Journal of Food Science and Technology

, Volume 52, Issue 1, pp 430–436

Comparative study of antioxidant compounds and antiradical properties of the fruit extracts from three varieties of Crataegus pinnatifida

Authors

  • Dequan Dou
    • Department of Landscape ArchitectureBeijing University of Agriculture
  • Pingsheng Leng
    • Department of Landscape ArchitectureBeijing University of Agriculture
  • Yuehua Li
    • Department of Landscape ArchitectureBeijing University of Agriculture
  • Yan Zeng
    • Tianjin Institute of Industrial BiotechnologyChinese Academy of Sciences
    • Tianjin Institute of Industrial BiotechnologyChinese Academy of Sciences
Original Article

DOI: 10.1007/s13197-013-0954-6

Cite this article as:
Dou, D., Leng, P., Li, Y. et al. J Food Sci Technol (2015) 52: 430. doi:10.1007/s13197-013-0954-6

Abstract

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.

Keywords

Crataegus PinnatifidaAntioxidantFree radicalPolyphenolTannin

Introduction

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

In the DPPH method (Shimada et al. 1992), 2.0 ml extract sample was mixed with 0.5 ml of 1 mM DPPH solution in ethanol. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min, and then the absorbance (A) was measured at 517 nm. For all experiments, the 75 % (v/v) aqueous ethanol was used as a control to instead of the sample solution. The DPPH free radical scavenging effect was calculated as:
$$ {\rm{Scavenging}}\:{\rm{activity}}\:{\rm{for}}\:{\rm{DPP}}{{{\rm{H}}}^{\cdot }}\:\left( \% \right) = \left( {{{{\rm{A}}}_{{{\rm{control}}}}} - {{{\rm{A}}}_{{{\rm{sample}}}}}} \right)/{{{\rm{A}}}_{{{\rm{control}}}}} \times 100\:\% $$
(1)

Determination of the scavenging activity of ABTS˙+ free radical

The ABTS reagent was prepared according to the method of Pellegrini et al. (1999). To determine the scavenging activity, 0.9 ml of ABTS reagent was mixed with 0.1 ml of the sample and the absorbance was measured at 734 nm after 6 min of incubation at room temperature. The ABTS˙+ scavenging effect (%) was calculated as:
$$ {\rm{Scavenging}}\:{\rm{activity}}\:{\rm{for}}\:{\rm{ABT}}{{{\rm{S}}}^{{\cdot + }}}\:\left( \% \right) = \left( {{{{\rm{A}}}_{{{\rm{control}}}}} - {{{\rm{A}}}_{{{\rm{sample}}}}}} \right)/{{{\rm{A}}}_{{{\rm{control}}}}} \times 100\:\% $$
(2)

TEAC assay

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).

Statistical analysis

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.

As listed in Table 1, the recovery of dried pulps and their extracts had a significant difference (p < 0.05) among the three varieties of C. pinnatifida. In the case of methanol extraction, the recovery of SLH was the highest (66.5 %), followed by SZ (60.7 %) and VSZ (50.9 %). However, in the water-based extractions, the recovery of SZ was the highest [76.9 % for the hot water extract and 70.3 % for the 70 % (v/v) aqueous methanol extract]. This result showed that the three varieties of C. pinnatifida possessed different amounts of chemical constituents, and the yield of these compounds was affected by the extraction method.
Table 1

Recovery of dried pulps and their extracts from the three varieties of C. pinnatifida.

Sample

Dry pulps/Fresh pulps (wt/wt, %)

Recovery (wt/wt, %)

70 % (v/v) Methanol

Methanol

Hot water

SLH

21.6 ± 0.47a

67.8 ± 2.31a

66.5 ± 2.24a

73.6 ± 4.70a

SZ

26.1 ± 0.23b

70.3 ± 3.06b

60.7 ± 1.76b

76.9 ± 3.21b

VSZ

28.6 ± 0.71c

51.5 ± 0.13c

51.0 ± 1.90c

56.2 ± 1.12c

The results were given as mean values ± standard deviation (n = 3)

Different small superscripts (a–c) denoted the significant difference (p < 0.05) of the varieties of C. pinnatifida in the test

SLH: Shanihong, C. pinnatifida Bge. var. major N.E.Br.; SZ: Shanzha, C. pinnatifida Bge; VSZ: Original Variety Shanzha, C. pinnatifida Bge. var C. pinnatifida

Evaluation of antioxidant content in the extracts

The polyphenolic compounds containing flavonoids, procyanidins (dimers to hexamers) and phenolic acid were probably the active constituents of C. pinnatifida and were associated with the antioxidative effect (Chu et al. 2003; Zhang et al. 2001; Yang and Liu 2012). For the three varieties of C. pinnatifida, their total contents of polyphenolics and condensed tannin in 70 % (v/v) aqueous methanol extracts were higher than that in hot water and methanol extracts (Table 2). The finding indicated that the yield of antioxidant substances was strongly affected by the extraction solvents (Sasidharan and Menon 2011; Annegowda et al. 2012). Different solvents including ethanol, methanol, water, acetone, or their combinations were used to extract total polyphenolics and condensed tannin. Some reports demonstrated that the polyphenolic extraction from the plant material with a mixture of methanol and water (7/3, v/v) was more efficient than that with water, ethanol, or methanol (Vierling et al. 2003; Genovese and Lannes 2009).
Table 2

Total polyphenolic and condensed tannin contents in the different solvent extracts of SLH, SZ and VSZ from the three varieties of C. pinnatifida

Extract

Total polyphenols* (mg g−1)

Condensed tannins** (mg g−1)

70 % (v/v) Methanol

Methanol

Hot water

70 % (v/v) Methanol

Methanol

Hot water

SLH

104.6 ± 4.16a

97.7 ± 2.88a

91.0 ± 1.57a

41.1 ± 1.20a

37.4 ± 2.04a

32.9 ± 2.37a

SZ

43.5 ± 2.17b

31.4 ± 1.68b

37.7 ± 1.04b

13.4 ± 1.32b

9.2 ± 1.33b

9.9 ± 1.40b

VSZ

73.9 ± 2.18c

67.5 ± 1.19c

59.0 ± 1.31c

27.2 ± 2.10c

24.8 ± 1.96c

18.7 ± 2.45c

*Expressed as mg gallic acid equivalent/g; **Expressed as mg catechin equivalent/g

The results were given as mean values ± standard deviation (n = 3)

Different small superscripts (a–c) denoted the significant difference (p < 0.05) of the three varieties of C. pinnatifida in the test

Among the extracts, the amount of total polyphenolics in SLH, expressed as gallic acid equivalents, was the richest (91.0–104.6 mg/g), followed by that in VSZ (59.0–73.9 mg/g) and in SZ (31.4–43.5 mg/g). On the other hand, the content of condensed tannin, expressed as catechin equivalents, was 32.9–41.1, 18.7–21.8 and 9.2–11.4 mg/g in SLH, VSZ and SZ, respectively (Table 2). The result was in accordance with some reports of other varieties of C. pinnatifida and other species of hawthorn. After being extracted with methanol, the catechin equivalents in the extracts of C. pinnatifida Bunge var. typica Schneider and C. pinnatifida Bunge were measured as 11.3 mg/g and 44.5 mg/g, respectively (Li et al. 2010). The total phenolic content in the 70 % (v/v) aqueous methanol extract of C. monogyna Jacq was 93.4 mg gallic acid equivalents per gram (Öztük and Tuncel 2011). Moreover, although the respective determination of polyphenolics and condensed tannin using Folin-Ciocalteu and Vanillin reagent was based on the different reaction mechanisms, and the reactants exhibited different affinities to individual substrates, a significant correlation (r = 0.997, p < 0.05) was observed between the total polyphenolic and condensed tannin content (Fig. 1a).
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Fig. 1

Correlation between (a) total polyphenolic contents and condensed tannins (r = 0.997, p < 0.05), b total polyphenolic contents and IC50 values for DPPH˙ radical (r = 0.973, p < 0.05), and (c) total polyphenolic contents and TEAC values (r = 0.993, p < 0.05). The samples were extracted from the three varieties of C. pinnatifida (SLH, SZ and VSZ) with hot water, 70 % (v/v) aqueous methanol and methanol

Comparing antioxidant capacities of the extracts from the three varieties of C. pinnatifida

Based on the above results, polyphenolic compounds and condensed tannins were abundant in C. pinnatifida, and the contents of these compounds were significantly different in the different varieties. To investigate the effect of these compounds on the antiradical activity of the extracts, spectrophotometric methods with stable DPPH˙ radical were used to evaluate the free radical scavenging properties. The decrease in the absorbance of DPPH˙ radical brought by antioxidants was due to the scavenging of the radical by hydrogen donation, and the effect was expressed as a percentage inhibition of the radical formation. As illustrated in Table 2, the content of the antioxidant compounds was the highest in the 70 % (v/v) methanol extracts. Therefore, these extracts were used as examples to determine the antiradical activity (Fig. 2a). The 70 % (v/v) methanol extracts all demonstrated concentration dependence in the DPPH˙ radical scavenging activity at the low range of concentration. For example, the DPPH˙ radical scavenging effect of SLH increased sharply with increase in the concentration up to 0.4 mg/ml, after which the effect increased slowly, until a plateau was finally reached. At the concentration of 0.4 mg/ml, the antiradical effect of SLH was 80 %, while that of SZ and VSZ was 47 % and 60 %, respectively.
https://static-content.springer.com/image/art%3A10.1007%2Fs13197-013-0954-6/MediaObjects/13197_2013_954_Fig2_HTML.gif
Fig. 2

Concentration-dependent scavenging activity on (a) DPPH˙radical and (b) ABTS˙+ radical of the 70 % (v/v) methanol extracts from SLH, SZ and VSZ. The results were given as mean values ± standard deviation (n = 3)

At a low concentration, the DPPH˙ radical scavenging activities of the 70 % (v/v) methanol extracts were significantly different (p < 0.05). However, when the concentration was higher than 1.2 mg/ml, the extracts had almost the same activity (Fig. 2a). To investigate the effects of extraction solvents, the DPPH˙ radical scavenging activities of the extracts from hot water, 70 % (v/v) methanol and methanol were all evaluated by IC50 value, which was widely used to measure the antiradical efficiency (Kanner et al. 1994; Kedare and Singh 2011). Figure 3a showed that no matter which solvent was used, the IC50 value of SLH was at a lower concentration of about 0.2 mg/ml, followed by the values of more than 0.3 mg/ml for VSZ and 0.4 mg/ml for SZ. The data indicated that among the three varieties of C. pinnatifida, SLH exhibited the greatest activity in quenching DPPH˙ radical. Compared with fruits (Lim and Tee 2007) like orange (IC50 = 5.4 mg/ml), papaya (IC50 = 3.5 mg/ml) and star fruit (IC50 = 3.8 mg/ml), SLH showed a better DPPH˙ radical scavenging activity. However, this antiradical activity was still lower than that of C. pinnatifida Bunge var. typica Schneider, whose IC50 value in the water and 70 % (v/v) methanol extracts was found as 0.2 and 0.1 mg/ml, respectively (Li et al. 2010). According to Egea et al. (2010), the difference of hawthorn fruits in the DPPH˙ scavenging capacity could be explained by many factors, such as the extraction procedure, ripening state of the fruits or genotypic and environmental differences.
https://static-content.springer.com/image/art%3A10.1007%2Fs13197-013-0954-6/MediaObjects/13197_2013_954_Fig3_HTML.gif
Fig. 3

Comparison of (a) IC50 values for DPPH˙ radical and (b) TEAC values of different extracts from SLH, SZ and VSZ. The samples were extracted with hot water, 70 % (v/v) aqueous methanol and methanol. The results were given as mean values ± standard deviation (n = 3), and the significant difference (p < 0.05) of the three varieties of C. pinnatifida was denoted by different small superscripts (ac)

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).

Conclusions

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.

Acknowledgments

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).

Supplementary material

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© Association of Food Scientists & Technologists (India) 2013