Studies on Antioxidative Activities of Methanol Extract from Murraya Paniculata

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Food Science and Human Wellness 4 (2015) 108–114

Studies on antioxidative activities of methanol extract from Murraya paniculata

∗

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

Received 5 May 2015; received in revised form 25 June 2015; accepted 2 July 2015

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 ﬂavonoids

•

(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 signiﬁcantly 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 plant are utilized in folk medicine to treat stomach ache, tooth

ache, gout, diarrhea, dysentery, rheumatism, cough and hysteria

Murraya paniculata (L.) belongs to the Rutaceae family. [4,5]. The use of M. paniculata has also been recommended for

This plant is native to areas in Southeast Asia [1], such as the treatment of cuts, joint pains and body aches [6].

Southern China, Vietnam and Malay Peninsula. In China, the A few researches have extensively investigated the antioxi-

ornamental plant M. paniculata is widely cultivated on the dant of M. paniculata. For examples, Chen and co-workers [7]

sides of the road for decorative purposes. In Southeast Asia, have reported that MPE is a stronger antioxidant than vitamin E.

it has been used for topical medicinal applications as health During a preclinical study, Gautam and collaborators [8] have

food [2]. For example, a solution of the bark of M. paniculata, found that MPE decreases the free radical level in diabetic rats

when mixed with other ingredients, is used as an antidote signiﬁcantly. Rodríguez and collaborators [9] have reported the

for snake bites. The leaves of M. paniculata are known to antioxidant activity of M. paniculata essential oil is stronger than

possess antibiotic activity against Mycococcus pyogenes and that butylated hydroxyanisole and butylated hydroxytoluene.

Escherichia coli [3]. Furthermore, the leaves and roots of this 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 ﬂavonoids) have been

Corresponding author at: Key Laboratory of Protection and Development

Utilization of Tropical Crop Germplasm Resources, Ministry of Education, Col- isolated from the leaves and roots of M. paniculata, Present in

lege of Environment and Plant Protection, Hainan University, Haikou, Hainan several medical plants, ﬂavonoids [11–16], with their hydroxyl

570228, PR China. Tel.: +86 898 66192915; fax: +86 898 66192915.

groups and unsaturation, are widely recognized for their natu-

E-mail addresses: [email protected] (C.-h. Zhu),

ral antioxidant properties. Frequently, the antioxidant activity

[email protected] (Z.-l. Lei), [email protected] (Y.-p. Luo).

of an extract correlates linearly with the total ﬂavonoid content

Peer review under responsibility of Beijing Academy of Food Sciences.

http://dx.doi.org/10.1016/j.fshw.2015.07.001

C.-h. Zhu et al. / Food Science and Human Wellness 4 (2015) 108–114 109

of the extracts [17,18]. In this study, we have evaluated the 2.4. Determination of reducing activity

methanol extract of M. paniculata (MPE) for its reducing activ-

ity, lipid peroxidation inhibition, radical scavenging activity Reducing activity exhibited by the extract was measured

•

(for radicals such as 11-diphenyl-2-picryl-hydrazil (DPPH ), based on a modiﬁcation of a previously reported method [20].

−• •

superoxide anion (O2 ), and hydroxyl (OH )), and hydrogen Samples solutions (1.0 mL) containing different concentrations

peroxide (H2O2) scavenging activity. 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 hexacyano-

ferrate (1.5 mL, 1.0%) and sodium phosphate buffer (1.0 mL,

2. Materials and methods

0.2 mol/L, pH 6.6), This mixture was subsequently incubated

◦

in a water bath (50 C) for 20 min, this reaction was termi-

2.1. Chemicals

nated by the addition of trichloroacetic acid (1.0 mL, 10%).

The reasulting mixture was centrifuged at 3000 × g for 10 min.

Rutin (C27H30O16), water-soluble vitamin E (Trolox),

• The supernatant (2.5 mL) was mixed with a solution of FeCl3

1,1-diphenyl-2-picrylhydrazyl (DPPH ), linoleic acid

(0.5 mL, 0.1%) and distilled water (2 mL). The mixture was

(C18H32O2), potassium nitro blue tetrazolium (NBT), N-

incubated at RT for 10 min, following which the absorbance

methyl phenazinemethosulfate (PMS), nicotinamide adenine

700 nm was measured. A higher absorbance of the reaction

dinucleotide (NADH), D-2-deoxyribose (C4H9O3CHO),

mixture indicates a stronger reducing activity. 50% methanol

thiobarbituric acid (TBA), horseradish peroxidase (HRP)

was used as the blank control, and a solution of trolox was

were obtained from sigma chemicals. All other chemicals and

used as the positive control [21,22]. Each experiment was per-

reagents were procured from commercial sources in China and

formed in triplicate and the average of three measurements was were analytical grade.

recorded.

2.2. Plant material and extraction

2.5. Determination of lipid peroxidation

Whole plant samples of M. paniculata were picked from

Lipid peroxidation was determined using the linoleic acid

Hainan University campus during May, 2012. These plants were

model system described previously [23]. Sample solution con-

washed with fresh water, and then dried in the shade for sev-

taining different concentrations of MPE (0.02, 0.04, 0.08, 0.16,

eral days. The dried plant samples were ground into powder

0.32, 0.64 and 1.28 mg/mL) were prepared in 50% methanol.

through 40 meshes, and then stored at room temperature (RT)

Equal quantities of linoleic acid and Tween-20, was mixed

for subsequent use.

in sodium phosphate buffer (0.05 mol/L, pH 7.4) to prepare

To extract the compounds soluble in methanol, the ground

0.02 mol/L linoleic acid emulsion. The sample solution (0.1 mL)

plant samples (20 g) were soaked in methanol at room tempera-

and linoleic acid emulsion (1.25 mL) were mixed in a gradu-

ture, Sufﬁcient quantity of methanol, i.e., enough to completely

ated test tube. Phosphate buff was added to make the volume

submerge the power, was used and the samples was extracted

2.50 mL, following which the test tube was stored in dark at

three times, with fresh methanol each time, over period of 3 ◦

37 C. The above solution (0.1 mL) was taken in 75% ethanol

days. All the extracts were ﬁltered. The combined ﬁltrates were

(4.7 mL), 30% ammonium thiocyanate (0.1 mL) and FeCl2

concentrated on a rotary evaporator (Heidolph, Germany) under

◦ (0.1 mL, 0.02 mol/L, dissolved in 3.5% HCl) solution were

vacuum at 40 C and dried to yield the MPE.

added. After 3 min, the thiocyanate value was determined by

measuring the absorbance at 500 nm. Absorbances were mea-

2.3. Determination of total ﬂavonoids content sured at 24 h intervals until a maximum absorbance of negative

control was achieved. 50% methanol was selected as the negative

The total ﬂavonoids content of the MPE was determined control, and Trolox was used as the positive control. The inhi-

following a protocol described previously, but with a few mod- bition rate of linoleic acid peroxidation by MPE was calculated

iﬁcations [19]. The extract (1.6 mg) was dissolved in a mixture using the following equation [24–26]:

of 50% methanol (2.5 mL) and 5% NaNO2 (1.0 mL) solutions.

Lipid peroxidation inhibition effect(%)

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 mix- − A /A ×

(1 Sample 500 Control 500) 100

ture. The mixture was diluted to 25 mL with 50% methanol and

allowed to stand for 15 min at RT. The absorbance of the solu-

tion was measured at 500 nm, the absorbance of 50% methanol 2.6. Radical scavenging activity

was used as the blank control. Based on the above methods,

•

serial dilutions of rutin (2–6 and 8 mL samples, 0.2 mg/mL, stock 2.6.1. Determination of DPPH scavenging activity

samples) in 50% were used to create standard curve. The total The radical scavenging activity of the MPE was determined

ﬂavonoids content was calculated based on the linear equation by measuring the decrease in absorbance of methanolic solu-

•

obtained from the standard curve with rutin (RE, mg/g extract). tion of DPPH in the presence of the extract. Samples solutions

The average of six replicate measurements for each experiment containing different concentrations (0.02, 0.04, 0.08, 0.16, 0.32,

was recorded. 0.63, 1.25, 2.50 and 5.00 mg/mL) of the MPE were prepared in

110 C.-h. Zhu et al. / Food Science and Human Wellness 4 (2015) 108–114

•

50% methanol. A solution of DPPH (0.1 mg/L) was prepared 2.7. Determination of H2O2 scavenging activity

•

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 HRP-phenol red reaction solution was prepared (HRP

for 30 min. Absorbance at 517 nm was montiored and compared 24 U/mL, phenol red 0.2 mg/mL) in sodium phosphate buffer

with that of the blank. The 50% methanol solution was selected (0.1 mol/L, pH 7.4). Samples containing different concentra-

as the negative control and trolox was used as the positive control tions (0.02, 0.04, 0.08, 0.16, 0.32, 0.63 and 1.25 mg/mL) of the

[27,28]. MPE were prepared in 50% methanol. Aliquots of the sam-

•

The capability to scavenge DPPH was calculated using the ple solution (0.1 mL) and H2O2 solution (0.4 mL, 4 mmol/L)

following equation: 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 mix-

• ◦

= − A /A

DPPH scavenging effect(%) (1 )

Sample 517 Control 517 ing at 37 C for 20 min. Horseradish peroxide enzyme (1 mL)

◦

␮

×100 was added, and incubated at 37 C for 10 min. NaOH (50 L,

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 scaveng-

2.6.2. Determination of O2 scavenging activity

ing of hydrogen peroxide was calculated by using the following

The superoxide anion radical scavenging activity was

equation:

measured according to the method reported by Nishikimi

[29], However, with a slight modiﬁcation. Sodium phosphate

= − A /A ×

Scavenging effect(%) (1 Sample 610 Control 610) 100

buffer (100 mmol/L, pH 7.4) containing a solution of NADH

(468 ␮mol/L), NBT (156 ␮mol/L) and PMS (60 ␮mol/L) was

prepared. Sample solutions containing different concentrations 2.8. Statistical analysis

(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 All data were analyzed by ANOVA and expressed as

formed in the PMS–NADH system containing NADH (1 mL), mean ± SD. Analysis of variance and the difference between

NBT (1 mL), PMS (0.1 mL) and sample solution (0.1 mL). These the samples were determined by Duncan’s multiple range test,

reaction mixtures were incubated at RT for 5 min, and then and p < 0.05 was used to determine statistical signiﬁcance.

the absorbance at 560 nm was measured relative to the blank.

An aliquot of phosphate buffer was used as the blank con-

3. Results and discussion

trol. A decrease in the intensity of absorbance at 560 nm by

−•

the reaction mixture indicated the increase in O2 scavenging

3.1. Total ﬂavonoids content

activity. EC50 value (mg/mL) were reported relative to the con-

centration of the sample inducing 50% scavenging activity. The

Absorbance of the solution at 500 nm was measured by

scavenging activity was calculated according to the following

UV–vis spectrophotometer using aluminum nitrate as the chro-

equation:

mogenic agent. The calibration curve prepared using different

concentrations of the standard rutin yielded the following equa-

= − A /A ×

Scavenging effect(%) (1 Sample 560 Control 560) 100 2

tion: y = 6.8250 × −0.0498 (R = 0.9261). The absorbance of the

extract was determined easily and the total ﬂavonoids content

• of MPE was calculated based on the above equation ␮g/g of

2.6.3. Determination of HO scavenging activity

extracts. The total ﬂavonoids content in MPE was determined

Hydroxyl radical scavenging activity was evaluated using

to be 375 ± 12.7 mg/g. The color of the MPE was dark green.

a modiﬁed version of the methods described by Elizabeth

On dilution, the extract appeared to be a lighter shade of green.

et al. [30]. Samples solution containing different concentra-

In order to reduce any interference in the absorbance due to the

tions (0.02, 0.04, 0.08, 0.16, 0.32, 0.63, 1.25 and 2.50 mg/mL)

color, we performed six replicates of the experiment.

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,

3.2. Reducing activity

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

◦

The dose-response curve for the reducing activity of the

added and incubated at 37 C for 2 h. Subsequently, a mixture of

MPE was determined using the Prussian blue method (Fig. 1).

TBA (1.0%) and NaOH (50 mmol/L, 0.75 mL) was added, fol-

In this method, higher antioxidative activity is indicated by

lowed by the addition of trichloroacetic acid (0.75 mL, 2.8%).

stronger reducing power. Therefore, we estimated the antiox-

The mixture was incubated in a boiling water bath for 10 min.

idation ability based on the observed reducing power. Trolox, a

After cooling, the absorbance at 520 nm was measured relative

water-soluble vitamin E, which exhibits excellent anti-oxidative,

to the blank solution. Trolox was used as the positive control.

was used as the positive control and reference antioxidant

All experiments were performed in triplicate. The scavenging

[31]. At concentration <0.16 mg/mL, the absorbance of trolox

activity was evaluated using the following equation:

remained unchanged, Meanwhile the absorbance of the sam-

= − A /A ×

Scavenging effect(%) (1 Sample 520 Control 520) 100 ple increased sharply when the concentration increased from

C.-h. Zhu et al. / Food Science and Human Wellness 4 (2015) 108–114 111

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.

0.04 mg/mL to 0.08 mg/mL; any further increase in the con- higher (p < 0.05) than of trolox (EC50 = 0.70 0.01). A higher

centration did not lead to a change in the absorbance. At absorbance implieds a relative stronger reducing activity, there-

concentration >0.16 mg/mL, the absorbance of both the MPE fore, the assay showed that the extract possessed a higher

sample and trolox increased rapidly with the increase in concen- reducing activity.

tration. At 1.28 mg/mL, reducing activity of MPE reached 75%,

while the reducing activity of trolox was 62.5%. Comparison

3.3. Inhibition of lipid peroxidation

of EC50 values of samples and trolox (Table 1), the reduc-

ing activity of the extract (EC50 = 0.26 ± 0.01) was signiﬁcantly

The lipid peroxidation inhibition activity of the MPE was

Table 1 evaluated by the ferric thiocyanate method (FTC) [32]. Lipid

In vitro

EC50 values of MPE and trolox. peroxidation was inhibited by trolox and the MPE in a strongly

dose-dependent manner. The rate of inhibition increased with

Antioxidative activity MPE (mg/mL) Trolox (mg/mL)

b b increase in the concentration of trolox or the sample. It is

Reducing activity 0.26 ± 0.01 0.70 ± 0.01

b likely that the total ﬂavonoids inhibited the auto-oxidation of

Lipid peroxidation 0.023 ± 0.03 0.052 ± 0.02

2+ 3+

inhibitionb linoleic acid, which in turn reduces Fe to Fe , therefore, lead-

DPPH radical scavenging 0.93 ± 0.02 1.319 ± 0.03 ing to a decreased absorbance and, consequently, indicating an

activity increased inhibition. Higher inhibition of lipid peroxidation was

Superoxide anion radical 0.581 ± 0.01 0.747 ± 0.01

observed with MPE than that with trolox. At 0.02 mg/mL, the

scavenging activity

b extract inhibited only 49.0%of the lipid peroxidation activity. At

Hydroxyl radical 0.302 ± 0.05 0.576 ± 0.04

b the highest investigated concentration (1.28 mg/mL), the extract

scavenging activity

Hydrogen peroxide 0.470 ± 0.02 0.583 ± 0.02 inhibited 87.0% of the lipid peroxidation activity: at the same

Scavenging activity concentration, trolox inhibited 81.0% of the peroxidation activ-

a ity (Fig. 2). When compared to the uniform structure of trolox,

EC50 values determined the concentration inhibited 50% of the scavenging

activity. the stronger reducing activity of the MPE could be attributed

Statistically signiﬁcant difference ( = 0.05). to the diverse structure of its constituent ﬂavonoids. The EC50

112 C.-h. Zhu et al. / Food Science and Human Wellness 4 (2015) 108–114

• −•

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

•

value of MPE was determined to be 0.023 ± 0.03 mg/mL, while 3.4. Radical scavenging activity

that of the positive control was 0.052 ± 0.02 mg/mL (p < 0.05).

•

Statistical analysis revealed that the inhibition of lipid peroxi- 3.4.1. DPPH scavenging activity

•

dation by the MPE was signiﬁcantly higher than that by trolox DPPH accepts hydrogen radicals or electrons to generate sta-

(Table 1). ble diamagnetic molecules. Based on their hydrogen-donating

C.-h. Zhu et al. / Food Science and Human Wellness 4 (2015) 108–114 113

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

ability, the antioxidative properties of proton-donating sub- scavenging activity of MPE was marginally, however, statisti-

•

stances was demonstrated using DPPH based assay. The cally insigniﬁcant (p < 0.05).

antioxidative activity of the MPE could be evaluated based on its

•

ability to reduce DPPH , which was determined by monitoring

3.4.3. Hydroxyl radical scavenging activity

• •

the absorbance at 517 nm [33,34]. When DPPH is scavenged

The HO scavenging activity of MPE was determined using

•

by the MPE, the absorbance of the solution decreases; the extent

Fenton’s method. The results showed that the HO scavenging

of the decrease in absorbance indicates the strength of the scav-

• activity by MPE displayed a dose–response curve; scavenging

enging activity. Fig. 3(A) shows the DPPH scavenging activity

effect became stronger with increase in concentration of MPE in

of the MPE and trolox. From Fig. 3(A) that the scavenging activ-

the sample (Fig. 3(C)). The scavenging effect could be attributed

ity is strengthened with an increase the concentration of either

to the ability of MPE to inhibit the activity radical, which resulted

MPE or trolox. At 5.00 mg/mL, the MPE scavenged 68% of

• a decreased the absorbance. It was observed that MPE exhibited

the DPPH , while the positive control (trolox, scavenged only

• an activity that was higher than that of trolox. At 2.50 mg/mL,

62%). Intriguingly, the DPPH scavenging activity of trolox

MPE scavenged 71.0% of the radical activity, while trolox at the

was a marginally higher than that of MPE at 0.32 mg/mL. As

same concentration, only inhibited 63.0% of the radical activ-

•

shown in Table 1 the EC50 values for MPE and trolox were

ity. The EC50 value of MPE for HO scavenging activity was

0.93 ± 0.02 mg/mL and 1.319 ± 0.01 mg/mL respectively. The

• 0.302 0.05 mg/mL, which was signiﬁcantly higher than that

differences in the DPPH scavenging activities of MPE and

of Trolox (0.576 ± 0.04 mg/mL) (Table 1).

trolox were statistically insigniﬁcant.

3.5. Hydrogen peroxide scavenging activity

3.4.2. Superoxide anion radical scavenging activity

−•

Dose-dependent H2O2 scavenging activity of the MPE

Superoxide anions radicals (O2 ) play a critical role in the

is shown in Fig. 4. At concentrations < 0.16 mg/mL, the

formation of reactive oxygen species (ROS), such as hydroxyl

H2O2 scavenging activity of trolox and MPE increased

radical, hydrogen peroxide and singlet oxygen, which induce

rapidly with increase in concentration. However, at concentra-

oxidative damage in lipids, proteins and DNA [35,36]. The

−•

tion > 0.16 mg/mL, the rate of increase in the scavenging activity

ability of MPE to transform O2 can indicate that MPE has

−•

with increase in concentration was relatively slower. It is likely

a stronger scavenging activity. In our assay, O2 scavenging

that at low concentrations both MPE and trolox scavenged H2O2

activity of methanol extract was determined by monitoring the

−•

efﬁciently. At higher concentrations, the absorbance decreased

consumption of O2 auto-oxidation by hydroxylamine in the

slowly indicating that the scavenging effect increased slowly. At

presence of NBT and the absorbance at 560 nm was measured.

the highest evaluated concentration (1.25 mg/mL), the MPE was

As the data of Fig. 3 (B) shows, samples containing different

found to scavenge 57.0% of the hydrogen preoxide, while trolox

concentrations of MPE (0.02–2.56 mg/mL) exhibited different

−•

was 54.0% of the peroxide at the same concentration. The mea-

O2 scavenging activities. With an increase in the concentra-

sured EC50 value of MPE and trolox were 0.470 ± 0.02 mg/mL

tions of MPE, the activities became stronger. At the highest

and 0.583 ± 0.02 mg/mL, respectively.

evaluated concentration (2.56 mg/mL), MPE transformed 67%

−•

of O2 , an activity comparable to that of trolox (64%) at a

similar concentration. The EC50 value of MPE for scaveng- 4. Conclusion

ing superoxide anion radical was 0.581 ± 0.01 mg/mL, whereas

the EC50 value of trolox was 0.747 ± 0.01 mg/mL (Table 1). In this study, the antioxidative activities of the MPE was

•

When compared to that of trolox, the superoxide anion radical evaluated by determining its reducing, DPPH scavenging, lipid

114 C.-h. Zhu et al. / Food Science and Human Wellness 4 (2015) 108–114

−• •

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