Antioxidant capacity of extracts from calyx fruits of roselle (Hibiscus sabdariffa L.)

1 School of Chemistry and Chemical Engineering, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China. 2 School of Pharmacy, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China. 3 School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China. 4 School of the Environment, Jiangsu University, 301 Xuefu Rd., 212013 Zhenjiang, Jiangsu, China.

oxygen species can induce many kinds of diseases. Nevertheless, natural antioxidants protect against these oxidative stresses and associated diseases. They have played an important role in the health care industry (López et al., 2007). Studies demonstrated that the calyx of roselle (Mourtzinos et al., 2008) was rich in phenolic compounds and anthocyanins. It was reported that those compounds could be considered as a great source of natural antioxidants. Those compounds were extracted from roselle in different studies using water (Mojiminiyi et al., 2007;Tsai et al., 2002), acidified methanol (Kao et al., 2009) and absolute ethanol (Tseng, 1998) as extract dissolvent, or sequentially with hexane, ethyl acetate and acidified methanol (Christian et al., 2006). However, to the best of our knowledge, there were no studies reported about the antioxidant activities of the aqueous extract or extracts with different concentrations of ethanol from roselle. In the present study, the antioxidant capacities of the different extracts of roselle were studied by 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH), hydroxyl (OH) and 2-2'azino-bis-(3-ethyl-benzthia-zoline-6-sulfonic acid) (ABTS) assays. The contents of total phenols (TP), anthocyanins and total saccharides (TS) were also determined. Furthermore, the correlations between antioxidant capacities and contents of total phenols, anthocyanins and total saccharides were analyzed.

MATERIALS AND METHODS
Roselle (H. sabdariffa L.) was obtained from Danyang, Zhenjiang, Jiangsu Province in November 2008, and identified by Chen Li (Chief Pharmacist, Food and Drug Administration Bureau of Zhenjiang).

Chemicals and reagents
DPPH, ABTS and Folin-Ciocalteu reagent were obtained from Sigma-Aldrich (Milwaukee, WI, USA). All solvents and chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Preparation of roselle extracts
The calyx and fruit were dried in a hot-air dryer (Yi-heng Science and Technique Co. Ltd., China) at 70°C and triturated before solvent extraction. Fifteen grams of roselle calyx and fruit powders were extracted with 300 ml of water or 30% ethanol, 60% ethanol, 95% ethanol at 75°C for 1 h, followed by filtration with filter paper (30 to 50 µM, Hangzhou Xinhua Paper Industry Co., Ltd., Hangzhou, Zhejiang, China), respectively, and then the residues were re-extracted with 300 ml of the same solvent as described above. The combined supernatants were concentrated in a rotary evaporator at 55°C, and lyophilized to afford the various extracts. The extraction yield was calculated as: Sample extract weight Extraction yield (%) = × 100 Total sample weight

DPPH assay
The DPPH free radical-scavenging activity of each sample was determined according to the method described by Scherer and Godoy. (2009). Briefly, 2.0 ml of roselle extract samples or ascorbic acid standards at different concentrations were added to 2.0 ml DPPH dehydrated ethanol solution (0.2 mM) separately. After a 30min incubation at room temperature in the dark, the absorbance was measured at 517 nm. The radical scavenging activity was calculated as follows: Where, A0 is the absorbance of the blank sample, Ai is the absorbance in the presence of the test compound at different concentrations and Aj is the absorbance of the blank reagent. The IC50 (concentration providing 50% inhibition) was calculated graphically using a calibration curve in the linear range by plotting the extract concentration vs. the corresponding scavenging effect, and ascorbic acid was used for comparison.

Hydroxyl radical-scavenging assay
According to the method described by Su et al. (2009), 2.0 ml FeSO4 (6 mM) aqueous solution was added to 2.0 ml sample extracts, and then 2.0 ml H2O2 (6 mM) was added. After a 10-min incubation period at room temperature, 2.0 ml salicylic acid (6 mM) were added, and incubated for 30 min. The absorbance was measured at 510 nm. The radical scavenging activity was calculated as above. All tests were carried out in triplicate.

ABTS assay
The ABTS assay was performed according to the method described by Li et al. (2009) with slight modification. ABTS radical cation (ABTS + ) was produced by mixing the 7 mM ABTS solution with 2.45 mM potassium persulfate aqueous solution leaving them in the dark at room temperature for 12 to 16 h. The ABTS + solution was diluted with phosphate buffer to the absorbance of 0.70 ± 0.02 at 734 nm. Then 6 min after adding 0.2 ml of sample aqueous solution to 3.8 ml of diluted ABTS + solution, the A734 absorbance was measured. For the blank, 0.2 ml water was used instead of the sample. The radical scavenging activity was calculated as above. All tests were carried out in triplicate.

Determination of total phenols
Total phenols were determined by the Folin-Ciocalteu method of Chew et al. (2009). Briefly, 1.5 ml Folin-Ciocalteu reagent (10%, v/v) was mixed with 1.2 ml 7.5% (w/v) Na2CO3 solution, then 0.3 ml sample solution was added. After a 30-min incubation at room temperature in dark, the absorbance was measured at 765 nm. Gallic acid was used as a standard compound for the standard curves. The results were presented in mg gallic acid equivalent (GAE)/g extract. All the experiments were carried out in triplicate.

Determination of anthocyanins
Anthocyanins were determined by the pH differential method of Chew et al. (2009) with slight modifications. Briefly, 1.0 ml 0.2 mol/L potassium chloride solution (pre-adjusted to pH 1.0 with 1.0 mol/L HCl) or 1.0 ml 1 mol/L sodium acetate buffer (pre-adjusted to pH 4.5 with 1.0 mol/L HCl) was added into 2.0 ml of extracts respectively. The absorbances were then measured at 517 and 700 nm. The sample absorbance equals A517 value minus A700 value. The concentration of anthocyanins was calculated with the following formula: Concentration of anthocyanins (mg/L) = A / εL × MW × 10 3 × Dilution factor Where, A is the difference of sample absorbance between pH 1.0 and 4.5, ε is molar extinction coefficient for cyanidin-3-glucoside (26,900); L is the path length of the spectrophotometer cell (1.0 cm), and MW is molecular weight of cyanidin-3-glucoside (449.2 g/mol). The result was expressed as mg cyanidin-3-glucoside (cy-3glu) equivalent/g extract. All the experiments were carried out in triplicate

Determination of total saccharides
Total saccharides were determined by the phenol-sulfuric method of Chinese Pharmacopoeia (2005). Sixty milligrams of anhydrous dextrose was dissolved in 100 ml water (0.6 mg anhydrous dextrose /ml) and it was then used as standard solution. Next, 1.0, 1.5, 2.0, 2.5 and 3.0 ml of standard solution were diluted to 50 ml, respectively, and then 2.0 ml aliquot of each solution was immediately mixed with 1.0 ml 4% phenol and 7.0 ml sulfuric acid. Aqueous extract 30% Alcohol extract 60% Alcohol extract 95% Acohol extract DPPH radical-scavenging inhibition (%)
After a 30-min-incubation at 40°C, the mixture was left in an icebath for 5 min and then the absorbance was measured at 490 nm.
The tests were carried out in triplicate. The total saccharides of sample were quantified with the standard curve mentioned above. The total saccharide values were presented in milligram (mg) anhydrous dextrose equivalent / g extract.

Statistical analysis
Data were reported as mean ± standard deviation (SD) of triplicate determinations. Statistical calculations by SPSS version 16.0 software (SPSS Inc, Chicago, USA) were carried out. One way analysis of variance was applied for determining differences between results of samples. Values of p < 0.05 were considered as significantly different.

Antioxidant capacity
In this study, the antioxidant properties of the aqueous and different ethanol extracts of calyx and fruit of roselle were tested, and then compared with the activity of the well known antioxidants ascorbic acid. Moreover, the IC 50 values, which refer to the smallest concentration of the antioxidants necessary for 50% of inhibition, were calculated. The sequence for DPPH radical-scavenging abilities were shown in Figure 1: 30% ethanol extract > 60% ethanol extract > aqueous extract > 95% ethanol extract. The IC 50 values of DPPH radicals scavenging for the aqueous and different ethanol extracts from calyx and fruit of roselle were shown in Table 1. The IC 50 values of these four extracts were 486.52 ± 10.56, 289.01 ± 16.68, 465.88 ± 36.94 and 1051.72 ± 184.20 µg/ml, respectively. In the hydroxyl radical-scavenging assay, the antioxidant activities were in the similar order: 30% ethanol extract > aqueous extract > 95% ethanol extract > 60% ethanol extract (Figure 2), and IC 50 values were 1097. 76 ± 237.63, 1238.50 ± 108.40, 1268.66 ± 71.24 and 1540.83 ± 140.97 µg/ml, respectively (Table 1). The scavenging effects on the ABTS radical also showed that the 30% ethanol extract had the lowest IC 50 value (423.25 ± 31.38 µg/ml) and highest antioxidant activity (Figure 3 and Table 1). Meanwhile the 95% ethanol extract has the highest IC 50 value (2087.08 ± 201.55 µg/ml) in ABTS assay (Table 1).

Correlations between antioxidant activity and contents of total phenols, anthocyanins and total saccharides
Correlations were studied between the antioxidant activities and contents of total phenols, anthocyanins and total saccharides. It was observed that 30% ethanol extract had the highest radical-scavenging activities among the four extracts, while the 95% ethanol extract had much lower results. Correlation analysis (Table 3) showed that the higher activities of 30% ethanol extract were probably based on its higher contents of total phenols (R 2 = 0.9615, 0.9089 and 0.9771, respectively).

Conclusion
Our results clearly show that 30% ethanol extract of calyx and fruit of roselle had strong antioxidant activities. Our study suggests that this extract could be used as an effective and safe antioxidant source. Notably, our correlation study indicates that the total phenol contents of this extract played major roles in the antioxidant activities.