药学学报

No.	Medicinal part	Collecting site		No.	Medicinal part	Collecting site
T1-G	Root	Zhuoni County, Gansu Province		P6-G	Root	Lichuan Town, Tanchang County, Gansu Province
T1-GJ	Rhizome	Zhuoni County, Gansu Province	P6-GJ	Rhizome	Lichuan Town, Tanchang County, Gansu Province
T2-G	Root	Hezuo City, Gansu Province	P7-G	Root	Guanting Town, Tanchang County, Gansu Province
T2-GJ	Rhizome	Hezuo City, Gansu Province	P7-GJ	Rhizome	Guanting Town, Tanchang County, Gansu Province
T3-G	Root	Yangyong Town, Lintan County, Gansu Province	P8-G	Root	Qiaotou Town, Li County, Gansu Province
T3-GJ	Rhizome	Yangyong Town, Lintan County, Gansu Province	P8-GJ	Rhizome	Qiaotou Town, Li County, Gansu Province
T4-G	Root	Tuanjie Village, Ruoergai County, Sichuan Province	P9-G	Root	Mazichuan Town, Min County, Gansu Province
T4-GJ	Rhizome	Tuanjie Village, Ruoergai County, Sichuan Province	P9-GJ	Rhizome	Mazichuan Town, Min County, Gansu Province
P1-G	Root	Shizi Township, Tanchang County, Gansu Province	P10-G	Root	Caoping Township, Li County, Gansu Province
P1-GJ	Rhizome	Shizi Township, Tanchang County, Gansu Province	P10-GJ	Rhizome	Caoping Township, Li County, Gansu Province
P2-GJ	Rhizome	Taoping Town, Li County, Gansu Province	O1-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province
P3-G	Root	Hadapu Town, Tanchang County, Gansu Province	O2-G	Root	Shimiaozi Village, Lichuan City, Hubei Province
P3-GJ	Rhizome	Hadapu Town, Tanchang County, Gansu Province	O2-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province
P4-GJ	Rhizome	Caoping Township, Li County, Gansu Province	O3-G	Root	Shimiaozi Village, Lichuan City, Hubei Province
P5-G	Root	Caoping Township, Li County, Gansu Province	O3-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province
P5-GJ	Rhizome	Awu Town, Tanchang County, Gansu Province	O4-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province

No.	Medicinal part	Collecting site		No.	Medicinal part	Collecting site
T1-G	Root	Zhuoni County, Gansu Province		P6-G	Root	Lichuan Town, Tanchang County, Gansu Province
T1-GJ	Rhizome	Zhuoni County, Gansu Province	P6-GJ	Rhizome	Lichuan Town, Tanchang County, Gansu Province
T2-G	Root	Hezuo City, Gansu Province	P7-G	Root	Guanting Town, Tanchang County, Gansu Province
T2-GJ	Rhizome	Hezuo City, Gansu Province	P7-GJ	Rhizome	Guanting Town, Tanchang County, Gansu Province
T3-G	Root	Yangyong Town, Lintan County, Gansu Province	P8-G	Root	Qiaotou Town, Li County, Gansu Province
T3-GJ	Rhizome	Yangyong Town, Lintan County, Gansu Province	P8-GJ	Rhizome	Qiaotou Town, Li County, Gansu Province
T4-G	Root	Tuanjie Village, Ruoergai County, Sichuan Province	P9-G	Root	Mazichuan Town, Min County, Gansu Province
T4-GJ	Rhizome	Tuanjie Village, Ruoergai County, Sichuan Province	P9-GJ	Rhizome	Mazichuan Town, Min County, Gansu Province
P1-G	Root	Shizi Township, Tanchang County, Gansu Province	P10-G	Root	Caoping Township, Li County, Gansu Province
P1-GJ	Rhizome	Shizi Township, Tanchang County, Gansu Province	P10-GJ	Rhizome	Caoping Township, Li County, Gansu Province
P2-GJ	Rhizome	Taoping Town, Li County, Gansu Province	O1-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province
P3-G	Root	Hadapu Town, Tanchang County, Gansu Province	O2-G	Root	Shimiaozi Village, Lichuan City, Hubei Province
P3-GJ	Rhizome	Hadapu Town, Tanchang County, Gansu Province	O2-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province
P4-GJ	Rhizome	Caoping Township, Li County, Gansu Province	O3-G	Root	Shimiaozi Village, Lichuan City, Hubei Province
P5-G	Root	Caoping Township, Li County, Gansu Province	O3-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province
P5-GJ	Rhizome	Awu Town, Tanchang County, Gansu Province	O4-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province

No.	Retention time/min	Relative molecular mass	Compound	Molecular formula	VIP
1	14.75	488	Rhein-O-acetyl-glucose_1	C₂₃H₂₀O₁₂	1.213 4
2	12.65	862	Rhein-rhein (C10-C10′) type aglycone_17	C₄₂H₃₈O₂₀	1.187 4
3	10.52	446	Rhein-8-O-β-D-glucopyranoside	C₂₁H₁₈O₁₁	1.178 0
4	15.94	948	Rhein-rhein (C10-C10′) type aglycone_16	C₄₅H₄₀O₂₃	1.168 3
5	10.68	862	Sennoside B	C₄₂H₃₈O₂₀	1.163 1
6	13.32	862	Sennoside A	C₄₂H₃₈O₂₀	1.142 5
7	4.49	594	Rheinosides C	C₂₇H₃₀O₁₅	1.131 7
8	14.87	474	Aloe-emodin-O-acetyl-glucopyranoside	C₂₃H₂₂O₁₁	1.111 6
9	12.92	948	Rhein-rhein (C10-C10′) type aglycone_14	C₄₅H₄₀O₂₃	1.106 2
10	5.93	594	Rheinosides D	C₂₇H₃₀O₁₅	1.092 6
11	13.88	948	Rhein-rhein (C10-C10′) type aglycone_15	C₄₅H₄₀O₂₃	1.085 4
12	15.40	432	Emodin-O-glucose_1	C₂₁H₂₀O₁₀	1.083 0
13	22.48	446	Physcion-8-O-β-D-glucopyranoside_1	C₂₂H₂₂O₁₀	1.070 9
14	19.44	416	Chrysophanol-1-O-glucose	C₂₁H₂₀O₉	1.062 0
15	13.42	488	Rhein-O-acetyl-glucose_2	C₂₃H₂₀O₁₂	1.059 0
16	15.81	848	Aloe-emodin-rhein (C10-C10′) type aglycone/emodin-rhein (C10-C10′) type aglycone_23	C₄₂H₄₀O₁₉	1.058 2
17	13.14	518	Carboxyl-emodin-8-O-(6′-O-acetyl)-glucose	C₂₄H₂₂O₁₃	1.056 1
18	16.89	832	Chrysophanol-rhein (C10-C10′) type aglycone_16	C₄₂H₄₀O₂₁	1.051 0
19	14.07	474	Aloe-emodin-8-O-β-D-(6′-O-acetyl)-glucopyranoside	C₂₃H₂₂O₁₁	1.049 2
20	21.47	568	Chrysophanol-O-galloyl-O-glucose_2	C₂₈H₂₄O₁₃	1.039 9
21	19.23	862	Physcion-rhein (C10-C10′) type aglycone_7	C₄₃H₄₂O₁₉	1.011 9
22	19.48	862	Physcion-rhein (C10-C10′) type aglycone_8	C₄₃H₄₂O₁₉	1.010 7
23	3.17	610	Rheinosides A	C₂₇H₃₀O₁₆	1.006 9

No.	Retention time/min	Relative molecular mass	Compound	Molecular formula	VIP
1	14.75	488	Rhein-O-acetyl-glucose_1	C₂₃H₂₀O₁₂	1.213 4
2	12.65	862	Rhein-rhein (C10-C10′) type aglycone_17	C₄₂H₃₈O₂₀	1.187 4
3	10.52	446	Rhein-8-O-β-D-glucopyranoside	C₂₁H₁₈O₁₁	1.178 0
4	15.94	948	Rhein-rhein (C10-C10′) type aglycone_16	C₄₅H₄₀O₂₃	1.168 3
5	10.68	862	Sennoside B	C₄₂H₃₈O₂₀	1.163 1
6	13.32	862	Sennoside A	C₄₂H₃₈O₂₀	1.142 5
7	4.49	594	Rheinosides C	C₂₇H₃₀O₁₅	1.131 7
8	14.87	474	Aloe-emodin-O-acetyl-glucopyranoside	C₂₃H₂₂O₁₁	1.111 6
9	12.92	948	Rhein-rhein (C10-C10′) type aglycone_14	C₄₅H₄₀O₂₃	1.106 2
10	5.93	594	Rheinosides D	C₂₇H₃₀O₁₅	1.092 6
11	13.88	948	Rhein-rhein (C10-C10′) type aglycone_15	C₄₅H₄₀O₂₃	1.085 4
12	15.40	432	Emodin-O-glucose_1	C₂₁H₂₀O₁₀	1.083 0
13	22.48	446	Physcion-8-O-β-D-glucopyranoside_1	C₂₂H₂₂O₁₀	1.070 9
14	19.44	416	Chrysophanol-1-O-glucose	C₂₁H₂₀O₉	1.062 0
15	13.42	488	Rhein-O-acetyl-glucose_2	C₂₃H₂₀O₁₂	1.059 0
16	15.81	848	Aloe-emodin-rhein (C10-C10′) type aglycone/emodin-rhein (C10-C10′) type aglycone_23	C₄₂H₄₀O₁₉	1.058 2
17	13.14	518	Carboxyl-emodin-8-O-(6′-O-acetyl)-glucose	C₂₄H₂₂O₁₃	1.056 1
18	16.89	832	Chrysophanol-rhein (C10-C10′) type aglycone_16	C₄₂H₄₀O₂₁	1.051 0
19	14.07	474	Aloe-emodin-8-O-β-D-(6′-O-acetyl)-glucopyranoside	C₂₃H₂₂O₁₁	1.049 2
20	21.47	568	Chrysophanol-O-galloyl-O-glucose_2	C₂₈H₂₄O₁₃	1.039 9
21	19.23	862	Physcion-rhein (C10-C10′) type aglycone_7	C₄₃H₄₂O₁₉	1.011 9
22	19.48	862	Physcion-rhein (C10-C10′) type aglycone_8	C₄₃H₄₂O₁₉	1.010 7
23	3.17	610	Rheinosides A	C₂₇H₃₀O₁₆	1.006 9

基于拟靶向代谢组学探究三种基原大黄泻下药效差异及其药效物质基础

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唐进程 ¹ , 赵江怡 ¹ , 周易 ¹ , 彭博杨 ¹ , 陈欣欣 ¹ , 张媛 ¹^,² , 刘凤波 ¹^,² , 赵婷 ¹^,²^,^* , 魏胜利 ¹^,²^,^*

药学学报 | 研究论文 2025,60(6): 1886-1895

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药学学报 |研究论文 2025 , 60 (6) : 1886 -1895

基于拟靶向代谢组学探究三种基原大黄泻下药效差异及其药效物质基础

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唐进程¹, 赵江怡¹, 周易¹, 彭博杨¹, 陈欣欣¹, 张媛¹^,², 刘凤波¹^,², 赵婷¹^,²^,^*, 魏胜利¹^,²^,^*

作者信息

¹北京中医药大学中药学院, 北京 102488

²中药材规范化生产教育部工程研究中心, 北京 100102

通讯作者:

赵婷, E-mail: wsl7491@126.com

魏胜利, E-mail: 19053181@qq.com

作者简介:

作者贡献:

唐进程负责整体实验设计、实施、数据分析及论文撰写工作; 赵江怡参与动物实验、拟靶向代谢组学部分工作及论文修改; 周易、彭博扬、陈欣欣参与拟靶向代谢组学部分工作; 刘凤波参与部分样品前处理工作; 张媛、赵婷、魏胜利对实验设计、论文撰写及修改进行指导。

Exploring the differences in purgative efficacy among three origins of rhubarb and active components of purgative efficacy based on pseudo-targeted metabolomics

Jin-cheng TANG¹, Jiang-yi ZHAO¹, Yi ZHOU¹, Bo-yang PENG¹, Xin-xin CHEN¹, Yuan ZHANG¹^,², Feng-bo LIU¹^,², Ting ZHAO¹^,²^,^*, Sheng-li WEI¹^,²^,^*

Affiliations

¹School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China

²Engineering Research Center of Good Agricultural Practice for Chinese Crude Drugs of Ministry of Education, Beijing 100102, China

出版时间: 2025-06-12 doi: 10.16438/j.0513-4870.2024-1042

文章导航

摘要

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大黄是一种多基原药材, 三种基原大黄泻下药效存在着差异, 且发挥泻下药效的物质基础尚不明确。本研究采用拟靶向代谢组学结合药理实验(获得北京中医药大学动物伦理委员会批准, 批准号: BUCM-4-2022080301-3026) 的研究方法揭示三种基原大黄泻下药效差异, 并筛选出大黄泻下药效物质基础。通过动物药理实验分析三种基原大黄泻下药效的差异; 后通过文献查找大黄潜在泻下药效成分并结合裂解规律及软件优化得到相应离子对, 利用三重四极杆(QQQ-MS) 的动态多反应监测(dynamic multiple reaction monitoring, dyMRM) 模式获取各成分的峰面积, 采用多元统计分析方法对三种基原大黄潜在泻下药效化学成分进行分析, 从而实现拟靶向代谢组学分析, 并结合药理实验结果筛选出大黄泻下药效物质基础。药理实验结果表明, 唐古特大黄泻下药效显著高于药用大黄和掌叶大黄; 主成分分析(principal components analysis, PCA) 结果表明, 三种基原大黄潜在泻下药效成分存在明显差异, 通过正交偏最小二乘判别分析(orthogonal partial least squares-discriminant analysis, OPLS-DA) 结合Kruskal-Wallis检验筛选出97个差异潜在泻下药效活性成分; 结合药理实验结果筛选出泻下药效成分23个, 其中化学结构明确的化合物有番泻苷A、番泻苷B、大黄酸苷A、大黄酸苷C、大黄酸苷D、大黄素甲醚-8-O-葡萄糖苷、大黄酸-8-O-葡萄糖苷、大黄酚-1-O-葡萄糖苷。综上, 采用拟靶向代谢组学结合药理实验, 明确了三种基原大黄泻下药效的强弱, 揭示了潜在泻下药效成分(蒽醌、蒽酮类成分) 在三种基原大黄中的相对含量分布情况, 并筛选出了大黄的泻下药效物质基础, 不仅为大黄药材质量评价和临床精准用药提供一定的理论依据, 也为中药寻找功效标志物提供新的思路。

关键词

大黄 / 三种基原 / 拟靶向代谢组学 / 泻下 / 药效物质基础

Abstract

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Rhubarb is a Chinese medicinal herb of multiple origins, there are differences in the purgative efficacy of three origins of rhubarb, and the active components of the purgative efficacy of rhubarb is not yet clear. The study reveals the differences in the purgative efficacy of three origins of rhubarb by method of pseudo-targeted metabolomics combined with pharmacological experiments (approval number: BUCM-4-2022080301-3026), and screens out the active components of the purgative efficacy of rhubarb. Analyzing the differences in the purgative efficacy of three origins of rhubarb through animal pharmacological experiments. Subsequently, potential active components of purgative efficacy were identified through literature review, and corresponding ion pairs were obtained by fragmentation pathways and software optimization, and the peak areas of each component were obtained by using dyMRM mode of QQQ-MS. Multivariate statistical analysis was used to analyze the potential active components of purgative efficacy in rhubarb with different origins, thereby achieving pseudo-targeted metabolomics analysis, and the active components can be screened with pharmacological experimental results. The pharmacological experimental results indicate that the purgative efficacy of Rheum tanguticum Maxim. ex Regel is significantly higher than that of Rheum palmatum L. and Rheum officinale Baill. The results of PCA showed that there were significant differences in the potential active components of purgative efficacy among three origins of rhubarb. 97 differential potential active components of purgative efficacy were screened through OPLS-DA combined with Kruskal Wallis test; combined with the results of metabolomics, 23 active components of purgative efficacy were screened, among which the compounds with clear chemical structures were sennoside A, sennoside B, rheinosides A, rheinosides C, rheinosides D, physcion-8-O-glucoside, rhein-8-O-glucoside, chrysophanol-1-O-glucoside. The above results suggest that the method of pseudo-targeted metabolomics combined with pharmacological experiments has been used to clarify the relationship of purgative efficacy between the three origins of rhubarb, reveal the distribution of potential active components of purgative efficacy in three origins of rhubarb, and screen out the active components of purgative efficacy. This not only provides a theoretical basis for the quality evaluation and precise clinical use of rhubarb, but also provides a new idea for traditional Chinese medicine to search for efficacy markers.

Key words

rhubarb / three origins / pseudo-targeted metabolomics / purgative efficacy / pharmacological substance basis

引用本文

唐进程, 赵江怡, 周易, 彭博杨, 陈欣欣, 张媛, 刘凤波, 赵婷, 魏胜利. 基于拟靶向代谢组学探究三种基原大黄泻下药效差异及其药效物质基础. 药学学报, 2025 , 60 (6) : 1886 -1895 . DOI: 10.16438/j.0513-4870.2024-1042

Jin-cheng TANG, Jiang-yi ZHAO, Yi ZHOU, Bo-yang PENG, Xin-xin CHEN, Yuan ZHANG, Feng-bo LIU, Ting ZHAO, Sheng-li WEI. Exploring the differences in purgative efficacy among three origins of rhubarb and active components of purgative efficacy based on pseudo-targeted metabolomics[J]. Acta Pharmaceutica Sinica, 2025 , 60 (6) : 1886 -1895 . DOI: 10.16438/j.0513-4870.2024-1042

正文

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大黄是典型的多基原、多功效中药, 为蓼科植物掌叶大黄Rheum palmatum L.、唐古特大黄Rheum tanguticum Maxim. ex Regel和药用大黄Rheum officinale Baill.的根和根茎, 具有泻下攻积、清热泻火、凉血解毒、逐瘀通经、利湿退黄等功效^[1]。大黄中所含的化学成分是其发挥相应药效的物质基础, 研究表明, 三种基原大黄所含化学成分种类和含量存在差异^{[2, 3]}, 这将导致三种基原大黄药效上的差异。因此, 对多基原药材药效差异及其对应药效物质的研究是十分必要的。目前, 三种基原大黄化学成分的研究多为对三种基原大黄某几种常见化学成分在三种基原中的分布进行分析^{[4, 5]}, 而对发挥某功效的化学成分在三种基原大黄中的分布情况研究鲜有报道。

拟靶向代谢组学方法首先通过Q-TOF、Orbitrap等高分辨质谱中非靶向地获得样品的整体代谢物图谱; 然后, 通过软件包或人工处理获得每个母离子与其特征子离子组成的离子对; 最后, 将特征离子对应用于QQQ-MS, 对样品中的每种代谢物进行dyMRM分析。该方法结合了非靶向代谢组学和靶向代谢组学的特点, 具有高通量、无偏向检测、高特异性、定量准确、线性范围广、稳定性好、灵敏度高、数据处理简单、成分覆盖面广等优点^[6], 适用于药效物质基础的筛选。目前, 该方法在疾病诊断指标及植物代谢成分方面的研究均有较好的应用^{[6, 7]}。

因此, 本研究以4年生、加工方法等条件一致的三种基原大黄样品作为研究对象, 通过拟靶向代谢组学的方法, 分析三种基原大黄潜在泻下药效成分(研究表明大黄中发挥泻下药效作用的成分为蒽醌、蒽酮类成分^[8-10]) 的差异特征; 通过药理实验研究三种基原大黄泻下药效的差异, 并结合拟靶向代谢组学分析和药理实验结果筛选泻下药效物质基础。

材料与方法

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仪器分析天平(瑞士梅特勒-托利多仪器有限公司, 型号: AL204); 三重四极杆液质联用系统(美国Agilent公司, 型号: 6475); 数控超声波清洗器(昆山市超声仪器有限公司, 型号: KQ-500DE); 高速万能粉碎机(天津市泰斯特仪器有限公司, 型号: FW100); C₁₈色谱柱(美国Waters公司, 型号: ACQUITYTM UPLC BEH, 2.1 mm × 50 mm, 1.7 μm); 全波段酶标仪(美国伯腾仪器有限公司, 型号: Synergy Ⅱ); 离心机(德国西格玛实验室离心机公司, 型号: 3K15); 三用恒温水箱(中仪国科科技有限公司, 型号: DK-S600); 电热鼓风干燥箱(天津市泰斯特仪器有限公司, 型号: 101-1A)。

试药番泻苷A (上海源叶生物科技有限公司, P01S8F42887, 纯度≥ 98%); 乙腈(美国赛默飞世尔科技有限公司, A955-4, 质谱级); 质谱级甲酸、戊巴比妥钠(德国Merck公司, 20444904723、57-33-0); 甲醇(北京化工厂, 分析级); 屈臣氏纯净水(广州屈臣氏食品饮料有限公司, 20191028H); 复方地酚诺酯片(常州康普药业有限公司, 2204006); 水通道蛋白3 (aquaporin 3, AQP3)、5-羟色胺(5-HT) 酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA) 试剂盒(上海酶联生物科技有限公司, YJ001869、20230217); PBS缓冲液(北京索莱宝科技有限公司, 20221010, pH 7.2~7.4); 生理盐水(石家庄四药有限公司, 2209082005)。

动物 SPF级雄性ICR小鼠, 体重16~17 g (适应性饲养至体重20 g)。由斯贝福(北京) 生物技术有限公司提供[许可证号: SCXK (京) 2019-0010]。饲养于北京中医药大学良乡校区动物房, 食水自由摄入, 动物房温度20~24 ℃, 自由光照, 湿度44%~55%, 定时通风换气。本实验获得北京中医药大学动物伦理委员会批准, 批准号: BUCM-4-2022080301-3026。

大黄供试品 大黄药材均为2021年10月中旬至11月下旬, 从甘肃、四川、湖北采挖4年生(移栽后3年) 的三种基原大黄, 经北京中医药大学魏胜利教授鉴定, 编号T1~T4为蓼科植物唐古特大黄(R.tanguticum Maxim. ex Balf.)、编号P1~P10为掌叶大黄(Rheum palmatum L.)、编号O1~O4为药用大黄(R.officinale Baill.)。大黄样品采收后, 将药材的根和根茎分开(部分掌叶、药用大黄的根因未达到直径 > 3 cm的要求, 故未列出), 分别切成4 mm左右的厚片, 60 ℃烘干2天后粉碎, 4号筛滤过, 混匀, 四分法缩分取样(样品信息见表1)。留样药材存放于北京中医药大学中药学院中药资源样本室。

样品制备

造模剂配制取复方地芬诺酯片50 mg (20片), 用研钵研磨成粉末后全部转移至50 mL量瓶, 加适量水溶解, 用水稀释至刻度线, 摇匀, 即得质量浓度为1 mg·mL^-1, 临用前配制。

阳性药配制准确称取番泻苷A5 mg, 用水定容至5 mL, 即得1 mg·mL^-1番泻苷A溶液。

大黄提取液准确称取大黄粉末0.5 g (过四号筛), 置于25 mL锥形瓶中, 加入10 mL 70%甲醇溶解, 超声处理提取30 min。旋干溶剂(70%甲醇), 精密加10 mL水溶解, 得到浓度为0.05 g·mL^-1的大黄提取液。

拟靶向代谢组学分析供试品溶液制备精密称取大黄粉末(过四号筛) 0.25 g, 置于具塞三角瓶内, 精密加入70%甲醇25 mL, 称定重量。超声(250 W, 40 kHz) 提取30 min, 放冷, 称重, 用70%甲醇补足损失的重量, 混匀, 过滤, 取续滤液过滤(0.22 μm微孔滤膜), 即得。

质控(quality control, QC) 样品溶液制备等量称取每批样品适量, 混匀后作为QC样品。分别精密称量0.5和0.25 g QC样品, 分别置于50 mL具塞三角瓶内, 精密加入70%甲醇25 mL, 称定重量, 超声提取30 min, 放冷, 称重, 用70%甲醇补足失重。混匀, 0.22 μm微孔滤膜滤过, 取续滤液, 得QC母液和QC溶液。QC样品覆盖了三种基原大黄所含的化合物, 因此可以用于方法学考察, 其中QC母液用于线性考察, QC溶液用于重复性和精密度考察。

大黄泻下药效测定

分组、造模、给药及取材将60只健康的雄性ICR小鼠随机平均分成6组, 在测试1 h前保持自由饮食饮水。第1组作为空白组(不建模不给药); 第2组作为模型组(仅建模不给药); 第3组作为阳性药对照组(番泻苷A); 第4组作为唐古特大黄给药实验组; 第5组作为掌叶大黄给药实验组; 第6组作为药用大黄给药实验组。每只小鼠按照0.01 mg·g^-1的剂量给予复方地酚诺酯溶水溶液灌胃^[11]。30 min后, 实验组小鼠按照0.5 mg·g^-1 (中国药典2020年版规定大黄的用量为3~15 g·d^-1)。按60 kg体重计, 即50~250 mg·kg^-1·d^-1, 剂量换算系数取8.65, 则小鼠剂量为432.5~2 162.5 mg·kg^-1·d^-1, 因该提取液为醇提液, 取剂量为0.5 mg·g^-1 (预实验表明该给药剂量合适) 给予三种基原大黄的提取液灌胃; 对照组小鼠按照0.01 mg·g^-1剂量给予番泻苷A水溶液灌胃; 空白组小鼠给予相应体积的水灌胃。给药30 min后, 收集每只小鼠8 h内排出的粪便, 及时将小鼠粪便放入对应编号的EP管中, 并盖紧盖子。采用小鼠眼球取血法取血, 在室温下将全血标本静置2 h后于4 ℃离心机中以3 000 r·min^-1分离血清10 min, 将上清液转移至干净的EP管中。采血完成后, 将结肠组织剪下, 并用生理盐水将肠道内的粪便冲洗干净。

小鼠粪便粒数、湿重及干湿比的测定将收集得到的小鼠的粪便放入已称重的EP管中, 每次收集粪便时记录粪便粒数, 得到小鼠给药后8 h的排便粒数; 将收集完粪便的EP管称重后减去管重, 得到每只小鼠的粪便湿重; 将装有小鼠粪便的EP管口打开, 放入盘中置于烘箱中60 ℃烘干后, 称量装有小鼠干粪便的EP管重, 用装有小鼠干粪便的EP管重减去管重, 得到每只小鼠粪便的干重, 干湿比计算公式为干湿比(%) =干重/湿重× 100%。

小鼠血清中5-HT和结肠中AQP3水平测定取小鼠血清和结肠组织适量, 按照对应试剂盒说明书测定小鼠血清中5-HT及结肠中AQP3的水平。

拟靶向代谢组学分析

通过文献查找大黄潜在泻下药效活性成分(蒽醌、蒽酮类), 并结合裂解规律确定其碎片离子, 使用Agilent MassHunter Qualitative Analysis软件的Optimizer功能获取每个离子对的裂解电压和碰撞能并选取各化合物丰度最大的碎片离子与母离子作为该化合物的定量离子对, 这样便得到了化合物的离子对、裂解电压、碰撞能及保留时间。采用dyMRM模式测定各样本中潜在泻下药效活性成分的相对含量, 后通过OPLS-DA结合Kruskal-Wallis检验筛选出差异潜在泻下药效活性成分。

色谱条件色谱柱为ACQUITY BEH C₁₈ (2.1 mm × 50 mm, 1.7 μm)。流动相为乙腈(A)-0.1%甲酸溶液(B) 梯度洗脱, 洗脱程序为: 0~2.5 min, 5%~11% A; 2.5~5.5 min, 11%~13% A; 5.5~7.5 min, 13%~15% A; 7.5~13 min, 15%~20% A; 13~21.5 min, 20%~33% A; 21.5~24 min, 33%~50% A; 24~29 min, 50%~100% A; 29~32 min, 100% A; 32~33 min, 100%~5% A; 33~35 min, 5% A。柱温: 40 ℃, 流速: 0.3 mL·min^-1, 进样量: 2 μL。

质谱条件质谱条件离子源为安捷伦喷射流电喷雾离子源(AJS ESI), 采用负离子检测模式; 鞘气温度250 ℃, 鞘气流速11.0 L·min^-1; 喷嘴电压500 V; 雾化气压力35 psi (1 psi ≈ 6.9 kPa); 毛细管电压3 500 V, 干燥气温度300 ℃, 干燥气流速5.0 L·min^-1; 扫描方式为dyMRM模式。

离子对的确定现代研究表明蒽醌、蒽酮类化合物是大黄发挥泻下作用的主要化学物质^[8-10]。通过查找文献^{[10, 12-14]}, 结合蒽醌、蒽酮类化合物的质谱裂解规律得到离子碎片。通过Agilent MassHunter Qualitative Analysis软件的Optimizer功能在负离子模式下对每个离子对的裂解电压和碰撞能进行优化并选取各化合物丰度最大的碎片离子与母离子作为该化合物的定量离子对。

拟靶向代谢组学分析方法学考察

线性关系考察分别精密吸取QC母液0.2、0.5、1、2、5 mL, 用70%甲醇稀释至10 mL, 即得5个不同质量浓度的QC稀释液, 0.22 μm微孔滤膜滤过。分别吸取上述QC稀释液和母液各2 μL, 按“色谱条件”及“质谱条件”项进行测定。以各离子对峰面积(Y) 对QC样本质量浓度(X) 进行线性回归, 计算Pearson相关系数, 用于评价线性的好坏。

重复性考察平行配制的6份QC溶液, 测定每个离子对的峰面积并计算其RSD值。

精密度考察连续平行进样, 测定6针QC溶液中各离子对的峰面积并计算其RSD值。

数据分析 三种基原大黄泻下药效差异实验数据以均值±标准差(

$ \overline{x} $

± s) 的方式表示, SPSS Statistics 25.0统计软件(美国IBM公司) 进行单因素方差分析(analysis of variance, ANOVA) 和LSD多重比较, P < 0.05则认为具有统计学显著差异, P < 0.01则认为具有统计学极显著差异。三种基原大黄泻下药效成分数据分析, 利用Qualitative Analysis 10.0 (美国Agilent公司) 软件对动态多反应监测模式下测定的各化合物峰面积进行积分, 并进行归一化处理; SPSS Statistics 25.0统计软件进行Pearson相关性分析、Kruskal-Wallis单因素方差检验; SIMCA version14.1 (瑞典Umea公司) 进行主成分分析和正交偏最小二乘法判别分析。

结果

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1　方法学

1.1　线性关系

98.4%和99.5%离子对的Pearson相关系数大于0.9和0.8, 表明该拟靶向代谢组学方法线性关系良好。

1.2　重复性

94.6%的峰(占总峰面积的99%)RSD值小于20%, 表明该拟靶向代谢组学方法具有良好的重复性。

1.3　精密度

92.4%的峰(占总峰面积的99%)RSD值小于20%, 表明该拟靶向代谢组学方法具有良好的精密度。

2　三种基原大黄泻下药效差异

2.1　三种基原大黄对便秘小鼠排便量、粒数及干湿比的影响

结果表明(图1A~C), 与空白组相比, 模型组8 h内排便湿重(下简称排便量) 和粒数显著降低(P < 0.05), 干湿比显著升高(P < 0.05); 药用大黄、掌叶大黄组排便量和粒数显著或极显著降低(P < 0.05或P < 0.01), 干湿比显著或极显著升高(P < 0.05或P < 0.01); 唐古特大黄组排便量、粒数和干湿比均无显著性差异。与模型组相比, 唐古特大黄组排便量和排便粒数显著提高(P < 0.05), 干湿比极显著降低(P < 0.01); 药用大黄、掌叶大黄组排便量、粒数和干湿比均无显著性差异。综上可知, 唐古特大黄能够显著增加小鼠排便量、粒数及显著降低干湿比, 具有明显泻下作用, 而药用大黄、掌叶大黄无明显泻下作用。

2.2　三种基原大黄对便秘小鼠血清中5-HT和结肠中AQP3含量的影响

结果表明(图1D、E), 与空白组相比, 模型组小鼠血清中5-HT含量(下简称5-HT含量) 显著降低(P < 0.05), 结肠组织中AQP3 (下简称AQP3含量) 显著升高(P < 0.05); 药用大黄、掌叶大黄组5-HT含量均极显著降低(P < 0.01), AQP3含量显著或极显著升高(P < 0.05或P < 0.01); 唐古特大黄组AQP3含量显著降低(P < 0.05)。与模型组相比, 药用大黄、掌叶大黄组5-HT含量无显著性差异, 药用大黄组AQP3含量极显著升高(P < 0.01); 唐古特大黄组5-HT含量极显著升高(P < 0.01), AQP3含量极显著降低(P < 0.01)。综上可知, 唐古特大黄通过提高小鼠血清中的5-HT含量, 5-HT作用于相应的受体, 增强肠道平滑肌收缩, 起到泻下作用; 同时降低结肠组织中AQP3蛋白的表达, 减少肠道内水分吸收, 增加肠道内水分, 起到泻下作用。

3　三种基原大黄泻下药效成分分析

3.1　离子对的确定

以大黄素甲醚-8-O-葡萄糖苷和番泻苷A/B为例, m/z 283.06 [M-H-Glc]^-、m/z 268.04 [M-H-Glc-CH₃]^-、m/z 240.04 [M-H-Glc-CH₃-CO]^-、m/z 212.05 [M-H-Glc-CH₃-2CO]^-、m/z 184.05 [M-H-Glc-CH₃-3CO]^-等均可以作为大黄素甲醚-8-O-葡萄糖苷的特征离子对; m/z 699.13 [M-H-Glc]^-、m/z 537.08 [M-H-2Glc]^-、m/z 817.20 [M-H-CO₂]^-、m/z 655.14 [M-H-CO₂-Glc]^-、m/z 493.09 [M-H-CO₂-2Glc]^-等均可作为番泻苷A/B的特征离子, 此外C10-C10′键容易断裂, 形成m/z 386.10、m/z 224.45的特征离子, 见图2。得到潜在泻下药效成分(蒽醌、蒽酮类化合物) 的母离子与碎片离子后, 通过Agilent MassHunter Qualitative Analysis软件的Optimizer功能在负离子模式下对每个离子对的裂解电压和碰撞能进行优化并选取各化合物丰度最大的碎片离子与母离子作为该化合物的定量离子对。最终获得185个潜在泻下成分的离子对、保留时间、裂解电压及碰撞能。

3.2　基于PCA分析三种基原大黄潜在泻下药效成分差异

通过SPSSStatistics 26.0对185个化合物的峰面积进行标准化处理后, 将数据导入SIMCA version 14.1进行PCA分析。PCA得分散点图如图3A, PCA模型的稳定性(RX_cum²) 为0.881, 模型预测率(Q²) 为0.741, 表明该模型具有良好的解释和预测能力。由图3A可知, QC样本紧密聚类到图中心, 表明所建立的拟靶向代谢组学分析方法稳定可靠; 三种基原大黄在图中能够明显分开, 这表明三种基原大黄泻下药效成分存在明显差异。

3.3　三种基原大黄潜在泻下药效差异成分分析

为了筛选三种基原大黄潜在泻下药效差异成分, 建立了OPLS-DA模型。R²X和R²Y通常用于分别评估模型的拟合度和可靠性, Q²通常用于评估模型的预测能力^[15]。这些参数的值介于0和1之间, 越接近1, 模型的可预测性或解释性就越高^[16]。在本研究中, R²X_cum为0.832, R²Y_cum为0.973, Q²为0.947, 表明模型具有良好的解释率和预测性。由图3B可以看出三种基原大黄中蒽酮、蒽醌类化合物(潜在泻下药效成分) 存在显著差异。图3C为载荷图, 离原点越远表示该成分在三种基原中分布差异越大, 越能区分三种基原大黄。采用OPLS-DA模型的VIP > 1和Kruskal-Wallis检验(P < 0.05), 寻找差异性表达代谢物^[17]。最终筛选出97个差异化合物, 其中蒽醌类化合物22个, 蒽酮类化合物75个。潜在泻下药效差异成分在三种基原大黄中的分布见热图(图4)。

4　大黄泻下药效物质基础的探究

将三种基原大黄泻下药效结果与拟靶向代谢组学分析结果相匹配, 有23个差异成分(表2) 在泻下药效最强的唐古特大黄中含量最高, 其中少部分成分在掌叶大黄和药用大黄之间互有高低, 这与药理结果(泻下作用药用大黄和掌叶大黄之间无显著差异) 相一致。上述23个差异成分均为结合蒽醌、双蒽酮、蒽酮苷类, 蒽醌苷类10个、双蒽酮苷类10个、蒽酮苷类3个, 其中化学结构式明确的化合物有番泻苷A、番泻苷B、大黄酸苷A、大黄酸苷C、大黄酸苷D、大黄素甲醚-8-O-葡萄糖苷、大黄酸-8-O-葡萄糖苷、大黄酚-1-O-葡萄糖苷, 除大黄酚-1-O-葡萄糖苷外, 上述化合物泻下作用均有明确的报道^{[14, 18-20]}。综上结果表明, 大黄发挥泻下药效的物质基础(母核见图5) 均为蒽醌苷、双蒽酮苷及蒽酮苷类物质, 这些物质影响着大黄泻下药效的强弱。

讨论

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泻下作为大黄的重要功效之一, 已有大量相关研究。其中很多研究对大黄泻下药理作用机制进行了探索, 结果表明大黄通过胆碱样作用、抑制肠细胞膜上Na⁺, K⁺-ATP酶活性、抑制AQPs表达、提高5-HT的表达等多途径产生泻下作用^[21-24]。大黄泻下的药理作用较为明确, 但是发挥泻下作用的物质基础却尚未有系统的研究。大部分研究对大黄中某些物质的泻下药效进行探究, 如番泻苷类、芦荟大黄素、大黄酸-8-O-β-D-葡萄糖苷等^{[9, 25, 26]}, 这些化合物仅仅是大黄所含物质的一小部分, 其他化合物是否与泻下有关不得而知。因此, 通过对大黄潜在的泻下药效成分进行系统地研究, 不仅能揭示三种基原大黄潜在泻下药效成分的差异, 还能全面地了解发挥大黄泻下功效的物质基础。

本实验采用拟靶向代谢组学的方法对大黄中的潜在泻下药效成分(蒽醌、蒽酮类化合物) 进行检测。利用每个化合物的特征离子对在QQQ-MS的dyMRM模式下各化合物的峰面积进行准确确定, 比较三种基原大黄潜在泻下药效成分差异。PCA分析表明三种基原大黄潜在泻下药效成分具有明显差异, 从化学方面说明三种基原大黄泻下药效存在着差异; 采用OPLS-DA结合Kruskal-Wallis检验数据分析策略, 挖掘三种基原大黄潜在泻下药效的差异成分。通过药理实验测定三种基原大黄实际泻下药效, 并结合拟靶向代谢组学分析结果, 筛选出大黄泻下药效物质基础。本研究通过拟靶向代谢组学结合药理实验的方法, 对多基原药材药效差异进行研究, 并能够有效确定相应功效的物质基础, 为进一步提升中药材质量, 提供一定的理论依据; 为实现临床精准用药提供一定的指导作用。

此外, 拟靶向代谢组学在探究和寻找疾病的生物标志物应用较多, 但在中药领域应用较少。目前, 在中药中质量评价中存在着质量评价指标与其药效不相关的问题, 而该方法为此类问题研究提供了新的思路。不仅可以对多基原药材进行研究, 也可以用于探究不同药材加工方法、饮片炮制方法、煎煮方法对药效的影响并寻找其发挥药效的关键化合物, 进而改进工艺, 提升药效。

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2025年第60卷第6期

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doi: 10.16438/j.0513-4870.2024-1042

接收时间：2024-10-24
首发时间：2026-09-20
出版时间：2025-06-12

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收稿日期：2024-10-24
修回日期：2025-03-04

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¹北京中医药大学中药学院, 北京 102488

²中药材规范化生产教育部工程研究中心, 北京 100102

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赵婷, E-mail: wsl7491@126.com

魏胜利, E-mail: 19053181@qq.com

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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.	Medicinal part	Collecting site		No.	Medicinal part	Collecting site
T1-G	Root	Zhuoni County, Gansu Province		P6-G	Root	Lichuan Town, Tanchang County, Gansu Province
T1-GJ	Rhizome	Zhuoni County, Gansu Province	P6-GJ	Rhizome	Lichuan Town, Tanchang County, Gansu Province
T2-G	Root	Hezuo City, Gansu Province	P7-G	Root	Guanting Town, Tanchang County, Gansu Province
T2-GJ	Rhizome	Hezuo City, Gansu Province	P7-GJ	Rhizome	Guanting Town, Tanchang County, Gansu Province
T3-G	Root	Yangyong Town, Lintan County, Gansu Province	P8-G	Root	Qiaotou Town, Li County, Gansu Province
T3-GJ	Rhizome	Yangyong Town, Lintan County, Gansu Province	P8-GJ	Rhizome	Qiaotou Town, Li County, Gansu Province
T4-G	Root	Tuanjie Village, Ruoergai County, Sichuan Province	P9-G	Root	Mazichuan Town, Min County, Gansu Province
T4-GJ	Rhizome	Tuanjie Village, Ruoergai County, Sichuan Province	P9-GJ	Rhizome	Mazichuan Town, Min County, Gansu Province
P1-G	Root	Shizi Township, Tanchang County, Gansu Province	P10-G	Root	Caoping Township, Li County, Gansu Province
P1-GJ	Rhizome	Shizi Township, Tanchang County, Gansu Province	P10-GJ	Rhizome	Caoping Township, Li County, Gansu Province
P2-GJ	Rhizome	Taoping Town, Li County, Gansu Province	O1-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province
P3-G	Root	Hadapu Town, Tanchang County, Gansu Province	O2-G	Root	Shimiaozi Village, Lichuan City, Hubei Province
P3-GJ	Rhizome	Hadapu Town, Tanchang County, Gansu Province	O2-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province
P4-GJ	Rhizome	Caoping Township, Li County, Gansu Province	O3-G	Root	Shimiaozi Village, Lichuan City, Hubei Province
P5-G	Root	Caoping Township, Li County, Gansu Province	O3-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province
P5-GJ	Rhizome	Awu Town, Tanchang County, Gansu Province	O4-GJ	Rhizome	Shimiaozi Village, Lichuan City, Hubei Province

No.

Medicinal part

Collecting site

No.

Medicinal part

Collecting site

T1-G

Root

Zhuoni County, Gansu Province

P6-G

Root

Lichuan Town, Tanchang County, Gansu Province

T1-GJ

Rhizome

Zhuoni County, Gansu Province

P6-GJ

Rhizome

Lichuan Town, Tanchang County, Gansu Province

T2-G

Root

Hezuo City, Gansu Province

P7-G

Root

Guanting Town, Tanchang County, Gansu Province

T2-GJ

Rhizome

Hezuo City, Gansu Province

P7-GJ

Rhizome

Guanting Town, Tanchang County, Gansu Province

T3-G

Root

Yangyong Town, Lintan County, Gansu Province

P8-G

Root

Qiaotou Town, Li County, Gansu Province

T3-GJ

Rhizome

Yangyong Town, Lintan County, Gansu Province

P8-GJ

Rhizome

Qiaotou Town, Li County, Gansu Province

T4-G

Root

Tuanjie Village, Ruoergai County, Sichuan Province

P9-G

Root

Mazichuan Town, Min County, Gansu Province

T4-GJ

Rhizome

Tuanjie Village, Ruoergai County, Sichuan Province

P9-GJ

Rhizome

Mazichuan Town, Min County, Gansu Province

P1-G

Root

Shizi Township, Tanchang County, Gansu Province

P10-G

Root

Caoping Township, Li County, Gansu Province

P1-GJ

Rhizome

Shizi Township, Tanchang County, Gansu Province

P10-GJ

Rhizome

Caoping Township, Li County, Gansu Province

P2-GJ

Rhizome

Taoping Town, Li County, Gansu Province

O1-GJ

Rhizome