INTRODUCTION:

The importance of antioxidants has attracted increasing attention over the last decade. Free radicals are defined as any atom or molecule having unpaired electrons. They are involved in various physiological processes (initiation of per oxidation of the membrane lipids, inhibition of mitochondrial respiratory chain enzymes, etc.) and play a major role in the inception of many diseases and ultimately lead to cell death¹. They also affect the food’s sensory quality- color, taste and texture, which also shorten the shelf life and can result in rejection on the part of consumers.

Natural antioxidants are obtained from plants, fruits and vegetables and are often called as dietary antioxidants. Dietary antioxidant is defined as -“a substance in foods that significantly decreases the adverse effects of reactive species, such as reactive oxygen and nitrogen species, on normal physiological function in humans”. Natural antioxidants have gained increasing interest among consumers and the scientific community because they are economic, does not reduce food quality and do not exert side-effects like synthetic antioxidants.

Epidemiological studies have indicated that the dietary intake of fruits and vegetables helps in digestion², reduces the risk of chronic diseases and provide protection against cellular damage caused by exposure to high levels of free radicals^3,4.

This is attributed to the fact that these foods provide an optimal mix of antioxidants such as vitamin C and E, polyphenols, carotenoids^5,6, and complex carbohydrates, yet these are not sufficient in critical situations (oxidative stress, contamination, UV-exposure etc,) where the production of free radicals significantly increases⁷.

Health benefits of fruits and vegetables are mediated through their antioxidants content, so optimizing antioxidant intake may be useful in planning diets for health promotion. Therefore, the present study was conducted to investigate the antioxidant potential of some commonly consumed fruits and vegetables in Rajasthan. This study did not investigate the effect of cooking or storage on antioxidant content.

MATERIALS AND METHOD:

Plant materials:

The following samples of fruits and vegetables were collected from the market:

Coccinia grandis (Cucurbitaceae), Momordica dioica (Cucurbitaceae) Coriandrum sativum (Apiaceae), Psidium guajava (Myrtaceae), Punica granatum (Puniacaceae), Mangifera indica (Anacardiaceae), Carissa congesta/C. carandas (Apocynaceae), Capparis deciduas/C. aphylla (Capparidaceae), Prosopis cinneraria (Fabaceae), Acacia senegal (Fabaceae), Cordia myxa (Boraginaceae), Syzygium cumuni (Myrtaceae).

Chemicals:

2, 2’-Diphenyl-1-picryl hydrazyl (DPPH), gallic acid, Reduced nicotinamide adenine dinucleotide Sodium salt (NADH), Phenazine methosulphate (PMS) and Nitroblue tetrazolium (NBT) were obtained from Himedia, India. All other reagents and chemicals used were of analytical grade procured from local sources.

Sample collection and extraction:

A total of 13 fruits and vegetables samples commonly consumed in Rajasthan were purchased from the local markets. The fresh pulp or other part used of the sample was dried in an oven and this dried material was used for extraction. Each sample was extracted and analyzed in triplicates. Methanol extraction was done as 500 mg of powered sample was extracted for 4 hr at room temperature by shaking vigorously with 5 ml of 60% methanol containing 0.1% HCl. The sample suspensions were centrifuged (10,000 g for 15 min at 100C) and supernatant filtered through Whatman no.1 filter paper and the filtrate stored at -20⁰C till analysis.

Determination of Total Phenolic Content:

The plant samples were analyzed for total phenolic content using spectrophotometer (Specord 200, Analyte zena, Germany) by the method of⁸. Gallic acid was used as the reference standard. To 1ml of alcoholic extract, 1ml of commercial Folin-Ciocalteau reagent (50% diluted) and 2 ml of 20% sodium carbonate was added. The tubes were shaken and heated in the water bath (1min, 90oC), cooled under running tape water and centrifuged. The absorbance of supernatant was read against a blank without plant extract at 650nm. The unknown concentration was read from a standard curve made from different concentrations of gallic acid. All determinations were carried out in triplicate and the results are expressed as mg gallic acid equivalent per gram (mg GAE/g) of extract.

DPPH radical scavenging activity:

DPPH radical scavenging activity was determined according to methods described previously⁹. This method is based on the ability if the antioxidant to scavenge the DPPH cation radical. The DPPH method can be used for solid or liquid samples and is not specific to any particular antioxidant component, but applies to the overall antioxidant capacity of the sample. Briefly, 100μl aliquot of sample extract or standard was added to 2.9 ml of DPPH reagent (0.1 mM in methanol) and vortexed vigorously. It was incubated in dark for 30 min. at room temperature and the discoloration of DPPH was measured against blank at 517 nm. Percentage inhibition by the sample extract was calculated.

Superoxide radical scavenging activity (PMS/NADH System):

Superoxide anions were generated using PMS/NADH system. The superoxide anions are subsequently made to reduce nitroblue tetrazolium which yields a chromogenic product, which is measured at 560 nm. Briefly, ten milligrams of the extract was dissolved in 0.1mL methanol and made up the volume to 10 mL with 0.1M phosphate buffer pH7.4. The test sample / positive control (62.5 μl) in 0.1M phosphate buffer pH 7.4, 62.5 μl of 468 μM NADH solution, 62.5 μl of 150 μM NBT solution and 62.5 μl of 60μM PMS solution were added to a microwell plate and incubated at room temperature for 5 min. The absorbance was read at 560 nm. Percent inhibition was calculated¹⁰.

Statistical analysis:

All the data were recorded in triplicate (n=3) and results have been expressed as mean ± standard deviation. To correlate the results obtained with different methods, a regression analysis was performed and correlation coefficients were calculated.

RESULTS AND DISCUSSIONS:

Total Polyphenolic Content:

Total polyphenol content of different fruits and vegetables are shown in table 1. There was no significant variation in the phenol content of different samples. Maximum polyphenol content was observed in Prosopis cinneraria (12.53 mg GAE/gram), followed by Carissa congesta (10.46 mg GAE/gram).

Radical Scavenging Capacity:

Radical scavenging capacities of different fruits and vegetables were determined using DPPH and SOD assay, and are shown in Table 1.

DPPH radical scavenging capacity:

Maximum DPPH inhibition of ~91% was recorded in Mangifera indica followed by Psidium guajava and Prosopis cinnneraria (~90%). The lowest DPPH inhibition of ~15.62% was recorded in Acacia senegal which was ~9 fold lesser than the inhibition recorded in Mangifera indica.

Table 1 Radical Scavenging capacity and total phenolic content of selected fruits and vegetable used in diet in Rajasthan.

Plant name	Local name	Part used	Total polyphenols (mg GAE/gram)	DPPH % inhibition	SOD % inhibition
Acacia Senegal	Kumti	Dried seeds	2.93 ± 0.11	15.62 ± 0.22	1.70 ± 0.16
Capparis deciduas	Kair	Fruits	6.13 ± 0.23	82.13 ± 1.62	16.38 ± 0.58
Carissa congesta	Karanda, Karonda	Fruits	10.46 ± 0.21	87.57 ± 0.24	66.22 ± 1.14
Coccinia grandis	Tindori, Kundri	Fruits	6.93 ± 0.12	53.33 ± 0.02	23.99 ± 0.71
Cordia myxa	Lasora, Gunda	Fruit	7.80 ± 0.20	89.45 ± 0.11	61.99 ± 1.78
Coriandrum sativum	Dhaniya	Whole plant	4.06 ± 0.46	80.98 ± 2.42	37.83 ± 0.52
Mangifera indica	Aam	Unriped fruit	7.10 ± 0.14	91.51 ± 2.42	56.21 ± 1.01
Mangifera indica	Aam	Riped fruit	4.86 ± 0.12	86.78 ± 1.85	11.05 ± 0.66
Momordica dioica	Kinkoda	Fruits	4.20 ± 0.11	25.77 ± 0.56	22.92 ± 0.10
Prosopis cinneraria	Khejri (Sangri)	Pods	12.53 ± 0.83	90.33 ± 1.18	37.27 ± 0.22
Psidium guajava	Amrud	Fruits	6.20 ± 0.20	90.33 ± 0.46	35.55 ± 0.58
Punica granatum	Anar	Seeds	8.46 ± 0.46	88.36 ± 0.21	40.25 ± 1.60
Syzygiumcumuni	Jamun	Fruits	6.53 ± 1.15	89.87 ± 0.79	42.94 ± 0.19

Superoxide radical scavenging capacity:

Superoxide radical scavenging capacity of different samples varied from 1.70% to 66%. Maximum SOD inhibition was observed in Carissa congesta (~66%). This was followed by Cordia myxa (~62%).

Acacia Senegal showed the lowest antioxidant activity on all the assays, as the phenol content was low while Cordia myxa showed a good amount of radical’s inhibition in all the assays.

Correlation between assays:

To correlate the results obtained with the different methods, a regression analysis was performed (correlation coefficient, R). The results have been shown in figures 1-3 and table 2. The maximum correlation was found between SOD and DPPH followed by SOD and TPC (R=0.6198 and R=0.6094, respectively). The lowest correlation was found between TPC and DPPH (R=0.5718).

Figure 1. Correlation between TPC and SOD assays. Correlation coefficient R= 0.6094

Figure 2. Correlation between TPC and DPPH assays. Correlation coefficient R= 0.5718

Figure 3. Correlation between SOD and DPPH assays. Correlation coefficient R= 0.6198

Table 2 Correlation coefficient (R) between assays

	SOD	DPPH
DPPH	0.6198
TPC	0.6094	0.5718

The data from this study indicate that consumption of above mentioned fruits and vegetables may deliver healthful benefits by supplying natural antioxidants that are protective against cellular damage, improve digestion and maintain normal metabolism. Fruits and vegetables contain variety of such nutrients that maintains the normal functioning of the body. Vitamin C reduces the risk of hypertension¹¹. Phenolics are ubiquitous secondary metabolites in plants and possess a wide range of therapeutic uses such as antioxidant, antimutagenic, anticarcinogenic, free radical scavenging activities and also decrease cardiovascular complications¹². Flavonoids are polyphenols and they are abundantly found in fruits, vegetables, grains, bark, root, stem, flowers and tea. They possess anti-inflammatory, anti-allergic, antiviral and anti-carcinogenic properties due to their antioxidant potential¹³. Measurements of total antioxidant capacity could be a valuable tool in food technology, as the effect of growing conditions, seasonability, storage, processing, presentation techniques, cooking and genetic modifications of plant based foods could be determined¹⁴.

It was observed that no significant correlation was observed between TPC and antioxidant activity. Some researchers¹⁵ pointed out that there was a correlation between antioxidant capacity and phenolics content. However, some research groups¹⁶ stated that antioxidant activity does not necessarily correlate with high amounts of phenolics. The lack of correlation between TPC and antioxidant capacity could be due to the fact that Folin-Ciocalteau reagent used in TPC estimation, also reacts with sugars and ascorbic acid present in plants extracts and besides them, some simple phenols also react with the reagent, although they are not effective radical scavenging antioxidants¹⁷. The other probable reason for antioxidant capacity does not necessarily correlate the TPC is that the molecular antioxidant responses of phenolic compounds vary remarkably, depending on their chemical structure¹⁸. The results from different antioxidant assays are even difficult to compare because of the difference in substrates, probes, reaction conditions and quantification methods¹⁹.

CONCLUSION:

This study showed the antioxidant potential of some non-conventionally consumed fruits and vegetables of Rajasthan. This study can be helpful in providing information to the people regarding the increased uptake of these antioxidants and phenolics rich fruits and vegetables. Optimizing antioxidant intake by increased consumption of fruits and vegetables may be useful in planning diets for health promotion.

ACKNOWLEDGMENTS:

This work was supported by financial assistance from UGC-DRS under a special assistance program for medicinal plant research to KGR and JRF to NJ from UGC, New Delhi.

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Received on 17.11.2011 Modified on 02.12.2011

Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 121-124