MATERIALS AND METHODS

Materials
Dried plant materials were obtained from experimental station of Faculty of Pharmacy, Cairo University during 2008. Ultrapure water was prepared using a Millipore Milli-RO 12 plus system (Millipore Corp., Bedford, MA). All reagents and solvents were either of analytical or HPLC grade and were obtained from Sigma Chemical Co., (St. Louis, MO).

Preparation of Freeze-Dried Hydrodistilled Extracts (FDH Extract)
Hundred gram of ground plant material were suspended in 500 mL ultrapure water and hydrodistilled for 3 h in a hydrodistillation glass apparatus. This process was repeated for 2 h with fresh solvent and the combined aqueous extracts were then filtered, reduced in volume in vacuo (-45°C), freezedried and stored at -4°C. The extracts were dissolved in ultrapure water prior to use.

Determination of Total Phenols Content
The total phenols were estimated according to the Folin-Ciocalteu method (Singleton et al., 1999). To 100 μL sample were added 250 μL of undiluted Folin-Ciocalteu-reagent. After 1 min, 750 μL of 20% (w/v) aqueous Na₂CO₃ were added and the volume was made up to 5.0 mL with H₂O. The control contained all the reaction reagents except the extract. After 2 h of incubation at 25°C, the absorbance was measured at 760 nm and compared to a gallic acid calibration curve. Total phenols were determined as gallic acid equivalents (mg gallic acid/g extract) and the values are presented as means of triplicate analysis.

Measurement of Iron (III) to Iron (II)-Reducing Activity
The ability of the extracts to reduce iron (III) was assessed by the method of Benjakul et al. (2005). Three milliliter aliquot of each extract, was dissolved in water, was mixed with 5 mL of phosphate buffer (0.2 M, pH 6.6) and 5 mL of a 1% potassium hexacyanoferrate [K₃Fe(CN)₆] solution. After a 30 min incubation at 50°C, 5 mL of 10% trichloroacetic acid were added and the mixture was centrifuged for 10 min. Five milliliter aliquot of the upper layer was mixed with 5 mL of water and 1 mL of 0.1% aqueous FeCl₃ and the absorbance was recorded at 700 nm. Iron (III) reducing activity was determined as ascorbic acid equivalents (mmol ascorbic acid/g extract). The values are presented as the means of triplicate analysis.

Study the Inhibition of Linoleic Acid Peroxidation
The antioxidant activity was determined as the degree of inhibition on the haemoglobin-catalysed peroxidation of linoleic acid according to Kuo et al. (1999). Sample (20 μL) were added to 0.5 mL of 0.05 M phosphate buffer (pH 7.0), containing 0.14% Tween 20 and 4 mM linoleic acid and then equilibrated at 37°C for 3 min. The peroxidation of linoleic acid in the above reaction mixture was initiated by adding 30 μL of 0.035% haemoglobin (in water), followed by incubation at the same temperature for 10 min and stopped by adding 5 mL of 0.6% HCl (in ethanol). The hydroperoxide formed was assayed according to a ferric thiocyanate method with mixing, in order, of 30% ammonium thiocyanate (0.2 mL) and 0.02 M ferrous chloride (0.1 mL). The absorbance (A_s) was measured at 480 nm after 5 min. The absorbance blank (A₀) was obtained without adding haemoglobin to the above reaction mixture; the absorbance of the control (A₁₀₀) was obtained by replacing the sample by buffer.

(1)

The antioxidant activities of the samples were calculated as trolox equivalents (mg trolox/g extract) according to Eq. 1. The values are presented as the means of triplicate analysis.

Measurement of Iron (II) Chelation Activity
The chelation of iron (II) ions by the different extracts was carried out as described by Carter (1971). Two hundred microlitres of each extract were added to 100 μL of 2.0 mM aqueous FeCl₂ and 900 μL methanol. The controls contained all the reaction reagents except the extract or positive control substance. After a 5 min incubation, the reaction was initiated by added 400 μL of 5.0 mM ferrozine. After a 10 min equilibrium period, absorbance at 562 nm was recorded. The iron chelation activities were calculated from the absorbance of the control (A_c) and of the sample (A_s) using Eq. 2 and expressed as Na₂EDTA equivalents (mg Na₂EDTA/g extract). The values are presented as the means of triplicate analysis.

(2)

Preparation of Butter Oil
Fresh buffalo milk was pasteurized and separated into cream and skimmed milk using an Alfa Laval separator (AESC, Sweden). The cream was churned to obtain unsalted butter. The resultant butter was melted using moderate heat (55°C) and the clear butter oil layer was carefully decanted.

Chemical Analysis
Acid and peroxide values were determined according to the methods of the Association of Official Analytical Chemists (1980). The thiobarbituric acid (TBA) test was carried out according to the method described by Patton (1973).

Design of Oxidation System
The Butter Oil (BO) was distributed into eight portions (200 g each) in sterilized glass bottles. The first portion served as a control. BHT at rate of 200 ppm was added to the second portion. To the 3rd and 4th portions, FDH extract of clove was added at a rate of 200 and 400 ppm, respectively. To the 5th and 6th, FDH extract of caraway was added at a rate of 200 and 400 ppm, respectively. In the 7th and 8th, FDH extract of coriander was added at a rate of 200 and 400 ppm, respectively. Control and treated samples were placed in an incubator at 60 ± 1°C to accelerate the auto-oxidation of butter oil, (Thampson, 1960). The experimental period was terminated when an objectional odour and high lipid rancidity values were obtained with the samples after 28 days.

Statistical Analysis
All statistical analysis were carried out using iner-STAT-a (Vargas, Mexico) as add-in for Microsoft Excel 2007. Analysis of Variance (ANOVA) was followed by Tukeys’ pairwise comparison test at a level of p<0.05 for the determination of significant differences between treatments and control.

RESULTS AND DISCUSSION

Extraction Yield and Total Phenols
Table 1 shows the extraction yields and total phenols, for the FDH extract of selected herbs. The extraction yields was 245; 210 and 130 mg g^-1 for clove; coriander and caraway, respectively. Significant association found between the extraction yields and total phenols and the results from the different antioxidant assays. Phenolic substances which investigated by the Folin-Ciocalteu method (mg gallic acid/g extract) have been shown to be responsible for the antioxidant activity of plant materials. Phenolic substances were found in the order clove>coriander>caraway (p<0.05). The content of total phenolic shows a good correlation with the iron reduction assay (R² = 0.8871, p<0.05) and inhibition of lipid peroxidation assay (R² = 0.7327, p<0.05), while no correlation with the iron (III) to iron (II)-reducing assay or iron (II) chelation assay. The total phenols/extractable compounds ratio was 33.7% for clove; 28.6% for coriander and 18.4% for caraway.

Iron (III) to Iron (II)-Reducing Activity
Fe (III) reduction is often used as an indicator of electron-donating activity, which is an important mechanism of phenolic antioxidant action (Yildirim et al., 2001). The iron (III) to iron (II)-reducing activity is expressed as ascorbic acid equivalents (mmol ascorbic acid/g sample). The ascorbic acid equivalent in clove extract was significantly higher than for the other extracts (p<0.05). Ascorbic acid equivalents was 0.78 mmol ascorbic acid/g for clove extract; 0.32 mmol ascorbic acid/g for coriander extract and 0.13 mmol ascorbic acid/g for caraway extract.

Peroxidation of Linoleic Acid
In the haemoglobin-catalysed peroxidation of linoleic acid assay, linoleic acid served as a lipid model. Peroxidation was induced by haemoglobin and the damage was assayed following the thiocyanate and Trolox was used as reference substance (Kuo et al., 1999). Results indicated that clove was significantly better inhibitors of lipid peroxidation than the other extracts (p<0.05), Trolox equivalents ranked from 443 ± 12.11 mg trolox/g extract for clove to 93 ± 4.21 mg trolox/g extract for caraway. The antioxidant activity in this assay correlated well only with iron reduction (R² = 0.8958, p<0.05).

Iron (II) Chelation
Foods contaminated with transition metal ions which may be introduced by processing methods, these ions play an important role as catalysts of oxidative processes, leading to the formation of hydroxyl radicals and hydroperoxide decomposition reactions via., Fenton chemistry (Halliwell, 1997). These processes can be delayed by iron chelation and deactivation. Therefore, the ability of the extracts to chelate iron (II) ions was evaluated and expressed as mg Na₂EDTA/g extract. Clove show the significantly (p<0.05) best iron chelation (234 ± 5.21 mg Na₂EDTA/g extract), followed by coriander (143 ± 2.71 mg Na₂EDTA/g extract) and (87 ± 1.43 mg Na₂EDTA/g extract) for caraway. No significant correlation was found between the iron chelation ability of the extracts and the other antioxidant activities.

Effect of FDH Extract of Different Plants on Lipid Rancidity
In the present study the simple model system consists of butter oil mixed with FDH extract of selected herbs were designed to study oxidative process during storage. BHT and BHA are added as common effective synthetic antioxidants to food products in the range of 100-400 ppm (Allen and Hamiliton, 1999).

Table 1:	Extraction yields and total phenols for hydrodistilled extracts

Table 2:	Acid values for Butter Oil (BO) mixed with BHT and different FDH extract during storage
Acid value is expressed as mg KOH /1 g butter oil

Fig. 1:	Effect of different FDH extract on Butter Oil (BO) oxidative rancidity during storage

Experiments were conducted using BHT at 200 ppm in order to compare the anti-oxidative activity of different FDH extract towards butter oil rancidity. A high storage temperature (60 ± 1°C) was chosen to accelerate of BO oxidation due to a low degree of unsaturation of butter oil (Thampson, 1960). The experimental period was terminated when an objectional odour and high lipid rancidity values were obtained with the samples after 28 days.

Butter Oil System
Acid Values (AV) of butter oil (control) and mixed with different FDH extract at different rate and BHT during storage is shown in Table 2 and Fig. 1. The AV results indicate that different FDH extract exhibited antioxidant effect specially at rate of 400 ppm from different extracts with order of clove> coriander>caraway. Changes in the PV of butter oil mixed with different FDH extract at different rate and BHT during storage are shown in Fig. 1. The PV values of BO (control) and mixed with different extracts were very low at zero time and gradually increased with storage time. BHT produced significantly lower PV of butter oil when compared with other treatments. The addition of different extracts was effective as antioxidant when compared with the control experiment and in the same order; clove>coriander>caraway. The changes in the secondary oxidation products of BO are shown in Fig. 2, a result of the addition of different FDH extract based on the dada of TBA values. Addition of BHT at 200 ppm to butter oil caused very low increase in TBA values over time. On the other hand, the systems containing different FDH extract at different rate significantly linear reduce the formation of secondary oxidation products in comparison with the control experiment. The inhibitory effect of antioxidants against oxidation process in butter oil has been attributed to their donation of electrons or hydrogen atoms from phenolic hydroxyl groups to butter oil containing free radicals and to the formation of stable free radicals which do not initiate or propagate further oxidation of oils (Farag et al., 1993;Wollgast and Anklam, 2000).

Fig. 2:	Effect of different FDH extract on Butter Oil (BO) secondary oxidation products

The good correlation between the results from total phenols analysis and the antioxidative assays has been earlier reported by Zheng and Wang (2001) and Dorman et al. (2003, 2004).

In complex systems, such as food and food preparations, various different mechanisms may contribute to oxidative processes, such as in Fenton reactions, where transition metal ions play a vital role, different reactive oxygen species might be generated and various target structures such as lipids, proteins and carbohydrates, can be affected. Therefore, it is important to characterize the extracts by a variety of antioxidant assays (Halliwell, 1997; El-Ghorab et al., 2008; Ollanketo et al., 2002).

CONCLUSION

The result of the present study showed that the different FDH extracts which contain highest amount of phenolic compounds, exhibited the greatest antioxidant activity. All of the extracts in this research exhibited different extent of antioxidant activity. The catalytic effectiveness of the added materials can be ranked according to inhibition power as BHT (200 ppm)>clove>coriander>caraway> control. Plant extracts might substitute synthetic food antioxidants, which may influence human health when consumed chronically (Martinez-Tome et al., 2001).

ACKNOWLEDGMENT

This study funded by Taif University, Faculty of Science, Chemistry Department, Kingdom of Saudi Arabia.