ANTIOXIDANT EFFECT OF SPICES AND THEIR MIXTURE IN REFERENCE TO CUMIN, GARLIC, GINGER, MUSTARD, RED CHILLI AND TURMERIC

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1 J. Dairying, Foods & H.S., 27 (2) : , 2008 ANTIOXIDANT EFFECT OF SPICES AND THEIR MIXTURE IN REFERENCE TO CUMIN, GARLIC, GINGER, MUSTARD, RED CHILLI AND TURMERIC Vrinda Sikri and J.S. Berwal Dept. of Animal Products Technology, College of Animal Science, C.C. S. Haryana Agricultural University,Hisar , India ABSTRACT Present study revealed the antioxidant traits of spices and their mixture at varied concentration using β - carotene bleaching test both by Agar Diffusion and Spectrophotometric method. BHT (butylated hydroxytoluene), a potent synthetic antioxidant was taken as reference. The A.I.(Antioxidant Index) of ASE (Aqueous Spice Extract) by âcbt-adm were in order of spice mix (3.59) > ginger (3.40) > turmeric (3.27) > cumin (2.77) and red chilli (2.72). A.I. of BHT (200ppm) on third day was While the antioxidant activity resulted in the pattern of spice mix>ginger>turmeric>cumin>redchilli>garlic>mustard by âcbt- SP method. When the results of âcbt-adm and âcbt- SP were compared, ginger and spice mix at 4 percent concentration were inferred as potent antioxidant by both methods, which were comparable to BHT (200 ppm concentration). INTRODUCTION The paleolithic man started learning the Preservation techniques, which have been improved upon by man in different eras by employing empirical methods. Spices need fewer introductions as is well known all over the world. It constitutes an important group of agricultural commodities, which are virtually indispensable in the culinary art. According to International Organization for Standardization the term Spices and condiments applies to natural plant or vegetable products or mixtures thereof, in whole or ground form are used for imparting flavour, aroma, piquacy and for seasoning of foods. Antioxidant activity : Besides diagnostic and antimicrobial characteristics of spices, antioxidant properties had for long remained the interest of scientists. Antioxidant activity is defined as an activity of an antioxidant as any substance that when present at low concentration compared to those of an oxidizable substrate, substantially delays or prevent oxidation of that substrate (Halliwell 1990). Many plant tissue contain compound possessing antioxidant activity such as flavonoid and ascorbic acid (Haldeman et al., 1987).Spices pertaining to antioxidant traits (Jalay et al.1987) had concluded that clove followed by black pepper, ginger and rose petals were potent inhibitors. Shogaol and zingerone found in ginger (Kikuzake and Nakatini 1993), capsaicin and dihydrocapsiacin of red pepper (Nakatini et al. 1986) exhibited highest antioxidant properties. However antioxidant activity displayed by spices depends on several factors which include the chemical nature of the food, and medium to which they are added. To evaluate the same, a desirable method should be rapid, reproducible, should require small amount of chemicals and should not be influenced by the physical properties of the compound. Keeping this as base agar diffusion and spectrophotometric method of Taga et al. (1984) and Chevolleau et al. (1992), which was adopted by Mallet et al. (1994) and by Kumar (1999) was followed. This method was based on the ability of different extracts to decrease oxidative losses of â-carotene in a â-carotene linoleic acid emulsion. To have more elaborative view on the same an attempt has been made for evaluating

2 Vol. 27, No. 2, antioxidant characteristics of Spices and their mixtures, which are in common use in Indian recipes. MATERIAL AND METHODS The plan of work was been materialized in the Department of Animal Products Technology, CCSHAU Hisar with following material and methods Source of materials : Spices : Cumin, garlic, ginger, mustard, red chilli, turmeric and spice mixtures were used as spices. All these spices were purchased from local market of Hisar(India). Source of Chemicals : b-carotene and linoleic acid (C 18 H 32 O 2 ) from Merck (Dramstadt, Germany); Tween-40, Polyoxyethylene Sorbitane Monopalmitate and Bactoagar (Agar No.1) from Sigma Chemicals Co. (St. Louis, USA); Tween- 80 (Polyoxyethylene (20) Sorbitane Mono-oleate) from SD s Labchem Industry, Mumbai; Acetone, Chloroform, Ethanol ACS grade. BHT (Butylated hydroxyl toluene) (C 15 H 29 O) from Labchem Pvt. Ltd., Mumbai. Composition of nutrient media used : Czapek yeast extract agar (CYEA) Czapek concentrate* 10.0ml K 2 HPO 4 1.0g Sucrose 30.0g Agar 15.0g Yeast extracts 5.0g Distilled water 1.0litre ph 6.8 Czapek concentrate* NaNo g Kcl 5.0g FeSo 4. 7H 2 O 0.1g MgSo 4.7H 2 O 5.0g Distilled water 100.0ml (MTCC 1998) Preparation of aqueous spice extract (ASE) for β carotene bleaching test : The test procedure was similar as described by Godow et al. (1997) for plant materials. 10,20,40and 50g. of finely ground spices were poured with 1000 ml distilled water individually and mixed by stirring for 10 minutes and additional steeping for 30 minutes at room temperature. The extracts were filtered through muslin cloth followed by filter paper (Whatman No.1) and cooled to room temperature. Aliquots of the extracts were kept frozen (-18 o C) till further use. Antioxidant activity : Antioxidant activity of each ASE was determined by β-carotene bleaching test by both spectrophotometric and agar diffusion methods as described by Taga et al. (1984). This being based on the ability of the extracts to decrease losses of β-carotene in a β- carotene linoleic acid model system. β -carotene bleaching test agar diffusion method (β-cbt ADM) : The β-carotene bleaching test, diffusion method (β-cbt ADM) was selected because of its simplicity and visual evidence of results.three grams of bacto agar was dissolved in 200 ml of boiling distilled water and cooled at 50 0 C. To this was added 4 ml linoleic acid in ethanol at a concentration of 5 g. litre -1 and 20 ml acetone solution of β-carotene at a concentration of 1 g. litre -1. The model substrate so obtained was poured into a 8.5 cm diameter petridishes and allowed to solidify for 30 minutes. Three wells of approximately 50 μl capacity were punched in the agar of each petridishes using cork borer and a drop (approximately μl) of liquid agar was placed with pasteur pipette into each well. This helped for uniform diffusion of the test solutions in the agar. 20 μl of ASE after thawing was injected into each well and the wells were then closed with a microscopy cover glass and the petridishes were left at ambient temperature, until the background colour bleached. Each ASE was analysed in duplicate. The intensity of the colour remaining around each sample well was measured in mm, which represents the

3 122 J. DAIRYING, FOODS & H.S. substraction of the well diameter (5mm) and the results were expressed as protective efficiency. The bleaching time (hours) of β carotene surrounding each test spot was calculated and compared with the bleaching time (hours) of control test spot containing distilled water to determine anti oxidative index (A.I.)3 The intensity and persistence of the carotene colour was proportional to antioxidant activity, which was expressed as A.I. The values obtained by calculating A.I. were used to determine the minimum inhibitory concentration (MIC) of spices. β-carotene bleaching test by spectrophotometric method (β-cbt SP) : The spectrophotometric method (β-cbt SP) used to monitor instrumentally the bleaching of the β-carotenelinoleate model solution was almost similar to that described by Taga et al. (1984). The model test mixture for β-carotene bleaching test (Spectrophotometric method) was prepared by dissolving 1.0 mg. of β-carotene in 1 ml chloroform and by adding 20 mg. of linoleic acid and 200 mg. of Tween-40. Chloroform from model test mixture was removed using a rotary evaporator at 50 0 C. 50 ml of distilled water saturated with oxygen were added and the mixture was shaken vigorously. 5 ml of this mixture was poured to 0.2 ml of aqueous spice extract in test tubes. Absorbance measurement was made immediately at 470 nm. Control consisted of 5 ml of mixture and 0.2 ml of distilled water. The blank made with 20 mg. linoleic acid, 200 mg. Tween-40 and 50 ml oxygenated distilled water was adjusted to bring the spectrophotometer to zero. The tubes were placed in an agitating water bath kept at 50 0 C and absorbance measurement were made at 60 min intervals until the absorbance of the control read below Six replicates were prepared for each of the treatments. RESULTS AND DISCUSSION β-carotene bleaching test agar diffusion method (β-cbt ADM) : Results pertaining to the antioxidant activity of different spices are shown in Fig.1 and 2. BHT (butylated hydroxytoluene) was taken as reference amongst four most widely used synthetic antioxidants i.e. BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), PG (propyl gellate) and TBHQ (tertiary butylated hydroquinone). The phenolic compounds present in it reacts with free radicals, thereby terminate the chain reaction. The results for its antioxidant activity are shown in Fig. 1. The aqueous spice extract of spices at different concentration differed in their protective efficiency against bleaching of â-carotene (Fig.2). ASE of ginger followed by spice mix prevented the oxidation of β-carotene to significant (P<0.05) level. At 1 percent concentration spice mix (11.66mm) followed by ginger (10.33 mm) showed significantly (P<0.05) high protective efficiency while same two were significantly (P<0.05) similar at 2 and 4 percent level of concentration. At 5 percent concentration ginger exhibited (24.33 mm) maximum protective efficiency. Similar findings were reported by Arouma et al. (1997) and El. Alim et al. (1999) where ginger powder at 5 mg/ml concentration strongly inhibited the peroxidation of phospholipids liposomes. Antioxidant effect of spice mix in the present study confirms the findings of Madsen et al. (1997) and Aguirrezabal et al. (2000) who observed similar effects in inhibiting lipid oxidation when compared with nitrate and ascorbic acid. Arouma et al. (1997) explained that reaction of ginger with trichloromethylperoxyl radicals offers greater protection against lipid oxidation. They have also found that 6-gingerol and Zingerone are among the active components of ginger while Aeschbach et al. (1994) showed former (6-gingerol) as a powerful inhibition of phospholipids peroxidation. The ASE of cumin, turmeric and red chili showed less antioxidant activity while garlic and

4 Vol. 27, No. 2, mustard showed no activity, as it could not prevent the discoloration process. Except these two all the spices (ASE) showed increased protective efficiency with increasing concentration (Fig. 2). The bleaching time (hours) of â-carotene surrounding each test spot, that contained ASE of spices were also determined and the antioxidant activity of spices were calculated by the following equation and expressed as antioxidant index (A.I.). Bleaching time (hours) of β-carotene surrounding test spot (well) A.I. = Bleaching time (hours) of β-carotene surrounding control spot (well) When BHT was considered as reference antioxidant it was found that protective efficiency of BHT at 200 ppm concentration on 3 rd day (19.0mm) was similar to 4 percent concentration of ginger and spice mix. While on 7 th day protective efficiency of of BHT at same concentration level was comparable to protective efficiency of cumin, turmeric and red chilli. The A.I. of ASE were in order of spice mix (3.59) > ginger (3.40) > turmeric (3.27) > cumin (2.77) and red chilli (2.72). A.I. of BHT (200ppm) on third day was The said concentration and period was selected because of its protective efficiency, which was found similar to 4 percent concentration of ginger and spice mix. Bleaching time (hrs.) taken by garlic and mustard was near to or equal to that of control i.e.(22 hrs.). The A.I. of turmeric was next to ginger which was in conformity with findings of Jalay et al. (1987) who reported the activity of curcumin (the major phenolic pigment in turmeric) equal to three fourth activity of BHT at 5 percent concentration. Aguirrezabal et al. (2000) reported highest A.I. (4.55) of spice mix amongst spices while Jalay et al. (1987) were of the opinion that cumin and garlic also possess some antioxidant activity at higher concentration. Later is contrary to present findings, as ASE of garlic and mustard could not prevent discolouration. It might be due to use of fresh garlic instead of powder and insolubility of mustard seed in distilled water extract. Similar result for garlic was reported by Yang et al. (1993) and Kumar (1999). They proposed garlic as an effective hydroxyl radical scavenger substance such as allilin, diallyl sulfide, allyl sulfide and propyl sulfide accounting for an antioxidant effect. β-carotene bleaching test by spectrophotometric method (β-cbt SP) : The idea of this method first came in the mind of Marco (1968) who when realized that a compound with 0.29 μg/ml satntoquin showed decrease in Vt. A after 60 seconds, followed by an increase in optical density at 30 minutes. The increase in colour development must be owing to oxidation products. The highest level of linoleic acid showed the greatest relative increase at 30 minutes suggesting linoleic acid oxidation products as the major sources of the interfering colour. Later on Taga et al. (1984), Chevolleau et al. (1992), Mallet et al. (1994) and by Kumar (1999) measured the antioxidant activity of ASE on spectrophotometer at wave length of 470 nm. Results pertaining to antioxidant activity of ASE of cumin, garlic, ginger, mustard, red chilli, spice mix and turmeric are presented from Fig. 4 to 10. Similar to βcbt-adm, BHT was measured as reference (Fig. 3). The absorbance values of control i.e. without ASE reached to 0.0 at the end of 52 hours. A reading between 0.6 to 0.9 on spectrophotometer at 470 nm indicated workable concentration of â- carotene. The initial absorbance value was 1.47, 1.59, 1.67 and 1.75 when BHT was used as test solution at 100, 200, 500 and 1000 ppm concentration respectively. Effectiveness of this synthetic antioxidant increased with increasing concentration expressing higher optical density on spectrophotometer. Finally at he end of 52 hours it reached to 0.11, and 0.33 respectively with the same level of concentrations. The results of previous

5 124 J. DAIRYING, FOODS & H.S. experiments (βcbt ADM) displayed ginger and of 17, 32 and 52 hours using following expression: spice mix as potent antioxidants. Antioxidant Absorbance of sample activity of ginger was compared with BHT by RAA = Absorbance of BHT (200 ppm) βcbt- SP method. The initial absorbance values RAA values showed that initial of ginger (ASE) (Fig.6) were found similar to performance of ginger (0.94) and spice mix (0.94) absorbance values of BHT at 200ppm were quite similar. At the end of 32 hours RAA concentration (Fig. 3). The initial optical density of ginger (1.05) was higher than spice mix (0.91) for ginger (ASE) 1.50, 1.59, 1.62 and 1.70 at 1, while at the end of experiment i.e. 52 nd hour, 2, 4 and 5 percent concentration respectively RAA of both were (3.27) similar. However was reduced to 0.31, 0.38, 0.46 and 0.51 absorbance value of BHT (200 ppm) was found respectively at the end of experiment. 2.2 and 3.4 times lower to ginger and spice mix The antioxidant activity resulted in the at 4 percent concentration respectively. pattern of spice mix > ginger > turmeric > When the results of βcbt-adm and cumin > redchilli > garlic > mustard by βcbt- βcbt- SP were compared, ginger and spice mix SP method. Spice mix had initial optical density at 4 percent concentration were inferred as potent values 1.41, 1.49, 1.59 and 1.62 at their antioxidant by both method, which was respective concentration, which was reduced to comparable to BHT (200 ppm concentration). 0.42, 0.51, 0.03 and 0.66 at the end of However increase protective efficiency of BHT experiment. Red chilli, garlic and mustard did was noticed in βcbt-adm. Volatile nature of not show any significant (P<0.05) difference. this antioxidant could be the reason for its less As the effectiveness of ginger and spice performance by βcbt-sp as the sample was kept mix at 4 percent concentration and BHT (200 at 50 0 C. Because of this reason it is considered ppm) was found as potent antioxidant by βcbt less suitable in frying oils and baked product. ADM. Therefore it was selected to calculate BHT at low concentration (>200 ppm) is Relative antioxidant activity (RAA) in βcbt SP permissible (PFA 1993) as at this level it acts as method. RAA activity was calculated at the end preservative and antioxidant. REFERENCES Aeschbach,R. et al. (1994) Food Chem. Toxicol.,32: Aguirrezabal,M.M. et al. (2000) Meat Sci., 54: Arouma,O.I. et al. (1997) Food Chem., 60(2): Chevolleau,S. et al. (1992) Fr Crops. Gras, 39:3-8. El. Alim,SSLA. et al. (1999) J. Sc. Food & Agric., 79(2): Godow,A.V. et al. (1997) Food Chem., 60(1): Halliwell,B. (1990) Res. Commun., 9(1): 32. Haldeman,J.D. et al. (1987) J. Food Protect., 50(5): Jalay,A.B. et al (1987) J. Food. Protect., 50(1): Kikuzake,H. and Nakatini,N. (1993) J. Food Sci.., 58(6): Kumar,V. (1999) M.V.Sc. Thesis, CCS Haryana Agriculture University, Hisar, India. Madsen,H.L. et al. (1997) Food. Chem., 57(2): Mallet,J.F. et al. (1994) Food. Chem., 49: Marco J.G. (1968) J. Am. Oil Chem. Soc., 45: M.T.C.C. (1998) Communication of Institute of Microbial Technology, Chandigarh, India. Nakatini,N. et.al (1986) Environ. Health Perspect., 67: 135. PFA (1993) Prevention of Food Adulteration Act (Seventh Amendment). 15 th edition, Eastern Book Company, India. Taga,S.M. et al.(1984) J. Am. Oil Chem. Soc., 61(5): Yang,G.C. et al.(1993) J. Food Drug Analysis, 1(4),