Difference analysis in the content of the total phenols, flavonoid, and anthocyanin of 149 accessions of hawthorn gremplasm resources

[Objective] Hawthorn (Crataegus L.), a plant belonging to the Rosaceae family and the apple tribe of the subfamily Maloideae, is renowned for its dual utility as both food and medicinal herb. Its historical use in medicine and food processing underscores its significance in both areas. The bioactive components present in hawthorn, particularly total phenols, total flavonoids, and anthocyanins, are known to contribute significantly to its health benefits, exhibiting a range of effects including anti-inflammatory, anti-diabetic, and anti-cancer properties. However, the diversity among hawthorn species is vast, leading to substantial variations in the contents of these active components. A systematic evaluation method is needed for the diversity of bioactive components content in hawthorn fruit. This study aimed to offer a basis for the selection of new varieties of hawthorn, and improve the utilization of these resources in the development of functional foods and other areas. [Methods] 149 accessions of hawthorn germplasm resources from the National Hawthorn Germplasm Repository in Shenyang were used in this research. The fruits of these resources were meticulously processed to ensure the integrity of the total phenols, total flavonoids, and anthocyanins. The process began with cutting the hawthorn fruits in-to pieces, which were then subjected to liquid nitrogen treatment to preserve their freshness and potency. The treated fruits were subsequently stored at -80 ℃ to maintain their quality for later analysis. The samples were ground using a grinder to obtain a fine powder, from which 0.5 grams of freeze-dried hawthorn powder was weighed for each variety. A small amount of pre-cooled 1% HCl methanol solution was added to the powder, and the mixture was diluted to 10 mL. The mixture was homogenized and extracted in the dark at 4 ℃ for 20 minutes, with occasional shaking to ensure thorough mixing. After the extraction process, the samples were centrifuged at 12 000 r·min-1 for 10 minutes at 4 ℃ to separate the supernatant from the solid residue. The absorbance values at wavelengths of 280 nm, 325 nm, 600 nm, and 530 nm were measured for each variety using a 1% HCl-methanol solution as the blank reference. All data were processed using Excel 2016 for calculations of mean values, standard deviations, coefficients of variation, and membership functions. Descriptive statistics and frequency analysis were performed using SPSS v26.0 software, and cluster analysis was conducted using TBtools-Ⅱ software to classify the resources based on the content of total phenols, total flavonoids, and anthocyanins. [Results] The results of this study revealed significant variations in the content of total phenols, total flavonoids, and anthocyanins among the 149 accessions of hawthorn germplasm resources. Yepu No.8 was identified as the highest total phenol content (1.531 mg·g-1), while Huairen Xiangyang had the lowest (0.110 mg·g-1). Similarly, Yepu No.8 also exhibited the highest total flavonoid content (56.434 mg·g-1), Zizhenzhu No.1 was the lowest (19.763 mg·g-1). In terms of anthocyanin content, Yesheng Shanlihong No.3 had the highest (720.218 μg·g-1), and Huangguo had the lowest (19.187 μg·g-1). A preliminary grading standard for these compounds was established using percentile analysis. The total phenol content below 0.260 mg·g-1 was regarded as low, 0.260-0.587 mg·g-1 considered as medium, and above 0.587 mg·g-1 was high. The total flavonoid content below 22.242 mg·g-1 was regarded as low, 22.242-28.103 mg·g-1 considered as medium, and above 28.103 mg·g-1 was high. The anthocyanin content below 99.875 μg·g-1 was regarded as low, 99.875-249.819 μg·g-1 considered as medium, and above 249.819 μg·g-1 was high. The cluster analysis revealed five distinct groups, with the first group including Yepu No.8, Wuming Hawthorn-4, and Yesheng Shanlihong-3, having the highest average content of total phenols (1.531-0.557 mg·g-1), total flavonoids (56.434-28.848 mg·g-1), and anthocyanins (720.218-293.449 μg·g-1), with coefficients of variation of 35.1%, 23.4%, and 30.1%, respectively. The second group included Yu8002, Baiquan7807, and Muhuli-1, with total phenol content (0.980-0.327 mg·g-1), total flavonoid content (41.841-22.796 mg·g-1), and anthocyanin content (353.637-19.318 μg·g-1), with coefficients of variation of 24.7%, 11.5%, and 34.0%, respectively. The third group included Zizhenzhu-1, Chaojinxing-1, and 795507, with total phenol content (0.526-0.180 mg·g-1), total flavonoid content (26.533-19.763 mg·g-1), and anthocyanin content (193.442-19.187 μg·g-1), with coefficients of variation of 23.0%, 7.14%, and 45.4%, respectively. The fourth group included Xifenghong, Wangbaodi Dajinxing-2, and Lulong Dashanzha, with total phenol content (0.596-0.365 mg·g-1), total flavonoid content (26.861-21.795 mg·g-1), and anthocyanin content (256.720-110.848 μg·g-1), with coefficients of variation of 12.6%, 5.1%, and 19.7%, respectively. The fifth group included Feixian Zirou-1, Xifen No.5, and Songshancun Shisheng, with total phenol content (0.332-0.110 mg·g-1), total flavonoid content (27.198-21.870 mg·g-1), and anthocyanin content (299.691-105.526 μg·g-1), with coefficients of variation of 25.8%, 6.20%, and 26.1%, respectively. [Conclusion] Based on the membership function values and cluster analysis, 15 accessions of excellent hawthorn germplasm resources were selected. These results would provide the basis for the selection of suitable varieties for fresh consumption, processing, and the future hawthorn breeding. The grading standards established through percentile analysis would provide a systematic evaluation of the total phenol, total flavonoid, and anthocyanin content in mature fruits of hawthorn germplasm resources. This study would not only contribute to the understanding of the diversity of bioactive compounds in hawthorn, but also would serve as a valuable resource for the development of functional foods and the advancement of hawthorn cultivation and utilization.