中国药学杂志

No.	Herbal name	Habitat (in Chinese)	Acquisition time	Lot number
S1	E. brevicornu Maxim.	Zhang county, Gansu province(甘肃漳县)	2021.6	202106001
		Weiyuan county, Gansu province(甘肃渭源县)	2021.6	202106002
		Wudu district, Gansu province(甘肃武都区)	2021.6	202106003
		Li county, Gansu province(甘肃礼县)	2021.6	202106004
		Li county, Gansu province(甘肃礼县)	2021.6	202106005
		Qinzhou district, Gansu province(甘肃秦州区)	2021.6	202106006
		Lintan county, Gansu province(甘肃临潭县)	2021.6	202106007
		Jishishan county, Gansu province(甘肃积石山县)	2021.6	202106008
		Huating city, Gansu province(甘肃华亭市)	2021.6	202106009
		Yangcheng county, Shanxi province(山西阳城县)	2021.8	202108001
		Pinglu county, Shanxi province(山西平陆县)	2021.7	202107001
S2	E. sagittatum (Sieb. et Zucc.) Maxim.	Liuba county, Shaanxi province(陕西留坝县)	2021.12	202112001
		Liuba county, Shaanxi province(陕西留坝县)	2021.12	202112002
		Rongjiang county, Guizhou province(贵州榕江县)	2021.12	202112003
		Anhua county, Hunan province(湖南安化县)	2021.12	202112004
		Nanzhang county, Hubei province(湖北南漳县)	2021.12	202112005
		Sansui county, Guizhou(贵州三穗县)	2022.1	202201001
		Leishan county, Guizhou province(贵州雷山县)	2022.1	202201002
		Leishan county, Guizhou province(贵州雷山县)	2022.1	202201003
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201004
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201005
S3	E. pubescens Maxim.	Shawan district, Sichuan province(四川沙湾区)	2021.10	202110001
		Shawan district, Sichuan province(四川沙湾区)	2021.10	202110002
		Bazhong city, Sichuan province(四川巴中市)	2022.4	202204001
		Bazhong city, Sichuan province(四川巴中市)	2022.4	202204002
		Ningqiang county, Shaanxi province(陕西宁强县)	2021.12	202112006
		Ningqiang county, Shaanxi province(陕西宁强县)	2021.12	202112007
		Lüeyang county, Shaanxi province(陕西略阳县)	2021.11	202111001
		Lüeyang county, Shaanxi province(陕西略阳县)	2021.11	202111002
		Xingwen county, Sichuan province(四川兴文县)	2022.2	202202001
		Xingwen county, Sichuan province(四川兴文县)	2022.2	202202002
S4	E. koreanum Nakai	Fengcheng city, Liaoning province(辽宁凤城市)	2021.8	202108002
		Fengcheng city, Liaoning province(辽宁凤城市)	2021.8	202108003
		Hengren county, Liaoning province(辽宁恒仁县)	2021.8	202108004
		Hengren county, Liaoning province(辽宁恒仁县)	2021.8	202108005
		Kuandian county, Liaoning province(辽宁宽甸县)	2021.8	202108006
		Kuandian county, Liaoning province(辽宁宽甸县)	2021.8	202108007
		Qingyuan county, Liaoning province(辽宁清原满族自治县)	2021.8	202108008
		Qingyuan county, Liaoning province(辽宁清原满族自治县)	2021.8	202108009
		Fusong county, Jilin province(吉林抚松县)	2021.6	202106010
		Fusong county, Jilin province(吉林抚松县)	2021.6	202106011
S5	E. wushanense T.S. Ying	Zhenping county, Shaanxi province(陕西镇坪县)	2021.11	202111003
		Zhenping county, Shaanxi province(陕西镇坪县)	2021.11	202111004
		Zhenping county, Shaanxi province(陕西镇坪县)	2021.11	202111005
		Dazhou city, Sichuan province(四川达州市)	2021.7	202107002
		Dazhou city, Sichuan province(四川达州市)	2021.7	202107003
		Dazhou city, Sichuan province(四川达州市)	2021.7	202107004
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201006
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201007
		Badong county, Hubei province(湖北巴东县)	2022.4	202204003
		Badong county, Hubei province(湖北巴东县)	2022.4	202204004
S6	E. myrianthum Steam	Yongchuan district, Chongqing(重庆永川区)	2022.1	202201008
		Yongchuan district, Chongqing(重庆永川区)	2022.1	202201009
		Jianhe county, Guizhou province(贵州剑河县)	2022.4	202204005
		Jianhe county, Guizhou province(贵州剑河县)	2022.4	202204006
		Baojing county, Hunan province(湖南保靖县)	2022.4	202204007
		Baojing county, Hunan province(湖南保靖县)	2022.4	202204008
S7	E. lishihchenii Stearn	Dayu county, Jiangxi province(江西大余县)	2022.1	202201010
		Dayu county, Jiangxi province(江西大余县)	2022.1	202201011
		Dayu county, Jiangxi province(江西大余县)	2022.1	202201012
S8	E. acuminatum Franch.	Nanming district, Guizhou province(贵州南明区)	2022.5	202205001
		Nanming district, Guizhou province(贵州南明区)	2022.5	202205002
		Nanchuan district, Chongqing(重庆南川区)	2022.4	202204009
		Nanchuan district, Chongqing(重庆南川区)	2022.4	202204010
S9	E. leptorrhizum Stearn	Nanming district, Guizhou province(贵州南明区)	2022.5	202205003
		Nanming district, Guizhou province(贵州南明区)	2022.5	202205004
		Songtao county, Guizhou province(贵州松桃县)	2022.5	202205005
		Songtao county, Guizhou province(贵州松桃县)	2022.5	202205006

No.	Herbal name	Habitat (in Chinese)	Acquisition time	Lot number
S1	E. brevicornu Maxim.	Zhang county, Gansu province(甘肃漳县)	2021.6	202106001
		Weiyuan county, Gansu province(甘肃渭源县)	2021.6	202106002
		Wudu district, Gansu province(甘肃武都区)	2021.6	202106003
		Li county, Gansu province(甘肃礼县)	2021.6	202106004
		Li county, Gansu province(甘肃礼县)	2021.6	202106005
		Qinzhou district, Gansu province(甘肃秦州区)	2021.6	202106006
		Lintan county, Gansu province(甘肃临潭县)	2021.6	202106007
		Jishishan county, Gansu province(甘肃积石山县)	2021.6	202106008
		Huating city, Gansu province(甘肃华亭市)	2021.6	202106009
		Yangcheng county, Shanxi province(山西阳城县)	2021.8	202108001
		Pinglu county, Shanxi province(山西平陆县)	2021.7	202107001
S2	E. sagittatum (Sieb. et Zucc.) Maxim.	Liuba county, Shaanxi province(陕西留坝县)	2021.12	202112001
		Liuba county, Shaanxi province(陕西留坝县)	2021.12	202112002
		Rongjiang county, Guizhou province(贵州榕江县)	2021.12	202112003
		Anhua county, Hunan province(湖南安化县)	2021.12	202112004
		Nanzhang county, Hubei province(湖北南漳县)	2021.12	202112005
		Sansui county, Guizhou(贵州三穗县)	2022.1	202201001
		Leishan county, Guizhou province(贵州雷山县)	2022.1	202201002
		Leishan county, Guizhou province(贵州雷山县)	2022.1	202201003
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201004
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201005
S3	E. pubescens Maxim.	Shawan district, Sichuan province(四川沙湾区)	2021.10	202110001
		Shawan district, Sichuan province(四川沙湾区)	2021.10	202110002
		Bazhong city, Sichuan province(四川巴中市)	2022.4	202204001
		Bazhong city, Sichuan province(四川巴中市)	2022.4	202204002
		Ningqiang county, Shaanxi province(陕西宁强县)	2021.12	202112006
		Ningqiang county, Shaanxi province(陕西宁强县)	2021.12	202112007
		Lüeyang county, Shaanxi province(陕西略阳县)	2021.11	202111001
		Lüeyang county, Shaanxi province(陕西略阳县)	2021.11	202111002
		Xingwen county, Sichuan province(四川兴文县)	2022.2	202202001
		Xingwen county, Sichuan province(四川兴文县)	2022.2	202202002
S4	E. koreanum Nakai	Fengcheng city, Liaoning province(辽宁凤城市)	2021.8	202108002
		Fengcheng city, Liaoning province(辽宁凤城市)	2021.8	202108003
		Hengren county, Liaoning province(辽宁恒仁县)	2021.8	202108004
		Hengren county, Liaoning province(辽宁恒仁县)	2021.8	202108005
		Kuandian county, Liaoning province(辽宁宽甸县)	2021.8	202108006
		Kuandian county, Liaoning province(辽宁宽甸县)	2021.8	202108007
		Qingyuan county, Liaoning province(辽宁清原满族自治县)	2021.8	202108008
		Qingyuan county, Liaoning province(辽宁清原满族自治县)	2021.8	202108009
		Fusong county, Jilin province(吉林抚松县)	2021.6	202106010
		Fusong county, Jilin province(吉林抚松县)	2021.6	202106011
S5	E. wushanense T.S. Ying	Zhenping county, Shaanxi province(陕西镇坪县)	2021.11	202111003
		Zhenping county, Shaanxi province(陕西镇坪县)	2021.11	202111004
		Zhenping county, Shaanxi province(陕西镇坪县)	2021.11	202111005
		Dazhou city, Sichuan province(四川达州市)	2021.7	202107002
		Dazhou city, Sichuan province(四川达州市)	2021.7	202107003
		Dazhou city, Sichuan province(四川达州市)	2021.7	202107004
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201006
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201007
		Badong county, Hubei province(湖北巴东县)	2022.4	202204003
		Badong county, Hubei province(湖北巴东县)	2022.4	202204004
S6	E. myrianthum Steam	Yongchuan district, Chongqing(重庆永川区)	2022.1	202201008
		Yongchuan district, Chongqing(重庆永川区)	2022.1	202201009
		Jianhe county, Guizhou province(贵州剑河县)	2022.4	202204005
		Jianhe county, Guizhou province(贵州剑河县)	2022.4	202204006
		Baojing county, Hunan province(湖南保靖县)	2022.4	202204007
		Baojing county, Hunan province(湖南保靖县)	2022.4	202204008
S7	E. lishihchenii Stearn	Dayu county, Jiangxi province(江西大余县)	2022.1	202201010
		Dayu county, Jiangxi province(江西大余县)	2022.1	202201011
		Dayu county, Jiangxi province(江西大余县)	2022.1	202201012
S8	E. acuminatum Franch.	Nanming district, Guizhou province(贵州南明区)	2022.5	202205001
		Nanming district, Guizhou province(贵州南明区)	2022.5	202205002
		Nanchuan district, Chongqing(重庆南川区)	2022.4	202204009
		Nanchuan district, Chongqing(重庆南川区)	2022.4	202204010
S9	E. leptorrhizum Stearn	Nanming district, Guizhou province(贵州南明区)	2022.5	202205003
		Nanming district, Guizhou province(贵州南明区)	2022.5	202205004
		Songtao county, Guizhou province(贵州松桃县)	2022.5	202205005
		Songtao county, Guizhou province(贵州松桃县)	2022.5	202205006

No.	Herbal name	Angle of thorns at leaf edge/° ($\bar{x}±s$)	F test	Comparison of multiple means
S1	E. brevicornu	56.66	±11.35	11.12	<0.001	P<0.05,when compared with S4 and S5
S2	E. sagittatum	48.65	±14.39			P<0.05,when compared with S3 and S4
S3	E. pubescens	64.54	±13.12			P<0.05,when compared with S2,S4,S5,S7,S8 and S9
S4	E. koreanum	36.32	±10.41			P<0.05,when compared with S1,S2,S3,S6,and S8
S5	E. wushanense	43.17	±9.83			P<0.05,when compared with S1,S3,and S6
S6	E. myrianthum	58.04	±8.78			P<0.05,when compared with S4,S5,and S9
S7	E. lishihchenii	47.99	±13.41			P<0.05,when compared with S3
S8	E. acuminatum	48.97	±10.41			P<0.05,when compared with S3 and S4
S9	E. leptorrhizum	45.56	±9.86			P<0.05,when compared with S3 and S6

No.	Herbal name	Angle of thorns at leaf edge/° ($\bar{x}±s$)	F test	Comparison of multiple means
S1	E. brevicornu	56.66	±11.35	11.12	<0.001	P<0.05,when compared with S4 and S5
S2	E. sagittatum	48.65	±14.39			P<0.05,when compared with S3 and S4
S3	E. pubescens	64.54	±13.12			P<0.05,when compared with S2,S4,S5,S7,S8 and S9
S4	E. koreanum	36.32	±10.41			P<0.05,when compared with S1,S2,S3,S6,and S8
S5	E. wushanense	43.17	±9.83			P<0.05,when compared with S1,S3,and S6
S6	E. myrianthum	58.04	±8.78			P<0.05,when compared with S4,S5,and S9
S7	E. lishihchenii	47.99	±13.41			P<0.05,when compared with S3
S8	E. acuminatum	48.97	±10.41			P<0.05,when compared with S3 and S4
S9	E. leptorrhizum	45.56	±9.86			P<0.05,when compared with S3 and S6

No.	Herbal name	The portion of wave depth of epidermal cells in the leaves/%($\bar{x}±s$)	The P-value of the Welch test	Comparison of multiple means
S1	E. brevicornu	33.17±4.35	<0.001	P<0.05,when compared with S4 and S5
S2	E. sagittatum	28.41±5.49		P<0.05,when compared with S4 and S8
S3	E. pubescens	34.08±6.86		P<0.05,when compared with S5
S4	E. koreanum	38.56±4.14		P<0.05,when compared with S1,S2,S5,S6,S7 and S9
S5	E. wushanense	26.01±5.57		P<0.05,when compared with those of S1,S3,S4,and S8
S6	E. myrianthum	29.73±4.41		P<0.05,when compared with S4
S7	E. lishihchenii	27.37±6.16		P<0.05,when compared with S4 and S8
S8	E. acuminatum	34.02±4.31		P<0.05,when compared with those of S2,S5 and S7
S9	E. leptorrhizum	31.07±4.05		P<0.05,when compared with S4

No.	Herbal name	The portion of wave depth of epidermal cells in the leaves/%($\bar{x}±s$)	The P-value of the Welch test	Comparison of multiple means
S1	E. brevicornu	33.17±4.35	<0.001	P<0.05,when compared with S4 and S5
S2	E. sagittatum	28.41±5.49		P<0.05,when compared with S4 and S8
S3	E. pubescens	34.08±6.86		P<0.05,when compared with S5
S4	E. koreanum	38.56±4.14		P<0.05,when compared with S1,S2,S5,S6,S7 and S9
S5	E. wushanense	26.01±5.57		P<0.05,when compared with those of S1,S3,S4,and S8
S6	E. myrianthum	29.73±4.41		P<0.05,when compared with S4
S7	E. lishihchenii	27.37±6.16		P<0.05,when compared with S4 and S8
S8	E. acuminatum	34.02±4.31		P<0.05,when compared with those of S2,S5 and S7
S9	E. leptorrhizum	31.07±4.05		P<0.05,when compared with S4

No.	Herbal name	The density of the mastoid/%($\bar{x}±s$)	The P-value of the Welch test	Comparison of multiple means
S2	E. sagittatum	9.08±2.64	<0.001	P<0.05,when compared with S6,S7 and S8
S5	E. wushanense	11.09±2.94		P<0.05,when compared with S6,S7 and S8
S6	E. myrianthum	15.74±2.87		P<0.05,when compared with S2,S5,S7 and S8
S7	E. lishihchenii	34.09±3.94		P<0.05,when compared with S2,S5,S6 and S8
S8	E. acuminatum	39.71±5.35		P<0.05,when compared with S2,S5,S6,and S7

No.	Herbal name	The density of the mastoid/%($\bar{x}±s$)	The P-value of the Welch test	Comparison of multiple means
S2	E. sagittatum	9.08±2.64	<0.001	P<0.05,when compared with S6,S7 and S8
S5	E. wushanense	11.09±2.94		P<0.05,when compared with S6,S7 and S8
S6	E. myrianthum	15.74±2.87		P<0.05,when compared with S2,S5,S7 and S8
S7	E. lishihchenii	34.09±3.94		P<0.05,when compared with S2,S5,S6 and S8
S8	E. acuminatum	39.71±5.35		P<0.05,when compared with S2,S5,S6,and S7

9种淫羊藿叶的微性状与显微鉴别研究

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邹立思 ¹ , 易东阳 ² , 潘雅楠 ³ , 林丽群 ³ , 肖承鸿 ⁴ , 裴高升 ⁵ , 薛佳 ¹ , 刘训红 ¹^,^*

中国药学杂志 | 论著 2024,59(9): 776-788

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中国药学杂志 | 论著 2024, 59(9): 776-788

9种淫羊藿叶的微性状与显微鉴别研究

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邹立思¹, 易东阳², 潘雅楠³, 林丽群³, 肖承鸿⁴, 裴高升⁵, 薛佳¹, 刘训红¹^,^*

作者信息

¹ 南京中医药大学, 南京 210023

² 重庆三峡医药高等专科学校, 重庆 404120

³ 南京传统中医门诊部, 南京 210023

⁴ 贵州中医药大学, 贵阳 550025

⁵ 国药集团同济堂(贵州)制药有限公司, 贵阳 550200

邹立思,男,高级实验师研究方向:中药栽培、鉴定及品质评价研究

通讯作者:

*刘训红,男,硕士,教授研究方向:中药鉴定与品质评价研究 Tel: (025)85811511

Micro-Characteristics and Microscopic Identification of 9 Kinds of Epimedium Leaves

Lisi ZOU¹, Dongyang YI², Yanan PAN³, Liqun LIN³, Chenghong XIAO⁴, Gaosheng PEI⁵, Jia XUE¹, Xunhong LIU¹^,^*

Affiliations

¹ Nanjing University of Traditional Chinese Medicine, Nanjing 210023, China

² Chongqing Three Gorges Medical College, Chongqing, 404120, China

³ Nanjing Traditional Chinese Medicine Outpatient Department, Nanjing 210023, China

⁴ Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China

⁵ Sinopharm Tongjitang (Guizhou) Pharmaceutical Co., Ltd., Guiyang 550200, China

出版时间: 2024-05-08 doi: 10.11669/cpj.2024.09.003

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摘要

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目的系统研究市售9种淫羊藿叶的微性状及显微特征,归纳其专属且实用的鉴别特征,为市售常见淫羊藿药材的有效鉴别提供参考依据。方法采用性状鉴别、微性状鉴别及显微鉴别方法,结合景深合成技术,得到9种淫羊藿叶的高清特征图片;将部分特征图进行数字化提取,并用SPSS 26.0软件进行统计学分析。结果微性状鉴别特征中叶柄毛被、叶缘刺、叶下表面乳突和毛被、叶基部主脉横切面木心数、叶上表面角质层,显微鉴别特征中叶上表皮细胞波状深度、非腺毛、乳突表面及侧面观形态等作为9种淫羊藿叶分类鉴别的专属性特征;不同品种淫羊藿叶边缘刺角度、叶上表皮细胞波状深度占比和乳突密度等特征值的差异具有统计学意义(P<0.05)。结论微性状和显微鉴别均可区分9种淫羊藿叶,微性状特征的量化及其统计学分析弥补了传统经验鉴别受主观因素影响的不足,为市售常见淫羊藿药材流通、检验、临床用药及标准起草等工作提供参考。

关键词

淫羊藿叶 / 微性状 / 显微鉴别 / 偏振光 / 统计分析

Abstract

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OBJECTIVE To systematically study the micro-characteristics and microscopic characteristics of 9 commercially available Epimedium leaves, summarize more exclusive and practical features and provide reference for effective identification of Epimedii Folium on market. METHODS Using the character identification, micro-character identification, microscopic identification and combining with the technique of depth synthesis, high definition feature images of 9 species of Epimedium leaves were obtained. Some of the feature maps were extracted digitally and analyzed by SPSS 26.0 software. RESULTS The micro-characteristics of petiole hair, leaf margin spines, nipple and hairs on lower surface, parenchyma number on the base of leaf main vein transverse section, cuticle on the surface of leaf, as well as the microscopical characters of upper epidermal cell wave depth, non-glandular hair and nipple surface morphology, all of that can be regarded as specific features of 9 species of Epimedium leaves in classification and identification. There was statistically significant difference in the angle of thorns at the edge of leaves, the proportion of wave depth in upper epidermal cells and the density of nipple among all samples (P<0.05). CONCLUSION Nine kinds of Epimedium leaves could be distinguished through the micro-characteristics and micro-identification methods. The quantitative analysis and statistical analysis of micro-property will make up for the deficiency of traditional experience identification by subjective factors. The results of this study will provide a reference for the circulation, inspection, clinical medication and standard drafting and so on.

Key words

Epimedium leaves / micro-characteristics / micro-identification / polarized light / statistical analysis

引用本文

邹立思, 易东阳, 潘雅楠, 林丽群, 肖承鸿, 裴高升, 薛佳, 刘训红. 9种淫羊藿叶的微性状与显微鉴别研究. 中国药学杂志, 2024 , 59 (9) : 776 -788 . DOI: 10.11669/cpj.2024.09.003

Lisi ZOU, Dongyang YI, Yanan PAN, Liqun LIN, Chenghong XIAO, Gaosheng PEI, Jia XUE, Xunhong LIU. Micro-Characteristics and Microscopic Identification of 9 Kinds of Epimedium Leaves[J]. Chinese Pharmaceutical Journal, 2024 , 59 (9) : 776 -788 . DOI: 10.11669/cpj.2024.09.003

正文

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淫羊藿之名,始见于《神农本草经》,列为中品。味辛,性温,具有补肝肾、强筋骨、助阳益精、祛风除湿等功能,为常用中药,药用历史悠久^[1]。现代研究表明,淫羊藿具有促进性腺功能、促进骨形成、改善血液流变学指标、保护心肌缺血、抗肿瘤、免疫调节、抗抑郁等药理作用,临床用于骨质疏松、冠心病、神经衰弱、慢性气管炎、病毒性心肌炎、白细胞减少症、妇女更年期综合征及高血压等病治疗^[2]。2020年版《中国药典》收载淫羊藿(Epimedium brevicornu Maxim.)、箭叶淫羊藿[E. sagittatum (Sieb. et Zucc.) Maxim.]、柔毛淫羊藿(E. pubescens Maxim.)、朝鲜淫羊藿(E. koreanum Nakai)、巫山淫羊藿(E. wushanense T. S. Ying)共5个基原,前4者叶入药称“淫羊藿”,后者叶入药称“巫山淫羊藿”^[3];《贵州省中药材民族药材质量标准》2003年版收载粗毛淫羊藿(E. acuminatum Franch.)、天平山淫羊藿(E. myrianthum Steam)、黔岭淫羊藿(E. leptorrhizum Stearn),其地上部分入药称“黔淫羊藿”^[4];商品药材常出现混淆品,如时珍淫羊藿(E. lishihchenii Stearn)等。淫羊藿商品药材以野生资源为主,目前仅箭叶淫羊藿、柔毛淫羊藿、天平山淫羊藿有小规模的人工栽培,其基原十分复杂,品种混杂现象较为严重^[5-7],又因入药时以叶或地上部分为主,花和花序等淫羊藿属植物分类鉴定的重要特征难以见到,导致其品种鉴定历来是中药生产、检验和科研中的一个难点。叶是淫羊藿药材鉴别研究的主要内容,现有关于淫羊藿叶微性状和显微鉴别多集中在2020年版《中国药典》收载的基原且不够系统,更未结合淫羊藿属其他常见种类进行系统的对比鉴别^[8-11]。

本研究在已有研究基础上^[12-18],深入主产区调研收集以上9种淫羊藿叶样品,采用性状、微性状和显微鉴别方法对其进行系统的鉴别研究,归纳了较为专属且实用的鉴别特征,结合景深扩展技术进行拍摄,并将部分特征量化,结合统计学分析,从而为市售淫羊藿药材的准确鉴定提供参考资料和科学依据。

1 仪器、试剂及样品

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1.1 仪器

EOS 70D型单反相机、EF 100 mm f/2.8 USM型微距镜头、EF 24~70 mm 1:4 L USM型广角镜头(日本Canon公司);FF 25 mm f/2.8 2.5~5×超微放大镜头(安徽长庚光学科技有限公司);Eclipse E100型生物显微镜(日本Nikon公司);RM2135型切片机(德国LEICA公司);Helicon Focus Pro 7.7.0专业版聚焦堆叠软件(Heliconsoft公司);Adobe Photoshop图像处理软件(Adobe公司);Fiji Is Just ImageJ图像处理软件(NIH公司)。

1.2 试剂

无水乙醇(天津市德恩化学试剂有限公司)、二甲苯(烟台市双双化工有限公司)、石蜡(上海华永石蜡有限公司)、番红(德国RUIBIO公司)等。

1.3 样品

实验所用淫羊藿的样品均为笔者在各主产地收集,均由南京中医药大学中药鉴定教研室刘训红教授做品种鉴定,确定为正品。样品信息见表1。

2 方法

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2.1 性状及微性状鉴别

对同一品种不同产地的淫羊藿干燥叶分别进行3次取样并观察。将样品表面清理干净,选取所需部位备用,在叶片基部徒手制作主脉的横剖面。将叶片与主脉横剖面分别放置于拍摄台上,使用单反相机和微距镜头,并结合景深扩展技术^[19],收集实验样品同一位置、不同景深影像数据(每个位置30~50张图片),再使用Helicon Focus Pro 7.7.0专业版聚焦堆叠软件得到高清全息彩色图像数据。

2.2 显微鉴别

2.2.1 表皮制片

取完整叶片参照2020年版《中国药典》四部通则 2001显微鉴别法^[3],采取定位取材、徒手切片法获取观察切片,于显微镜下观察,并用相机收集图像数据。

2.2.2 横切面制片

分别选取淫羊藿叶片的上、中、下3部分,用剪刀分割成小块,于酒精乙酸福尔马林混合液(FAA)固定液中固定48 h后漂洗,体积分数1%番红(体积分数50%乙醇配制)染色,乙醇和二甲苯进行脱水透化,经二甲苯溶液透明后浸蜡包埋;蜡块用纯水浸泡后切片,郝氏粘贴剂贴片,50 ℃下保存2 d,用二甲苯进行脱蜡处理;体积分数0.5%固绿染液(体积分数95%乙醇配制)染色,中性树胶封片。分别置于显微镜下,采用正常光明场和偏振光暗场对比观察法,并收集图像数据。

2.2.3 粉末制片

样品粉碎过筛,参照2020年版《中国药典》四部通则 2001显微鉴别法制片^[3],于显微镜下观察,并收集显微图像数据。

2.2.4 样品测量

将样品置于显微物镜下,用相机收集清晰的完整叶片图像,利用ImageJ的角度测量工具测量叶边缘刺的角度;选取叶上表皮细胞制片,用目镜测微尺测量叶上表皮细胞波状深度占比(上表皮细胞边缘两波峰中间点至波谷的距离,除以两波峰中间点经过波谷延伸至另一边细胞边缘的距离);选取有乳突的淫羊藿叶下表皮制片,用相机收集清晰的显微图片,利用Adobe Photoshop截取像素大小相同的区域,将图片导入ImageJ,调整阈值,去除背景后,利用Analyze Particles插件自动分析,得到乳突密度(淫羊藿叶下表皮单位面积内乳突面积/淫羊藿叶下表皮单位面积×100%);以上数据,不同品种均随机选取测量20次。

2.3 统计学方法

采用SPSS 26.0统计软件分别对不同品种淫羊藿叶边缘刺的角度、叶上表皮细胞波状深度占比、叶下表皮乳突密度等数据进行统计学分析。

3 结果

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3.1 性状鉴别

9 种淫羊藿叶的性状特征可通过对叶形状、质地、毛被、颜色、顶端、基部等形态的多点对比进行区分。首先按叶质地将淫羊藿、朝鲜淫羊藿归为纸质叶组,其余品种均为革质叶。淫羊藿与朝鲜淫羊藿相比,淫羊藿叶片小且较圆、质地较硬、呈黄绿或绿色;朝鲜淫羊藿叶片大而呈薄纸质,绿色,顶端骤尖。革质叶组中柔毛淫羊藿叶背面密被长柔毛,时珍淫羊藿叶背面明显呈苍白色;其余品种按毛被可分为伏毛组的天平山淫羊藿、粗毛淫羊藿,柔毛组的箭叶淫羊藿、巫山淫羊藿、黔岭淫羊藿。伏毛组中天平山淫羊藿伏毛细小,粗毛淫羊藿粗短;柔毛组中黔岭淫羊藿叶顶端骤尖、基部圆钝、薄革质,箭叶淫羊藿、巫山淫羊藿叶顶端渐尖、基部常呈尖三角形、革质,箭叶淫羊藿叶下表面为短柔毛,而巫山淫羊藿为长柔毛。见图1。

3.2 微性状鉴别

淫羊藿:叶柄光滑无毛。叶边缘刺较细,与叶片呈30°~60°夹角。叶下表面被长柔毛或短柔毛;无乳突。长柔毛长450~1 100 μm,结节状,节间常有缢缩,上部较细,弯曲;短柔毛长250~460 μm,直或略弯曲,节间不缢缩,上部渐狭。叶基部主脉横剖面具木心(下文简称“木心”)3;角质层薄,膜质。

箭叶淫羊藿:叶柄光滑无毛。叶边缘刺较粗,与叶片呈30°~60°夹角。叶下表面被伏毛或短柔毛;乳突疏散分布,呈圆锥状,不明显。伏毛长230~500 μm,明显对折或垂直弯曲,基部节间缢缩,上部常扭曲;短柔毛长160~200 μm,直或略弯曲,节间不缢缩。木心5;角质层厚。

柔毛淫羊藿:叶柄密被毛。叶边缘刺较粗,与叶片呈60°~80°夹角。叶下表面被长柔毛,长800~1 200 μm,卷曲状,无节间,不缢缩,上部渐尖;无乳突。叶基部主脉横剖面具木心5;角质层厚。

朝鲜淫羊藿:叶柄光滑无毛。叶边缘刺较细,与叶片呈30°~45°夹角。叶下表面被长柔毛或短柔毛;无乳突。长柔毛长800~1 200 μm,节间缢缩,明显呈念珠状,基部直立,基部以上扭转弯曲;短柔毛长200~600 μm,直或略弯,较细,分节较少,节间不缢缩或稍缢缩,上部渐狭。木心3;角质层薄,膜质。

巫山淫羊藿:叶柄近无毛。叶边缘刺较粗,与叶片呈45°~60°夹角。叶下表面被长柔毛,长180~800 μm,蛛丝状,基部结节状,直立,节间缢缩或不明显缢缩,基部以上扭转弯曲;乳突疏散分布,呈圆锥状,不明显。木心7;角质层厚。

天平山淫羊藿:叶柄近无毛。叶边缘刺较粗,与叶片呈30°~80°夹角。叶下表面被伏毛,长160~410 μm,上部直或略弯曲,淡黄棕色,节间颜色较深;乳突稍密集分布,呈球状。木心7;角质层厚。

时珍淫羊藿:叶柄疏被毛。叶边缘刺较细,与叶片呈30°~60°夹角。叶下表面被长柔毛,长800~1 500 μm,弯曲,结节状,节间不缢缩,上部渐狭;乳突密集分布,呈球状。木心5;角质层厚。

粗毛淫羊藿:叶柄光滑无毛。叶边缘刺较粗,与叶片呈30°~60°夹角。叶下表面被伏毛,长150~450 μm,基部明显垂直折弯,半透明状,有时略带淡黄色;乳突密集分布,呈球状。木心3~5;角质层厚。

黔岭淫羊藿:叶柄疏被毛。叶边缘刺较粗,与叶片呈30°~60°夹角。叶下表面被长柔毛,长630~1 200 μm,结节状,节间有时缢缩,基部直立,基部以上扭转弯曲;无乳突。木心5;角质层厚。

9种淫羊藿叶的微性状特征在叶柄毛被、叶缘锯齿、叶下表面毛被及乳突、叶基部主脉横剖面木心数、叶上表面角质层厚度等方面有明显区别,且具有种的专属性特征。淫羊藿、柔毛淫羊藿、朝鲜淫羊藿、黔岭淫羊藿均无乳突,其余品种有乳突。箭叶淫羊藿、巫山淫羊藿乳突分布均稀疏,皱缩细小,但箭叶淫羊藿毛茸有2种,巫山淫羊藿仅1种。时珍淫羊藿、天平山淫羊藿、粗毛淫羊藿乳突分布均密集,但时珍淫羊藿叶背为长柔毛,天平山淫羊藿、粗毛淫羊藿为短伏毛,天平山淫羊藿的短伏毛上部直或略弯曲,粗毛淫羊藿的短伏毛基部垂直折弯。无乳突组中,淫羊藿、朝鲜淫羊藿叶边缘刺较细,木心3,角质层薄,柔毛淫羊藿、黔岭淫羊藿叶边缘刺较粗,木心5,角质层厚。淫羊藿和朝鲜淫羊藿区别在于后者长柔毛节间明显缢缩呈念珠状。柔毛淫羊藿和黔岭淫羊藿区别在于前者叶柄密被毛,叶边缘缺刻半圆形,长柔毛卷曲状。见图2~6。

3.3 显微鉴别

显微鉴别包括叶上、下表皮制片、叶主脉横切片和粉末的显微特征。

叶的上下表皮制片可观察到非腺毛形态、乳突表面观、表皮细胞形态等特征,非腺毛较完整,表皮细胞边界清晰,可以估算非腺毛长度及上表皮细胞波状深度占比。见图7~9。

叶基部主脉横切面石蜡切片用于补充观察乳突侧面形状、主脉维管束数目、表皮细胞形态、厚壁组织等。偏振光显微镜下,木质部、厚壁组织、导管、纤维、草酸钙方晶偏振光现象明显,具有强烈彩色光泽,乳突、表皮细胞、非腺毛光泽不明显,其他部分无偏振光现象。见图10。

叶的粉末制片可观察到非腺毛、乳突侧面观、草酸钙柱晶形态等特征,非腺毛结构较表皮制片清晰,可以进一步测量非腺毛细胞数、壁厚等数值。见图11。

淫羊藿:上表皮细胞波状深度占比30%~40%。2种非腺毛,一种长度550~750 μm,基部细胞壁厚2.5~6.5 μm,顶端细胞占非腺毛总长度占比(下文简称“非腺毛占比”)30%~60%,细胞壁未增厚;另一种长度310~350 μm,基部细胞壁厚2.5~5 μm,非腺毛占比50%~60%,细胞壁未增厚。无乳突。基部主脉横切面维管束(下文简称“主脉维管束”)3,下表皮细胞大小不一,排列不整齐,上下表皮内各1~2列细胞明显增厚。

箭叶淫羊藿:占比20%~30%。两种非腺毛,一种长度260~580 μm,基部细胞壁厚5~10 μm,非腺毛占比20%~30%,壁厚2.5~5 μm;另一种长度150~340 μm,基部细胞壁厚4~7 μm,非腺毛占比60%,壁厚2.5~5 μm。乳突表面观边界不明显。主脉维管束5,下表皮细胞大小均匀,排列整齐,上下表皮内各3~4列细胞明显增厚。

柔毛淫羊藿:占比30%~40%。非腺毛长度900~1 200 μm,基部细胞壁厚2.5~5 μm,非腺毛占比90%以上,壁厚3~10 μm。无乳突。主脉维管束4~5,下表皮细胞大小不一,排列略整齐,上下表皮内各1~2列细胞明显增厚。

朝鲜淫羊藿:占比35%~40%。2种非腺毛,一种长度800~1 100 μm,基部细胞壁厚5~7 μm,非腺毛占比20%,细胞壁未增厚;另一种长度280~350 μm,基部细胞壁厚2.5~5 μm,非腺毛占比60%~80%,壁厚2.5~5 μm。无乳突。主脉维管束3~5,下表皮细胞大小不一,排列不整齐,上下表皮内各1~2列细胞略增厚。

巫山淫羊藿:上表皮细胞波状深度占比20%~30%。非腺毛长度150~730 μm,基部细胞壁厚2.5~3 μm,非腺毛占比20%~80%,细胞壁未增厚。乳突表面观边界不明显。主脉维管束7,下表皮细胞大小不一,排列不整齐,上下表皮内各2~3列细胞明显增厚。

天平山淫羊藿:上表皮细胞波状深度占比20%~30%。非腺毛长度150~400 μm,基部细胞壁厚2.5~5 μm,非腺毛占比30%~50%,壁厚2.5~5 μm。乳突表面观双圆圈形,侧面观长乳头状。主脉维管束5~7,下表皮细胞大小不一,排列不整齐,上下表皮内各3~4列细胞明显增厚。

时珍淫羊藿:上表皮细胞波状深度占比20%~30%。非腺毛长度700~1 300 μm,基部细胞壁厚2.5~5 μm,非腺毛占比20%~50%,壁厚5~8 μm。乳突表面观双圆圈形,侧面观长乳头状。主脉维管束5~7,下表皮细胞大小不一,排列不整齐,上下表皮内各4~5列细胞明显增厚。

粗毛淫羊藿:上表皮细胞波状深度占比30%~40%。非腺毛长180~340 μm,基部细胞壁厚2~5 μm,非腺毛占比80%~90%,壁厚5~13 μm。乳突表面观多边形,侧面观短乳头状。主脉面维管束3~5,下表皮细胞大小不一,排列不整齐,上下表皮内各4~7列细胞明显增厚。

黔岭淫羊藿:上表皮细胞波状深度占比30%~35%。非腺毛长550~1 400 μm,基部细胞壁厚5~8 μm,非腺毛占比30%~40%,壁厚5~10 μm。无乳突。主脉维管束4~5,下表皮细胞大小均匀,排列整齐,上下表皮内各2~3列细胞明显增厚。

通过3部分显微特征相结合,可明显区分9种淫羊藿。不同淫羊藿品种根据上表皮细胞波状深度占比和叶基部主脉横切面维管束可分为两组,天平山淫羊藿、时珍淫羊藿、箭叶淫羊藿和巫山淫羊藿的上表皮细胞波状深度占比多小于30%,叶基部主脉横切面维管束多为5~7个,与其余品种不同。天平山淫羊藿、时珍淫羊藿乳突表面观明显呈双圆圈形,但天平山淫羊藿乳突侧面观明显较时珍淫羊藿短,箭叶淫羊藿、巫山淫羊藿乳突表面观均边界不明显,但箭叶淫羊藿有2种非腺毛,且叶上表皮细胞大小均匀,排列整齐,巫山淫羊藿则不同。剩下5种淫羊藿中,仅粗毛淫羊藿有乳突;淫羊藿非腺毛顶端细胞壁无明显增厚,朝鲜淫羊藿、柔毛淫羊藿、黔岭淫羊藿均增厚;朝鲜淫羊藿有2种非腺毛,黔岭淫羊藿与柔毛淫羊藿仅1种;柔毛淫羊藿与黔岭淫羊藿相比,非腺毛顶端细胞较长。

3.4 统计学分析

3.4.1 叶边缘刺的角度

结果表明,不同淫羊藿品种的叶边缘刺角度存在显著区别,F=11.12,P<0.001。Bonferroni多重均数比较结果显示,S1明显大于S4、S5,S3明显大于S2、S4、S5、S7、S8、S9,S6明显大于S4、S5、S9,S2、S8明显大于S4,以上差异均具有统计学意义(P<0.05),其他品种之间无显著性差异(P>0.05)。见表2。

聚类分析结果发现,当类间距离大于13时,9种淫羊藿可分为3类:S3与其他种明显不同,聚为一类;S1、S6、S2聚为一类;其他聚为一类见图12。

3.4.2 叶上表皮细胞波状深度占比

Welch检验结果显示,不同淫羊藿品种的叶上表皮细胞波状深度占比存在显著区别(P<0.001)。Tamhane's T2多重均数比较结果显示,S1、S3明显大于S5,S4明显大于S1、S2、S5、S6、S7、S9,S8明显大于S2、S5、S7,以上差异均具有统计学意义(P<0.05),其他品种之间无显著性差异(P>0.05)(表3)。

聚类分析结果发现,当类间距离大于18时,9种淫羊藿可以分为2个类群,S3、S8、S4聚为一类;其他聚为一类(图13)。

3.4.3 叶下表皮乳突密度

Welch检验结果显示,5种淫羊藿的乳突密度存在显著区别(P<0.001)。Tamhane's T2多重均数比较结果显示,S8、S7明显大于S2、S5、S6,S8明显大于S7,S6明显大于S2、S5,以上差异均具有统计学意义(P<0.05),S2、S5之间无显著性差异(P>0.05)。见表4。

聚类分析结果发现,当类间距离大于3时,5种淫羊藿可以分为2个类群,S7、S8聚为一类;S2、S5、S6聚为一类。结果见图14。

4 讨论

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4.1 创新性分析

与已有研究相比,本实验采用了性状、微性状、常规显微鉴别及偏振光显微鉴别方法,利用大图影像拼接技术与实时景深扩展技术,获得了9种淫羊藿叶的高清全息影像图,并分别对微性状及显微特征区别点进行总结。首次将叶缘刺及叶尖形态描述具象化,提出乳突形态与分布密度、叶基部主脉横剖面木心数、叶肉横剖面的角质层厚度、叶柄毛被等新的重要鉴别特征;利用正常光明场与偏振光暗场对比观察法,能够快速定位具有双折射性的组织,淫羊藿叶主脉木质部、厚壁组织细胞壁增厚程度、导管、纤维、草酸钙方晶清晰可见;并创新性结合Adobe Photoshop和ImageJ将部分性状特征进行数字化提取,弥补了传统经验鉴别中受主观因素影响的不足。本实验中淫羊藿叶品种较为全面,包含了国家和地方中药标准收载品种,均为市场流通中常见品种及易混淆品;同一品种不同产地多次取样,保证了实验结果的稳定性。

4.2 不同鉴别方法适用性及优缺点讨论

在传统鉴别中,宏观的性状鉴别最为方便快捷,但较依赖个人经验且需要样品保持完整;而微性状鉴别和显微鉴别则更为精准且适用于切制或破碎的叶片,但均需要借助显微镜,其中微性状鉴别不同于显微鉴别在观察前不需要繁琐的制片。本研究中微性状鉴别适合从非腺毛的形态、乳突密度值、叶基部主脉横剖面木心数、叶肉横剖面的角质层厚度、叶缘锯齿等方面进行鉴别;显微鉴别适合从叶上表皮细胞波状深度、非腺毛和乳突的形态特征、横切面维管束及表皮细胞壁角质层厚度等方面进行鉴别。微性状与显微鉴别在结果上基本一致,但也有细微差异,如实验发现,箭叶淫羊藿与巫山淫羊藿叶下表皮乳突分布较少,在对完整叶片进行微性状鉴别时观察到有零散分布,而在粉末的显微观察中未发现,猜测可能与粉末制片时取样不均有关,或在粉碎过程中破坏了乳突结构。

4.3 特征量化可行性分析

在微性状和显微性状鉴别中对可量化特征进行多样品的测量并得到数值范围,当不同品种该特征的数值相差较大且没有交叉时,考虑其是否可作为分类鉴别的依据。鉴于此,本实验研究将不同品种淫羊藿叶边缘刺角度、叶上表皮细胞波状深度占比和乳突密度三者特征量化,并进行统计学分析,结果表明,三者特征值的差异均具有统计学意义;聚类分析结果与微性状鉴别、显微鉴别结果基本一致。但统计学分析中具有差异性和分类学上具有鉴别意义是两个不同概念,如柔毛淫羊藿叶边缘刺角度均大于60°,与其他品种数值无交叉,聚类分析结果中单独聚为一类,且在不同产地的多批次样品观察中属于稳定特征,可以作为其种的鉴别依据;其他品种淫羊藿叶边缘刺角度数值范围均有交叉,虽然统计学分析中具有差异性,但聚类分析结果中仅能判断某几种亲缘关系较近,不能作为种间鉴别依据,该特征对其他淫羊藿品种是否具有鉴别意义还有待商榷。

基金

收起

江苏高校品牌专业建设工程项目资助(PPZY2015A070)
南京市2023年“科技三峡行”重庆万州区对口支援项目资助(2023101S-01)

参考文献

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2024年第59卷第9期

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doi: 10.11669/cpj.2024.09.003

接收时间：2023-03-08
首发时间：2025-11-25
出版时间：2024-05-08

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收稿日期：2023-03-08

基金

江苏高校品牌专业建设工程项目资助(PPZY2015A070)

南京市2023年“科技三峡行”重庆万州区对口支援项目资助(2023101S-01)

作者信息

¹ 南京中医药大学, 南京 210023

² 重庆三峡医药高等专科学校, 重庆 404120

³ 南京传统中医门诊部, 南京 210023

⁴ 贵州中医药大学, 贵阳 550025

⁵ 国药集团同济堂(贵州)制药有限公司, 贵阳 550200

通讯作者:

*刘训红,男,硕士,教授研究方向:中药鉴定与品质评价研究 Tel: (025)85811511

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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No.	Herbal name	Habitat (in Chinese)	Acquisition time	Lot number
S1	E. brevicornu Maxim.	Zhang county, Gansu province(甘肃漳县)	2021.6	202106001
		Weiyuan county, Gansu province(甘肃渭源县)	2021.6	202106002
		Wudu district, Gansu province(甘肃武都区)	2021.6	202106003
		Li county, Gansu province(甘肃礼县)	2021.6	202106004
		Li county, Gansu province(甘肃礼县)	2021.6	202106005
		Qinzhou district, Gansu province(甘肃秦州区)	2021.6	202106006
		Lintan county, Gansu province(甘肃临潭县)	2021.6	202106007
		Jishishan county, Gansu province(甘肃积石山县)	2021.6	202106008
		Huating city, Gansu province(甘肃华亭市)	2021.6	202106009
		Yangcheng county, Shanxi province(山西阳城县)	2021.8	202108001
		Pinglu county, Shanxi province(山西平陆县)	2021.7	202107001
S2	E. sagittatum (Sieb. et Zucc.) Maxim.	Liuba county, Shaanxi province(陕西留坝县)	2021.12	202112001
		Liuba county, Shaanxi province(陕西留坝县)	2021.12	202112002
		Rongjiang county, Guizhou province(贵州榕江县)	2021.12	202112003
		Anhua county, Hunan province(湖南安化县)	2021.12	202112004
		Nanzhang county, Hubei province(湖北南漳县)	2021.12	202112005
		Sansui county, Guizhou(贵州三穗县)	2022.1	202201001
		Leishan county, Guizhou province(贵州雷山县)	2022.1	202201002
		Leishan county, Guizhou province(贵州雷山县)	2022.1	202201003
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201004
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201005
S3	E. pubescens Maxim.	Shawan district, Sichuan province(四川沙湾区)	2021.10	202110001
		Shawan district, Sichuan province(四川沙湾区)	2021.10	202110002
		Bazhong city, Sichuan province(四川巴中市)	2022.4	202204001
		Bazhong city, Sichuan province(四川巴中市)	2022.4	202204002
		Ningqiang county, Shaanxi province(陕西宁强县)	2021.12	202112006
		Ningqiang county, Shaanxi province(陕西宁强县)	2021.12	202112007
		Lüeyang county, Shaanxi province(陕西略阳县)	2021.11	202111001
		Lüeyang county, Shaanxi province(陕西略阳县)	2021.11	202111002
		Xingwen county, Sichuan province(四川兴文县)	2022.2	202202001
		Xingwen county, Sichuan province(四川兴文县)	2022.2	202202002
S4	E. koreanum Nakai	Fengcheng city, Liaoning province(辽宁凤城市)	2021.8	202108002
		Fengcheng city, Liaoning province(辽宁凤城市)	2021.8	202108003
		Hengren county, Liaoning province(辽宁恒仁县)	2021.8	202108004
		Hengren county, Liaoning province(辽宁恒仁县)	2021.8	202108005
		Kuandian county, Liaoning province(辽宁宽甸县)	2021.8	202108006
		Kuandian county, Liaoning province(辽宁宽甸县)	2021.8	202108007
		Qingyuan county, Liaoning province(辽宁清原满族自治县)	2021.8	202108008
		Qingyuan county, Liaoning province(辽宁清原满族自治县)	2021.8	202108009
		Fusong county, Jilin province(吉林抚松县)	2021.6	202106010
		Fusong county, Jilin province(吉林抚松县)	2021.6	202106011
S5	E. wushanense T.S. Ying	Zhenping county, Shaanxi province(陕西镇坪县)	2021.11	202111003
		Zhenping county, Shaanxi province(陕西镇坪县)	2021.11	202111004
		Zhenping county, Shaanxi province(陕西镇坪县)	2021.11	202111005
		Dazhou city, Sichuan province(四川达州市)	2021.7	202107002
		Dazhou city, Sichuan province(四川达州市)	2021.7	202107003
		Dazhou city, Sichuan province(四川达州市)	2021.7	202107004
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201006
		Xuanhan county, Sichuan province(四川宣汉县)	2022.1	202201007
		Badong county, Hubei province(湖北巴东县)	2022.4	202204003
		Badong county, Hubei province(湖北巴东县)	2022.4	202204004
S6	E. myrianthum Steam	Yongchuan district, Chongqing(重庆永川区)	2022.1	202201008
		Yongchuan district, Chongqing(重庆永川区)	2022.1	202201009
		Jianhe county, Guizhou province(贵州剑河县)	2022.4	202204005
		Jianhe county, Guizhou province(贵州剑河县)	2022.4	202204006
		Baojing county, Hunan province(湖南保靖县)	2022.4	202204007
		Baojing county, Hunan province(湖南保靖县)	2022.4	202204008
S7	E. lishihchenii Stearn	Dayu county, Jiangxi province(江西大余县)	2022.1	202201010
		Dayu county, Jiangxi province(江西大余县)	2022.1	202201011
		Dayu county, Jiangxi province(江西大余县)	2022.1	202201012
S8	E. acuminatum Franch.	Nanming district, Guizhou province(贵州南明区)	2022.5	202205001
		Nanming district, Guizhou province(贵州南明区)	2022.5	202205002
		Nanchuan district, Chongqing(重庆南川区)	2022.4	202204009
		Nanchuan district, Chongqing(重庆南川区)	2022.4	202204010
S9	E. leptorrhizum Stearn	Nanming district, Guizhou province(贵州南明区)	2022.5	202205003
		Nanming district, Guizhou province(贵州南明区)	2022.5	202205004
		Songtao county, Guizhou province(贵州松桃县)	2022.5	202205005
		Songtao county, Guizhou province(贵州松桃县)	2022.5	202205006

No.

Herbal name

Habitat (in Chinese)

Acquisition time

Lot number

E. brevicornu Maxim.

Zhang county, Gansu province(甘肃漳县)

2021.6

202106001

Weiyuan county, Gansu province(甘肃渭源县)

2021.6

202106002

Wudu district, Gansu province(甘肃武都区)

2021.6

202106003

Li county, Gansu province(甘肃礼县)

2021.6

202106004

Li county, Gansu province(甘肃礼县)

2021.6

202106005

Qinzhou district, Gansu province(甘肃秦州区)

2021.6

202106006

Lintan county, Gansu province(甘肃临潭县)

2021.6

202106007

Jishishan county, Gansu province(甘肃积石山县)

2021.6

202106008

Huating city, Gansu province(甘肃华亭市)

2021.6

202106009

Yangcheng county, Shanxi province(山西阳城县)

2021.8

202108001

Pinglu county, Shanxi province(山西平陆县)

2021.7

202107001

E. sagittatum (Sieb. et Zucc.) Maxim.

Liuba county, Shaanxi province(陕西留坝县)

2021.12

202112001

Liuba county, Shaanxi province(陕西留坝县)

2021.12

202112002

Rongjiang county, Guizhou province(贵州榕江县)

2021.12

202112003

Anhua county, Hunan province(湖南安化县)

2021.12

202112004

Nanzhang county, Hubei province(湖北南漳县)

2021.12

202112005

Sansui county, Guizhou(贵州三穗县)

2022.1

202201001

Leishan county, Guizhou province(贵州雷山县)

2022.1

202201002

Leishan county, Guizhou province(贵州雷山县)

2022.1

202201003

Xuanhan county, Sichuan province(四川宣汉县)

2022.1

202201004

Xuanhan county, Sichuan province(四川宣汉县)

2022.1

202201005

E. pubescens Maxim.

Shawan district, Sichuan province(四川沙湾区)

2021.10

202110001

Shawan district, Sichuan province(四川沙湾区)

2021.10

202110002

Bazhong city, Sichuan province(四川巴中市)

2022.4

202204001

Bazhong city, Sichuan province(四川巴中市)

2022.4

202204002

Ningqiang county, Shaanxi province(陕西宁强县)

2021.12

202112006

Ningqiang county, Shaanxi province(陕西宁强县)

2021.12

202112007

Lüeyang county, Shaanxi province(陕西略阳县)

2021.11

202111001

Lüeyang county, Shaanxi province(陕西略阳县)

2021.11

202111002

Xingwen county, Sichuan province(四川兴文县)

2022.2

202202001

Xingwen county, Sichuan province(四川兴文县)

2022.2

202202002

E. koreanum Nakai

Fengcheng city, Liaoning province(辽宁凤城市)

2021.8

202108002

Fengcheng city, Liaoning province(辽宁凤城市)

2021.8

202108003

Hengren county, Liaoning province(辽宁恒仁县)

2021.8

202108004

Hengren county, Liaoning province(辽宁恒仁县)

2021.8

202108005

Kuandian county, Liaoning province(辽宁宽甸县)

2021.8

202108006

Kuandian county, Liaoning province(辽宁宽甸县)

2021.8

202108007

Qingyuan county, Liaoning province(辽宁清原满族自治县)

2021.8

202108008

Qingyuan county, Liaoning province(辽宁清原满族自治县)

2021.8

202108009

Fusong county, Jilin province(吉林抚松县)

2021.6

202106010

Fusong county, Jilin province(吉林抚松县)

2021.6

202106011

E. wushanense T.S. Ying

Zhenping county, Shaanxi province(陕西镇坪县)

2021.11

202111003

Zhenping county, Shaanxi province(陕西镇坪县)

2021.11

202111004

Zhenping county, Shaanxi province(陕西镇坪县)

2021.11

202111005

Dazhou city, Sichuan province(四川达州市)

2021.7

202107002

Dazhou city, Sichuan province(四川达州市)

2021.7

202107003

Dazhou city, Sichuan province(四川达州市)

2021.7

202107004

Xuanhan county, Sichuan province(四川宣汉县)

2022.1

202201006

Xuanhan county, Sichuan province(四川宣汉县)

2022.1

202201007

Badong county, Hubei province(湖北巴东县)

2022.4

202204003

Badong county, Hubei province(湖北巴东县)

2022.4

202204004

E. myrianthum Steam

Yongchuan district, Chongqing(重庆永川区)

2022.1

202201008

Yongchuan district, Chongqing(重庆永川区)

2022.1

202201009

Jianhe county, Guizhou province(贵州剑河县)

2022.4

202204005

Jianhe county, Guizhou province(贵州剑河县)

2022.4

202204006

Baojing county, Hunan province(湖南保靖县)

2022.4

202204007

Baojing county, Hunan province(湖南保靖县)

2022.4

202204008

E. lishihchenii Stearn

Dayu county, Jiangxi province(江西大余县)

2022.1

202201010

Dayu county, Jiangxi province(江西大余县)

2022.1

202201011

Dayu county, Jiangxi province(江西大余县)

2022.1

202201012

E. acuminatum Franch.

Nanming district, Guizhou province(贵州南明区)

2022.5

202205001

Nanming district, Guizhou province(贵州南明区)

2022.5

202205002

Nanchuan district, Chongqing(重庆南川区)

2022.4

202204009

Nanchuan district, Chongqing(重庆南川区)

2022.4

202204010

E. leptorrhizum Stearn

Nanming district, Guizhou province(贵州南明区)

2022.5

202205003

Nanming district, Guizhou province(贵州南明区)

2022.5

202205004

Songtao county, Guizhou province(贵州松桃县)

2022.5

202205005

Songtao county, Guizhou province(贵州松桃县)

2022.5

202205006

No.	Herbal name	Angle of thorns at leaf edge/° ($\bar{x}±s$)	F test	Comparison of multiple means
S1	E. brevicornu	56.66	±11.35	11.12	<0.001	P<0.05,when compared with S4 and S5
S2	E. sagittatum	48.65	±14.39			P<0.05,when compared with S3 and S4
S3	E. pubescens	64.54	±13.12			P<0.05,when compared with S2,S4,S5,S7,S8 and S9
S4	E. koreanum	36.32	±10.41			P<0.05,when compared with S1,S2,S3,S6,and S8
S5	E. wushanense	43.17	±9.83			P<0.05,when compared with S1,S3,and S6
S6	E. myrianthum	58.04	±8.78			P<0.05,when compared with S4,S5,and S9
S7	E. lishihchenii	47.99	±13.41			P<0.05,when compared with S3
S8	E. acuminatum	48.97	±10.41			P<0.05,when compared with S3 and S4
S9	E. leptorrhizum	45.56	±9.86			P<0.05,when compared with S3 and S6

No.

Herbal
name

Angle of thorns at leaf edge/°
($\bar{x}±s$)

F test

Comparison of multiple means

E. brevicornu

56.66

±11.35

11.12

<0.001

P<0.05,when compared with S4 and S5

E. sagittatum

48.65

±14.39

P<0.05,when compared with S3 and S4

E. pubescens

64.54

±13.12

P<0.05,when compared with S2,S4,S5,S7,S8 and S9

E. koreanum

36.32

±10.41

P<0.05,when compared with S1,S2,S3,S6,and S8

E. wushanense

43.17

±9.83

P<0.05,when compared with S1,S3,and S6

E. myrianthum

58.04

±8.78

P<0.05,when compared with S4,S5,and S9

E. lishihchenii

47.99

±13.41

P<0.05,when compared with S3

E. acuminatum

48.97

±10.41

P<0.05,when compared with S3 and S4

E. leptorrhizum

45.56

±9.86

P<0.05,when compared with S3 and S6

No.	Herbal name	The portion of wave depth of epidermal cells in the leaves/%($\bar{x}±s$)	The P-value of the Welch test	Comparison of multiple means
S1	E. brevicornu	33.17±4.35	<0.001	P<0.05,when compared with S4 and S5
S2	E. sagittatum	28.41±5.49		P<0.05,when compared with S4 and S8
S3	E. pubescens	34.08±6.86		P<0.05,when compared with S5
S4	E. koreanum	38.56±4.14		P<0.05,when compared with S1,S2,S5,S6,S7 and S9
S5	E. wushanense	26.01±5.57		P<0.05,when compared with those of S1,S3,S4,and S8
S6	E. myrianthum	29.73±4.41		P<0.05,when compared with S4
S7	E. lishihchenii	27.37±6.16		P<0.05,when compared with S4 and S8
S8	E. acuminatum	34.02±4.31		P<0.05,when compared with those of S2,S5 and S7
S9	E. leptorrhizum	31.07±4.05		P<0.05,when compared with S4

No.

Herbal
name

The portion of wave depth of
epidermal cells in the leaves/%($\bar{x}±s$)

The P-value
of the Welch test

Comparison
of multiple means

E. brevicornu

33.17±4.35

<0.001

P<0.05,when compared with S4 and S5

E. sagittatum

28.41±5.49

P<0.05,when compared with S4 and S8

E. pubescens

34.08±6.86

P<0.05,when compared with S5

E. koreanum

38.56±4.14

P<0.05,when compared with S1,S2,S5,S6,S7 and S9

E. wushanense

26.01±5.57

P<0.05,when compared with those of S1,S3,S4,and S8

E. myrianthum

29.73±4.41

P<0.05,when compared with S4

E. lishihchenii

27.37±6.16

P<0.05,when compared with S4 and S8

E. acuminatum

34.02±4.31

P<0.05,when compared with those of S2,S5 and S7

E. leptorrhizum

31.07±4.05

P<0.05,when compared with S4

No.	Herbal name	The density of the mastoid/%($\bar{x}±s$)	The P-value of the Welch test	Comparison of multiple means
S2	E. sagittatum	9.08±2.64	<0.001	P<0.05,when compared with S6,S7 and S8
S5	E. wushanense	11.09±2.94		P<0.05,when compared with S6,S7 and S8
S6	E. myrianthum	15.74±2.87		P<0.05,when compared with S2,S5,S7 and S8
S7	E. lishihchenii	34.09±3.94		P<0.05,when compared with S2,S5,S6 and S8
S8	E. acuminatum	39.71±5.35		P<0.05,when compared with S2,S5,S6,and S7

No.

Herbal name

The density of the mastoid/%($\bar{x}±s$)

The P-value of the Welch test

Comparison of multiple means

E. sagittatum

9.08±2.64

<0.001

P<0.05,when compared with S6,S7 and S8

E. wushanense

11.09±2.94

P<0.05,when compared with S6,S7 and S8

E. myrianthum

15.74±2.87

P<0.05,when compared with S2,S5,S7 and S8

E. lishihchenii

34.09±3.94

P<0.05,when compared with S2,S5,S6 and S8

E. acuminatum

39.71±5.35

P<0.05,when compared with S2,S5,S6,and S7