The constituents of garlic is not the mirror of the intact garlic, it varies from whole to crushed and processed garlic. The constituents of garlic preparations differ considerably based on the subjective treatment conditions⁵. The antioxidant properties of garlic are well documented and still explored since, many of its therapeutic benefits are attributed to antioxidant effect. Many studies have been carried out to explore antioxidant activity of garlic and its various preparations. The benefits of crude extracts or fractions of garlic of different varieties are scarce. The present research paper deals with evaluation of the methanol extracts of three garlic varieties for antioxidant activity using various in vitro methods.

2. MATERIALS AND METHODS:

2.1. Plant material:

Three varieties of garlic (Allium sativum) namely, Multiple clove garlic (G1), Single bulb garlic (G2) and Himalayan garlic (G3) were chosen for the study. G1 was procured from farmers from Satara district in Maharashtra, G2 was obtained from local garlic vendor and G3 was purchased from Dadar ayurvedic pharmacy, Mumbai. Procured plant materials were authenticated by botanist from Botanical Survey of India, Pune and the voucher specimens (ACPL1N, ACPL2 and NID-31 respectively) were deposited for future use.

2.2. Chemicals and Instruments:

1, 1-Diphenyl-2 picryl hydrazyl (DPPH) from Sigma Chemicals, USA. Ascorbic acid, Thiobarbituric acid (TBA), Trichloro acetic acid (TCA), Potassium chloride from Hi media laboratories Ltd., Mumbai, Folin–Ciocalteu (FC) reagent, K₃Fe(CN)_6,aluminium trichloride and other chemicals were procured from spectrochem (India) Ltd., Mumbai. All the chemicals and solvents used were of analytical grade.

The instruments used for the study were UV spectrophotometer (Jasco, V-630), laboratory centrifuge (Remi motors, R4C) and digital pH meter (Equip-tronics, EQ-610).

2.3. Preparation of extracts:

Fresh Garlic bulbs were peeled and chopped to fine pieces of 3 mm thickness. The cut pieces were soaked in methanol for overnight at room temperature. Next day, the contents were refluxed for 2 hrs at temperature not exceeding 60⁰ C, cooled and filtered. Filtrate was evaporated to dryness at reduced temperature. Percentage yield was calculated and was found to be 16, 12.6 and 13 % respectively for G1, G2 and G3 varieties. The extracts (ME-G1, ME-G2 and ME-G3) obtained were then stored in a desiccator and used for further study.

2.4. Phytochemical analysis:

Phytochemical screeningof extracts was carried out for presence or absence of various constituents⁶. TLC profiles using pre-coated silica gel plates as stationary phase (GF254, Merck) was developed. The mobile phase used was n-butanol: water: acetic acid: formic acid (28:8:9:2) and ninhydrin as spraying agent for visualization of spots⁷.

2.4.1. Quantification of total phenolics – by Folin-Ciocalteu method:

The amount of total phenolic compounds in the extract was determined colorimetrically with the Folin–Ciocalteu (FC) reagent. The reaction mixture contained 50 µl of the sample, 250 µl of FC reagent, 750 µl of sodium carbonate solution. The volume was made upto 5ml with water and was incubated in dark under ambient conditions for 2 hrs to complete the reaction. The absorbance of the resulting solution was measured at 760nm in a UV spectrophotometer. The concentration of total phenolic compounds was expressed as mg of gallic acid equivalents (GAE) per g of dried extract, using a standard curve of gallic acid. The linearity obtained in the range of 6-20 µg/ml. All the measurements were carried out in triplicates^8,9.

2.4.3. Quantification of total flavonoids:

The flavonoids content was determined by aluminium chloride method using rutin as a reference compound¹⁰. This method based on the formation of a complex flavonoid-aluminum having the absorption maximum at 415 nm. The extract in methanol was mixed with 20% aluminium trichloride in methanol and a drop of acetic acid, and then diluted with methanol to 5 ml. The absorption at 415 nm was read after 40 min. The absorption of standard rutin solution (0.5 mg/ml) in methanol was measured under the same conditions. The amount of flavonoids in plant extracts in rutin equivalents (RE) was calculated.

2.5. In vitro antioxidant studies:

The antioxidant activity of the extracts was evaluated using simpler and rapid scavenging assays viz, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical assay, total reducing power and TBARS (Thiobarbituric acid reactive substance) assay.

2.5.1. DPPH (1, 1, diphenyl 2-picryl hydrazyl) assay:

Radical scavenging activity of the compounds against the stable DPPH radical is determined spectrophotometrically. The various concentrations of each extract prepared in methanol were added to 1ml of 0.1 mM DPPH solution. An equal amount of methanol and DPPH are added to the control. After 30 minutes of incubation in dark at room temperature, absorbance was recorded at 517 nm. The percent DPPH radical scavenging was calculated with the equation: % DPPH radical scavenging = [(control absorbance - sample absorbance) / control absorbance] × 100. Ascorbic acid was used as the standard for comparison. The experiment was carried out in triplicate and IC₅₀ values were calculated¹¹.

2.5.2. Total Reducing power:

The antioxidant compounds form a colored complex with potassium ferricyanide, trichloro acetic acid (TCA) and ferric chloride that was measured at 700 nm. Different concentrations of the extract mixed with 2.5 ml of phosphate buffer (pH 6.6), and 2.5 ml of 1% K₃Fe(CN)₆. The mixture was incubated at 50⁰C for 20 min, 2.5 ml of 10% TCA was added to the mixture and centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%), and the absorbance was measured at 700 nm. Rutin was used as standard. The graph was plotted with the average of the three determinations ¹².

2.5.3. TBARS assay:

The incubation mixture contained potassium chloride (150mM), 0.3 ml of 10% mouse brain homogenate as lipid source and various concentrations of each crude extract in a volume of 0.5 ml. Peroxidation is initiated by adding 0.1 ml ferrous sulphate. After incubating for 20 minutes at 37°C, reaction was stopped by adding 1 ml TBA/TCA/BHT solution, followed by heating at 80°C for 15 minutes and centrifuged at 1000 rpm for 10 minutes and absorbance of the supernatant liquid was recorded at 535 nm. Blank was carried out without drug. The percentage of anti-lipid peroxidation effect (% ALP) or % inhibition was calculated with the equation: % Inhibition = [absorbance of Ferrous sulphate – absorbance of Test / absorbance of Ferrous sulphate– absorbance of Control] × 100. The experiment was carried out in triplicate and IC₅₀ values were calculated^13,14.

2.6. Statistics:

All the experiments were carried out in triplicate. The results of all experiments were expressed as mean ± S.E.M. The concentration of the extracts that cause 50% of inhibition (IC₅₀) were determined by the linear regression analysis using Microsoft Excel programme for Windows, v.XP.

3. RESULTS:

Phyochemical investigation of the extracts revealed the presence of organosulfur compounds, saponins especially of furostanol type, phenolics and steroids. Total phenolics and flavonoid contents were determined for all the varieties as presented in Table 1. The higher amount total phenolics were found in the ME-G3 variety. But, the higher amount of flavonoids was found in the ME-G1 variety.

Table 1: Total phenolic and flavonoid contents of the extracts of three garlic varieties

S.NO	Garlic Varieties	Total phenolics (mg GAE/G)	Total flavonoids (µg RE/G)
1	ME-G1	0.32 ± 0.006	3.51 ± 0.07
2	ME-G2	0.33 ± 0.006	2.43 ± 0.04
3	ME-G3	0.47 ± 0.011	1.21 ± 0.08

The values are expressed in Mean ± SEM; N=3; N= Number of replicates

Antioxidant activity: In DPPH assay, all the extracts were able to reduce DPPH radical in concentration dependent manner. The values obtained by DPPH assay was comparable to the results reported by other authors^8. The IC₅₀values of the extracts are expressed in Table 2. The single bulb variety of garlic exhibited better radical scavenging capacity as compared to other varieties. The IC₅₀of ascorbic acid was found to be 3.74 µg/ml.

In TRP, there was concentration dependent increase in the absorbance of reaction mixture indicates the increase in the reducing power of the extract. The results are shown in Figure 1. In garlic extracts the concentration ranged from 200-1000µg/ml and in standard rutin where the concentration ranged from 3-15 µg/ml.

Figure: 1 Total reducing power of extracts of three garlic varieties. Plot of absorbance Vs concentration. The values are expressed in Mean ± SEM; n =3

In TBARS assay, methanol extract of all garlic varieties prevented ferrous sulphate – induced lipid peroxidation in concentration - dependent manner in mice liver homogenate. The single bulb garlic extract showed better activity in protection against lipid peroxidation compared with other garlic varieties. The IC₅₀values of the extracts are presented in Table 2. The IC₅₀ values were comparable to that of standard ascorbic acid of IC₅₀ 24.50 µg/ml.

Table 2: IC ₅₀values of extracts of three garlic varieties.

S.NO	Garlic Varieties	DPPH Assay IC ₅₀ (mg/ml)	TBARS Assay IC ₅₀(µg/ml)
1	ME-G1	6.35	90.40
2	ME-G2	2.91	80.42
3	ME-G3	6.4	90.21
4	Ascorbic acid	0.0037	24.50

IC ₅₀ (concentrations of the extracts that cause 50% of inhibition); N=3; N= Number of replicates

4. DISCUSSION:

Plants with antioxidant potential play an important role in protecting living organisms from the oxidative damage and act as life style enhancers. Garlic is one of the well known miraculous spices and considered as one of the best disease preventive foods in modern medicine. By considering its benefits and research proved its prophylactic and therapeutic uses in various chronic conditions like cancer, diabetes, atherosclerosis, inflammation, where free radicals play a major causative factor. Several constituents in garlic and garlic preparations can scavenge free radicals, protect membranes from damage, maintains cell integrity and it has the potency to enhance the activity of the some antioxidant enzymes^15,16.

The study showed that methanol extracts of all the garlic varieties showed a good antioxidant activity in DPPH assay. Peroxidation of lipids is involved in many disease conditions like atherosclerosis, diabetes etc. Antioxidants play a key role by inhibiting lipid peroxidation. All extracts of garlic showed good activity against ferrous sulphate induced lipid peroxidation and the results were comparable with the standard antioxidant ascorbic acid.

The presence of phenolics and flavonoids in all varieties may contribute to antioxidant activity of garlic. The extract with high phenolics or with high flavonoids did not show maximum antioxidant activity. The extract ME-G2 variety showed potent antioxidant activity among all three varieties, contained appreciable amounts of both flavonoids and phenolics. This may indicate that both phenolics and flavonoids contribute to antioxidant activity of single bulb garlic.

The phytoconstituents like terpenoids, steroids and phenolic compounds such as tannins, coumarins and flavonoids have protective effects due to its antioxidant properties. Natural antioxidants, especially phenolics and flavonoids, are safe and also bioactive. A number of scientific papers indicate the phenolics as effective contributor to antioxidant activity and inhibit oxidative mechanisms. Hence, the antioxidant activity could be attributed at least in part, to the presence phenolic compounds and flavonoids^17,18,19.

In conclusion, the present study reveals good antioxidant activity of three varieties of garlic which are commonly used in India. The single bulb garlic showed better activity compared to other garlic varieties.

5. ACKNOWLEDGEMENTS:

The authors are grateful to the Indian council of medical research (ICMR), Government of India for financial support.

6. REFERENCES:

1. Halliwell B. Free radicals, antioxidants and human diseases; curiosity, cause, or consequence? Lancet. 1994; 334: 721-724.

2. Sharma A, Bhardwaj S, Mann AS, Jain A and Kharya MD. Screening Methods of Antioxidant Activity: An Overview. Phcog Rev. 2007; l 1: 232-238.

3. Koleva II, Beek TAV, Linssen JPH, Groot A, Evstatieva LN. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochem.Anal. 2002; 13: 8-17.

4. Banerjee SK, Maulik SK. Effects of garlic on cardiovascular disorders: a review. Nutr J.2002; 1:1-14.

5. Lawson LD, Koch HP. The science and therapeutic applications of Allium sativm L and related species. Williams and Wilkins. A Waverly company, 1996, 2 nd edition.

6. Kokate CK. Phyto chemical screening. In: Practical pharmacognosy, Vallabha prakshan, New Delhi, pp.103-107.

7. Keusgen M. TLC analysis of Allium sativum constituents. Planta med.1997; 63: 93-94.

8. Bozin B, Mimica-Dukic N,Samojlik I,Goran A,Igic R. Phenolics as antioxidants in garlic (Allium sativum L.,Alliaceae). Food Chem. 2008; 111:925-929.

9. Srinivasan R, Chandrasekar MJN, Nanjan MJ, Suresh B. Antioxidant activity of Caesalpinia digyna root. J Ethnopharmacol. 2007; 113:284-291.

10. Abdel-Hameed ES. Total phenolic contents and free radical scavenging activity of certain Egyptian Ficus species leaf samples. Food Chem. 2009; 114:1271–1277.

11. Ansari NM, Houlihan L, Hussain B, Pieroni A. Antioxidant Activity of Five Vegetables Traditionally Consumed by South-Asian Migrants in Bradford, Yorkshire, UK Phytotherapy research. 2005; 19: 907-911.

12. Rajeshwar Y, Kumar GPS, Gupta M, Mazumder UK. Studies on in vitro antioxidant activities of methanol extract of Mucuna pruriens (fabaceae) seeds. European bulletin of drug research. 2005; 13:31-39.

13. Narla RS, Rao MNA. Scavenging of free radicals and inhibition of lipid peroxidation by 3-phenylsydnone. J Pharm Pharmacol. 1995; 47: 623-625.

14. Sreejayan, Rao MNA. Curcuminoids as potent inhibitors of lipid peroxidation. J Pharm Pharmacol. 1994; 46:1013-1016.

15. Chandra A, Mahdi AA, Ahmad S, Singh RK. Indian herbs result in hypoglycemic responses in streptozotocin-induced diabetic rats. Nutr Res. 2007; 27: 161– 168.

16. Lee YM, Gweon OC, Seo YJ, Im J, Kang MJ, Kim MJ, Kim JI. Antioxidant effect of garlic and aged black garlic in animal model of type 2 diabetes mellitus. Nutr Res Pract. 2009; 3:156-161.

17. Mallurwar VR, Pathak AK. Hepatoprotective and antioxidant activity of fresh aerial roots of Ficus bengalensis. Ind J.Nat.Prod. 2006; 23: 8-12.

18. Bors W, Michel C. Chemistry of the antioxidant effects of polyphenols. Ann N Y Acad Sci. 2002; 957: 57-69.

19. Queiroz YS, Ishimoto EY, Bastos DHM, Sampaio GR, Torres EAFS. Garlic (Allium sativum L.) and ready-to-eat garlic products: In vitro antioxidant activity. Food Chem.2009; 115:371–374.

Received on 05.04.2011

Accepted on 24.05.2011

Research Journal of Pharmacognosy and Phytochemistry. 3(4): July- August 2011, 162-165