Comparative study of antioxidant compounds and antiradical properties of the fruit extracts from three varieties of Crataegus pinnatifida
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- Dou, D., Leng, P., Li, Y. et al. J Food Sci Technol (2015) 52: 430. doi:10.1007/s13197-013-0954-6
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Crataegus pinnatifida (C. pinnatifida) is widely cultivated in China as an edible fruit and a traditional herb medicine. In this study, we determined the total polyphenolic and condensed tannin content of the fruit extracts from three varieties of C. pinnatifida, and assessed the antioxidant activities in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) decolorization assays. Compared with the extracts from C. pinnatifida Bge. (Shanzha, SZ) and C. pinnatifida Bge. var. pinnatifida (an original variety of Shanzha, VSZ), the extracts of C. pinnatifida Bge. var. major N. E. Br. (Shanlihong, SLH) showed a higher polyphenolic and condensed tannin content, as well as a higher radical scavenging activity with a Trolox equivalent antioxidant capacity (TEAC) value of approximately 50 mg/g. Furthermore, the total polyphenolic content in the extracts was significantly correlated with the IC50 value for DPPH˙ (r = −0.986, p < 0.05) and TEAC value (r = 0.997, p < 0.05). These results demonstrated that the three varieties of C. pinnatifida, especially SLH, had a potential application as antioxidants in the research of human nutrition and medicine.
KeywordsCrataegus PinnatifidaAntioxidantFree radicalPolyphenolTannin
Hawthorn has been long planted in China as an edible fruit, and among the 18 reported species of Chinese hawthorn Crataegus pinnatifida (C. pinnatifida) is the most important one (Cui et al. 2006). In the northeastern China, three closely related varieties of C. pinnatifida, namely Shanlihong (SLH, C. pinnatifida Bge. var. major N.E.Br.), Shanzha (SZ, C. pinnatifida Bge.) and the original variety of Shanzha (VSZ, C. pinnatifida Bge. var. pinnatifida) are widely cultivated because of their good taste and nutritional value. Meanwhile, their fruits are widely processed into many kinds of snacks, jams, jellies, juices, and alcoholic beverages.
Besides its application in food products, C. pinnatifida has been used as an important traditional Chinese medicine material in the treatment of indigestion, dyspnea and kidney disorders. Furthermore, C. pinnatifida also has several other active medical therapeutic effects, including cardiotonic, anti-inflammation, antitumor, blood pressure regulation, and immunomodulation (Kao et al. 2007; Kwok et al. 2010; Yang and Liu 2012). Although the mechanisms require further investigations, the antioxidants are involved in the health-promoting effects of C. pinnatifida, because these compounds can reduce initiation and propagation of free radicals, which are closely related to the pathogenesis of a wide number of diseases (Yang and Liu 2012).
With the increasing focus on antioxidant activity of polyphenolics in fruits and berries, total levels of polyphenolics, rather than those of individual compounds, have aroused many research interests recently (Meda et al. 2005). In this regard, it is advisable to assess the total content of antioxidant compounds and their antiradical activity, and then establish the correlation between them, which is very important for health claims in nutriceutical products. Although the health-promoting effects of C. pinnatifida have been well described, there is still a limitation on the information concerning the comparison of the contents of total polyphenolic compounds, condensed tannin, as well as the antiradical effect among the three varieties of C. pinnatifida (SLH, SZ and VSZ).
In this study, we presented the analysis of the total polyphenolic and condensed tannin content in the three varieties of C. pinnatifida (SLH, SZ and VSZ), and the determination of the antiradical activities in DPPH and ABTS decolorization assays. Furthermore, the potential antioxidant properties of the fruit extracts were evaluated by TEAC value. The relationship between the total polyphenolic content and the antiradical capacity was also investigated.
Materials and methods
Materials and chemicals
Gallic acid, catechin, vanillin, Trolox, DPPH and ABTS were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemical reagents used were of analytical grade. SLH and SZ were collected from Chengde County of Hebei Province, and VSZ was collected from Pinggu district of Beijing, China. The pulps of SLH, SZ and VSZ were freeze-dried after removing the seeds, and then were ground into powder in a coffee grinder, respectively.
Preparation of extracts
The dried pulps were extracted with hot water, 70 % (v/v) aqueous methanol and methanol, at a solid-to-solvent ratio of 1:50 (w/v) in a sonication bath for 20 min. After centrifugation the supernatant was collected. The procedure was repeated twice. Then the supernatant was pooled, evaporated to dryness under vacuum and lyophilized as the extract. As a stock solution for measurement, the lyophilized hot water or organic soluble-extracts were dissolved in 100 ml of distilled water or 75 % (v/v) aqueous ethanol at an extract concentration of 50 mg/ml, respectively,
Measurement of total polyphenolic and condensed tannin content
Using the Folin-Ciocalteau method (Yen and Hsieh 1998), the total polyphenolic content in the extract was measured by the absorbance at 765 nm. The results were expressed as milligrams of gallic acid equivalents per gram from a calibration curve of gallic acid (0–8 μg/ml). The soluble-condensed tannin was assayed colorimetrically by the modified Vanillin method (Price et al. 1978). The total volume of the reaction medium was fixed at 6 ml by 1 ml of sample, 2.5 ml of reagent A (1 % vanillin solution in methanol), and 2.5 ml of reagent B (8 % HCl in methanol). The absorbance of the solution was measured at 500 nm, after the incubation in dark at room temperature for 20 min. The condensed tannins content in the extract was expressed as milligrams of catechin equivalents per gram from a calibration curve, in which the concentration of catechin was in a range of 0–150 μg/ml.
Determination of the scavenging activity of DPPH˙ free radical
Determination of the scavenging activity of ABTS˙+ free radical
A calibration curve was prepared with concentrations ranging from 0 to 5 μg/ml of Trolox for scavenging activities on ABTS˙+ radicals. The TEAC value was defined as the concentration of Trolox having an antioxidant activity equivalent to 1 g of the extract. To calculate the TEAC value, the gradient of the plot of the percentage inhibition of absorbance vs. the concentration plot for the antioxidant in question was divided by the gradient of the plot for Trolox (Re et al. 1999).
All determinations were conducted in triplicate and all results were expressed as mean ± standard deviation (SD). The differences were calculated by one-way analysis of variance (ANOVA) using the statistical software (SPSS, version 14.0, SPSS Inc., Chicago, USA). Correlations between different parameters were calculated as Pearson’s correlation coefficient (r) using Excel for Windows 2000. Differences or correlations were considered significant at p < 0.05.
Results and discussion
Characterization of dried pulp-extract from the three varieties of C. pinnatifida
The recovery of dried pulps and the extracts was determined by weight. The ratio of dry pulps to fresh pulps was measured as 21.6 % for SLH, 26.1 % for SZ, and 28.6 % for VSZ. This finding indicated that SLH had the highest moisture content. In addition, compared with SZ and VSZ, SLH had a better taste and a larger size. These physical characteristics of SLH could result from its longer period of cultivation and domestication in China.
Recovery of dried pulps and their extracts from the three varieties of C. pinnatifida.
Dry pulps/Fresh pulps (wt/wt, %)
Recovery (wt/wt, %)
70 % (v/v) Methanol
21.6 ± 0.47a
67.8 ± 2.31a
66.5 ± 2.24a
73.6 ± 4.70a
26.1 ± 0.23b
70.3 ± 3.06b
60.7 ± 1.76b
76.9 ± 3.21b
28.6 ± 0.71c
51.5 ± 0.13c
51.0 ± 1.90c
56.2 ± 1.12c
Evaluation of antioxidant content in the extracts
Total polyphenolic and condensed tannin contents in the different solvent extracts of SLH, SZ and VSZ from the three varieties of C. pinnatifida
Total polyphenols* (mg g−1)
Condensed tannins** (mg g−1)
70 % (v/v) Methanol
70 % (v/v) Methanol
104.6 ± 4.16a
97.7 ± 2.88a
91.0 ± 1.57a
41.1 ± 1.20a
37.4 ± 2.04a
32.9 ± 2.37a
43.5 ± 2.17b
31.4 ± 1.68b
37.7 ± 1.04b
13.4 ± 1.32b
9.2 ± 1.33b
9.9 ± 1.40b
73.9 ± 2.18c
67.5 ± 1.19c
59.0 ± 1.31c
27.2 ± 2.10c
24.8 ± 1.96c
18.7 ± 2.45c
Comparing antioxidant capacities of the extracts from the three varieties of C. pinnatifida
The antioxidant activity of the 70 % (v/v) methanol extracts was also assessed using ABTS˙+ radical decolorization assay. The reaction time of Trolox (4 μg/ml) and 70 % (v/v) methanol extracts at the same concentration level (0.1 mg/ml) was measured. Trolox, as a standard, exhibited an immediate fast radical-trapping rate, and the reaction was completed within 30 s. However, the extracts had a slower onset of radical-trapping than Trolox, showing an inhibitory effect up to 6 min of reaction (see Supplementary material). Therefore, after incubation in the dark at room temperature for 6 min, the ABTS˙+ radical scavenging activities of the 70 % (v/v) methanol extracts were measured, in the concentration range of 0.025−0.300 mg/ml (Fig. 2b). The result indicated that the scavenging activity pattern in the ABTS assay was similar to that in the DPPH assay, and the extract from SLH had the strongest ABTS˙+ radical scavenging effect.
Furthermore, the antioxidant activities of all extracts were calculated in terms of TEAC value and a higher TEAC value represented a more potent radical scavenging capacity. The TEAC value of the extracts from SLH was in the range of 49.9−53.3 mg/g (Fig. 3b). After conversion, the data meant that the ABTS˙+ scavenging capacity of the fresh fruit of SLH was equivalent to approximately 55 μM Trolox per gram. Fan et al. (2011) reported a similar TEAC value of SZ. After extracted with 80 % (v/v) acetone, the TEAC value of the fresh SZ fruit was measured as 45 μM Trolox equivalents per gram. In agreement with the observation in DPPH assay, the TEAC values of the VSZ and SZ also followed with that of SLH, suggesting that in our study SLH possessed the most remarkable antioxidant capacity. Moreover, the TEAC values of the fresh SLH, SZ and VSZ were all higher than that of C. monogyna (8.4 μM Trolox/g) and C. azarolus (4.1 μM Trolox/g) (Egea et al. 2010).
Relationship between antioxidant substance and antiradical activity
Polyphenolic compounds were abundant in C. pinnatifida and their application could improve the health properties associated with radical scavenging activity in the human body. However, the relationship between the total polyphenolic content and antiradical activity of the three varieties of C. pinnatifida has not yet been studied. In our experiment, the highest polyphenolic content and antiradical activity were both found in SLH, while the lowest values were observed in SZ. Furthermore, in the extracts a strong correlation (p < 0.05) was found between the total polyphenolic content and the antiradical activity. For example, the linear regression coefficient (R2) of the TEAC value on the total polyphenolic content was calculated as 0.9933 (Fig. 1c). Although in several hawthorn fruits of Turkey, the antioxidant capacity for DPPH˙ poorly correlated with the total phenolics (Çaliskan et al. 2012), a linear correlation between antioxidant activities and polyphenolic contents was found in many plant extracts, fruits, vegetables, and beverages (Meda et al. 2005; Buratti et al. 2007; Jagdish et al. 2009).
In the present study, we investigated the content of total polyphenolics and condensed tannins in the extracts from three varieties of C. pinnatifida (SLH, SZ and VSZ). The results indicated that SLH, particularly its 70 % (v/v) methanol extract, possessed numerous amounts of antioxidant substances. The results also showed that in DPPH and ABTS assays, the antiradical capacities of the three varieties of C. pinnatifida were in the descending order of SLH > VSZ > SZ. Furthermore, a significant linear correlation was found between the total polyphenolic content and the antioxidative potencies against DPPH˙and ABTS˙+ radicals. Therefore, considering their abundant polyphenolics and good antioxidant properties, the three varieties of C. pinnatifida, especially SLH, could be applied as good sources of cultivated berries for medicine and food industrial application.
This work was supported by the Science and Technology Planning Project of Beijing (KM201110020012), National Natural Science Foundation of China (20972181), and the Science and Technology Planning Project of Tianjin (11ZCKFNC02000).