药学学报

Sample No.	Geographical origin	Supplier
S1	Sichuan	Beijing Tong Ren Tang Co., Ltd., Beijng
S2	Sichuan	Herb Pharmacies of Chinese PLA general hospital, Beijng
S3	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S4	Sichuan	Local herb market in Guigang, Guangxi
S5	Sichuan	Beijing Tong Ren Tang Co., Ltd.(Jixi shop), Heilongjiang
S6	Sichuan	Local herb market in Dingzhou, Hebei
S7	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S8	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S9	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S10	Sichuan	Local herb market in Chengdu, Sichuan
S11	Yunnan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S12	Yunnan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S13	Yunnan	Anguo Chinese Herbal Medicine Market, Hebei
S14	Yunnan	Anguo Chinese Herbal Medicine Market, Hebei
S15	Yunnan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S16	Gansu	Anguo Chinese Herbal Medicine Market, Hebei
S17	Gansu	Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu
S18	Gansu	Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu
S19	Gansu	Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu
S20	Unknown	National Institutes for Food and Drug Control

Sample No.

Geographical origin

Supplier

Sichuan

Beijing Tong Ren Tang Co., Ltd., Beijng

Sichuan

Herb Pharmacies of Chinese PLA general hospital, Beijng

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

Sichuan

Local herb market in Guigang, Guangxi

Sichuan

Beijing Tong Ren Tang Co., Ltd.(Jixi shop), Heilongjiang

Sichuan

Local herb market in Dingzhou, Hebei

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S10

Sichuan

Local herb market in Chengdu, Sichuan

S11

Yunnan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S12

Yunnan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S13

Yunnan

Anguo Chinese Herbal Medicine Market, Hebei

S14

Yunnan

Anguo Chinese Herbal Medicine Market, Hebei

S15

Yunnan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S16

Gansu

Anguo Chinese Herbal Medicine Market, Hebei

S17

Gansu

Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu

S18

Gansu

Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu

S19

Gansu

Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu

S20

Unknown

National Institutes for Food and Drug Control

Sample No.	Geographical origin	Supplier
S1	Sichuan	Beijing Tong Ren Tang Co., Ltd., Beijng
S2	Sichuan	Herb Pharmacies of Chinese PLA general hospital, Beijng
S3	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S4	Sichuan	Local herb market in Guigang, Guangxi
S5	Sichuan	Beijing Tong Ren Tang Co., Ltd.(Jixi shop), Heilongjiang
S6	Sichuan	Local herb market in Dingzhou, Hebei
S7	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S8	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S9	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S10	Sichuan	Local herb market in Chengdu, Sichuan
S11	Yunnan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S12	Yunnan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S13	Yunnan	Anguo Chinese Herbal Medicine Market, Hebei
S14	Yunnan	Anguo Chinese Herbal Medicine Market, Hebei
S15	Yunnan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S16	Gansu	Anguo Chinese Herbal Medicine Market, Hebei
S17	Gansu	Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu
S18	Gansu	Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu
S19	Gansu	Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu
S20	Unknown	National Institutes for Food and Drug Control

Sample No.

Geographical origin

Supplier

Sichuan

Beijing Tong Ren Tang Co., Ltd., Beijng

Sichuan

Herb Pharmacies of Chinese PLA general hospital, Beijng

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

Sichuan

Local herb market in Guigang, Guangxi

Sichuan

Beijing Tong Ren Tang Co., Ltd.(Jixi shop), Heilongjiang

Sichuan

Local herb market in Dingzhou, Hebei

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S10

Sichuan

Local herb market in Chengdu, Sichuan

S11

Yunnan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S12

Yunnan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S13

Yunnan

Anguo Chinese Herbal Medicine Market, Hebei

S14

Yunnan

Anguo Chinese Herbal Medicine Market, Hebei

S15

Yunnan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S16

Gansu

Anguo Chinese Herbal Medicine Market, Hebei

S17

Gansu

Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu

S18

Gansu

Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu

S19

Gansu

Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu

S20

Unknown

National Institutes for Food and Drug Control

Sample No.	Similarity
S1	0.967
S2	0.947
S3	0.964
S4	0.974
S5	0.968
S6	0.978
S7	0.966
S8	0.935
S9	0.986
S10	0.963
S11	0.923
S12	0.975
S13	0.962
S14	0.861
S15	0.957
S16	0.866
S17	0.882
S18	0.916
S19	0.890
S20	0.975

Sample No.

Similarity

0.967

0.947

0.964

0.974

0.968

0.978

0.966

0.935

0.986

S10

0.963

S11

0.923

S12

0.975

S13

0.962

S14

0.861

S15

0.957

S16

0.866

S17

0.882

S18

0.916

S19

0.890

S20

0.975

Sample No.	Similarity
S1	0.967
S2	0.947
S3	0.964
S4	0.974
S5	0.968
S6	0.978
S7	0.966
S8	0.935
S9	0.986
S10	0.963
S11	0.923
S12	0.975
S13	0.962
S14	0.861
S15	0.957
S16	0.866
S17	0.882
S18	0.916
S19	0.890
S20	0.975

Sample No.

Similarity

0.967

0.947

0.964

0.974

0.968

0.978

0.966

0.935

0.986

S10

0.963

S11

0.923

S12

0.975

S13

0.962

S14

0.861

S15

0.957

S16

0.866

S17

0.882

S18

0.916

S19

0.890

S20

0.975

Peak No.	MS and MS/MS information	Molecular weight	Constituent	Reference
8	349 [M-H]^-, 373 [M+Na]⁺, 368 [M+NH₄]⁺	350	Toosenoside A	11
11	531 [M-H]^-, 578 [M+HCOO]^-, 533 [M+H]⁺, 555 [M+Na]⁺, 515 [M+H-H₂O]⁺	532	Meliatoosenin D	12
15	573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺	574	Toosendanin^*	13, 14
16	573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺	574	Toosendanin^* (tautomeric form)	13, 14
20	557 [M+H]⁺, 579 [M+Na]⁺, 539 [M+H-H₂O]⁺, 574 [M+NH₄]⁺	556	Toosendanal	15
21	613 [M+H]⁺, 595 [M+H-H₂O]⁺, 635 [M+Na]⁺, 630 [M+NH₄]⁺, 1 247 [2M+Na]⁺	612	Meliatoosenin Q	16

Peak No.

MS and MS/MS information

Molecular weight

Constituent

Reference

349 [M-H]^-, 373 [M+Na]⁺, 368 [M+NH₄]⁺

350

Toosenoside A

531 [M-H]^-, 578 [M+HCOO]^-, 533 [M+H]⁺, 555 [M+Na]⁺, 515 [M+H-H₂O]⁺

532

Meliatoosenin D

573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺

574

Toosendanin^*

13, 14

573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺

574

Toosendanin^* (tautomeric form)

13, 14

557 [M+H]⁺, 579 [M+Na]⁺, 539 [M+H-H₂O]⁺, 574 [M+NH₄]⁺

556

Toosendanal

613 [M+H]⁺, 595 [M+H-H₂O]⁺, 635 [M+Na]⁺, 630 [M+NH₄]⁺, 1 247 [2M+Na]⁺

612

Meliatoosenin Q

Peak No.	MS and MS/MS information	Molecular weight	Constituent	Reference
8	349 [M-H]^-, 373 [M+Na]⁺, 368 [M+NH₄]⁺	350	Toosenoside A	11
11	531 [M-H]^-, 578 [M+HCOO]^-, 533 [M+H]⁺, 555 [M+Na]⁺, 515 [M+H-H₂O]⁺	532	Meliatoosenin D	12
15	573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺	574	Toosendanin^*	13, 14
16	573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺	574	Toosendanin^* (tautomeric form)	13, 14
20	557 [M+H]⁺, 579 [M+Na]⁺, 539 [M+H-H₂O]⁺, 574 [M+NH₄]⁺	556	Toosendanal	15
21	613 [M+H]⁺, 595 [M+H-H₂O]⁺, 635 [M+Na]⁺, 630 [M+NH₄]⁺, 1 247 [2M+Na]⁺	612	Meliatoosenin Q	16

Peak No.

MS and MS/MS information

Molecular weight

Constituent

Reference

349 [M-H]^-, 373 [M+Na]⁺, 368 [M+NH₄]⁺

350

Toosenoside A

531 [M-H]^-, 578 [M+HCOO]^-, 533 [M+H]⁺, 555 [M+Na]⁺, 515 [M+H-H₂O]⁺

532

Meliatoosenin D

573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺

574

Toosendanin^*

13, 14

573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺

574

Toosendanin^* (tautomeric form)

13, 14

557 [M+H]⁺, 579 [M+Na]⁺, 539 [M+H-H₂O]⁺, 574 [M+NH₄]⁺

556

Toosendanal

613 [M+H]⁺, 595 [M+H-H₂O]⁺, 635 [M+Na]⁺, 630 [M+NH₄]⁺, 1 247 [2M+Na]⁺

612

Meliatoosenin Q

HPLC-CAD结合化学计量学的川楝子饮片指纹图谱研究

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张春泥 ¹ , 王英姿 ¹^,^* , 孙欣光 ² , 赵阳 ² , 郑伟 ³ , 李文华 ¹ , 龙珍 ⁴ , 马百平 ²^,^*

药学学报 | 研究论文 2017,52(3): 456-461

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药学学报 | 研究论文 2017, 52(3): 456-461

HPLC-CAD结合化学计量学的川楝子饮片指纹图谱研究

全屏

张春泥¹, 王英姿¹^,^*, 孙欣光², 赵阳², 郑伟³, 李文华¹, 龙珍⁴, 马百平²^,^*

作者信息

1.北京中医药大学中药学院, 北京 100102

2.军事医学科学院放射与辐射医学研究所, 北京 100850

3.天津中医药大学研究生院, 天津 300193

4.赛默飞科技有限公司, 北京 100080

通讯作者:

* 王英姿, Tel:86-10-84738615, E-mail:wangyzi@sina.com;

马百平, Tel:86-10-66930265, E-mail:mabaiping@sina.com

Chromatographic fingerprint analysis of Toosendan Fructus by HPLC-CAD coupled with chemometrics methods

Chun-ni ZHANG¹, Ying-zi WANG¹^,^*, Xin-guang SUN², Yang ZHAO², Wei ZHENG³, Wen-hua LI¹, Zhen LONG⁴, Bai-ping MA²^,^*

Affiliations

1. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China

2. Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China

3. Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China

4. ThermoFisher Scientific Corporation, Beijing 100080, China

出版时间: 2017-03-12 doi: 10.16438/j.0513-4870.2016-0965

文章导航

摘要

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建立川楝子饮片的高效液相串联电雾式检测器（HPLC-CAD）指纹图谱。采用Agilent ZOBAX SB C₁₈（4.6 mm×250 mm，5 μm）色谱柱；以乙腈-0.1%甲酸水溶液为流动相，梯度洗脱，柱温30℃，进样量5 μL，流速为1 mL·min^-1；CAD雾化气为氮气，雾化气压力为35 psi，雾化室温度35℃。采用相似度评价、聚类分析及主成分分析等方法，对不同来源、不同产地的20批次饮片进行系统比较与归类。结果确定了28个共有峰，并对其中的6个色谱峰进行了指认和鉴定，将不同产地的样品分为3类。方法学考察结果表明，该方法精密度、重复性以及稳定性良好，为市售川楝子饮片的质量评价和控制提供了更加全面科学的依据。

关键词

川楝子 / HPLC-CAD / 指纹图谱 / 相似度分析 / 聚类分析 / 主成分分析

Abstract

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A new method was developed for the chromatographic fingerprint analysis of Toosendan Fructus by HPLC coupled with the charged aerosol detector (CAD) in the present study. Samples were well separated on an Agilent ZOBAX SB C₁₈ column (4.6 mm×250 mm, 5 μm) by gradient elution using acetonitrile and water containing 0.1% formic acid (v/v) at the flow rate of 1.0 mL·min^-1. The column temperature was 30℃ and the injection volume was 5 μL. The nitrogen inlet pressure of the charged aerosol detector (CAD) was 35 psi, and the nebulizer chamber temperature was 35℃. In addition, the method of the chromatographic fingerprints combined with multivariate statistical analysis was effective and reasonable lead to an accurate classification of 20 batches of samples from different locations. The results showed that 28 common peaks were observed in the fingerprint and the samples were classified into three clusters. The established method was well validated, and showed high precision, good repeatability, and satisfactory stability. It may serve in the quality control and evaluation of Toosendan Fructus.

Key words

Toosendan Fructus / HPLC-CAD / fingerprint / similarity evaluation / hierarchical cluster analysis / principle components analysis

引用本文

张春泥, 王英姿, 孙欣光, 赵阳, 郑伟, 李文华, 龙珍, 马百平. HPLC-CAD结合化学计量学的川楝子饮片指纹图谱研究. 药学学报, 2017 , 52 (3) : 456 -461 . DOI: 10.16438/j.0513-4870.2016-0965

Chun-ni ZHANG, Ying-zi WANG, Xin-guang SUN, Yang ZHAO, Wei ZHENG, Wen-hua LI, Zhen LONG, Bai-ping MA. Chromatographic fingerprint analysis of Toosendan Fructus by HPLC-CAD coupled with chemometrics methods[J]. Acta Pharmaceutica Sinica, 2017 , 52 (3) : 456 -461 . DOI: 10.16438/j.0513-4870.2016-0965

正文

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川楝子为楝科植物川楝 (Melia toosendan Sieb.et Zucc.) 的干燥成熟果实, 是传统的理气、驱虫药, 始载于《神农本草经》, 迄今已有2 000余年的用药历史, 临床上具有广泛的应用。现代药理学研究表明川楝子具有驱虫、镇痛、抗肿瘤、抗病毒、抑制破骨细胞等作用。川楝子中含有多种化学成分, 主要包括萜类、挥发油、黄酮、酚酸等^{[1, 2]}。2015版《中国药典》中收载了川楝子和炒川楝子两个饮片品种, 并规定以三萜类成分川楝素的含量作为质量评价指标^[3]。然而仅仅采用单一成分进行检测, 往往难以全面系统反映川楝子饮片的内在质量。指纹图谱针对中药多组分、多靶点的特点, 从“全成分”的角度出发进行中药的质量控制, 具有系统性、特征性等特点, 能较全面的反映中药中所含化学成分的种类, 目前已在中药质量评价和控制研究中得到了较好的应用。

截至目前, 关于川楝子的指纹图谱研究已有部分报道, 但其检测方法大多为高效液相串联紫外检测器 (HPLC-UV)^[4-6], 少数采用高效液相串联蒸发光散射检测器^[7] (HPLC-ELSD)。川楝子中川楝素等三萜类化合物如采用HPLC-UV分析很难达到理想的基线分离; 蒸发光散射检测器虽然对如川楝素等化合物的检测灵敏度高于UV的末端波长检测, 但由于其对含量较低成分的检测不够灵敏, 限制了其进一步的应用。电雾式检测器 (CAD) 已在液相色谱分析中得到了较好的应用。由于其不依赖于化合物的分子结构, 尤其适用于紫外吸收较弱的化合物, 且该检测器较ELSD具有更高的灵敏度^[8-10]。因此, 本实验将HPLC-CAD方法用于川楝子饮片指纹图谱的研究, 并通过3种检测器 (UV、ELSD和CAD) 的对比, 进一步证实了CAD检测器用于川楝子指纹图谱研究的优势。同时, 本研究采用多种化学计量学数据处理方法, 对20批次不同来源、不同产地的川楝子饮片进行分析评价, 以期为川楝子饮片的质量控制提供依据。

材料与方法

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仪器 Waters 2695型高效液相色谱仪 (Waters Corporation, Milford, MA, USA), 包含四元溶剂洗脱系统, 自动进样器, 柱温箱; Corona Ultra型电雾式检测器 (Corona Ultra, ThermoFisher, USA); 2000 ES型蒸发光散射检测器 (2000 ES, Alltech Associates, USA); 2487型紫外检测器 (2487, Waters, USA); 色谱柱: Agilent ZOBAX SB C₁₈ (4.6 mm × 250 mm, 5 μm)、TGL-16A台式高速冷冻离心机 (长沙平凡仪器仪表有限公司); 超高效液相: Waters ACQUITYTM超高效液相色谱系统 (Waters Corporation, Milford, MA, USA); 质谱: SYNAPT G1质谱系统 (Waters Corporation, Manchester, UK); 色谱柱: Acquity HSS T3 C₁₈ (100 mm × 2.1 mm, 1.8 μm, Waters, Ireland); Sartorius BP211D十万分之一电子天平; 天鹏TP600超声波清洗器; 0.22 μm微孔滤膜 (上海兴亚净化材料厂)。

试剂与样品 甲醇 (色谱纯, ThermoFisher, USA); 乙腈 (色谱纯, ThermoFisher, USA); 甲酸 (色谱纯, Acros Co., Ltd., NJ, USA); 蒸馏水 (广州屈臣氏蒸馏水有限公司); 19批次市售川楝子饮片购自全国不同地区, 经北京中医药大学中药鉴定系刘春生教授鉴定为楝科植物川楝 (Melia toosendan Sieb.et Zucc.) 的干燥成熟果实, 1批次川楝子标准药材购自于中国食品药品检定研究院 (批号: 121464-201102), 详见表 1; 川楝素对照品购自于北京四面体生物科技有限公司 (批号: 150408);其他试剂均为市售分析纯。

高效液相色谱条件 色谱柱: Agilent ZOBAX SB C₁₈ (4.6 mm × 250 mm, 5 μm), 柱温30 ℃; 进样量5 μL, 流速1 mL·min^-1; 紫外检测波长270 nm; 蒸发光散射检测器:漂移管温度110.0 ℃, 载气流量2.4 L·min^-1; Corona Ultra电雾式检测器雾化气为氮气, 雾化气压力为35 psi (1 psi ≈ 6.9 kPa), 雾化室温度35 ℃; 流动相为: A (乙腈)、B (0.1%甲酸-水), 梯度洗脱条件如下: 0~3 min, 5% A; 3~20 min, 5%~20% A; 20~35 min, 20%~32% A; 35~45 min, 32%~38% A; 45~60 min, 38%~50% A; 60~75 min, 50% A; 75~83 min, 50%~75% A; 83~98 min, 75%~90% A; 98~109 min, 90%~95% A。

UHPLC-Q-TOF/MS^E色谱及质谱条件 色谱柱: Waters Acquity UPLC HSS T3色谱柱 (2.1 mm × 100 mm, 1.8 μm) 柱温: 45 ℃; 进样量: 2 μL, 流速0.5 mL·min^-1; 流动相: A (乙腈)、B (0.1%甲酸-水), 梯度洗脱条件如下: 0~1.0 min, 5% A; 1.0~8.0 min, 5%~22% A; 8.0~12.0 min, 22%~38% A; 12.0~15.0 min, 38%~43% A; 15.0~17.0 min, 43%~45% A; 17.0~20.0 min, 45%~50% A; 20.0~23.0 min, 50%~60% A; 23.0~27.0 min, 60%~75% A; 27.0~28.0 min, 75%~77% A; 28.0~30.0 min, 77%~85% A; 30.0~32.0 min, 85% A; 32.0~32.1 min, 85%~95% A; 32.1~34.0 min, 95% A。

电喷雾电离离子源 (ESI), 离子化模式分为正、负离子, 离子源温度为100.0 ℃, 脱溶剂气体为氮气, 流速为900 L·h^-1, 温度为450 ℃。毛细管电压为3 kV, 锥孔电压为30 V, 扫描范围为m/z 100~1 500。低能量扫描时trap电压为6 eV, transfer电压为4 eV; 高能量扫描时, 负离子模式: trap电压为25~50 eV, transfer电压为20 eV, 正离子模式: trap电压为75~100 eV, transfer电压为20 eV。准确质量数用leucineenk ephalin作校正液。

对照品储备液的制备 精密称取川楝素对照品适量, 甲醇溶解并定容, 摇匀, 配制成1 mg·mL^-1的川楝素对照品溶液。

供试品溶液的制备 精密称取川楝子中粉粉末2.0 g, 加入70%甲醇溶液20 mL超声提取40 min, 离心机离心10 min (转速8 000 r·min^-1), 将上清液减压浓缩至干, 加入70%甲醇溶液1 mL复溶, 过微孔滤膜 (0.22 μm), 即得供试品溶液。

不同检测器指纹图谱的对比 按“供试品溶液制备方法”制备川楝子饮片供试品溶液, 取同一供试品溶液, 按“高效液相色谱条件”, 进相同体积5 μL, 分别采用CAD、ELSD及UV检测器建立川楝子饮片指纹图谱。

方法学考察

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精密度按“供试品溶液的制备”制备川楝子饮片供试品溶液, 取同一供试品溶液, 按“高效液相色谱条件”, 进样5 μL, 连续进样6次。以各共有峰相对峰面积和相对保留时间进行考察。

重复性按“供试品溶液的制备”平行制备6份川楝子饮片供试品溶液, 按“高效液相色谱条件”进样分析, 以各共有峰相对峰面积和相对保留时间进行考察。

稳定性按“供试品溶液的制备”制备川楝子饮片供试品溶液, 按“高效液相色谱条件”, 分别于0、2、4、6、8、10、12和24 h进样5 μL, 以各共有峰相对峰面积和相对保留时间进行考察。

HPLC-CAD指纹图谱的建立及相似度评价 取上述20批川楝子药材, 按“供试品溶液制备方法”制备供试品溶液, 分别精密吸取供试品溶液5 μL, 注入到高效液相色谱仪测定, 按“高效液相色谱条件”进样分析, 记录色谱图。将上述20批川楝子药材所得各色谱图导入2012版《中药色谱指纹图谱相似度评价系统》软件进行数据处理。以S20为参照图谱, 时间窗为0.1, 中位数法, 经多点校正自动匹配生成川楝子饮片色谱指纹图谱共有模式。

聚类分析 以20批次川楝子药材共有峰的峰面积作为变量, 运用SPSS 20.0统计软件, 采用组间平均数联结法, 以“Pearson相关性”作为样品相似度的距离计算公式, 对川楝子样品进行聚类分析。

主成分分析 将20批川楝子饮片指纹图谱中共有峰峰面积导入统计软件SPSS 20.0, 所有数据经过标准化处理后, 进行主成分分析, 计算贡献率及累计贡献率。

结果

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1 川楝子饮片HPLC-CAD、HPLC-ELSD及HPLC-UV指纹图谱对比

结果如图 1所示。川楝子饮片HPLC-CAD指纹图谱较HPLC-ELSD及HPLC-UV指纹图谱有更高的灵敏度和更多的色谱峰, 且指标性成分川楝素的色谱峰与其他色谱峰分离度良好, 因此CAD检测器更适用于川楝子饮片的指纹图谱分析。

2 HPLC-CAD指纹图谱的方法学考察

2.1 精密度

结果显示, 共有峰相对保留时间和相对峰面积的RSD分别为0.03%~1.03%和0.34%~2.03%, 说明各主要色谱峰保留时间与峰面积基本一致, 表明仪器精密度良好。

2.2 重复性

结果显示, 共有峰相对保留时间和相对峰面积的RSD分别为0.11%~1.75%和1.03%~2.64%, 表明该方法重复性良好。

2.3 稳定性

结果显示, 共有峰相对保留时间和相对峰面积的RSD分别为0.03%~1.01%和0.56%~2.22%, 表明该样品在24 h内稳定。

3 川楝子HPLC-CAD指纹图谱的建立及相似度评价

建立川楝子饮片色谱指纹图谱共有模式 (图 2), 确定28个峰为共有特征色谱峰, 并对20批川楝子饮片的相似度进行了评价, 结果见表 2。

此外, 通过液质联用分析以及与对照品和相关文献^[11-16]比对的方法, 对28个共有峰中的6个色谱峰进行了指认和鉴定。峰15、16经标准品比对后鉴定为川楝素 (toosendanin, 两个峰为川楝素的互变异构体^[14])。峰8、11、20、21经质谱数据分析和文献比对, 分别鉴定为toosenoside A、meliatoosenin D、toosendanal、meliatoosenin Q (表 3)。

4 川楝子样品聚类分析

聚类分析结果见图 3。从树状图可以看出, 当分类距离取21时, 20批次川楝子药材被分为3大类 (Ⅰ类、Ⅱ类和Ⅲ类), 分类结果与相似度结果一致。第Ⅰ类由相似度高于0.935的川楝子药材组成, 第Ⅱ类由相似度介于0.866~0.923之间的川楝子药材组成, 第Ⅲ类则由相似度最低的川楝子药材组成。

5 川楝子样品主成分分析

结果显示第1、2、3位的主成分方差贡献率分别为35.971%、17.378%和12.484%, 前3个主成分累计贡献率为65.833%, 因此前3个主成分的分析结果基本显示出了不同批次川楝子饮片之间的相似度和差异性。以3个主成分建立坐标系得到PCA得分分布图, 见图 4。由图 4可见, 不同批次的川楝子饮片在三维图上可分为3类, 所得结果与相似度评价结果和聚类分析结果一致。

讨论

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通过对比发现, CAD检测器有明显的优势: CAD指纹图谱较ELSD指纹图谱有着更高的灵敏度, 能够表征出川楝子饮片更多的色谱峰信息; CAD指纹图谱与UV指纹图谱相比, 更适用于川楝子药材中无紫外吸收或弱紫外吸收化合物的检测, 特别是指标性成分川楝素在CAD检测器下得到了良好的分离。与此同时, 通过系统的方法学考察, 结果显示采用HPLC-CAD方法所建立的川楝子指纹图谱, 精密性、重复性和稳定性良好, 表明该方法稳定、可靠。

本实验采用相似度评价、聚类分析和主成分分析等方法, 对不同来源、不同产地的川楝子饮片进行分析评价。20批次川楝子饮片均按2015版《中国药典》项下规定的川楝子饮片含量测定方法进行了含量测定, 结果显示20批次的饮片川楝素含量均符合药典规定。同时采用上述3种分析方法得到的分类结果具有良好的一致性。结果显示, 20批次川楝子饮片分为3大类, 第Ⅰ类由相似度高于0.935的川楝子饮片组成 (包括S10、S20、S2、S1、S6、S12、S4、S9、S5、S3、S13、S7、S15和S8), 第Ⅱ类由相似度介于0.866~0.923之间的川楝子饮片组成 (包括S11、S16、S17、S18和S19), 第Ⅲ类则由相似度最低的S14批次川楝子饮片组成。通过分析各饮片产地来源发现:第Ⅰ类主要由10批产自四川、3批产自云南的饮片和川楝子标准药材组成, 且各批次相似度结果高于0.935, 说明产自四川和大多数产自云南的川楝子饮片差异不明显, 且与标准药材有着良好的一致性。第Ⅱ类由4批产自甘肃的饮片和1批产自云南的饮片组成, 相似度介于0.866~0.923, 说明产自甘肃的川楝子饮片与产自四川和云南的饮片存在一定的差异性, 可能是由于甘肃与四川和云南的地理位置、生态环境等相差较大进而造成了饮片间的差异。第Ⅲ类由1批产自云南的相似度最低的川楝子饮片组成。与此同时, 发现产自云南的5批饮片聚类结果分为了3类, 且相似度介于0.861~0.975之间, 说明产自云南的川楝子饮片之间差异性较大。初步分析原因可能为采收地点、产地初加工方法及药材的生长周期等因素的不同导致了饮片间的差异。

基金

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国家科技基础性工作专项资助项目(2014FY111100-3)
北京中医药大学2016年度基本科研业务费项目(在读研究生项目：2016-JYB-XS118)

参考文献

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文献

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参考文献引证文献

排序方式：

[1]

Li ZH, Ju JM, Hua JL, et al. Research advances of Toosendan Fructus[J]. Chin J Exp Tradit Med Formula (中国实验方剂学杂志), 2015, 21:219-223.

[2]

Zhang Q, Li QS, Liang JY, et al. Limonoids from fruits of Melia toosendan[J]. Acta Pharm Sin (药学学报), 2010, 45:475-478.

[3]

Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (中华人民共和国药典)[S]. 2015 ed. Vol 1. Beijing: China Medical Science Press, 2015: 42-43.

[4]

Sheng YH, An R, Wang XH, et al. Chromatography finger-print of the constituents of Melia toosendan Sieb[J]. Chin Tradit Pat Med (中成药), 2009, 31:168-171.

[5]

Zhou N, Yang Q, Li YJ, et al. HPLC fingerprints of ethanolic part of Toosendan Fructus[J]. Chin J Mod Appl Pharm (中国现代应用药学), 2012, 29:814-818.

[6]

Li JY, Shen L. Study on HPLC fingerprints of Fructus Toosendan from different harvest times[J]. J Mod Med Health (现代医药卫生), 2013, 29:983-985.

[7]

Shen LQ, Peng F, Cai JN, et al. Fingerprint analysis of limonoids in Fructus Toosendan by HPLC-ELSD[J]. Chin J Pharm Anal (药物分析杂志), 2014, 34:1431-1434.

[8]

Cintrón JM, Risley DS. Hydrophilic interaction chromato-graphy with aerosol-based detectors (ELSD, CAD, NQAD) for polar compounds lacking a UV chromophore in an intravenous formulation[J]. J Pharm Biomed Anal, 2013, 78-79:14-18.

[9]

Eom HY, Park SY, Kim MK, et al. Comparison between evaporative light scattering detection and charged aerosol detection for the analysis of saikosaponins[J]. J Chromatogr A, 2010, 1217:4347-4354.

[10]

Wang L, He WS, Yan HX, et al. Performance evaluation of charged aerosol and evaporative light scattering detection for the determination of ginsenosides by LC[J]. Chroma-tographia, 2009, 70:603-608.

[11]

Wang L, Li F, Yang CY, et al. Neolignans, lignans and glycoside from the fruits of Melia toosendan[J]. Fitoterapia, 2014, 99:92-98.

[12]

Yi Z, Tang CP, Ke CQ, et al. Limonoids and triterpenoids from the stem bark of Melia toosendan[J]. J Nat Prod, 2010, 73:664-668.

[13]

Li ZH, Xu JD, Jiu JM, et al. Chemical constituents from water extract of Toosendan Fructus by UPLC-ESI-Q-TOF/MS[J]. Chin Tradit Herb Drugs (中草药), 2015, 46:496-501.

[14]

Zhang CN, Sun XG, Zhao Y, et al. Quantitative analysis of toosendanin in the fruit of Melia toosendan Sieb. Et Zucc (Meliaceae) by high-performance liquid chromatography coupled with charged aerosol detection[J]. Chromatographia, 79:1381-1386.

[15]

Tada K, Takido M, Kitanaka S. Limonoids from fruit of Melia toosendan, and their cytotoxic activity[J]. Phyto-chemistry, 1999, 51:787-791.

[16]

Zhang Y, Tang CP, Ke CQ, et al. Limonoids from the fruits of Melia toosendan[J]. Phytochemistry, 2012, 73:106-113.

2017年第52卷第3期

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文章信息

doi: 10.16438/j.0513-4870.2016-0965

接收时间：2016-09-30
首发时间：2026-01-14
出版时间：2017-03-12

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文章信息

作者

出版历史

收稿日期：2016-09-30
修回日期：2016-11-23

基金

国家科技基础性工作专项资助项目(2014FY111100-3)

北京中医药大学2016年度基本科研业务费项目(在读研究生项目：2016-JYB-XS118)

作者信息

1.北京中医药大学中药学院, 北京 100102

2.军事医学科学院放射与辐射医学研究所, 北京 100850

3.天津中医药大学研究生院, 天津 300193

4.赛默飞科技有限公司, 北京 100080

通讯作者:

* 王英姿, Tel:86-10-84738615, E-mail:wangyzi@sina.com;

马百平, Tel:86-10-66930265, E-mail:mabaiping@sina.com

参考文献

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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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TxT

Sample No.	Geographical origin	Supplier
S1	Sichuan	Beijing Tong Ren Tang Co., Ltd., Beijng
S2	Sichuan	Herb Pharmacies of Chinese PLA general hospital, Beijng
S3	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S4	Sichuan	Local herb market in Guigang, Guangxi
S5	Sichuan	Beijing Tong Ren Tang Co., Ltd.(Jixi shop), Heilongjiang
S6	Sichuan	Local herb market in Dingzhou, Hebei
S7	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S8	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S9	Sichuan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S10	Sichuan	Local herb market in Chengdu, Sichuan
S11	Yunnan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S12	Yunnan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S13	Yunnan	Anguo Chinese Herbal Medicine Market, Hebei
S14	Yunnan	Anguo Chinese Herbal Medicine Market, Hebei
S15	Yunnan	Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan
S16	Gansu	Anguo Chinese Herbal Medicine Market, Hebei
S17	Gansu	Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu
S18	Gansu	Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu
S19	Gansu	Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu
S20	Unknown	National Institutes for Food and Drug Control

Sample No.

Geographical origin

Supplier

Sichuan

Beijing Tong Ren Tang Co., Ltd., Beijng

Sichuan

Herb Pharmacies of Chinese PLA general hospital, Beijng

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

Sichuan

Local herb market in Guigang, Guangxi

Sichuan

Beijing Tong Ren Tang Co., Ltd.(Jixi shop), Heilongjiang

Sichuan

Local herb market in Dingzhou, Hebei

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

Sichuan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S10

Sichuan

Local herb market in Chengdu, Sichuan

S11

Yunnan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S12

Yunnan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S13

Yunnan

Anguo Chinese Herbal Medicine Market, Hebei

S14

Yunnan

Anguo Chinese Herbal Medicine Market, Hebei

S15

Yunnan

Chengdu Hehuachi Chinese Herbal Medicine Market, Sichuan

S16

Gansu

Anguo Chinese Herbal Medicine Market, Hebei

S17

Gansu

Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu

S18

Gansu

Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu

S19

Gansu

Lanzhou Huanghe Chinese Herbal Medicine Market, Gansu

S20

Unknown

National Institutes for Food and Drug Control

Sample No.	Similarity
S1	0.967
S2	0.947
S3	0.964
S4	0.974
S5	0.968
S6	0.978
S7	0.966
S8	0.935
S9	0.986
S10	0.963
S11	0.923
S12	0.975
S13	0.962
S14	0.861
S15	0.957
S16	0.866
S17	0.882
S18	0.916
S19	0.890
S20	0.975

Sample No.

Similarity

0.967

0.947

0.964

0.974

0.968

0.978

0.966

0.935

0.986

S10

0.963

S11

0.923

S12

0.975

S13

0.962

S14

0.861

S15

0.957

S16

0.866

S17

0.882

S18

0.916

S19

0.890

S20

0.975

Peak No.	MS and MS/MS information	Molecular weight	Constituent	Reference
8	349 [M-H]^-, 373 [M+Na]⁺, 368 [M+NH₄]⁺	350	Toosenoside A	11
11	531 [M-H]^-, 578 [M+HCOO]^-, 533 [M+H]⁺, 555 [M+Na]⁺, 515 [M+H-H₂O]⁺	532	Meliatoosenin D	12
15	573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺	574	Toosendanin^*	13, 14
16	573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺	574	Toosendanin^* (tautomeric form)	13, 14
20	557 [M+H]⁺, 579 [M+Na]⁺, 539 [M+H-H₂O]⁺, 574 [M+NH₄]⁺	556	Toosendanal	15
21	613 [M+H]⁺, 595 [M+H-H₂O]⁺, 635 [M+Na]⁺, 630 [M+NH₄]⁺, 1 247 [2M+Na]⁺	612	Meliatoosenin Q	16

Peak No.

MS and MS/MS information

Molecular weight

Constituent

Reference

349 [M-H]^-, 373 [M+Na]⁺, 368 [M+NH₄]⁺

350

Toosenoside A

531 [M-H]^-, 578 [M+HCOO]^-, 533 [M+H]⁺, 555 [M+Na]⁺, 515 [M+H-H₂O]⁺

532

Meliatoosenin D

573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺

574

Toosendanin^*

13, 14

573 [M-H]^-, 619 [M+HCOO]^-, 575 [M+H]⁺, 597 [M+Na]⁺, 557 [M+H-H₂O]⁺, 497 [M+H-H₂O-CH₃COOH]⁺, 537 [M+Na-CH₃COOH]⁺

574

Toosendanin^* (tautomeric form)

13, 14

557 [M+H]⁺, 579 [M+Na]⁺, 539 [M+H-H₂O]⁺, 574 [M+NH₄]⁺

556

Toosendanal

613 [M+H]⁺, 595 [M+H-H₂O]⁺, 635 [M+Na]⁺, 630 [M+NH₄]⁺, 1 247 [2M+Na]⁺

612

Meliatoosenin Q