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银杏(Ginkgo biloba L.)是中国特有的珍贵孑遗植物,具有很高的经济价值和药用价值。银杏叶片主要含黄酮类和萜类内酯类等药用成分,对心血管、神经系统均具有较强的药理活性[1-2],其中银杏萜类内酯主要包括银杏内酯A、银杏内酯B、银杏内酯C和白果内酯[3],具有抗氧化、抗炎、抗血小板聚集、保护神经等药理活性[4-9],其中银杏内酯B是在自然界中迄今为止发现的活性最强的血小板活化因子拮抗剂[10]。
目前,关于银杏黄酮和萜类内酯含量在不同品种、不同区域和不同环境间的差异性研究较多[11-15]。已有研究表明,黄酮和萜类内酯含量受遗传、立地条件和环境因子等多方面因素的影响,不同种源间其含量也存在一定差异[13,14]。而这些差异究竟是受遗传的影响还是环境因素长期综合作用所致,尚不明确。同质园试验是近几年用来研究遗传和环境因素对植物初生生长和次生代谢的影响的一种直接有效的方法[16],而同质园条件下银杏不同种源幼苗生长及药用成分含量差异方面的研究未见报道。因此,以4个不同种源地泸定(LDS)、青川(QCS)、苍溪(CXS)和开江(MCS)银杏种子为试验材料,进行同质园栽培,分析银杏对环境的适应性,研究同质园条件下银杏药用成分含量的差异,探讨遗传和环境长期作用对这些差异性的影响,为高效选育叶片药用含量高的种源和品种等提供依据。
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自2017年以来,通过对四川银杏种资源调查和叶用银杏产业发展的调研,在前期对银杏叶片黄酮含量的研究基础上,于2020年10月在泸定、青川、苍溪和开江四个区域(见表1),分别选取15株长势旺盛,无明显病虫害,结实量大的银杏雌株收集种子混匀,2020月12份进行同质园种植栽培,2021年8月对其一年生苗表型性状和叶片药用成分进行测定。
表 1 银杏种子种源收集地概况
Table 1. General situation of Ginkgo biloba seed provenance collection sites
种源 采集地 经度 纬度 海拔/m 年均温
/℃年降雨量/mm QCS 青川 105°21′ 32°26′ 998 13.7 1027.2 CXS 苍溪 106°04′ 31°49′ 441 16.7 1046.7 LDS 泸定 102°13′ 29°57′ 1247 16.5 664.4 MCS 开江 107°59′ 31°02′ 481 16.8 1237.4 -
不同种源采用相同的种植技术,均采用人工点播种植方式,其株行距为30 cm×10 cm。每个种源种植三个小区,小区面积为6 m×9 m,小区采用随机区组排列,小区间设保护行。
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种子横径、纵径、侧径采用电子游标卡尺测定,精确到小数点后2位,每个种源各测定种子数量50粒,重复3次。百粒重选用电子天平测定,精确到小数点后2位,随机选取100粒种子,重复3次。
采取等距抽样法每个小区抽取30株,每个种源三个小区共计90株。分别测定各样株的地径、株高、冠幅、单株叶片数量,随机摘取银杏苗上、中、下部位不同方向的叶片50片,测量其平均质量以及计算出单株叶重。
总黄酮醇苷和萜类内酯含量测定参照2020版《中国药典》一部银杏叶含量测定,高效液相色谱法(通则0512)测定,计算公式为:
总黄酮醇苷含量=(槲皮素含量+山柰酚含量+异鼠李素含量)×2.51,
含萜类内酯=银杏内酯A含量+银杏内酯B含量+银杏内酯C含量+白果内酯含量
其计算结果精确到小数点后2位,重复3次。
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利用Excel对银杏表型性状、叶片总黄酮醇苷和萜类内酯类含量进行整理分析及相关计算,采用SPSS 27软件进行多重比较和方差分析。
Variations of Seedling Growth and Medicinal Components Contents in Leaves of Ginkgo biloba from Different Provenances in Common Garden
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摘要: 开展不同种源银杏同质园试验,揭示环境对不同种源银杏的影响,为选育药用价值高的银杏种源和品种提供依据。收集了川内4个不同种源的银杏种子,经同质园栽培后,对其幼苗生长和叶片药用成分含量进行测定分析,并对各测定结果进行相关性分析。结果显示:(1)各种源间种子横径、纵径和百粒重均极显著差异(P<0.01),且均开江最大;(2)各种源间苗高、单株叶数、单叶重和单株叶重均极显著差异(P<0.01),冠幅显著差异(P=0.02)。其中苗高、单株叶数、单株叶重、冠幅均开江最大,单叶重泸定最大;(3)各种源间叶片萜类内酯、白果内酯含量呈极显著差异(P<0.01),黄酮、银杏内酯A和银杏内酯B含量均显著差异(P=0.02,P=0.03,P=0.01)。其中黄酮含量苍溪最高,萜类内酯、白果内酯、银杏内酯A、银杏内酯B含量均泸定最高;(4)苗高与单株叶重呈极显著正相关(r=0.912);萜类内酯与白果内酯和银杏内酯B含量(r=0.928和0.962)均呈极显著正相关。不同种源的银杏同质园栽培结果表明,由于亲代长期受种源地环境的影响,子代幼苗的生长以及药用成分含量具有显著差异。Abstract: The Common garden experiment of Ginkgo biloba from different provenances was carried out to reveal the influence of environment on different provenances of Ginkgo biloba, so as to provide basis for breeding the provenances and varieties of Ginkgo biloba with high medicinal value. In this study, four ginkgo seeds from different provenances in Sichuan were collected, and after being cultivated in common garden, the seedling growth and the content of medicinal components in leaves were measured and analyzed, and the correlation analysis of the results was carried out. The results were as follows: (1) There were significant differences in the transverse diameter, vertical diameter and 100-seed weight from different provenance (P<0.01), and Kaijiang were the largest. (2) There were significant differences in seedling height, leaf number per plant, individual leaf weight and leaf weight per plant from different provenances (P<0.01), and significant differences in crown width (P=0.02). Among them, seedling height, leaf number per plant, leaf weight per plant and crown width are the largest, and single leaf weight is the largest. (3) There were significant differences (P<0.01) in the contents of terpene lactones and ginkgolides in leaves from various sources, and significant differences in the contents of flavonoids, Ginkgolides A and Ginkgolides B (P=0.02, P=0.03, P=0.01). Among them, the content of flavonoids is the highest in Cangxi, and the contents of terpenoids, ginkgolides, Ginkgolides A and Ginkgolides B are the highest in Luding. (4) Seedling height was positively correlated with the individual leaf weight (r=0.912). Terpene lactones were positively correlated with the content of bilobalide and Ginkgolide B (r=0.928, 0.962). The results showed that Ginkgo biloba from different provenances had significant differences in the growth of offspring seedlings and the content of medicinal components after cultivation in common garden, which was due to the long-term influence of parents of the environment of the provenance.
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Key words:
- Ginkgo biloba;
- Medicinal Components;
- Common garden
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图 1 不同种源银杏种子表型性状
注:图中数值为平均值±标准误;同一行上不同小写字母表示不同种源之间有显著差异(P<0.05)下图同。Note: The values in the figure are average ± standard error; Different lowercase letters on the same line indicate that there are significant differences among different provenances (P<0.05).
Fig. 1 Phenotypic characters of Ginkgo biloba L. seeds from different provenances
图 4 银杏各性状间相关性分析
注:*、**分别表示差异显著水平为0.05和0.01。GW.百粒重;D.地径;MH.苗高;LW.单株叶重;FG.总黄酮苷;BBD.白果内酯;GL-A.银杏内酯A;GL-B.银杏内酯B;GL-C.银杏内酯C;TL.萜类内酯Note: * and * * indicate that the level of significant difference is 0.05 and 0.01 respectively. GW: 100-seed weight; D: Ground diameter; MH: Seedling height; LW: Leaf weight per plant; FG: Total flavone glycoside; BBD: Bilobalide; Gl-A: Ginkgolide A; Gl-B: Ginkgolide B; Gl-C: Ginkgolide C; TL: Terpenoid lactone
Fig. 4 Correlation analysis of various characters of Ginkgo biloba L.
表 1 银杏种子种源收集地概况
Tab. 1 General situation of Ginkgo biloba seed provenance collection sites
种源 采集地 经度 纬度 海拔/m 年均温
/℃年降雨量/mm QCS 青川 105°21′ 32°26′ 998 13.7 1027.2 CXS 苍溪 106°04′ 31°49′ 441 16.7 1046.7 LDS 泸定 102°13′ 29°57′ 1247 16.5 664.4 MCS 开江 107°59′ 31°02′ 481 16.8 1237.4 -
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