Antioxidant potential of various extracts of Cassia fistula was determined by the DPPH, FRAP, Fe3+ reducing power, and hydrogen peroxide scavenging assay. Methanolic extracts of Cassia fistula showed the highest amount of phenolic and flavonoid content and reducing capacity, whereas hexane extracts exhibited the lowest level of reducing capacity. The order of antioxidant activity in Cassia fistula extracts displayed from higher to lower level as methanolic extracts of pulp, methanolic extracts of seed, hexane extracts of pulp, and hexane extracts of seed. The antioxidant potential of Cassia fistula extracts significantly correlated (P<0.02) with the phenolic content of the methanolic extracts. Ascorbic acid taken as control showed highest antioxidant power in the present study.
1. Introduction
Cassia fistula Linn. (Caesalpiniaceae) has great therapeutic implication in Indian system of medicine and exerts an antipyretic, analgesic, antiinflammatory, and hypoglycemic effects [1, 2]. Over the past few years, there has been an exponential growth in study of pharmacological properties of this plant [3–5]. Antioxidant components are microconstituents that inhibit lipid oxidation by inhibiting the initiation or propagation of oxidizing chain reactions, and are also involved in scavenging of free radicals. Clinical approaches of antioxidants increased multifold during the recent time for the management and therapeutic implication of neurodegenerative disorders, aging, and chronic degenerative diseases. In view of the above, we designed the study to evaluate the antioxidant potential and phenolic content in Cassia fistula.
2. Materials and Methods2.1. Reagent and Chemicals
All the chemicals and solvents were of analytical grade and obtained from Merck and HiMedia, Mumbai, India.
2.2. Preparation of Cassia fistula Extracts
The fresh ripe fruits of Cassia fistula were collected in June from the campus of Delhi College of Pharmaceutical Science and Research (DIPSAR), New Delhi, India, and properly authenticated. A voucher of specimen (PM 21) was stored in the laboratory for further reference. The fruit pulp and seed were separated and grounded to coarse powder. It was extracted with the hexane for 72 hrs and the same materials were reextracted with methanol for 72 hrs in soxhlet apparatus. The extract was filtered and dried in rotavapour. Hence, we obtained hexane extract of seed (HES), hexane extract of pulp (HEP), methanolic extract of seed (MES), and methanolic extract of pulp (MEP).
2.3. Estimation of Total Phenolic Content
The total phenol content was estimated in the methanolic extracts of seed and pulp using Folin-Ciocalteu reagent (FCR) according to the procedure reported by Singleton et al. [6], using standard Gallic acid (Y=1.8478x+0.0702,R2=0.9971) and Tannic acid (Y=1.388x+0.0472,R2=0.9958), curve standardized in the lab for the calculation of Gallic acid equivalent (GAE) and Tannic acid equivalent (TAE) per gram of extracts, respectively. The blue complex was formed by the reduction of reagent by phenolic compounds in extract. Briefly, aliquot of 400 μL of extract was added to 1.6 mL of sodium carbonate (7.5% in deionised water) and 2 mL of Folin-Ciocalteu reagent (diluted 10-fold in deionised water). After incubation of 1 hr at room temperature, absorbance was measured at 525 nm using LaboMed Inc. Spectrophotometer (USA). All determination was carried out in triplicate. Total phenolic content was expressed in mg GAE and TAE per gram of extracts, using calibration curve.
2.4. Estimation of Flavonoid Content
Total flavonoids contents were estimated both in methanolic extracts of pulp and seed by method of Zhishen et al. [7], using Quercetin standard. Briefly, 0.5 mL of aliquot of extract was added to 75 μL of 5% NaNO2 solution. After 6 minutes, 150 μL of a 10% AlCl3 6H2O solution was added and the mixture was allowed to stand another 5 minutes. Then, 0.5 mL of 1 mol/l NaOH and 2.5 mL of distilled water was added. The solutions were mixed and absorbance was measured at 510 nm using LaboMed Inc. Spectrophotometer (USA). All experiments were carried out in triplicate. Total flavonoid content was calculated as mgQE/g, using the following equation based on the calibration curve:
(1)Y=2.234x+0.992,R2=0.9927,
where x was the absorbance and y was the mgQE/g.
2.5. DPPH Radicals Scavenging Activity
DPPH radicals scavenging of Cassia fistula extracts was estimated according to the method of Miliauskas [8, 9]. DPPH radicals absorbed maximum at 515 nm, which disappears with reduction by an antioxidant compound (s). Three milliliter (3 mL) DPPH solution in methanol (0.1 mM) was mixed with 100 μL of extracts (mg/mL). In control 100 μL methanol (without extracts) mixed with DPPH solution. The samples were incubated in a water bath for 20 min at 37°C, and the decrease in absorbance at 515 nm was measured. Ascorbic acid was used as positive reference. The experiment was carried out in triplicate. Radical scavenging activity was calculated using the following formula:
(2)%inhibition=[(AC-AE)AB]×100,
where AC= absorbance of the control and AE= absorbance of tested samples.
Ferric reducing power of Cassia fistula extracts were determined using FRAP assay [10, 11]. This method is based on the reduction of colourless ferric complex (Fe3+ tripyridyltriazine) to blue-colored ferrous complex (Fe2+ tripyridyltriazine) by the action of electron donating antioxidants at low pH. The reduction was monitored by measuring the change of absorbance at 593 nm. The working FRAP reagent was prepared by mixing 10 volumes of 300 mM acetate buffer, pH 3.6, with 1 volume of 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) in 40 mM HCl and with 1 volume of 20 mM ferric chloride. All the required solutions were freshly prepared before their uses. 100 μL of samples (mg/mL) were added to 3 mL of prepared FRAP reagent. The reaction mixture was incubated in a water bath for 30 min at 37°C. Then, the absorbance of the samples was measured at 593 nm. The difference between absorbance of sample and the absorbance of blank was calculated and used to calculate the FRAP value. FRAP value was expressed in terms of mmol Fe2+/g of sample using ferric chloride standard curve Y=1.7057x-0.2211,R2=0.9904. All measurements were calculated from the value obtained from triplicate assays.
2.7. Hydroxyl Radical Scavenging Activity Assay
The scavenging activity for hydroxyl radicals was measured with Fenton reaction [12, 13]. Reaction mixture contained 60 μL of 1.0 mM FeCl3, 90 μL of 1 mM 1,10-phenanthroline, 2.4 mL of 0.2 M phosphate buffer (pH 7.8), 150 μL of 0.17 M H2O2, and 1.5 mL of extract at various concentrations. After incubation at room temperature for 5 min, the absorbance of reaction mixture was noted at 560 nm. The hydroxyl radicals scavenging activity was calculated according to the following equation and compared with ascorbic acid as standard:
(3)%Inhibition=((AB-AE)AB×100),
where AB was the absorbance of blank (without extract) and AE was the absorbance of tested samples.
2.8. Reducing Power Assay
The Fe3+ reducing power of extracts was determined by the method of Oyaizu [9, 11, 14]. The extract (0.75 mL) of various concentrations was mixed with 0.75 mL of phosphate buffer (0.2 M, pH 6.6) and 0.75 mL of potassium hexacyanoferrate (K3Fe(CN)6) (1%, w/v), followed by incubation at 50°C in a water bath for 20 min. The reaction was stopped by adding 0.75 mL of trichloroacetic acid (TCA) solution (10%) and mixture centrifuged at 800 g for 10 min. 1.5 mL of the obtained supernatant was mixed with 1.5 mL of distilled water and 0.1 mL of ferric chloride (FeCl3) solution (0.1%, w/v) for 10 min. The absorbance of reaction mixture was taken at 700 nm. Higher value absorbance of the reaction mixture indicated greater reducing power.
3. Statistical Analysis
Results were expressed as mean value ± SD (n=3). Regression analysis was performed to calculate the dose-response relation between the extracts. Linear regression analysis was performed to find out the correlation coefficient. Statistical significance was evaluated employing t-test and P<0.05 which were considered to be significant.
4. Result and Discussion4.1. DPPH Radical Scavenging
DPPH (1,1-diphenyl-2-picrylhydrazyl) analysis is one of the best-known, accurate, and frequently employed methods for evaluating antioxidant activity [15]. It is a stable free radical because of its spare electron delocalization over the whole molecule. The donation of H+ to the DPPH radicals made a corresponding change from violet colour to pale yellow in the solution. The DPPH scavenging also made a proportionate decrease in its absorbance at 517 nm (see Scheme 1).
The order of DPPH scavenging against Cassia fistula extract was found to be in the order of MEP>MES>HEP>HES. Antioxidant activity of the methanol extracts of pulp and seed was also compared to total phenolic content and it was further found that radical scavenging effects of extracts were directly proportional to the phenolic content present in extracts (Table 1). Hence, methanol extract of pulp and seed showed the significant radical scavenging as the concentration increased, whereas hexane extract of pulp and seed showed low scavenging effect up to 1 mg/mL; thereafter, DPPH scavenging increased with the concentration of extract. The positive control, ascorbic acid showed maximum scavenging effect at very low concentration (Figure 1). A significant correlation coefficient (r, 0.978) was found between the antioxidant activity of the methanolic extract of pulp and seed. The proton radical scavenging action is known to be one of the important mechanisms for measuring antioxidant activity. This assay determines the scavenging of stable radical species DPPH by antioxidants compounds present in the extracts. The results showed the greater rate of DPPH scavenging activity of both the methanolic extracts as compared to thehexane extracts probably due to the presence of high content of phenolic compounds. EC50 value was determined from the plotted graph of scavenging activity against various concentrations of extracts, which is defined as the efficient concentration of antioxidant necessary to decrease the initial DPPH radicals concentration by 50% (Table 2). The lowest EC50 indicates the strongest ability of the extracts to act as DPPH radicals scavengers. Out of the all extracts, methanolic extract of pulp and seed showed the lowest EC50, 0.915 and 1.088 mg/mL, whereas hexane extracts of pulp and seed showed 1.865 and 2.239 mg/mL, respectively (Table 2). Ascorbic acid showed highest DPPH radicals scavenging with EC50 of 0.102 mg/mL. Sun and Ho reported a significant correlation between total phenolics and scavenging ability of buckwheat extracts on DPPH radicals [16]. Our study clearly indicated that the methanol extracts of pulp and seed of Cassia fistula exhibited high content of phenolic compounds which was significantly correlated with the DPPH radical scavenging activity (P<0.005).
Quantification of total phenolic and flavonoid contents in methanolic extract of pulp (MEP) and seed (MES).
EC50 value is defined as the effective concentration of antioxidant necessary to decrease the radical concentration by 50%. FRAP values represent as equivalent mmol of Fe2+/gram sample.
Cassia fistula extracts
EC50 (DPPH) mg/mL
EC50 (hydroxyl peroxide)mg/mL
FRAP value
MEP
0.915
0.889
136.05
MES
1.088
1.058
112.02
HEP
1.865
1.723
75.09
HES
2.239
2.075
69.02
Ascorbic acid
0.102
0.105
—
The graphical representation of DPPH scavenging activity of hexane extracts of seed (HES), pulp (HEP), methanolic extracts of seed (MES), and pulp (MEP). Values are expressed as mean ± standard deviation (n=3). Ascorbic acid (AA) was used as a standard.
4.2. Hydroxyl Radical Scavenging Activity
The hydroxyl radicals are extremely reactive oxygen species that can react with every possible molecule in living organisms, especially with proteins, DNA, and lipids [17]. Hydroxyl radicals are capable of rapid initiation of the lipid peroxidation process by extracting hydrogen atoms from unsaturated fatty acids [18]. The electron or proton donation capacities of Cassia fistula extracts were further confirmed by the Fenton reaction system. Under in vitro, condition very reactive OH- radicals were formed through the Fenton reaction. The reversible reaction between Fe2+ and 1,10-Phenathroline formed Fe(phen)22+, which react with H2O2 and formed OH- free radicals [19, 20]:
(4)Fe(phen)32+⇆Fe(phen)22++phen,Fe(phen)22++H2O2→Fe(phen)23++OH-+{OH●}OH●.
Among the reactive oxygen species (ROs), H2O2 is the most reactive and predominant radical generating molecule endogenously during aerobic metabolism [21]. The results showed that methanolic extracts of pulp and seed of Cassia fistula have scavenging ability of OH- free radicals in a dose-dependent manner (Figure 2). The highest activity was noted for the pulp extract of methanol followed by methanolic extract of seed at all concentrations. OH- free radical scavenging potential of all the extracts was found to be lower than that of the reference compound ascorbic acid. The EC50 values of MEP, MES, HES, HEP, and ascorbic acid were 0.889, 1.058, 2.075, 1.723, and 0.105 mg/mL, respectively (Table 2). The trend obtained in OH- free radical scavenging behaviour was similar to the DPPH free radical scavenging nature (Figure 2). The result of OH- free radical scavenging activity was significantly correlated with the DPPH free radical scavenging (P,0.05).
The graphical representation of hydroxyl radical scavenging activity of hexane extracts of seed (HES), pulp (HEP), methanolic extracts of seed (MES), and pulp (MEP). Values are expressed as mean ± standard deviation (n=3). Ascorbic acid (AA) was used as a standard.
4.3. Reducing Power
The reducing power assay is often used to evaluate the ability of an antioxidant to donate an electron [22]. In this assay, the ability of extracts to reduce Fe3+ to Fe2+ was determined. The presence of antioxidants in the extracts resulted into reduction of the ferric cyanide complex (Fe3+) to the ferrous cyanide form (Fe2+). In reducing power assay, antioxidants cause the reduction of the Fe3+ into Fe2+, thereby changing the solution into various shades from green to blue, depending on the reducing power of the compounds [23]. Strong reducing agents, however, formed Perl’s Prussian blue colour and absorbed at 700 nm. Figure 3 showed the reducing activities of various extracts of Cassia fistula in comparison with ascorbic acid as standard. The higher the absorbance of the reaction mixture, the higher would be the reducing power. Methanolic extract of pulp and seed showed some degree of electron donation. Reducing power of different extracts increased with the concentration of the extract. Hexane extract of seed and pulp showed less degree of Fe3+ reduction than the methanolic extracts. The reducing power was found to be in order of MEP>MES>HEP>HES. Interestingly, the rate of reducing power of methanolic extracts of both pulp and seed (MEP and MES) first increased rapidly with concentration but later it decreased. The reducing power of reference compound (Ascorbic acid) was found to be higher than all the tested extracts. It has been reported that the reducing power of substances is probably because of their hydrogen-donating ability [24]. Methanolic extracts of pulp and seed might, therefore, contain high amount of reductones than hexane extracts of pulp and seed. Hence, methanolic extracts of fruit pulp and seed may act as electron donors and could react with free radicals to convert them into more stable products and then terminate the free radical chain reactions.
The graphical representation of reducing power of hexane extracts of seed (HES), pulp (HEP), methanolic extracts of seed (MES), and pulp (MEP). Values are expressed as mean ± standard deviation (n=3). Ascorbic acid (AA) was used as a standard.
Bhalodia et al. [25] recently reported antioxidant activity of the Cassia fistula flower. They reported that the hydroalcoholic extract of flower demonstrated significant radical scavenging activity in ferric reducing power and DPPH assays. It was also correlated with the total phenols which could be responsible for the antioxidant activity [25].
The reducing power assay is often used to evaluate the ability of an antioxidant to donate an electron which is an important mechanism of phenolic antioxidant action [17]. Many reports have revealed that there is a direct correlation between antioxidant activities and reducing power of certain plant extracts [26, 27].
4.4. FRAP Assay
FRAP assay measures the reducing potential of an antioxidant reacting with a ferric tripyridyltriazine (Fe3+-TPTZ) complex and producing a coloured ferrous tripyridyltriazine (Fe2+-TPTZ) [28]. The reducing properties associated with the presence of compounds exert their action by breaking the free radical chain through donating a hydrogen atom [29, 30]. FRAP assay showed positive correlation between reducing power and phenolic content in Cassia fistula extracts (Table 2). Here also, methanolic extracts of pulp showed greater FRAP value as 136.05 equivalent mmol of Fe2+/g sample. The other extracts MES, HEP, and HES showed FRAP value 112.02, 75.09, and 69.02 equivalent mmol of Fe2+/g samples, respectively. The result was similar with Benzie and Szeto [27], who found a strong correlation between total phenolic content and FRAP assay. Rice-Evans et al. [30] reported that phenolic compounds have redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. The redox potential of phenolic compounds played an important role in determining the antioxidant potential.
5. Conclusion
Antioxidant potential of Cassia fistula extract demonstrated the highest reducing power in the methanolic extract of pulp and seed. It was concluded that the antioxidant activity of these extracts was directly proportional to the phenolic contents. The hexane extracts of seed and pulp, however, did not significantly reduce the free radicals under in vitro study.
Acknowledgment
The authors acknowledge Professor M. P. Sharma (Department of Botany, Jamia Hamdard, New Delhi, India) for the identification of plant materials.
PatelD. G.KarbhariS. S.GulatiO. D.GokhaleS. D.Antipyretic and analgesic activities of Aconitum spicatum and Cassia fistula1965157122272-s2.0-0013800943RizviM. M. A.IrshadM.El HassadiG.YounisS. B.Bioefficacies of Cassia fistula: an Indian labrum2009362872922-s2.0-77953675868BhaktaT.MukherjeeP. K.MukherjeeK.PalM.SahaB. P.Studies on in vivo wound healing activity of Cassia fistula linn. Leaves (Leguminosae) in rats19984284872-s2.0-0031878964IrshadM.SinghM.RizviM. M. A.Assessment of anthelmintic activity of Cassia fistula L20105346349IrshadM.ShreazS.ManzoorN.KhanL. A.RizviM. M. A.Anticandidal activity of Cassia fistula and its effect on ergosterol biosynthesis20114977277332-s2.0-7995816263910.3109/13880209.2010.544318SingletonV. L.OrthoferR.Lamuela-RaventósR. M.Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent19982991521782-s2.0-003179024210.1016/S0076-6879(99)99017-1ZhishenJ.MengchengT.JianmingW.The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals19996445555592-s2.0-003300472910.1016/S0308-8146(98)00102-2MiliauskasG.VenskutonisP. R.Van BeekT. A.Screening of radical scavenging activity of some medicinal and aromatic plant extracts20048522312372-s2.0-034684996710.1016/j.foodchem.2003.05.007BoonchumW.PeerapornpisalY.KanjanapothiD.PekkohJ.PumasC.JamjaiU.AmornlerdpisonD.NoiraksarT.VacharapiyasophonP.Antioxidant activity of some seaweed from the Gulf of Thailand201113195992-s2.0-79951934131DudonnéS.VitracX.CoutiéreP.WoillezM.MérillonJ. M.Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays2009575176817742-s2.0-6454916153810.1021/jf803011rLuqmanS.SrivastavaS.KumarR.MauryaA. K.ChandaD.Experimental assessment of Moringa oleifera leaf and fruit for its antistress, antioxidant, and scavenging potential using in vitro and in vivo assays201220125190842-s2.0-8486341131510.1155/2012/519084YuL.HaleyS.PerretJ.HarrisM.WilsonJ.QianM.Free radical scavenging properties of wheat extracts2002506161916242-s2.0-003707036110.1021/jf010964pGanuG. P.JadhavS. S.DeshpandeA. D.Antioxidant and antihyperglycemic potential of methanolic extract of bark of mimusops elengi l. In mice2010221161232-s2.0-7995214456510.5138/ijpm.2010.0975.0185.02018OyaizuM.Studies on products of browning reactions: antioxidant activities of products of browning reaction prepared from glucose amine198644307315ZhouK.YuL.Effects of extraction solvent on wheat bran antioxidant activity estimation20043777177212-s2.0-384309257310.1016/j.lwt.2004.02.008SunT.HoC. T.Antioxidant activities of buckwheat extracts20059047437492-s2.0-704422047810.1016/j.foodchem.2004.04.035MohamedH.OnsM.YosraE. T.RaydaS.NejiG.MoncefN.Chemical composition and antioxidant and radical-scavenging activities of Periploca laevigata root bark extracts20098958979052-s2.0-6384914883510.1002/jsfa.3532AruomaO. I.Free radicals, oxidative stress, and antioxidants in human health and disease19987521992122-s2.0-0031996717BurgessJ.PrinceR. H.1132. Kinetics of reactions of ligand-substituted tris-(2,2′- bipyridyl)iron(II) complexes1965606160662-s2.0-3704904822510.1039/JR9650006061CyfertM.Kinetics of reaction of Fephen32+ with hydrogen peroxide in neutral medium198598125282-s2.0-0344939532WallingC.Fenton's reagent revisited1975841251312-s2.0-1542789741YildirimA.MaviA.OktayM.KaraA. A.AlgurO. F.BilalogluV.Comparison of antioxidant and antimicrobial activities of Tilia (Tilia argentea Desf ex DC), sage (Salvia triloba L.), and Black tea (Camellia sinensis) extracts20004810503050342-s2.0-003378241710.1021/jf000590kFerreiraI. C. F. R.BaptistaP.Vilas-BoasM.BarrosL.Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: individual cap and stipe activity20071004151115162-s2.0-3374657017310.1016/j.foodchem.2005.11.043ShimadaK.FujikawaK.YaharaK.NakamuraT.Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion19924069459482-s2.0-33751390842BhalodiaN. R.NariyaP. B.AcharyaR. N.ShuklaV. J.Evaluation of in vitro antioxidant activity of flowers of Cassia fistula Linn20113589599KolevaI. I.Van BeekT. A.LinssenJ. P. H.De GrootA.EvstatievaL. N.Screening of plant extracts for antioxidant activity: a comparative study on three testing methods20021318172-s2.0-003616404110.1002/pca.611BenzieI. F. F.SzetoY. T.Total antioxidant capacity of teas by the ferric reducing/antioxidant power assay19994726336362-s2.0-003301047710.1021/jf9807768GordonM. H.HudsonB. J. F.The mechanism of antioxidant action in-vitro1990London, UKElsevier Applied Science118DuhP. D.DuP. C.YenG. C.Action of methanolic extract of mung bean hulls as inhibitors of lipid peroxidation and non-lipid oxidative damage19993711105510612-s2.0-003275226710.1016/S0278-6915(99)00096-4Rice-EvansC. A.MillerN. J.PagangaG.Antioxidant properties of phenolic compounds19974304309