Objective To estimate the free radical scavenging activity of the acetone extract/fractions of Acacia nilotica （L.） Willd.Ex Del.,in the 1’-1’ diphenylpicryl-hydrazyl （DPPH） free radical scavenging,reducing and chelating power and lipid peroxidation assays.Methods The bark powder of the plant was extracted using two orders of solvent polarities by maceration extraction method and then the acetone extract was partitioned with ethyl acetate and water.L-ascorbic acid and BHT were used as reference antioxidants for comparing the activity of plant extracts/fractions.Results The scavenging activity of crude extract was found to be less than the fractions isolated.The percent inhibition with water fraction of acetone extract was 71.2%,73.66%,and 83.37% in DPPH,chelating power and lipid peroxidation assays respectively at maximum concentration.Conclusions Studies are in progress to evaluate the effect of extracts/ fractions in other antioxidant assays and also for the identification of the factors responsible for their antioxidant activity.

【关键词】  free radicals;1’-1’ diphenylpicryl-hydrazyl;antioxidant test;lipid peroxidation;Acacia nilotica;phenols

　　Correspondence to Dr.（Mrs.）Saroj Arora （Reader），Department of Botanical and Environmental Sciences,Guru Nanak Dev University,Amritsar 143005 Punjab,India

   　　 E-mail:jrosh1@rediffmail.com

　　 INTRODUCTION

    Aerobic metabolism entails the production of reactive oxygen species （ROS）;hence,there is a continuous requirement of antioxidants for their inactivation.In various diseases,the steady state of prooxidants and antioxidants may be disrupted in favor of the former,leading to oxidative stress,which may affect all types of biological molecules,including DNA,lipids,proteins,and carbohydrates［1,2］.Thus,oxidative stress may be involved in processes such as mutagenesis,carcinogenesis,lipid peroxidation,oxidation,and fragmentation of proteins,as well as carbohydrate damage［3,4］.

    The protection of the organism against oxidative stress relies not only on endogenous antioxidants but also on exogenous compounds taken in food［5］.Natural antioxidants in leafy vegetables and seeds,such as ascorbic acid,vitamin E and phenolic compounds,possess the ability to reduce the oxidative damage associated with many diseases,including cancer,cardiovascular disease,cataracts,atherosclerosis,diabetes,arthritis,immune deficiency diseases and aging［6-8］.To characterize a substance as an antioxidant,its interaction against a wide range of species more directly responsible for oxidative damage should be assessed.In particular,a complete screening of antioxidative ability should include assessment on the capacity of a putative antioxidant to scavenge superoxide,hydroxyl radical,and ferryl species,because some of the therapeutic actions of herbal drugs,such as antimutagenicity,anticarcinogenicity,and antiaging,may be due to the antioxidant activity of their constituents［9,10］.

    The recent trend of the science is to explore the antioxidant potential of natural compounds.So,keeping in mind the objective of this work was to investigate the antioxidative activity of acetone extract/fractions of acacia nilotica Willd Ex Del employing the DPPH radical-scavenging,reducing power,Fe2+ chelating ability and lipid peroxidation assay.

    MATERIALS AND METHODS

    The Preparation of the Experiment

    Acacia nilotica （L.） Willd.Ex Del.,is a medicinal tree which is known to be rich in phenolics consisting of condensed tannin and phlobatannin,gallic acid,protocatechuic acid pyrocatechol,（+） -catechin,（-） epigallocatechin-7-gallate,and （-） epigallocatechin-5,7-digallate.Bark on is trunk tough,fissured blackish gray or brown,powdery and peeling and has been used for colds,bronchitis,diarrhoea,dysentery,biliousness,bleeding piles and leucoderma［11-13］.

    The bark used in the present study was collected in November from a tree growing in the Guru Nanak Dev University Campus,Amritsar.It was washed with tap water,dried in oven at 40 ℃ for 24 hours and then ground to a fine powder.

    All chemicals like potassium ferricyanide,trichloroacetic acid,ferric chloride,EDTA,hydrogen peroxide,L-ascorbic acid,sodium hydroxide,BHA,KCl,sodium carbonate and other solvents used for extract preparation were of analytical grade.1’-1’ diphenylpicryl-hydrazyl （DPPH） and 2-thiobarbituric acid were obtained from Sigma Aldrich USA.Positive control L-ascorbic acid,BHT and ferrozine were procured from CDH and SRL respectively.

    Maceration Extraction

    The extracts of plant material were made by maceration extraction method at room temperature （Flow Chart 1 and 2）.Briefly 1 kg of fine bark powder was soaked sequentially in 3,000 ml of solvents in the order i.e. hexane,chloroform,ethyl acetate,acetone,methanol and then in the reverse order to prepare respective extracts.After filtering through folded paper,the supernatant in different solvents was recovered.Now respective solvents from the supernatant were evaporated through vacuum rotary evaporator to obtain the crude extract （CE） in dry form.

    Partitioning of Extract

    The crude acetone extract was then fractionated by double distilled water and ethyl acetate to obtain water fraction （WF） and ethyl acetate fraction （EAF）.For checking the antioxidant activity,each extract/fraction was dried and redissolved in methanol.

    Antioxidant Testing Assays

    The hydrogen donating activity of extract/fractions was measured by using the stable radical DPPH［14］.The reaction mixture contained 300 ml of different extract/fraction concentrations （1～100 mg/ml.） and 2 ml of DPPH（0.1 mM in methanolic solution）.The bleaching of colour of reaction mixture was measured by spectrophotometer （Systronic 2202 UV-VIS spectrophotometer） against the blank,which did not contain the extract/ fraction at 517 nm.The absorbance measured after 5 min of the antioxidant-DPPH radical reaction was used to compare the DPPH radical scavenging capacity of each extract/fractions.The L-ascorbic acid was used as the positive control.The percent DPPH decolourization of the sample was calculated by the equation:

    % Inhibition=B0-B1/B0×100

    B0:is the absorbance of negative control.

    B1:is the absorbance of reaction mixture.

    The IC50 values （inhibitory concentration 50） which is the amount of sample necessary to decrease the absorbance of DPPH by 50% was measured by plotting sample extract concentration against the absorbance.

    Reducing Power Assay

    The reducing power of extract/fractions was determined by the method of Oyaizu （1986）［15］.Different concentrations of extract/fractions in 1 ml of distilled water were mixed with phosphate buffer （2.5 ml,0.2 M,pH 6.6） and potassium ferricyanide ［K3Fe（CN）6］ （2.5 ml,1%）.The mixture was incubated at 50℃ for 20 min.Aliquots （2.5 ml） of trichloroacetic acid （10%） were added to the mixture,which was then centrifuged for 10 min at 1,036 g.The upper layer of solution （2.5 ml） was mixed with distilled water （2.5 ml） and FeCl3 （2.5 ml,0.1%）,and the absorbance was measured at 700 nm in a spectrophotometer.Increased absorbance of the reaction mixture indicated the increased reducing power.

        Flow Chart 1  Maceration extraction of bark powder of Acacia nilotica by increasing order of solvent polarity

    Flow Chart 2  Maceration extraction of bark powder of Acacia nilotica by decreasing order of solvent polarity

    Chelating Effects on Ferrous Ions

    The chelating effect on ferrous ions was determined according to the method of Dinis［16］,with some modifications.The extracts/fractions （0.25 ml） were mixed with 1.75 ml of methanol and 0.25 ml of 250 mM FeCl2.This was followed by the addition of 0.25 ml of 2 mM ferrozine,which was left to react at room temperature for 10 min before determining the absorbance of the mixture at 562 nm.The chelating effect （%） was calculated from the formula as given for DPPH scavenging assay.

    Lipid Peroxidation by Thiobarbituric Acid （TBA） Assay

    TBA reacts with malondialdehyde （MDA） to form a diadduct,a pink chromogen,which can be detected spectrophotometrically at 532nm［17］.Normal male rats （250 g） were used for the preparation of liver homogenate.The perfused liver was isolated,and 10% （w/v） homogenate was prepared with homogenizer at -4 ℃ with 0.15 M KCl.The homogenate was centrifuged at 800 g for 15 minutes,and clear cell-free supernatant was used for the study of in vitro lipid peroxidation.Different concentrations （50～400 μg/ml） of extract/fractions dissolved in methanol were taken in test tubes.One milliliter of 0.15 M KCl and 0.5 ml of rat liver homogenates was added to the test tubes.Peroxidation was initiated by adding 100 μl of 0.2 mM ferric chloride.After incubation at 37℃ for 30 minutes,the reaction was stopped by adding 2 ml of ice-cold HCl （0.25 N） containing 15% trichloroacetic acid （TCA）,0.38% TBA,and 0.5% BHT.The reaction mixtures were heated at 80℃ for 60 minuets.The samples were cooled,centrifuged and the absorbance of the supernatants was measured at 532 nm.The percentage inhibition of lipid peroxidation is calculated by the formula:

    Inhibition of lipid peroxidation （%）=1 -（sample OD/blank OD） × 100

    Statistical Analysis

    All experiments were repeated at least three times.Results were reported as Mean ± SE.

    RESULTS

    Figure 1 shows the percent decrease of absorbance caused by the different concentrations of extract/ fractions in DPPH assay.It was observed that crude acetone extract and its fractions exhibited an inhibitory potential in the dose dependent manner in the concentration （1～50 mg/ml）.The effect of the extract/ fraction though increases in the concentration （50～ 100 mg/ml） yet the increase was very less.It was also observed that with the increase in concentration the speed of the reaction also increased.

    Furthermore it was noticed that the rate of reaction of fractions and standard antioxidant （L-ascorbic acid） with DPPH was comparatively faster than with the crude extract,showing the higher H-donor capability of fractions.The water fraction showed its maximum H-donor ability of 71.2 % at concentration 100 mg/ml.It was also observed that the concentration higher than 100 mg/ml did not cause further decrease in absorbance.

    When IC50 values of crude extract,fractions and standard were compared,it was observed that IC50 value of water fraction was lower than that of the ethyl acetate fraction and crude extract,even ascorbic acid （Table 1）.The IC50 values were found to be in the order:water fraction （31.83 μg/ml） > ethyl acetate fraction （38.62 μg/ml） > L-ascorbic acid （48.30 μg/ml）.The IC50 value of the crude extract was comparatively higher and could not be calculated from the plot.The lower IC50 values for the fractions as compared to the crude extract coupled with their higher free radical scavenging activity showed that the fractions have more bioactive compounds as compared to the crude acetone extract.In nutshell,the order of free radical scavenging potential was observed to be Water fraction > Ethyl acetate fraction > Crude fraction.

    Table 1  IC50 Value of Acetone Extract/fractions of Acacia Nilotica and Standard Antioxidant in Different Antioxidant Systems

    Figure 1，2  The DPPH radical and reducing power potential of acetone extract/fractions of Acacia nilotica

    Figure 2 depicts the reducing potential of acetone extract/fractions obtained with the solvents in their increasing and decreasing order of polarity.The relative reducing power of water fraction of acetone extract obtained by both the orders of polarities and standard （BHT） was found to be maximum at 150 μg/ml concentration.In case of BHT,which is used,as reference antioxidant showed maximum reducing power of 1.971 at 90 μg/ml and it became constant at concentrations higher than 90 μg/ml.The water and ethyl acetate fractions showed reducing power of 1.804%,1.757% for increasing and 1.593%,1.354% for decreasing order of solvent polarity respectively at 150 μg/ml concentration.

    Figure 3 shows the chelating effect of acetone extract/fractions of Acacia nilotica on ferrous ions.The ability of chelating ferrous ions increased with increasing extract/fractions concentration.The chelating effect was found to be water fraction （83.62%） > ethyl acetate fraction （71.38%） > crude extract （28.18%） in case of extracts obtained by the order of polarity starting with hexane at maximum extract/fraction concentration i.e.400 μg/ml in case of increasing order of solvent polarity.

    The inhibition of LDL oxidation by acetone extract/fractions of Acacia nilotica （increasing and decreasing polarity） is shown in Figure 4.The water fraction strongly protected LDL from oxidation,exhibiting 88.33% inhibition at 700 μg/ml concentration as compared to the ethyl acetate fraction and crude extract of the same plant in increasing and decreasing order of polarity.Extracts were obtained by increasing order of solvent polarity,whose sequence of inhibition was:water fraction （83.37%） > ethyl acetate fraction

    Figure 3，4  The chelating power and lipid peroxidation inhibitory potential of acetone extract/fractions of Acacia nilotica

    （76.40%） > crude extract （32.22%）.In case of decreasing order of solvent polarity the trend is same but the percent inhibition was less in ethyl acetate fraction （64.72%） and crude extract （29.22%） but high in water fraction （88.33%） at 700 μg/ml concentration.The BHT was used as a standard polyphenol,which exhibited very high percent inhibition （98.27%） at 200 μg/ml concentration.

    DISCUSSION

    A critical examination of the results revealed that though all the extracts/fractions exhibited inhibition in all the assays,the water fractions were comparatively more effective than others.On analyzing the results obtained in DPPH assay,it was noticed that extracts/fractions act as good hydrogen donating agent,thereby bleaches the DPPH absorbance.More over it was seen that the water fractions obtained by increasing as well as by decreasing order of solvent polarities showed 71.2% and 87.70% inhibition respectively at 100 μg/ml concentration in DPPH scavenging assay and the same fraction also showed maximum inhibition at 400 μg/ml and 700 μg/ml in chelating and lipid peroxidation assays respectively.The results further indicate that the extract/fractions contain polyphenolic compounds,which are known scavengers and inhibitors of free radicals［18,19］.

    The reducing capacity assessed during this investigation is thought to be linked to important mechanism of phenolic antioxidant activity.As some researchers［20］ report that the antioxidative effect is concomitant with the development of reducing power.Therefore,reducing power evaluation was taken as an important parameter for the assessment of antioxidant activity.

    Iron is known to generate free radicals through the Fenton and Haber-Weiss reaction［21］.Metal ion chelating activity of an antioxidant molecule prevents oxyradical generation and the consequent oxidative damage likewise it is reported that the metal ion chelating capacity of the phenols and polyphenols plays a significant role in reducing the concentration of the catalysing transition metal in lipid peroxidation and stabilizing the oxidized form of the metal ion［22,23］.Chelation is also an important parameter in the sense that iron is essential for life because it is required for oxygen transport,respiration and the activity of many enzymes.However,iron is an extremely reactive metal and catalyzes oxidative changes in lipids,proteins and other cellular components［24,25］.

    In the lipid peroxidation assay,it is reported that the extracts/fractions may inhibit the formation of peroxides and hydroperoxides.So,the antioxidants in the extracts/fractions are therefore able to slow the process of rancidity and greatly increase the shelf life of foods.Among the radical scavenging assays used in the present study,it was observed that DPPH scavenging assay is best in estimating the total antioxidant potential.It is pertinent to mention that the results obtained in the present study are in conformity with our previous results on antimutagenic activity against genotoxic injury by NPD,sodium azide and 2-Aminofluorene in the Ames Salmonella histidine reversion assay［26,27］.

    Nearly all the extracts/fractions and standard antioxidant used for comparison studies on antioxidant activity clearly showed differences from test to test due to different reaction principles.Thus,it is strongly recommended and should be state of the art to use more than one assay to determine the antioxidant potential of food extracts or single compound.The broad range of antioxidant activity of the extracts indicates the potential of the bark as a source of natural antioxidants or nutraceuticals with potential application to reduce oxidative stress with consequent health benefits.

    Though other antioxidants are probably present in these extract/fractions,phenolic compounds might have made a significant contribution to their bioactivity.The work further reveals that the Acacia species can be an interesting source of antioxidants with their potential use in different fields （food,cosmetics,and pharmaceuticals）.A detailed chemical investigation of this plant to identify the compounds responsible for the antioxidant activity is in progress.

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（Editor HOU）