Xu Jie, Yang Bohan, Li Mingxiao, Li Zixiang, Tu Yuting, Tang Liyun, He Guozhen
School of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China.
Guangdong Yifang Pharmaceutical Co., Ltd., Guangdong Provincial Key Laboratory of Traditional Chinese Medicine Formula Granule, Foshan, Guangdong, China.
PLoS One. 2022 Aug 31;17(8):e0268246. doi: 10.1371/journal.pone.0268246. eCollection 2022.
Genuine Chinese medicine is produced from medicinal plant cultivated in a specific region and is of better quality and efficacy, more consistently qualified and famous than that from the same medicinal plant cultivated in other regions. The cultivating region of genuine medicinal plant is known as the genuine producing area. Yangchun City, which is in Guangdong Province of China, is a genuine producing area for the famous Chinese medicine Amomi Fructus (also called Sharen). Amomi Fructus is the ripe and dry fruit of the Zingiberaceae plant A. villosum Lour.. A. villosum was introduced from the Persian Gulf region and has been cultivated in China for over 1000 years. Until now there are no reports on screening for good germplasm of A. villosum.
The contents of volatile oil and bornyl acetate of Amomi Fructus from 14 populations were determined with GC method, and the relative contents of the main chemical components in the volatile oils were determined with GC-MS method. Evaluation and variance analysis of the comprehensive quality of the 14 samples were conducted by means of a multi-indicator entropy-weight TOPSIS model (Technique for Order Preference by Similarity to an Ideal Solution) combined with OPLS-DA (Orthogonal Partial Least Squares Discrimination Analysis) and HCA (Hierarchical Clustering Analysis). The ISSR (Inter-Simple Sequence Repeat) molecular marker technique and the UPGMA (unweighted pair-group method with arithmetic means) were employed to analyze the genetic relationship among A. villosum populations.
The contents of volatile oil and bornyl acetate differed significantly among the different populations, but the main chemical component in the volatile oil was the same in all the samples, which was bornyl acetate. OPLS-DA results showed that 9 indicators were the main factors influencing the quality differences among the 14 populations. The entropy-weight TOPSIS results showed that there were significant differences in the comprehensive qualities of the 12 populations from the genuine producing area. The best quality of fruit was found in the genuine producing area of Chunwan Town; the qualities of 33% of genuine fruits were lower than that of non-genuine fruits. Twenty-three DNA fragments were obtained by ISSR-PCR amplification using four ISSR primers, eleven of which were polymorphic loci, which accounted for 47.8%. The similarity coefficients (GS) of different populations of A. villosum ranged from 0.6087 to 0.9565.
There are significant differences among different populations of A. villosum in terms of the kinds of major chemical components and their contents, comprehensive quality and genetic diversity. The germplasm resources of A. villosum are rich in the genuine producing area. It means superior germplasm could be selected in the area. The comprehensive quality of the fruit of A. villosum from the non-genuine producing area is better than some of that from genuine producing area, proving that the non-genuine producing area can also produce Amomi Fructus with excellent quality.
道地中药材是指在特定地域种植的药用植物所产的药材，其质量和药效更佳，质量稳定性和知名度高于其他地区种植的同一种药用植物所产的药材。药用植物的种植地域被称为道地产区。中国广东省阳春市是著名中药材阳春砂（又称砂仁）的道地产区。阳春砂是姜科植物阳春砂A. villosum Lour. 的成熟干燥果实。阳春砂从波斯湾地区引入，在中国已种植1000多年。目前尚无阳春砂优良种质筛选的相关报道。
采用气相色谱法测定14个种群阳春砂挥发油和乙酸龙脑酯的含量，采用气相色谱 - 质谱联用法测定挥发油中主要化学成分的相对含量。运用多指标熵权TOPSIS模型（逼近理想解排序法）结合OPLS - DA（正交偏最小二乘法判别分析）和HCA（层次聚类分析）对14个样品的综合品质进行评价和方差分析。采用ISSR（简单序列重复区间）分子标记技术和UPGMA（非加权组平均法）分析阳春砂种群间的遗传关系。
不同种群阳春砂的挥发油和乙酸龙脑酯含量差异显著，但所有样品挥发油中的主要化学成分均为乙酸龙脑酯。OPLS - DA结果表明，9个指标是影响14个种群质量差异的主要因素。熵权TOPSIS结果表明，道地产区12个种群的综合品质存在显著差异。春湾镇道地产区的果实品质最佳；33%的道地果实品质低于非道地果实。使用4条ISSR引物进行ISSR - PCR扩增，共获得23条DNA片段，其中11条为多态性位点，占47.8%。阳春砂不同种群间的相似系数（GS）范围为0.6087至0.9565。
阳春砂不同种群在主要化学成分种类及其含量、综合品质和遗传多样性方面存在显著差异。道地产区阳春砂的种质资源丰富，意味着可以在该地区筛选出优良种质。非道地产区阳春砂果实的综合品质优于部分道地产区的果实，证明非道地产区也能产出优质的阳春砂。
