枇杷属野生种及其种间杂种后代株系的三萜酸含量分析

Analysis of triterpene acid contents in Eriobotrya plants and their inter-specific hybrids

【目的】测定枇杷属25个种或变种以及椭圆枇杷、台湾枇杷等代表性种与普通枇杷杂交后代株系叶片的4种主要三萜酸的含量,为枇杷属植物资源的药物利用和枇杷遗传改良提供基础数据。【方法】以枇杷属25个种类/变型、7个野生-栽培枇杷杂交组合的21个后代株系为试料,以3个栽培品种及种内杂种作为对照,采用高效液相色谱法(HPLC)分析枇杷叶片的熊果酸(Ursolic acid,UA)、齐墩果酸(Oleanolic acid,OA)、山楂酸(Maslinic acid,MA)和科罗索酸(Corosolic acid,CA)等主要三萜酸物质含量。【结果】根据熊果酸和齐墩果酸总含量,筛选出了高于中国药典标准2~3倍以上的高含量种类。其中大于高含量栽培品种解放钟30%以上的种类有8种,包括台湾枇杷、大渡河枇杷、香花枇杷、解放钟×大红袍、解放钟×栎叶枇杷、解放钟×恒春变型、大红袍×武葳山变型、解放钟×台湾枇杷。特别是台湾枇杷为最高含量种类,成熟叶片超过药典规定3倍以上,老叶为4倍以上;筛选出富含山楂酸和科罗索酸的种间杂交后代材料,如山楂酸和科罗索酸分别可达解放钟3.22倍和2倍的解放钟×椭圆枇杷8号等。【结论】多种枇杷属野生种及其杂种后代株系叶片的三萜酸含量远高于普通枇杷,作为纯天然无污染的药用枇杷叶片来源潜力巨大。

【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 ac-ids 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 spe-cies(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 ma-jor triterpenoid acids including Ursolic acid(UA),Oleanolic acid(OA),Maslinic acid(MA)and Coro-solic 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 investigat-ed,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 8μL,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 Erio-botrya plants.【Results】Chinese Pharmacopoeia(2020)took UA and OA contents as the quality con-trol 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.De-flexa,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 Jiefang-zhong,including E.deflexa,E.prinoides var dadunensis,E.fragrans,E.japonica×E.japonica,E.ja-ponica×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 triter-penoid 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,including E.japonica×E.deflexa No.1,5,7 and 8,E.japonica×E.deflexa f.koshunensis No.1 and 12,E.ja-ponica×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.de-flexa 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.