Keywords

Ziziphus mauritiana, Eriobotrya japonica, Antioxidants, DPPH assay, Chelating power assay, Phenols.

Introduction

Human beings are subjected to oxidative stress from various pollutants, radiations, intake of oxidized foods and in vivo production of free radicals. Uncontrolled oxidation in biological systems can damage the vital cellular membranes, proteins and nucleic acids leading to various degenerative diseases. Many studies indicate that fruits are important sources of vital compounds and act as sources of antioxidants and can protect the human beings from oxidative stress related diseases [1-3]. It has been found that regular intake of fresh fruits and vegetables lowers the risk of diseases associated with oxidative stress by providing bioactive compounds like ascorbic acid, flavonoids, proteins, carbohydrates, vitamin C and polyphenols such as anthocyanins, phenolic acids, and tannins etc. [4-6]. These fruits are also referred as natural functional foods [7].

Currently, limited information is available about the antioxidant activities of major two fruits of India i.e. Ziziphus mauritiana and Eriobotrya japonica. The plant Ziziphus mauritiana belongs to family Rhamnaceae, while Eriobotrya japonica belongs to family Rosaceae. Both fruits are very nutritious and are eaten raw or cooked. Therefore, this study was planned to know the antioxidants properties of these fruits using aqueous extract and employing DPPH assay and chelating power assay.

Materials and Methods

Plant materials

Fresh fruits of Ziziphus mauritiana and Eriobotrya japonica were purchased from the local market of Amritsar, Punjab, India. Fruits were washed with tap water.

         (Ziziphus mauritiana)                    (Eriobotrya japonica)

Preparation of extract

One gram fruit (without seed) was ground in pestle and mortar using liquid nitrogen. The powder was homogenized in 5ml distilled water and centrifuged at 4°C at 10,000 rpm for 15 min and the supernatant (aqueous extract) was used for further analysis.

Total phenolic content (TPC)

Total phenolic content of aqueous extract was determined by using method given by Singleton and Rossi [8]. Fruit extract (100μl) was diluted with water (upto 3 ml) and FC reagent (0.5 ml) was added. 2 ml sodium carbonate (20%) was added after three minutes and the absorbance of solution was measured at 650 nm using UV-Visible spectrophotometer. The results were measured in terms of mg gallic acid/100g fresh weight material.

Total flavonoid content (TFC)

Total flavonoid content of aqueous extract of fruits was determined as per method suggested by Kim [9]. 1ml sample was diluted with 4ml distilled water and mixed with 300μl NaNO2 (5%). 300μl AlCl3 (10%) was added to the solution and kept undisturbed for 5 minutes. After addition of 2ml NaOH (1M) solution was diluted with distilled water to prepare10 ml of total solution and absorbance of solution was taken at 510 nm. The results were measured in mg rutin/100g fresh weight material.

Antioxidant activities

DPPH free radical scavenging assay

The antioxidant activities of plant extracts on DPPH radical was estimated according to Blois [10]. The reaction mixture contains 200 μl different extract concentrations (25-200 mg/ml) and 2 ml of DPPH (0.1 mM in methanolic solution). The absorbance of the solution was recorded at 517 nm. The radical scavenging activity was expressed as inhibition percentage (% inhibition) and calculated as follows:

% DPPH radical scavenging = (1- AS/AC)*100.

AC = Absorbance of control, AS = Absorbance of sample solution.

Chelating power assay

The metal chelating activity of extracts was measured by the method given by Dinis [11] with few modifications. 1 ml plant extract (25-200mg/ml) was mixed with 3.5ml methanol, 0.2 ml ferrous chloride (2 mM) and 0.2 ml ferrozine (1 mM) and the solution was kept for 10 min at room temperature. The absorbance of the solution was determined at 562 nm. The decrease in absorbance of solution was taken as increase in chelating power.

Results

Total phenolic content

Amount of total phenolic content in Z. mauritiana and E. japonica was calculated from the calibration curve of gallic acid prepared by taking different concentrations of gallic acid. The equation for gallic acid is given below:

y = 0.0025x – 0.0036 (R² = 0.9941). Where y is absorbance and x is concentration of gallic acid. From the results, it was observed that aqueous extract of Z. mauritiana and E. japonica showed significant amount of phenolic content in aqueous extracts i.e. 105.36 and 8.16 mg gallic acid/100 g fresh weight respectively.

Total flavonoid content

Total flavonoid content was determined as mg rutin/100 g fresh weight of material from the calibration curve of rutin. The curve between absorbance vs. concentration of rutin can be described by the following equation.

y = 0.0013x + 0.0004 (R² = 0.9902). Where, y is absorbance and x is concentration of rutin. Total flavonoid content of Z. mauriana was found to be 116.31 mg and of E. japonica it was found to be 20.92 mg rutin/100 g fresh weight. From the results it was observed that aqueous extract of Z. mauriana and E.japonica showed significant amount of phenolic and flavonoid content.

DPPH assay

In DPPH assay, the hydrogen donating ability of the fruit extracts was determined by converting the DPPH radical to non radical by reduction process. The DPPH radical scavenging activity of aqueous extracts of fruits is shown in Table 1. Significant DPPH radical scavenging activity was found at all the tested concentrations in both plants extracts. The dose response relationship was found upto 200 mg/ml concentration. The Z. mauritiana showed 74.89 % activity and E. japonica showed 51.07 % activity in this assay. The R² values for Z. mauritiana and E. japonica were found to be 0.8146 and 0.9701 respectively (Figure 1 and 2). From the results, it is clear that both fruit extracts showed significant amount of hydrogen donating activity. Z. mauritiana showed more activity in DPPH assay than E. japonica.

Table 1. Percent Inhibition vs. concentration values of Z. mauritiana and E. japonica fruits in DPPH assays

Figure 1: Percent Inhibition vs. concentration graph of Z. mauriana aqueous extract in DPPH assay

Figure 2: Percent Inhibition vs. concentration graph of E. japonica aqueous extract in DPPH assay

Chelating power assay

In chelating power assay, the ability of fruit extracts to chelate metal ion especially Fe²⁺ ion was observed. Antioxidants prevent the oxidative stress by chelating metal ions that are involved in free radical generation in biological systems. In this assay, Ferrozine was used as it form complex with Fe²⁺ and in the presence of other complexing agents in extract its colour intensity is reduced which is measured spectrophotometrically. As illustrated in Table 2 it was found that Z. mauritiana and E. japonica showed no significant chelating ability. With increase in concentration of extract the absorbance increased in both the plant extracts, which indicates absence of chelating ability of extracts (Figure 3 and 4).

Figure 3: Absorbance vs. concentration graph of Z. mauriana aqueous extract in chelating assay

Figure 4: Absorbance vs. concentration graph of E. japonica aqueous extract in chelating assay

Table 2. Absorbance vs. concentration values of Z. mauritiana and E. japonica fruits in chelating assays

Discussion

Many fruits are known for their medicinal properties and daily consumption of fruits in our diet can protect us from oxidative stress disorders. Fruits provide us antioxidants in the form of phenolic compounds, ascorbic acid, carotenoids, tocopherols etc. [12-14]. It is well known that phenolic compounds including flavonoids of plant origin are mostly responsible for radical scavenging [15-18]. From the results, it is clear that Z. mauritiana and E. japonica are rich in phenolic and flavonoid content. The activity of both fruit aqueous extracts in DPPH assay may be due to the presence of total phenols and flavonoids. Phenols neutralize the free radicals by donating hydrogen atoms [19]. As shown in the results, in DPPH assay the maximum percent inhibition in Z. mauritiana was found to be 74.89%, which may be due to the presence of more phenolic content i.e. 105.36 mg gallic acid/100 g fresh weight as compared to E. japonica which showed 51.07 % inhibition activity and having less phenolic content 8.16 mg gallic acid/100 g fresh weight. Transition metals have been proposed as the catalysts for the initial formation of radicals and chelating compounds present in the plant extracts chelate the transition metals in the living systems and inhibit the generation of free radicals and protect the vital biomolecules form the free radical mediated oxidative stress. In the present study, both fruit extracts did not showed any chelating activity.

Conclusion

From the results, it is concluded that Z. mauritiana and E. japonica are rich in phenols and flavonoids and their antioxidant activities may be due to their hydrogen donating abilities instead of metal chelation.

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