Analysis of triterpene acid contents in Eriobotrya plants and their interspecific hybrids

Abstract:【Objective】China is native to loquat and has abundant germplasm resources of Eriobotrya plants. At present, studies on the medicinal substance triterpenoid acids in loquat leaves are limited in cultivated loquat. However, there are still 19 wild species of Eriobotrya plants are native to China, and almost no studies have been conducted on triterpenoid acids. The contents of four major triterpenoid acids in the leaves of 25 species and varieties of Eriobotrya plants (containing those introduced from Southeast Asia) and the hybrids of common loquat (E. japonica) and representative Eriobotrya wild species (E. elliptica, E. deflexa and so on) were investigated to provide basic data for drug utilization and genetic improvement of Loquat.【Methods】HPLC method was used to analyze the contents of four major triterpenoid acids including Ursolic acid (UA), Oleanolic acid (OA), Maslinic acid (MA) and Corosolic acid (CA) in the leaves of 25 species/variants of Eriobotrya plants, 21 wild-cultivated hybrids and 2 cultivars. HPLC conditions for the determination of UA and OA were optimized, and the separation effects of different chromatographic columns and mobile phase systems on UA and OA were investigated, including chromatographic columns of Waters Sunfire C18, Kromasil C18, hypersil ODS2, Inertsil ODS- SP, and Sunfire C18. The optimal chromatographic conditions were: Sunfire C18 chromatographic column, Gradient elution was performed with 0.1% phosphoric acid aqueous solution and methanol as mobile phase. The procedure was listed below: Within 0 to 5 min, the proportion of methanol increased from 97% to 99.5%; and within 5 to 25 min, the content of methanol remained at 99.5%. The flow rate was 0.300 mL·min^-1, the sample size was 8 μL, and the column temperature was 20 ℃; The detection wavelength was 208 nm. The standard curve of UA content was drawn to calculate the total triterpene acid content in the leaves of above wild species/variants, interspecific hybrids, and cultivars of Eriobotrya plants.【Results】Chinese Pharmacopoeia (2020) took UA and OA contents as the quality control indexes of loquat leaves. We found that in addition to the young leaves of E. elliptica var. petelottii , with the old leaves of E. poilanei and the mature leaves of three interspecific hybrids E. japonica × E. elliptica No. 13, 14 and 160, the contents of UA and OA in all samples could meet the requirements of Chinese Pharmacopoeia (2020). These Eriobotrya plants could be used as qualified Chinese medicinal materials and had the potential for further medicinal development. According to the total contents of UA and OA, the high-content species were selected, whose UA and OA contents were over 2-3 times higher than the standard of Chinese Pharmacopoeia. Especially, the highest content species was E. Deflexa, which was 3 times more than the standard of Chinese Pharmacopoeia in mature leaves and 4 times more than that in old leaves. Among the leaf materials with different maturity, there were 17 kinds of young leaves, 16 kinds of mature leaves and 19 kinds of old leaves, whose total UA and OA contents were 2- 3 times more than the Chinese Pharmacopoeia requirements. There were fourteen species, whose UA and OA contents were 2-3 times more than the Chinese Pharmacopoeia requirements in all three maturity; Eight species or hybrids were over 30% larger than the high- content variety Jiefangzhong, including E. deflexa , E. prinoides var dadunensis , E. fragrans, E. japonica × E. japonica, E. japonica × E. prinoides, E. japonica ×E. deflexa f. koshunensis, E. japonica ×E. deflexa f. buisanensis, and E. japonica × E. deflexa; Six species or hybrids were over 20% larger than the high-content variety Jiefangzhong, including E. cavaleriei, E. deflexa f. buisanensis, E. prinoides var. laotica, E. deflexa f. koshunensis, E. japonica × E. prinoides, and E. japonica × E. deflexa f. buisanensis. Except UA and OA, MA and CA were also the main triterpenoid acids in loquat leaves. We found that these four triterpenoid acids accounted for more than 90% of the total triterpenoid acids in the leaves of loquat cultivar Jiefangzhong. Seven interspecific hybrids with UA and OA content reaching 2-3 times compared to the Chinese Pharmacopoeia requirements were screened out in the second batch of experiment, includingE. japonica × E. deflexa No. 1, 5, 7 and 8, E. japonica ×E. deflexa f. koshunensis No. 1 and 12, E. japonica × E. elliptica No. 15. This research also screened out five interspecific hybrids rich in MA and CA, including MA high-content hybrids E. japonica × E. elliptica No. 8 and 160, E. japonica × E. deflexa f. koshunensis No. 1, E. japonica × E. deflexa No. 1, and CA high-content hybrids E. japonica ×E. elliptica No. 7 and 8. Especially, E. japonica × E. elliptica No. 8 had the highest contents of both MA and CA, which were 3.22 and 2 times higher than Jiefangzhong, respectively.【Conclusion】The contents of triterpenoid acid in the leaves of many Eriobotrya plants and their hybrid progeny were much higher than those of common loquat, which has great potential as a natural and pollution- free source of medicinal-used loquat leaves.