Introduction
The role of free radicals in many disease conditions has been well established. Oxidative stress due to the production of free radicals such as superoxide radical (O
2•
−), hydroxyl radical (•OH), peroxide radical (ROO•), and nitric oxide radical, is the major cause of a variety of pathological conditions including coronary heart diseases, reperfusion injury, inflammation, diabetes, drug toxicity, carcinogenesis and neurodegenerative diseases such as Parkinson and Alzheimer diseases
[1].
Antioxidant substances can block the harmful action of the free radicals by scavenging the free radicals and detoxify the organism. Synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are commonly used in food processing and preservation but have been found to have side effects and have been proved carcinogenic
[2]. Thus there has been increased interest in natural antioxidants, especially those of plant origin. In the past decade there have been many reports of plant extracts and different types of phytochemicals particularly polyphenols, the secondary metabolites from plants, which were shown to have antioxidant activity
[3,4]. Therefore, phenolics and other natural compounds are capable of protecting against reactive oxygen species-mediated damage with possible application in avoidance and/or curing of diseases. As safe sources of antioxidants, fruits and vegetables have been investigated for their antioxidant properties, for example blueberry (
Vaccinium corymbosum L.)
[5] and
Launaea procumbens
[6]. Fruits have been also associated conversely with aging and mortality from cardiovascular and neurodegenerative diseases
[7].
Monotheca buxifolia is a broad-leaved evergreen small tree belonging to the family Sapotaceae. This species is found in the hilly regions of Afghanistan and in Northern Pakistan. It is used as fuel, fodder, wood lumber, roof thatching materials, and particularly used as hedge around cultivated fields due to its barbed nature. This species bears small fruits, locally called Gurgura, sold in the local markets as fresh and dried food
[8,9]. Medicinally, fruits have laxative and digestive properties, and are used in the treatment of urinary tract diseases. They are also used to reduce temperature in fevers and as a vermifuge
[8,10,11].
There have been no reports on the antioxidant activities of this plant previously in the literature. Hence, the present work investigates the potential antioxidant free radical scavenging effects of a methanol extract of the powder of dried fruit pulp of M. buxifolia and its derived solvent fractions, in order to understand the usefulness of this plant as a foodstuff as well as in medicinal preparations. The antioxidant capacity was evaluated for different in vitro models such as scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroxyl, hydrogen peroxide (H2O2), superoxide radicals, and total antioxidant activity by the phosphomolybdate method and reducing power. Because of the effectiveness of the phenolics and flavonoids as antioxidants, the amounts of total phenolics and total flavonoids in the extracts/fractions were also determined.
Materials and Methods
2.1 Chemicals
Aluminum chloride, ascorbic acid, ferric chloride (FeCl3), ABTS, potassium persulfate, gallic acid, rutin, linoleic acid, DPPH, Folin-Ciocalteu's phenol reagent, phenazine methosulfate (PMS), nitro blue tetrazolium (NBT), trichloroacetic acid (TCA), and thiobarbituric acid (TBA) were acquired from Sigma Co. (St. Louis, MO, USA). Riboflavin, sulfuric acid, deoxyribose, sodium hydroxide (NaOH), sodium carbonate (Na2CO3), disodium hydrogen phosphate (Na2HPO4), sodium nitrite (NaNO2) and H2O2 were purchased from Wako Co. (Osaka, Japan). Ferrous chloride (FeCl2), sodium dihydrogen phosphate (NaH2PO4), potassium ferricyanide (K3Fe(CN)6), and solvents used were of analytical grade were purchased from Merck Co. (Darmstadt, Germany). Distilled deionized water (dd. H2O) was prepared using the Ultrapure water purification system (Lotus Co., Ltd., Taipei, Taiwan).
2.2 Plant material and extract preparation
The fruit was collected in April 2010 from KPK province of Pakistan and the plant was identified by its local name and later identified by Dr Mir Ajab Khan, Department of Plant Sciences, Quaid-i-Azam University, Islamabad. A specimen was kept at the Herbarium of the Pakistan Museum of Natural History, Islamabad. The fruits (5.0 kg fresh) of uniform size at maturity were collected and dried under shade to obtain 1.0 kg dry sample, excluding the seeds. The dried samples were powdered in a Willy Mill to 60-mesh size and used for solvent extraction. For extract preparation, 1.0 kg of dried sample was extracted twice with 2.0 L of 95% methanol at 25 °C for 48 h. The extracts were filtered with Whatman No. 1 filter paper and evaporate to dry the filtrate by using rotary evaporator. The extract was suspended in distilled water and partitions were made with increasing polarity of solvents i.e., n-hexane, ethyl acetate, chloroform, butanol, and water. Then all fractions were dried by using rotary evaporator, and preserved at 4 °C. The dry extract was weighed and the yield was determined as the percentage of air-dried weight of plant material.
2.3 Estimation of total polyphenolic contents
The total polyphenolic content was estimated using Folin-Ciocalteu reagent
[12]. Folin-Ciocalteu reagent (400 μL) was mixed with 200 μL of fractions (1.0 mg/mL) in a volumetric flask. The solution was heated to 25 °C for 5–10 min and mixed with 0.2 mL of 7% Na
2CO
3 solution, and finally the mixture was diluted with deionized distilled water and made up to 10.0 mL in a volumetric flask. Before taking the absorbance at 725 nm, the mixture was held for 2 hours at 25 °C. A calibration curve was plotted for the standard of gallic acid. Total phenolics were calculated as equivalent of per mg gallic acid (GAE) per gram of dried sample (mg/g).
2.4 Estimation of total flavonoids
Total flavonoid content was estimated according to the method of Park et al
[13]. Fifty milligrams of each fraction was suspended in 10 mL of 80% methanol and filtered through Whatman filter paper No. 42 (125 mm). In a test tube (10 mL), 0.3 mL of extracts, 3.4 mL of 30% methanol, 0.15 mL of 0.5M NaNO
2 and 0.15 mL of 0.3M AlCl
3.6H
2O was mixed. Five minutes after the addition of 1 mL of 1M NaOH, the absorbance was measured at 506 nm. Rutin was used to plot the calibration curve. Total flavonoids were calculated as mg rutin equivalents per gram of dried sample (mg/g).
2.5 Antioxidant activity assays
For the antioxidant assays, all fractions were dissolved (1.0 mg/mL of fractions) in 95% methanol and a series of concentration-dependent dilutions were made. For all antioxidant assays standard chemicals were used for comparison.
2.5.1
2.5.1 DPPH assay for radical scavenging activity
The DPPH assay was performed following the method of Blois
[14]. DPPH (2.4 mg) was dissolved in 100 mL methanol to prepare the stock solution and then stored at 20 °C until needed. The DPPH solution was diluted with methanol to achieve an absorbance of 0.980 (±0.02) at 517 nm with the spectrophotometer. A 500-μL aliquot of the above mixture was mixed with 500 μL of the samples at different concentrations (25–250 μg/mL). The mixture was incubated in the dark for 15 minutes and the absorbance was measured at 517 nm. The DPPH scavenging activity of various fractions was calculated by the following equation:
EC50 values calculated to determine the 50% inhibition of DPPH radicals. Ascorbic acid and rutin were used as standards.
2.5.2
2.5.2 Superoxide radical scavenging activity
The scavenging activity assay for superoxide anion radical was performed according to the method of Beauchamp and Fridovich
[15]. The reaction mixture consisted of 500 μL of 50 mM PO
4 buffer (pH 7.6), 300 μL of 50 mM riboflavin, 250 μL of 20 mM phospho-methozine sulphate (PMS), and 100 μL of 0.5 mM nitroblue tetrazolium (NBT) before adding up of 1.0 mL of various fractions. Triggering of reaction was done by illuminating the above solutions using a fluorescent lamp. After 20 minutes the absorbance was measured at 560 nm. The percentage of scavenging superoxide anion generation was calculated as:
EC50 values calculated to determine the 50% inhibition of superoxide radicals. Ascorbic acid was used as standards.
2.5.3
2.5.3 Phosphomolybdate assay (total antioxidant capacity)
The total antioxidant capacity assay of samples was carried out by the phosphomolybdenum method
[16]. A 0.1-ml aliquot of the sample solution was shaken with 1 mL of reagent solution (0.6M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The test tubes were covered and incubated in a water bath at 95 °C for 90 min. After the samples were cooled, the absorbance of the mixture was measured at 765 nm. Ascorbic acid was used as standard. The antioxidant capacity was estimated using the following formula:
2.5.4 Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity of samples was determined according to the method of Halliwell and Gutteridge
[17]. The reactive mixture was consisted of: 100 μL of pre-mixed ferric chloride (100 mM), 250 μL of 2.8 mM 2-deoxyribose in 50 mM phosphate buffer (pH 7.4), and 100 mM EDTA solution (1:1; v/v); 200 mM H
2O
2 (50 μL) without or with the 50 μL extract solution. The reaction was triggered by adding 50 μL of ascorbate (300 mM) and heated for 60 min at 37 °C. A solution of 1% thiobarbituric acid (TBA) in 500 μL of NaOH (50 mM) and 500 μL of 2.8%TCA was added. Then the mixture was placed in a boiling water bath for 15 minutes. After cooling, the absorbance was taken at 532 nm. Hydroxyl radical scavenging was calculated as:
2.5.5 ABTS radical scavenging activity
The ABTS radical scavenging activity was determined by calculating the disappearance of the ABTS radical cation, following the method with of Re et al
[18]. ABTS (7 mM) and potassium persulfate (2.4 mM) was mixed to make the stock solution and placed in the dark for 12–16 hours at room temperature. Then the solution was diluted by mixing 1 mL of ABTS
+ solution with 60% methanol to obtain an absorbance of 0.708 ± 0.001 units at 734 using the spectrophotometer. For each assay ABTS
+ solution was made fresh. Then 1 mL of plant extracts was added to react with 1 mL of the ABTS
+ solution and the absorbance was measured at 734 nm. The decrease in absorbance was taken after 1 minute up to 6 minutes. Then the final absorbance was noted. The percentage inhibition was calculated using following formula:
The antioxidant capacity of test samples was given by EC50, the concentration necessary for a 50% reduction of ABTS.
2.5.6
2.5.6 H2O2-scavenging activity
The H
2O
2 radical scavenging activity of extracts was determined according to the method of Ruch et al
[19]. H
2O
2 solution (2 mM) was prepared in 50 mM phosphate buffer (pH 7.4). An aliquot (0.1 mL) of the extract sample was placed into a test tube and the volume made up to 0.4 mL with 50 mM phosphate buffer (pH 7.4). After addition of 0.6 mL H
2O
2 solution, tubes were mixed and the absorbance of the H
2O
2 at 230 nm was determined. The ability to scavenge the H
2O
2 was calculated using the following equation:
The EC50 value is the effective concentration that is required to scavenge 50% H2O2 radicals. EC50 values calculated to determine the 50% inhibition of DPPH radicals. Ascorbic acid and rutin were used as standards.
2.5.7
2.5.7 Reducing power assay
The reducing power of extracts was estimated following the method of Oyaizu
[20]. Extract solution (2 mL), phosphate buffer (2 mL, 0.2M, pH 6.6) and potassium ferricyanide (2 mL, 10 mg/mL) were added, and then kept at 45 °C for 30 minutes. TCA (2 mL, 100 mg/L) was added to the reaction mixture. A 2-mL aliquot of the above mixtures was added to 2 mL of distilled water and 0.4 mL of 0.1% (w/v) ferric chloride in a test tube, the absorbance was measured after 10 minutes, at 700 nm. Increased absorbance of the reaction mixture suggests a high reducing power.
2.6 Statistical analysis
Readings for all antiradical scavenging assays were taken in triplicate. Graph Pad Prism 5 software (H.J. Motulsky, Prism5 Statistics Guide, GraphPad Software Inc., San Diego, CA, USA,
www.Graph Pad.com) was used to calculate the EC
50 values. Standard deviation and ANOVA was employed to investigate the differences among EC
50 of different fractions for different antiradical assays. The Pearson correlation coefficient for phenolic and flavonoids was also employed. All the assays findings were subjected to the Student
t test (
p < 0.05;
p < 0.01) to determine their significance.
Results and Discussion
3.1 Extraction yield, total phenolics and flavonoid contents
The percentage yield, total phenolic content, and flavonoids of the methanol extract and solvent fractions obtained from
M. buxifolia fruits are shown in
Table 1. The recovery percentage of extractable compounds varied from 4.56 ± 1.25 to 24.18 ± 3.22. The highest yield was given by the methanol extract (24.18 ± 3.22), whereas the aqueous fraction gave the lowest (4.56 ± 1.25). Although the active components of the medicinal plants compounds are not known, polyphenols have received growing attention because of some exciting new findings concerning their biological activities. Pharmacologically, the antioxidant potential of polyphenolic compounds, particularly free radical scavenging and inhibition of lipid peroxidation, are the most important. The total phenolic compounds as recorded in
Table 1 in
M. buxifolia fruit fractions (determined as gallic acid equivalents or GAE), ranged between 59.13 ± 2.6 mg and 16.66 ± 1.3 mg/g dry weight of fraction. The butanol extract showed the highest total phenolics (59.13 ± 2.6 mg GAE/g fraction), whereas the phenolic contents of
n-hexane were much smaller (16.66 ± 1.3 mg GAE/g), which is in agreement with other similar reports
[4,21]. The antioxidant property of the compounds was well correlated with the content of their phenolic compounds
[22]. Phenols contain good antioxidant, antimutagenic, and anticancer properties
[23]. Flavonoids are the naturally occurring polyphenolic compounds representing one of the most prevalent classes of compounds in vegetables, nuts, fruits, and beverages such as coffee, tea, and red wine
[24]. The present study showed flavonoid contents in the range of 4.11 ± 0.51 to 48.68 ± 2.8 mg as rutin equivalents/g fraction. The highest amount was observed in the aqueous fraction (48.68 ± 2.8 mg/g) followed by the methanol extract (42.045 ± 3.1 mg/g). Halliwell
[25] reported that plants rich in flavonoids are potential sources of natural antioxidants that would add to the overall antioxidant capacity of an organism and inhibit lipid peroxidation. Therefore, the result suggested that phenolic acids and flavonoids may be the major contributors for the antioxidative properties and inhibitory actions toward the oxidative reaction
in vitro and
in vivo.
3.2 DPPH radical scavenging activity
The DPPH radical has been used widely to test the antioxidant activities of plant extracts and foods. This method is based on the reduction of DPPH in methanol solution in the presence of a hydrogen-donating antioxidant, bringing about a color change from purple to yellow, which is measured at 517 nm
[14].
Figure 1A shows the scavenging effect of plant fractions on DPPH radical was in the following order methanol > butanol > ethyl acetate > aqueous >
n-hexane fractions. From the analysis of EC
50 values (
Table 2), the DPPH radical scavenging activity of the butanol fraction (24.1 ± 1.02) was found to be significantly higher (
p < 0.001) followed by methanol extract (42.7 ± 1.62) and aqueous fractions (56.5 ± 3.82). The EC
50 value for
n-hexane fraction was found to be greater than 300 μg/mL. The EC
50 value for butanol fraction was close to that of ascorbic acid (20.4 ± 0.62) and rutin (19.5 ± 1.33) used as positive controls. It shows that the
M. buxifolia fruits act as antioxidants since they possess hydrogen-donating properties. The scavenging activity of the extract increased in a concentration-dependent manner.
3.3 Phosphomolybdate assay
This assay is based on the reduction of phosphomolybdate ion in the presence of an antioxidant resulting in the formation of a green phosphate/MoV complex which is measured spectrophotometrically
[26].
Figure 1C shows antioxidant capacity of fractions of
M. buxifolia fruit in the order of aqueous > methanol > butanol > ethyl acetate >
n-hexane fractions. The aqueous fraction with an EC
50 value of (45.2 ± 2.53) showed high antioxidant capacity followed by the methanol fraction (56.4 ± 2.06). However, the antioxidant activity of ascorbic acid, a known antioxidant used as the positive control, was comparatively more effective than that of
M. buxifolia fractions.
3.4 ABTS radical scavenging activity
The ABTS assay is an excellent tool to determine the antioxidant activity of hydrogen donating and chain-breaking antioxidants. Pietta et al
[27] investigated the antioxidant activity of frequently used medicinal plants and verified that the phenolic compounds are important scavengers of ABTS.
Figure 1B shows that all the fractions of
M. buxifolia fruit exhibited a strong scavenging activity against ABTS radicals. The butanol and aqueous fractions with respective EC
50 values of (52.2 ± 2.57) and (73.1 ± 3.26) exhibited higher antioxidant activity. The butanol and aqueous extracts of
M. buxifolia fruits indicate that they could cease the oxidation process by reducing free radicals. This is due to the presence of high content phenols in these extracts, as polyphenols play a vital role as antioxidants in living systems due to the presence of hydroxyl groups in
ortho- and
para- positions
[28].
3.5 Hydroxyl radical scavenging activity
The hydroxyl radical is one of the most reactive oxygen species in living systems. It damages the cell by reacting with the polyunsaturated fatty acid of cell membrane phospholipids
[25]. Thus, removing OH radicals is very important for the protection of biological systems. In this study all the samples generally registered good hydroxyl radical scavenging activity in a concentration-dependent manner (25–300 μg/mL). Among them, the butanol fraction showed the highest OH scavenging potential (EC
50 value of 64.1 ± 3.15). The capability of
M. buxifolia fractions to eliminate hydroxyl radicals appears to directly relate to the inhibition of lipid peroxidation, and acts as scavengers of active oxygen species by breaking free radical chains.
3.6 H2O2 radical scavenging activity
Our environment contains H
2O
2 at low concentration levels in the air, water, human body, plants, microorganisms, food, and beverages. It enters the human body through inhalation or skin contact. H
2O
2 is rapidly decomposed in the body into oxygen and water resulting in hydroxyl radicals (OH•) that can begin lipid peroxidation and cause damage to cell membrane and DNA. The scavenging ability of extracts of
M. buxifolia fruit extracts on H
2O
2 radicals was in the order of aqueous >
n-butanol > methanol > ethyl acetate >
n-hexane fractions. Data obtained from EC
50 values (
Table 2) shows that the fractions showed moderate H
2O
2-scavenging activity. The percentage scavenging activity increased with increasing concentration of the fractions. However, the scavenging activities of ascorbic acid and rutin, used as positive controls for comparison, were relatively more evident than those of the
M. buxifolia fractions.
3.7 Superoxide radical scavenging activity
Figure 1D shows the superoxide radical (O
2•
−) scavenging activity of the samples, as measured by the riboflavin–NBT–light system
in vitro. Reduction of flavins in the presence of light generates superoxide radicals which reduce NBT, forming a blue-colored formazan
[15]. The fractions evaluated were found to be potent scavengers of superoxide radicals produced in the riboflavin–NBT–light system. The fractions inhibited the formation of the blue formazan in a concentration-dependent pattern and the scavenging potential was in the following order aqueous >
n-butanol > ethyl acetate > methanol >
n-hexane fractions. The EC
50 values of aqueous and butanol fractions were 22.3 ± 1.35and 24.4 ± 0.89. These results indicated that
M. buxifolia fruit fractions had a notable effect on inhibition of superoxide when compared with ascorbic acid (EC
50 values 21.1 ± 0.86), which was used as positive control.
3.8 Reducing power activity
Figure 2 shows the reducing power of sample extract compared to ascorbic acid the reductive capability was investigated by measuring the Fe
3+–Fe
2+ transformation in the presence of
M. buxifolia fruit fractions, following the method of Oyaizu
[20]. The reducing power of extracts is usually associated with the occurrence of reductants, which exert antioxidant action by donating a hydrogen atom and breaking the free radical chain. In our study the aqueous fraction showed highest reductive ability followed by the
n-hexane fraction. The reducing power of
M. buxifolia fractions suggest that it is likely to add considerably towards the overall antioxidant effect. However, the antioxidant activity of plant extracts have been recognized to have various mechanisms of action, such as binding of heavy metal ion catalysts, breakdown of peroxides, inhibition of chain initiation, reductive capacity on metals, and radical scavenging
[29]. Like the antioxidant activity, the reducing power of
M. buxifolia fruit fractions increased with increasing concentration of extract. However, the reducing power of ascorbic acid was comparatively more effective than that of our fractions.
3.9 Correlation with EC50 values of antioxidant activities and phytochemical contents
Through correlation analysis for phytochemical contents with EC
50 values of radical scavenging activities of various soluble fractions of
M. buxifolia fruits, the contents of phenolics and flavonoids exhibited good correlation with phosphomolybdate assay, DPPH, superoxide, and H
2O
2 radical scavenging activities (
Table 3). However, correlation in the case of ABTS and hydroxyl radical scavenging activity was found to be nonsignificant. The results indicate that phenolic acids and flavonoids are the major contributors to the antioxidant and free radical scavenging activities of fractions of
M. buxifolia fruits, and enhanced the importance of phenolic compounds in the antioxidant property of plant extracts. The perceptible correlation among different investigations exhibited that the antioxidant assays selected in the present investigation are feasible and complementary to the antioxidant activities in natural environment. Our results are in agreement with other reports of a strong correlation of antioxidant activities and total polyphenols
[4,30].
