1.Introduction
Interesting role of antioxidants in human health has prompted researches in the fields of food science and horticulture to assess fruit and vegetable antioxidant content (Kalt et al, 1999). The majority of the antioxidant capacity of a fruit or vegetable may arise from compounds such as flavonoids, isoflavones, flavones, anthocyanins, catechins and isocatechins rather than from vitamins C, E or β-carotene (Wang, 1996, Kähkönen et al,1999). Many of these phytochemicals could be helpful in protecting cells against the oxidative damage of free radicals (Wada & Ou, 2002) Fruit and vegetable antioxidants play an important role in reducing the risk of degenerative diseases such as cardiovascular disease, various cancers and neurological diseases (Kalt et al, 1999). About 5000 plant phenolic compounds have been known and model studies have demonstrated that many of them have antioxidant activity (Robards t al, 1999). The antioxidant activity of phenolic compounds is mainly because of their redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelators (Rice-Evans et al, 1995) Their antioxidant activity is generally based on the number and location of their hydroxyl groups as well as the presence of a 2-3 double bond and 4-oxofunction (Rice-Evans & Miller, 1998) The flavonoids, a large family of low molecular weight polyphenolic compounds, include the flavones, flavonols, flavonones, isoflavones, flavan-3ols and anthocyanins (Stewart et al, 2000). Although flavonoids are generally considered as nonnutritive agents, interest in these substances has increased due to their possible effects on human health (Hertog et al, 1992). In addition to their antioxidant activities, flavonoids have inhibitory effects on enzymes such as prostaglandin synthase, lipoxygenase and cycloxygenase, which are closely related to tumorigenesis, and may induce detoxifying enzymes such as glutathione Stransferase (Lee et al, 1995). Numerous types of flavonoid have been reported in fruits and vegetables; however, their type and content vary with cultivar and maturation (Hertog et al, 1992). All of these aspects explain the increasing interest in fruit phenolic compounds that has been manifested in the past few years. In this context, a large number of plant sources including many fruits and vegetables have been explored for their antioxidant potential. Therefore, the main objective of this study was to determine the antioxidant activity of different fruits and vegetables grown in Iran. Another aim was to evaluate whether total phenolic and flavonoid contents of samples are correlated with their antioxidant activity. A diet rich in vegetables (more than 5 servings per day) is recommended along with fruits and whole grains. An epidemiological study found that a diet of with such composition shows a negative association with the risk of chronic diseases. Antioxidant vitamins in vegetables are some of the important nutrients besides other vitamins, minerals, flavonoids and phytochemicals, which have been reported to contribute to health. Our local markets offer a variety of vegetables ranging from leafy to tubers ones (Tee et al, 1997). Beside the conventionally grown vegetables, currently, organically grown foods are gaining popularity among consumers, health educators, farmers and food retailers. Many consumers believe that organically grown vegetables are of better quality, healthier and more nutritious as compared with conventionally grown ones. This is due to a number of reasons which may vary from country to country such as safety, effect of environment, flavor, freshness, health benefits and nutritional value (Bourn & Prescott , 2002)
2.MATERIALS AND METHODS
Four types of green vegetables were selected based on their popularity among Iranian consumers. Conventionally-grown vegetables (400 g) were purchased from a local market at Kerman. Convenience sampling was used to obtain the samples. Fresh vegetables (carrots, celery, lettuce and red cabbage) were purchased from local markets in Kerman in summer. All vegetables were washed and grated before extraction. Healthy vegetables were selected randomly to have samples with uniform shape and color. Preparation of samples Upon arrival at the Department of Nutrition and Health Sciences laboratory was as follows: the fresh and healthy vegetables were immediately washed under tap water and excessive water dripped off. Edible portions (100 g) of the vegetables were cut into small pieces and homogenized using a blender (National; model MX-291N) for 2 min. The homogenized sample was transferred into an airtight container and kept at -20°C before vitamin analysis.
Determination of Vitamin C and Phenolic acids in Green Vegetables
The vegetables juice was extracted by careful hand-squeezing to obtain the juice. The juice was passed through a strainer to remove pulp. The freshly-squeezed juice was twice centrifuged at 3000 rpm for 10 min, the supernatants were then diluted (1:5). The dilutions were membrane filtered (0.20 μm) before injection.
2.1Analysis by UPLC
Antioxidant content of the sample solutions were separated by reversed phase chromatography on a 150 mm×4.6 mm i.d., 5 μm particle ZORBAX Eclipse XDB-C18 analytical column, which was detected by absorbance and quantified with external calibration graph. For the simultaneous detection of four analytes, the detector was set at λ=254 nm for ascorbic acid and λ=214 nm for the other organic acids. This setting was chosen as ascorbic acid has its maximum optical absorbance at 254 nm. The UPLC analysis was performed with an Agilent 1200 series system. Integration, data storage and processing were performed by Chemstation software. The determinations were made in isocratic conditions, at ambient temperature, using a mobile phase made of 0.2 % acetonitrile and 50 mM phosphate solution (dissolve 6.8 g potassium dihydrogen phosphate in 900 ml water; the pH value should be adjusted to pH =2.8 with phosphoric acid and then filled to 1000 ml with water) filtered through a polyamide membrane (0.2 μm) and degassed in vacuum. The flow rate of the mobile phase was 1.2 ml/min for all the chromatographic separations. The separation column was balanced with mobile phase until the baseline was stabilized. Sample injections were made at this point. 5 μl of prepared sample or standard solution were injected.
2.2Determination of phenolic compounds
The phenolic compounds (Gallic, catechin, caffeic, chlorogenic, ocoumaric, p-coumaric, ferulic, ciringic, vanillic, quercetin, and rutin acids) were determined through HPLC separation method. In three replicas, about 100g of samples was fragmented and 5mL fruit juice from each sample was transferred to centrifuge tubes. The samples were homogenously mixed then diluted by distilled water at the ratio of 1:1. The samples were then centrifuged at 15000 g for 15 minutes. The supernatants were passed through 0.45 mm membrane filter (Millipore Millex-HV Hydrophilic PVDF, Millipore, USA), then injected into HPLC system. The Chromatographic separation in Agilent 1100 series HPLC took place in DAD detector (Agilent. USA) with 250 x 4.6 mm, 4μm ODS column (HiChrom, USA). The following solvents in water with a flow rate of 1 mL min-1 and 20 µL injection volume were used for spectral measurements at 254 and 280 nm: methanol-acetic acid-water (10:2:88) and methanol-acetic acid-water (90:2:8) were used as mobile phase solvent A and B, respectively (Table 1.).
2.3Determination of total antioxidant activity
For the standard trolox equivalent antioxidant capacity (TEAC) assay, ABTS [29, 2-azinobis-(3-ethylbenzothiazoline-6sulfonic acid)] was dissolved in acetate buffer and prepared with potassium persulfate, as described by RiceEvans et al. [6]. The mixture was diluted in acidic medium of 20 mM sodium acetate buffer (pH 4.5) to an absorbance of 0.700 ± 0.01 at 734 nm for better stability. For spectrophotometric assay, 3 mL of the ABTS+ solution and 20 µL of fruit extract were mixed and incubated for 10 min and its absorbance was determined at 734 nm.
3.RESULTS AND DISCUSSION
In Fig. 1 the chromatograms of ascorbic acid and naringin in the standard solution and real samples are shown. The linearity of the method was evaluated according to area response. Selected wavelength for ascorbic acid, retention time, and concentration ranges of linear response, correlation coefficients and detection limit are summarized in Tab. 1. The detection limit (LOD) can be defined as the smallest peak detected with a signal height three times higher than that of the baseline. The limit of quantification (LOQ) refers to the lowest level of determinable analyte with an acceptable degree of confidence. In the present work, detection limits were estimated according to the hypothesis that in order to detect a peak, it should have a signal-to-noise ratio higher than 3. Precision was tested on ten replicas of independent vegetable samples. The RSD value was 0.154% indicating that the method was precise with a high degree of repeatability. The recovery of ascorbic acid from vegetables ranged from 97.2 to 103.6%. The levels of ascorbic, ferulic acid and naringin of the vegetables were shown in Table. 2.
Figure 1. Chromatogram of Ascorbic acid in standard solution (a), Naringin standard (b), Red cabbage standard (c), and celery (d)
Table 1. Wavelength, retention time, concentration range of lineal response, correlation coefficient and detection limit for standards.
| Analyte | λnm | RT(min) | Concentration Range (mg/l) | Correlation Coefficient (r2) | Detection Limit (mg/l) |
| Ascorbate | 254 | 3.2 | 0.5-200 | 0.9990 | 0.20 |
| Ferulic Acid | 283 | 4.1 | 0.5-100 | 0.9994 | 0.04 |
| Naringin | 283 | 11.2 | 5-200 | 0.9995 | 0.07 |
Table 2. Ascorbic acid and phenolic acids content (gr/l) of vegetables.
| Vegetables | Ascobate | Ferulic Acid | Naringin |
| Carrots | 1.08 | 0.57 | 1.60 |
| Celery | 0.98 | 0.82 | 0.21 |
| Lettuce | 0.68 | 0.92 | 0.37 |
| Red cabbage | 0.18 | 0.47 | 0.09 |
4.CONCLUSION
The present study was the first comprehensive investigations to determine the phenolic and antioxidants contents of vegetables grown in Kerman ecological conditions. Therefore, this initial study would be helpful in organizing further researches in this field. Based on the results of the present study, application of the high phenolic substance-containing cultivars in vegetable processing technology might contribute to the effective outcomes. It was seen that the carrot was in the forefront due to its Naringin content with analgesic, anti-allergenic, anti-asthmatic, antibacterial, immune-stimulating, antiviral, antiseptic and cancer-preventive effects. Moreover, red cabbage had higher antioxidant activity. This work is a contribution to the development of a rapid and precise UPLC procedure for quantitative determination of total phenolic compound of vegetables. Ascorbic acids, ferulic acid and naringin have been eluted from the column within 4 minutes.
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