have been suspected to cause or prompt negative health effects. Hence strong restrictions have been placed on their application and there is a trend to substitute them with naturally occurring antioxidants. Recently there has been an upsurge of interest in the therapeutic potentials of medicinal plants as antioxidants in reducing such free radical induced tissue injury. Glycyrrhiza glabra (Licorice) root is one of the common traditional Chinese medicines and used as flavoring and sweetening agents for tobaccos, chewing gums, candies, toothpaste and beverages. Polysaccharides from Glycyrrhizia glabra are enriched with glucuronic acid which showed strong anti-adhesive properties against H. pylori and P. gingivitis, .The main components (glycyrrhizin, 18beta-glycyrrhetinic acid, isoliquiritin, and liquiritigenin) isolated from Glycyrrhizia glabra possess antiallergic effects, such as antiscratching behaviour and IgE production-inhibitory activity . Glabridin, a major active constituent of Glycyrrhiza glabra is commonly used in the treatment of cardiovascular and central nervous system (CNS) diseases . It could be an effective agent for treatment of atopic dermatitis and a promising memory enhancer in both exteroceptive and interoceptive behavioural models of memory . The extract possesses an antidepressant-like effect .The main bioactive constituents of licorice are triterpene saponins and various types of flavonoids . Here we report the antioxidant ability in sequential extracts based on polarity, in order to find a better application because; free radicals are of great concern in the food industry and among consumers of processed food. 97 Journal of Herbal Medicine & Toxicology MATERIALS AND METHODS Plant materials The roots of Glycyrrhiza glabra were procured from the reliable local herbal suppliers of Mysore, Karnataka, India. Chemicals and instruments Butylated hydroxyanisole (BHA), nitroblue tetrazolium (NBT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), Phenazine methosulphate (PMS), Nicotinamide adenine dinucleotide reduced (NADH), deoxyribose 4-dimethyl amino cinnamaldehyde, Catechin were purchased from M/s Sigma Chemicals Co. (St. Louis, MO). Thiobarbituric acid (TBA), gallic acid, trichloroacetic acid (TCA), Ethylenediamine tetraacetic acid (TCA), Ascorbic acid and other chemicals were purchased from M/s Sisco Research Laboratories, Mumbai, India. All reagents procured were analytical grade. Absorbance was measured using Shimadzu U V spectrophotometer, atomic absorption spectrophotometer (model AAS Vario 6, Shimadzu, Japan). Extraction of plant samples For antioxidant analysis, Glycyrrhiza glabra roots were washed, dried in hot air oven at 400C and then finely powdered. The powder was used for extraction. Sequential extraction of the powder was done with different solvents with increasing polarity i.e. hexane, chloroform, ethylacetate, acetone, methanol, ethanol and water. 30g of the powder were extracted in 300ml of the solvent in glass conical flask on a mechanical shaker for 24h at room temperature. The extract was filtered with Whatmann filter paper no. 1 and dried by flash evaporation/lyophilisation. Proximate Composition The proximate composition of Glycyrrhiza glabra was determined using the Association of Official Chemists (AOAC) method .The mineral content of the stem was analyzed by an atomic absorption spectrophotometer. The vitamin C content was estimated by titrimetric method using 1, 2dicholorophenol indophenol dye. Vitamin E and total carotenoids in the powdered extract of Glycyrrhiza glabra were estimated as prescribed by Sadasivam and Manickam . Antioxidant activity by free radical scavenging method The 1, 1-diphenyl 2-picrylhydrazyl (DPPH) radicalscavenging assay reported by Hatano et al.,  was adopted for the determination of the antioxidant activity of Glycyrrhiza glabra extract with modifications. Briefly, 3ml of the reaction mixture contained 0.1ml of different concentration of the extract (1-10mg/ml) and aliquot of DPPH solution (0.5ml, 50mg/100ml) in methanol. Reaction mixture without the extract was used as control. The mixture was shaken vigorously and allowed to stand in dark for 45min. The antioxidant scavenging activity was measured as the decrease in absorbance of DPPH at 515nm against a blank and calculated using the following formula. Scavenging Effect (%) = [(A0 - A1) / A0 ] X 100 Where, A0 is the absorbance of control and A1 is the absorbance of sample. All the values expressed are the mean values carried out in triplicates. BHA was used as a positive control. Superoxide anion scavenging activity Measurement of superoxide radical scavenging activity of the Glycyrrhiza glabra extracts was based on the method reported by Liu et al.  with modifications .The superoxide is generated in 3ml of Tris HCl (16mM pH8.0) containing 1ml of NBT (50µm) solution, 1ml NADH (78µM) solution and sample solution of different extracts in a concentration of 1-10µg/ml .The reaction was started by adding 1ml of PMS solution (10um) to the mixture. The reaction mixture was incubated at 25oC for 5 min. and the absorbance at 560nm was measured against the blank samples. L-Ascorbic acid was used as a control. BHA was used as positive control. % Inhibition = [( Acontrol - Asample ) / Acontrol ) x100] Where Acontrol is the absorbance of the L-Ascorbic acid and Asample is the absorbance of standards. The data at each point were the average of three measurements. Hydroxyl radical scavenging activity 1. Non-site specific hydroxyl radical mediated 2deoxy-D-ribose degradation The assay was performed as reported by Halliwell et al.,  with modifications. The reaction mixture contained 100µl of 28mM 2-deoxy D ribose (dissolved in KH2PO4- K2HPO4) phosphate buffer pH 7.4, 500µl solution of various concentration of the G.g extracts (100µg-1000µg/ml), 200µl of 200µM FeCl3 and 1.04mM EDTA (1:1 v/v), 100µl of H2O2 (1.0 mM) and 100µl ascorbic acid (1.0mM). After an incubation 98 Raghavan et al. period of 1hr. at 37oC the extent of deoxy ribose degradation was measured by the TBA reaction. 1ml of TBA (1% in 50mM NaOH) and 1ml of TCA were added to the reaction mixture, tubes were heated at 100o C for 20 min. After cooling the absorbance was read at 532 nm against a blank (containing only buffer and deoxy ribose). The % inhibition was calculated by the formula Inhibition (%) = [(A0 - A1) / A0 ] X 100 Where, A0 is the absorbance of control and A1 is the absorbance of sample. All the values expressed are the mean values carried out in triplicates. BHA was used as a positive control. 2. Site-specific hydroxyl radical mediated 2deoxy-D-ribose degradation The ability of the Glycyrrhiza glabra extracts to inhibit site-specific hydroxyl radical mediated degradation was also carried out to understand its role as a metal chelator. The method was the same as described above except that buffer replaced EDTA. Reducing Power The reducing power of the prepared Glycyrrhiza glabra extract was determined by the method of Oyaizu . Briefly various concentration of the extract (0-5mg/ml) and the standard compound were mixed with phosphate buffer (2.5ml, 0.2mol/L, pH 6.6) and potassium ferricyanide [K3 Fe(CN)6], (2.5ml, 1%). The mixture was incubated at 50oCfor 20min. To the mixture 10% TCA was added to the mixture, which was then centrifuged at 3000rpm for 10min. The upper layer of solution (2.5ml) was mixed with distilled water (2.5ml), FeCl3 (0.5ml, 0.1%) and the absorbance was measured at 700nm.Increased absorbance of the reaction mixture indicated increased reducing power. α- tocopherol was used as a positive control. Ferrous ion chelating activity The assay was determined as described by Dinis et al., . Briefly the extracts were added to a solution of 2mmol/L FeCl2 (.05ml). The reaction mixture was initiated by the addition of 5mmol/L ferrozine (0.2ml), the mixture was shaken vigorously and left at room temperature for 10 min. Absorbance of the solution was then measured spectrophotometrically at 562nm. The % of Inhibition of ferrozine-Fe2+ complex was given by % Inhibition = [(A0 - A1 ) / A0 ] X 100 Moisture % 06.50 ± 0.70 Where, A0 was the absorbance of the control & A1 was the absorbance in the presence of the samples of G.G extracts and standards. The ability of the extract to chelate ferrous ion was compared with the standard BHA. Protein % 07.17 ± 0.74 Total phenolic contents Fat % 03.50 ± 0.70 Carbohydrates % 63.6 ± 1.69 Ash % 05.05 ± 0.07 Crude Fiber % 21.75 ± 1.06 Vitamin C mg/100g 27.65 ± 1.20 Vitamin E mg/100g 98.21 ± 1.22 Total carotenoids g/L 72.22 ± 3.72 Iron mg/100g 08.08 ± 0.02 Zinc mg/100g 00.96 ± 0.01 Sodium mg/100g 63.20 ± 0.56 Total phenolic content was estimated spectrophotometrically by Folin-Ciocalteau method carried out according to Singleton and Rossi  with some modifications. The 0.1ml Glycyrrhiza glabra extract concentration was made up to 3ml with double distilled water, to this 0.5ml of folin-Ciocalteau reagent was added and allowed to stand at room temperature for 10min, to the mixture 2ml of 7% sodium carbonate was added and kept in boiling water bath for 1min, cooled, after which the absorbance was read at 650nm against blank. The concentration was calculated using gallic acid as standard, and the results were expressed as milligram gallic acid equivalents per gram extract. Potassium mg/100g 180.48 ± 0.12 Flavonoids Polyphenols mg/100g 28.62 ± 1.05 Flavonoids mg/100g 08.63 ± 0.98 Tannins mg/100g 06.82 ± 1.02 The determination of flavanoids was carried out according to Delcour and Varebeke  .To 1ml of Glycyrrhiza glabra extract, 5ml chromogen reagent (1.0 g 4-dimethyl amino cinnamaldehyde dissolved in Table 1: Proximate composition and phytoconsttuents of Glycyrrhiza glabra 99 Journal of Herbal Medicine & Toxicology a cooled mixture of 250ml of concentrated HCl and 750ml of methanol, made upto 1 L methanol) was added. After 10min, the absorbance was measured at 640nm against a blank consisting of water instead of extract and the flavanoids content was calculated with (±) catechin and the concentration was expressed as (±) catechin equivalents. 100 Raghavan et al. Tannins A 0.5g sample was weighed and boiled with 75ml of water for 30min. The contents were centrifuged at 800 x g for 20min and the supernatant was made upto 100ml in a volumetric flask using water. Aliquots were treated with Folin-Denis reagent and absorbance was measured at 700nm as described by Sadasivam and Manickam . Statistical analysis The statistical processing of the data obtained from all studies is expressed as mean ± standard deviation (SD) of three separate experiments using the computer programme Excel. RESULTS AND DISCUSSION DPPH free radical scavenging activity Scavenging the stable DPPH radical is widely used a method to evaluate the free radical scavenging ability of various samples . Antioxidants act mainly on DPPH radical by their hydrogen-donating ability and hence its scavenging activities by the extract are most widely known to assess the antioxidant potential antioxidants, where they reduce the stable radical DPPH to the yellow-coloured diphenylpicrylhydrazine. The reduction capability of DPPH radical is determined by the decrease in absorbance at 517nm. The scavenging effect of the extracts and standards with the DPPH radical is presented in Fig.1. The methanol extract showed better radical scavenging activity than the other extracts. Superoxide radical (O2.-) scavenging activity Superoxide radical is known to be produced in vivo and can result in the formation of H2O2 via dismutation reaction and is known to be a very harmful species to cellular components . Fig 2 shows the inhibitory effect of methanol on superoxide radical generation. The scavenging activity of methanol extracts on superoxide radicals was increased with increase in concentration. The data showed that the extract was markedly a more potent scavenger of superoxide anion than other solvent extracts. Hydroxyl (OH.) radical-scavenging activity Of all the oxygen free radicals, the hydroxyl radical is the most reactive which severely damages adjacent bimolecules such as proteins, DNA, PUFA and nucleic acid causing aging, cancer and several diseases , and also known to be one of the quick initiators of the lipid oxidation process, abstracting hydrogen atoms from unsaturated fatty acids . Therefore, the removal of hydroxyl radical is probably one of the most effective defences of a living body against various diseases. Fig 3 and 4 show the effect of various Glycyrrhiza glabra extracts in deoxyribose scavenging assays (both non-site and site-specific). It was observed that all the extracts were effective in scavenging the hydroxyl radicals in non site-specific assay as well as in site-specific assay. However, the change was comparatively greater in the non site specific than in the site-specific assay indicating their radical scavenging activity. Metal chelating activity Metal chelation indicates the effective capacity for iron binding, suggesting its action as an antioxidant relating to its iron binding capacity wherein ferrozine can quantitatively form complexes with Fe2+. In the presence of other chelating agents, the complex formation is disrupted with the result that the red colour of the complex is decreased. Measurement of the rate of colour of reduction therefore allows estimation of the chelating activity of the coexisting chelator . Fig.5 shows that the formation of the ferrozine- Fe 2+ complex is not complete in the presence of methanol, ethanol and water extracts indicating their capacity in chelating iron. However, the chelating ability was relatively lower than that of EDTA. In this assay the extracts and standard compounds interfered with the formation of ferrous complex with the reagent ferrozine, indicating their metal chelating activity. Reducing power In reducing power assay, the presence of antioxidants in the sample result in the reducing of Fe3+ to Fe2+ by donating an electron. The reducing properties are generally associated with the presence of reductones (Duh, 1998), which have been shown to exert antioxidant action by breaking the free radical chain by donating hydrogen atom . Measuring the formation of Perl’s blue at 700nm can then monitor amount of Fe2+ complex. Fig. 6 shows the reducing power of methanol, ethanol and water extracts in increasing order in a dose dependent manner in comparison with the reference compound átocopherol. Here, the methanol extract was shown more reducing power than ethanol and water extracts. 101 Journal of Herbal Medicine & Toxicology  Composition and phytoconstituents The Table 1 shows that the root is rich in fibre, vitamin E, potassium, polyphenols and flavonoids . It is well known that plant phenolics, in general, are highly effective free radical scavengers and antioxidants .It is known that hydrogen-donating property of the polyphenolic compounds is responsible for the inhibition of free radical induced lipid peroxidation . Consequently the antioxidant activities of the Glycyrrhiza glabra extracts may be explained with respect to their total phenolics and flavonoid contents, with good correlation. This work shows the effective antioxidative activity of methanol extract in scavenging free radical in comparison to other solvents. Nowadays, there is an increasing interest in detecting the natural antioxidants, which are safe and effective in order to replace the commercial synthetic antioxidants. Hence Glycyrrhiza glabra extracts can be a source of natural antioxidants in replacing the synthetic ones.       REFERENCES:         Gutteridge, .JM.C.:. Free radicals in disease processes: A compilation of cause and consequences. Free Radic. 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