Abstract: Antioxidant activities of Freeze-Dried Hydrodistilled (FDH) extract of three herbs; clove, (Syzygium aromaticum L.); caraway (Carum carvil L.) and coriander (Coriandum satvium L.) was evaluated in vitro by iron reduction; iron chelation and inhibition of lipid peroxidation methods. Also, total phenols content and the extraction yield was determined. Furthermore, simple model of butter oil was designed for evaluate the produced extracts as natural preservatives. Acid value, peroxide value and thiobarbituric acid (TBA) test were determined for oxidation system of butter oil. Antioxidant activity of clove FDH extract was significantly higher than caraway and coriander extracts when evaluated in iron reduction; iron chelation and inhibition of lipid peroxidation methods. Based on acid value, peroxide value and thiobarbaturic acid test, different FDH extracts exhibited antioxidant effect specially at rate of 400 ppm from individual extracts with order of clove>coriander >caraway. Also, different correlation relationships were recorded in present study.
INTRODUCTION
Food quality decreasing occurs during processing and storage is related to oxidative processes which cause degradation affects on lipids, carbohydrates and protein these are often catalyzed by e.g., ferrous or copper (Halliwell, 1997). Synthetic antioxidants such as BHA (butylated hydroxyl anisole) or BHT (butylated hydroxyl toluene) usually used to decelerate these processes. These antioxidants suffer from the disadvantage that they are volatile and easily decompose at high temperatures. Additionally, it is still unclear whether chronic consumption can lead to health risks (Martinez-Tome et al., 2001). Plant-derived food additives, especially polyphenolic compounds, have also been ascribed health-promoting properties, as for example in terms of prevention of chronic cardiovascular diseases (Harborne and Williams, 2000; Singh et al., 2008).
Such food additives are required to be odour free and tasteless. Many herbs and spices, usually used as an excellent source of phenolic compounds which have been reported to serve as natural food preservatives due to their good antioxidant activity (Rice-Evans et al., 1996; Zheng and Wang, 2001; Justescn and Knuthsen, 2001). However, herbs and spices usually contain essential oil which show antioxidant activity but also carry flavour. Thus, the preparation of hydrodistilled extracts is using to remove the intrinsic flavour from the plant material. Furthermore, use of an aqueous solvent prevent solubility problems and this avoids harmful residues from organic solvents. Also, hydrodistilled extracts may use in the functionalization of foods and beverages as source of phenolic compounds which has a health-promoting properties (Harborne and Williams, 2000; Hinneburg et al., 2006; Ruberto et al., 2000; Teissedre and Waterhouse, 2000).
Some studies concerned for evaluate antioxidant activity of FDH extracts of different herbs (Dorman et al., 2003; Hinneburg et al., 2006; El-Ghorab et al., 2008), but there is no study about clove, caraway and coriander FDH extracts. In present study, FDH extracts were prepared from three aromatic plants; clove, (Syzygium aromaticum L.); caraway (Carum carvil L.) and coriander (Coriandum satvium L.). Also, total phenols content and extraction yields were determined and in vitro antioxidant activity (iron reduction; iron chelation and inhibition of lipid peroxidation) was studied in producing extracts. Furthermore, simple model of butter oil was designed for evaluate the produced extracts as natural preservatives.
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 (As)
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 (Ac) and of the sample
(As) 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.