Chao-hua Zhu,Zhen-lin Lei,Yan-ping Luo

Key Laboratory of Protection and Development Utilization of Tropical Crop Germplasm Resources,Ministry of Education,College of Environment and Plant Protection,Hainan University,Haikou,Hainan 570228,PR China

Abstract Murraya paniculata(L.),a well-known medical plant,has widely been used to treat inflammation stomach ache,internal and external injuries,and for other purposes.In this study,we determined the reducing,lipid peroxidation inhibition,1,1-diphenyl-2-picryl-hydrazil radical(DPPH?)scavenging,superoxide anion radical(O2??)scavenging,hydroxyl radical(HO?)scavenging and hydrogen peroxide(H2O2)scavenging activities of the methanolic extract of M.paniculata (MPE) by UV–vis spectrophotometer.The results showed that M.paniculata was rich in fl vonoids(375 mg RE/g of extract).Reducing, lipid peroxidation inhibition and HO? scavenging activities of the extract were 0.26 mg/mL, 0.023 mg/mL and 0.302 mg/mL,respectively,these activities were significantl higher than those of trolox.Other antioxidative behavior indicators,i.e.,DPPH?scavenging, O2?? scavenging and H2O2 scavenging activities of MPE were 0.93 mg/mL, 0.581 mg/mL and 0.47 mg/mL, respectively, and were comparable to those exhibited by trolox.These results indicate that the methanolic extract of M.paniculata exhibited strong antioxidative and radical scavenging activities.

Keywords: Antioxidative activity;Flavonoids;Methanol extract;Murraya paniculata;Radical scavenging activities

1.Introduction

Murraya paniculata(L.) belongs to the Rutaceae family.This plant is native to areas in Southeast Asia [1], such as Southern China, Vietnam and Malay Peninsula.In China, the ornamental plantM.paniculatais widely cultivated on the sides of the road for decorative purposes.In Southeast Asia,it has been used for topical medicinal applications as health food[2].For example,a solution of the bark ofM.paniculata,when mixed with other ingredients, is used as an antidote for snake bites.The leaves ofM.paniculataare known to possess antibiotic activity againstMycococcus pyogenesandEscherichia coli[3].Furthermore, the leaves and roots of this plant are utilized in folk medicine to treat stomach ache,tooth ache,gout,diarrhea,dysentery,rheumatism,cough and hysteria[4,5].The use ofM.paniculatahas also been recommended for the treatment of cuts,joint pains and body aches[6].

A few researches have extensively investigated the antioxidant ofM.paniculata.For examples,Chen and co-workers[7]have reported that MPE is a stronger antioxidant than vitamin E.During a preclinical study, Gautam and collaborators [8] have found that MPE decreases the free radical level in diabetic rats significantl .Rodríguez and collaborators[9]have reported the antioxidant activity ofM.paniculataessential oil is stronger than that butylated hydroxyanisole and butylated hydroxytoluene.Mita and collaborators[10]have determined that total phenolic content of MPE is high and its free radical scavenging activity is excellent.

Several secondary metabolites (＞70 fl vonoids) have been isolated from the leaves and roots ofM.paniculata,Present in several medical plants,fl vonoids[11–16],with their hydroxyl groups and unsaturation, are widely recognized for their natural antioxidant properties.Frequently, the antioxidant activity of an extract correlates linearly with the total fl vonoid content of the extracts [17,18].In this study, we have evaluated the methanol extract ofM.paniculata(MPE)for its reducing activity, lipid peroxidation inhibition, radical scavenging activity(for radicals such as 11-diphenyl-2-picryl-hydrazil (DPPH?),superoxide anion(O2??),and hydroxyl(OH?)),and hydrogen peroxide(H2O2)scavenging activity.

2.Materials and methods

2.1.Chemicals

Rutin (C27H30O16), water-soluble vitamin E (Trolox),1,1-diphenyl-2-picrylhydrazyl (DPPH?), linoleic acid(C18H32O2), potassium nitro blue tetrazolium (NBT),Nmethyl phenazinemethosulfate (PMS), nicotinamide adenine dinucleotide (NADH),D-2-deoxyribose (C4H9O3CHO),thiobarbituric acid (TBA), horseradish peroxidase (HRP)were obtained from sigma chemicals.All other chemicals and reagents were procured from commercial sources in China and were analytical grade.

2.2.Plant material and extraction

Whole plant samples ofM.paniculatawere picked from Hainan University campus during May,2012.These plants were washed with fresh water, and then dried in the shade for several days.The dried plant samples were ground into powder through 40 meshes, and then stored at room temperature (RT)for subsequent use.

To extract the compounds soluble in methanol, the ground plant samples(20 g)were soaked in methanol at room temperature,Sufficien quantity of methanol,i.e.,enough to completely submerge the power, was used and the samples was extracted three times, with fresh methanol each time, over period of 3 days.All the extracts were filtered The combined filtrate were concentrated on a rotary evaporator(Heidolph,Germany)under vacuum at 40°C and dried to yield the MPE.

2.3.Determination of total flavonoids content

The total flvonoids content of the MPE was determined following a protocol described previously,but with a few modification [19].The extract(1.6 mg)was dissolved in a mixture of 50%methanol(2.5 mL)and 5%NaNO2(1.0 mL)solutions.After incubating the mixture at RT for 6 min, 10% Al(NO3)3(1.0 mL)and NaOH(10 mL,1 mol/L)were added into the mixture.The mixture was diluted to 25 mL with 50%methanol and allowed to stand for 15 min at RT.The absorbance of the solution was measured at 500 nm,the absorbance of 50%methanol was used as the blank control.Based on the above methods,serial dilutions ofrutin(2–6and8 mLsamples,0.2 mg/mL,stock samples)in 50%were used to create standard curve.The total fl vonoids content was calculated based on the linear equation obtained from the standard curve with rutin(RE,mg/g extract).The average of six replicate measurements for each experiment was recorded.

2.4.Determination of reducing activity

Reducing activity exhibited by the extract was measured based on a modificatio of a previously reported method [20].Samples solutions(1.0 mL)containing different concentrations of the MPE(0.04,0.08,0.16,0.32,0.64 and l.28 mg/mL)were added independently to a mixture of potassium hexacyanoferrate (1.5 mL, 1.0%) and sodium phosphate buffer (1.0 mL,0.2 mol/L, pH 6.6), This mixture was subsequently incubated in a water bath (50°C) for 20 min, this reaction was terminated by the addition of trichloroacetic acid (1.0 mL, 10%).The reasulting mixture was centrifuged at 3000×gfor 10 min.The supernatant (2.5 mL) was mixed with a solution of FeCl3(0.5 mL, 0.1%) and distilled water (2 mL).The mixture was incubated at RT for 10 min, following which the absorbance 700 nm was measured.A higher absorbance of the reaction mixture indicates a stronger reducing activity.50% methanol was used as the blank control, and a solution of trolox was used as the positive control[21,22].Each experiment was performed in triplicate and the average of three measurements was recorded.

2.5.Determination of lipid peroxidation

Lipid peroxidation was determined using the linoleic acid model system described previously[23].Sample solution containing different concentrations of MPE(0.02,0.04,0.08,0.16,0.32, 0.64 and 1.28 mg/mL) were prepared in 50% methanol.Equal quantities of linoleic acid and Tween-20, was mixed in sodium phosphate buffer (0.05 mol/L, pH 7.4) to prepare 0.02 mol/L linoleic acid emulsion.The sample solution(0.1 mL)and linoleic acid emulsion (1.25 mL) were mixed in a graduated test tube.Phosphate buff was added to make the volume 2.50 mL, following which the test tube was stored in dark at 37°C.The above solution (0.1 mL) was taken in 75% ethanol(4.7 mL), 30% ammonium thiocyanate (0.1 mL) and FeCl2(0.1 mL, 0.02 mol/L, dissolved in 3.5% HCl) solution were added.After 3 min, the thiocyanate value was determined by measuring the absorbance at 500 nm.Absorbances were measured at 24 h intervals until a maximum absorbance of negative controlwas achieved.50%methanol was selected as the negative control,and Trolox was used as the positive control.The inhibition rate of linoleic acid peroxidation by MPE was calculated using the following equation[24–26]:

2.6.Radical scavenging activity

2.6.1.Determination of DPPH?scavenging activity

The radical scavenging activity of the MPE was determined by measuring the decrease in absorbance of methanolic solution of DPPH?in the presence of the extract.Samples solutions containing different concentrations(0.02,0.04,0.08,0.16,0.32,0.63,1.25,2.50 and 5.00 mg/mL)of the MPE were prepared in 50%methanol.A solution of DPPH?(0.1 mg/L)was prepared in methanol.DPPH?solution(3.9 mL)and the sample solution(0.1 mL)were mixed,and then incubated in a water bath at 37°C for 30 min.Absorbance at 517 nm was montiored and compared with that of the blank.The 50%methanol solution was selected as the negative control and trolox was used as the positive control[27,28].

The capability to scavenge DPPH?was calculated using the following equation:

2.6.2.Determination of O2??scavenging activity

The superoxide anion radical scavenging activity was measured according to the method reported by Nishikimi[29], However, with a slight modification Sodium phosphate buffer (100 mmol/L, pH 7.4) containing a solution of NADH(468 μmol/L), NBT (156 μmol/L) and PMS (60 μmol/L) was prepared.Sample solutions containing different concentrations(0.02,0.04,0.08,0.16,0.32,0.64,1.28 and 2.56 mg/mL)of the MPE were prepared in 50%methanol.Superoxide radicals were formed in the PMS–NADH system containing NADH (1 mL),NBT(1 mL),PMS(0.1 mL)andsamplesolution(0.1 mL).These reaction mixtures were incubated at RT for 5 min, and then the absorbance at 560 nm was measured relative to the blank.An aliquot of phosphate buffer was used as the blank control.A decrease in the intensity of absorbance at 560 nm by the reaction mixture indicated the increase in O2??scavenging activity.EC50value(mg/mL)were reported relative to the concentration of the sample inducing 50%scavenging activity.The scavenging activity was calculated according to the following equation:

2.6.3.Determination of HO?scavenging activity

Hydroxyl radical scavenging activity was evaluated using a modifie version of the methods described by Elizabeth et al.[30].Samples solution containing different concentrations(0.02,0.04,0.08,0.16,0.32,0.63,1.25 and 2.50 mg/mL)of the MPE were prepared in 50% methanol.To phosphate buffer (0.95 mL, 0.2 mol/L, pH 7.0) FeSO4-EDTA (0.15 mL,10 mmol/L), 2-deoxy-2-ribose (0.15 mL, 10 mmol/L), H2O2(0.15 mL, 10 mmol/L), and the sample solution (0.1 mL) was added and incubated at 37°C for 2 h.Subsequently,a mixture of TBA(1.0%)and NaOH(50 mmol/L,0.75 mL)was added,followed by the addition of trichloroacetic acid (0.75 mL, 2.8%).The mixture was incubated in a boiling water bath for 10 min.After cooling,the absorbance at 520 nm was measured relative to the blank solution.Trolox was used as the positive control.All experiments were performed in triplicate.The scavenging activity was evaluated using the following equation:

2.7.Determination of H2O2 scavenging activity

HRP-phenol red reaction solution was prepared (HRP 24 U/mL, phenol red 0.2 mg/mL) in sodium phosphate buffer(0.1 mol/L, pH 7.4).Samples containing different concentrations(0.02,0.04,0.08,0.16,0.32,0.63 and 1.25 mg/mL)of the MPE were prepared in 50% methanol.Aliquots of the sample solution (0.1 mL) and H2O2solution (0.4 mL, 4 mmol/L)were added to a test tube and the volume adjusted to 1.5 mL with sodium phosphate buffer (0.1 mol/L, pH 7.4), after mixing at 37°C for 20 min.Horseradish peroxide enzyme (1 mL)was added, and incubated at 37°C for 10 min.NaOH (50 μL,1 mol/L)was added to terminate the reaction.The absorbance at 610 nm was measured relative to that of a blank solution [26].Trolox was selected as a positive control.Percentage scavenging of hydrogen peroxide was calculated by using the following equation:

2.8.Statistical analysis

All data were analyzed by ANOVA and expressed as mean±SD.Analysis of variance and the difference between the samples were determined by Duncan’s multiple range test,andp＜0.05 was used to determine statistical significance

3.Results and discussion

3.1.Total flavonoids content

Absorbance of the solution at 500 nm was measured by UV–vis spectrophotometer using aluminum nitrate as the chromogenic agent.The calibration curve prepared using different concentrations of the standard rutin yielded the following equation:y=6.8250×?0.0498(R2=0.9261).The absorbance of the extract was determined easily and the total fl vonoids content of MPE was calculated based on the above equation μg/g of extracts.The total fl vonoids content in MPE was determined to be 375±12.7 mg/g.The color of the MPE was dark green.On dilution,the extract appeared to be a lighter shade of green.In order to reduce any interference in the absorbance due to the color,we performed six replicates of the experiment.

3.2.Reducing activity

The dose-response curve for the reducing activity of the MPE was determined using the Prussian blue method(Fig.1).In this method, higher antioxidative activity is indicated by stronger reducing power.Therefore, we estimated the antioxidation ability based on the observed reducing power.Trolox,a water-soluble vitamin E,which exhibits excellent anti-oxidative,was used as the positive control and reference antioxidant[31].At concentration ＜0.16 mg/mL, the absorbance of trolox remained unchanged, Meanwhile the absorbance of the sample increased sharply when the concentration increased from 0.04 mg/mL to 0.08 mg/mL; any further increase in the concentration did not lead to a change in the absorbance.At concentration ＞0.16 mg/mL, the absorbance of both the MPE sample and trolox increased rapidly with the increase in concentration.At 1.28 mg/mL,reducing activity of MPE reached 75%,while the reducing activity of trolox was 62.5%.Comparison of EC50values of samples and trolox (Table 1), the reducing activity of the extract(EC50=0.26±0.01)was significantl higher(p＜0.05)than of trolox(EC50=0.70±0.01).A higher absorbance implieds a relative stronger reducing activity,therefore, the assay showed that the extract possessed a higher reducing activity.

Fig.1.A plot of the concentration-dependent absorbance used in the evaluation of reducing activity curve.

Fig.2.Dose dependent inhibition of lipid peroxidation activity.

Table 1 In vitro EC50 valuesa of MPE and trolox.

3.3.Inhibition of lipid peroxidation

The lipid peroxidation inhibition activity of the MPE was evaluated by the ferric thiocyanate method (FTC) [32].Lipid peroxidation was inhibited by trolox and the MPE in a strongly dose-dependent manner.The rate of inhibition increased with increase in the concentration of trolox or the sample.It is likely that the total fl vonoids inhibited the auto-oxidation of linoleic acid,which in turn reduces Fe2+to Fe3+,therefore,leading to a decreased absorbance and,consequently,indicating an increased inhibition.Higher inhibition of lipid peroxidation was observed with MPE than that with trolox.At 0.02 mg/mL, the extract inhibited only 49.0%of the lipid peroxidation activity.At the highest investigated concentration(1.28 mg/mL),the extract inhibited 87.0% of the lipid peroxidation activity: at the same concentration,trolox inhibited 81.0%of the peroxidation activity(Fig.2).When compared to the uniform structure of trolox,the stronger reducing activity of the MPE could be attributed to the diverse structure of its constituent fl vonoids.The EC50value of MPE was determined to be 0.023±0.03 mg/mL,while that of the positive control was 0.052±0.02 mg/mL(p＜0.05).Statistical analysis revealed that the inhibition of lipid peroxidation by the MPE was significantl higher than that by trolox(Table 1).

Fig.3.Dose dependent radical scavenging activity of MPE of M.paniculata and trolox(A)DPPH? scavenging activity,(B)O2?? radical scavenging activity and(C)HO? scavenging activity.

3.4.Radical scavenging activity

3.4.1.DPPH?scavenging activity

Fig.4.Dose-dependent H2O2 scavenging activity of MPE and trolox.

DPPH?accepts hydrogen radicals or electrons to generate stable diamagnetic molecules.Based on their hydrogen-donating ability, the antioxidative properties of proton-donating substances was demonstrated using DPPH?based assay.The antioxidative activity of the MPE could be evaluated based on its ability to reduce DPPH?,which was determined by monitoring the absorbance at 517 nm [33,34].When DPPH?is scavenged by the MPE,the absorbance of the solution decreases;the extent of the decrease in absorbance indicates the strength of the scavenging activity.Fig.3(A)shows the DPPH?scavenging activity of the MPE and trolox.From Fig.3(A)that the scavenging activity is strengthened with an increase the concentration of either MPE or trolox.At 5.00 mg/mL, the MPE scavenged 68% of the DPPH?, while the positive control (trolox, scavenged only 62%).Intriguingly, the DPPH?scavenging activity of trolox was a marginally higher than that of MPE at 0.32 mg/mL.As shown in Table 1 the EC50values for MPE and trolox were 0.93±0.02 mg/mL and 1.319±0.01 mg/mL respectively.The differences in the DPPH?scavenging activities of MPE and trolox were statistically insignificant

3.4.2.Superoxide anion radical scavenging activity

Superoxide anions radicals(O2??)play a critical role in the formation of reactive oxygen species(ROS),such as hydroxyl radical, hydrogen peroxide and singlet oxygen, which induce oxidative damage in lipids, proteins and DNA [35,36].The ability of MPE to transform O2??can indicate that MPE has a stronger scavenging activity.In our assay, O2??scavenging activity of methanol extract was determined by monitoring the consumption of O2??auto-oxidation by hydroxylamine in the presence of NBT and the absorbance at 560 nm was measured.As the data of Fig.3 (B) shows, samples containing different concentrations of MPE (0.02–2.56 mg/mL) exhibited different O2??scavenging activities.With an increase in the concentrations of MPE, the activities became stronger.At the highest evaluated concentration(2.56 mg/mL),MPE transformed 67%of O2??, an activity comparable to that of trolox (64%) at a similar concentration.The EC50value of MPE for scavenging superoxide anion radical was 0.581±0.01 mg/mL,whereas the EC50value of trolox was 0.747±0.01 mg/mL (Table 1).When compared to that of trolox,the superoxide anion radical scavenging activity of MPE was marginally, however, statistically insignifican (p＜0.05).

3.4.3.Hydroxyl radical scavenging activity

The HO?scavenging activity of MPE was determined using Fenton’s method.The results showed that the HO?scavenging activity by MPE displayed a dose–response curve; scavenging effect became stronger with increase in concentration of MPE in the sample(Fig.3(C)).The scavenging effect could be attributed to the ability of MPE to inhibit the activity radical, which resulted a decreased the absorbance.It was observed that MPE exhibited an activity that was higher than that of trolox.At 2.50 mg/mL,MPE scavenged 71.0%of the radical activity,while trolox at the same concentration, only inhibited 63.0% of the radical activity.The EC50value of MPE for HO?scavenging activity was 0.302±0.05 mg/mL, which was significantl higher than that of Trolox(0.576±0.04 mg/mL)(Table 1).

3.5.Hydrogen peroxide scavenging activity

Dose-dependent H2O2scavenging activity of the MPE is shown in Fig.4.At concentrations ＜0.16 mg/mL, the H2O2scavenging activity of trolox and MPE increased rapidly with increase in concentration.However,at concentration ＞0.16 mg/mL,the rate of increase in the scavenging activity with increase in concentration was relatively slower.It is likely that at low concentrations both MPE and trolox scavenged H2O2efficientl .At higher concentrations,the absorbance decreased slowly indicating that the scavenging effect increased slowly.At the highest evaluated concentration(1.25 mg/mL),the MPE was found to scavenge 57.0%of the hydrogen preoxide,while trolox was 54.0%of the peroxide at the same concentration.The measured EC50value of MPE and trolox were 0.470±0.02 mg/mL and 0.583±0.02 mg/mL,respectively.

4.Conclusion

In this study, the antioxidative activities of the MPE was evaluated by determining its reducing,DPPH?scavenging,lipid peroxidation inhibition,O2??scavenging,HO?scavenging and H2O2scavenging activities.The MPE possesses a considerable amount of fl vonoids and displays considerably strong antioxidative properties;its reducing,lipid peroxidation inhibition and HO?scavenging activities are higher than that observed with Trolox.The DPPH?, O2??and H2O2scavenging activities of MPE and trolox are comparable.Taken together, these results indicate that MPE with higher antioxidant potentials,can be an effective free radical inhibitor or scavenger.These results show thatM.Paniculataand bioactive compounds isolated therefrom warrants further investigation.

Acknowledgments

We are grateful for financia support from the National NSFC(No.21162007)and Special Funds to Enhance the Comprehensive Strength of Midwestern Universities.