基于体外补血活性测定的当归品质评价新方法探索

刘欢欢; 张婷; 王敏; 于银萍; 苏联麟; 季德; 陆兔林; 李林

doi:10.14148/j.issn.1672-0482.2022.1110

基于体外补血活性测定的当归品质评价新方法探索

doi: 10.14148/j.issn.1672-0482.2022.1110

刘欢欢^1,,
张婷¹,
王敏¹,
于银萍¹,
苏联麟¹,
季德²,
陆兔林²,
李林^1, ,

1.
南京中医药大学药学院, 江苏南京 210023
2.
国家教育部中药炮制规范化及标准化工程研究中心, 江苏南京 210023

基金项目:

国家重点研发项目 2018YFC1707000

详细信息

作者简介:
刘欢欢, 女, 硕士研究生, E-mail: 20190350@njucm.edu.cn

通讯作者:
李林, 男, 副教授, 主要从事中药饮片质量标准研究, E-mail: lilin@njucm.edu.cn

中图分类号: R283
计量
- 文章访问数: 33
- HTML全文浏览量: 1
- PDF下载量: 2
- 被引次数: 0
出版历程
- 收稿日期: 2022-05-19
- 网络出版日期: 2022-12-15
- 发布日期: 2022-12-10

Exploration of A New Method for Evaluating the Quality of Angelica Sinensis Based on the Determination of Blood Tonic Activity in vitro

1.
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing 210023, China

摘要

摘要: 目的建立一种基于补血活性的生物学评价方法, 通过与当归的主要药效相关联, 对其内在品质进行评价。方法以氯化血红素为标准品, 用联苯胺染色法测定当归作用于K562细胞后的血红蛋白含量, 通过量反应平行线测定法计算效价, 完成方法学考察并以此比较不同产地、不同炮制方法当归饮片的效价。结果当归样品可靠性检验显示, 回归项有显著差异(P＜0.01);方法学考察中重复性和精密度RSD分别为1.13%、4.73%;不同产地当归饮片补血效价为399.61~8 920.32 U·mg^-1, 不同炮制方法当归饮片补血效价排序为生当归＞土炒当归＞酒当归, 不同入药部位当归饮片补血效价排序为当归身＞当归头＞当归须。结论所建立的当归饮片补血生物效价方法稳定可靠, 方法学考察结果符合《中国药典》2020年版“中药生物活性测定指导原则”相关要求, 可用于不同产地、不同炮制方法、不同入药部位的当归饮片补血疗效的评价, 为进一步完善当归质量评价与控制提供了新思路和新方法。
- 当归 /
- 补血活性 /
- 生物效价 /
- 血红蛋白
Abstract: OBJECTIVE To develop a biological evaluation method based on the blood tonic activity, combined with the main medicinal effects of Angelica sinensis, to evaluated its internal quality. METHODS Hemin was used as standard substance, the relative content of hemoglobin after added Angelica sinensis to K562 cells was determined by the benzidine staining method and the biological titer was calculated by parallel-line assay. After methodological investigation, the biological titers of different production areas and processing of Angelica sinensis were compared. RESULTS The reliability test of Angelica sinensis showed that the regression items were significantly different (P < 0.01). The RSDs of repeatability and precision were 1.13% and 4.73%, respectively, in the methodological investigation. The blood-enriching titers of its pieces from different origins were in the range of 399.61-8 920.32 U·mg^-1. The rank of blood tonic effects of different processed Angelica sinensis products was raw > soil > wine, and that of different medicinal parts was body > head > tail. CONCLUSION The established biological titer method of blood tonic for Angelica sinensis is stable and reliable. The results of methodological investigation are in accord with the relevant requirements of the "Guidelines for the determination of biological activity of traditional Chinese medicines" in the Chinese Pharmacopoeia (2020 edition). It can be used to evaluate the blood tonic titer of Angelica sinensis from different areas, processing methods and medicinal parts, which provides a new idea for further study on the improving of Angelica sinensis quality control.
- Angelica sinensis /
- blood tonic activity /
- biological titer /
- hemoglobin

HTML全文

图 1 Hemin(A)和当归饮片(B)补血活性量效曲线图

Figure 1. Dose-effect curves of Hemin (A) and angelica sinensis decoction pieces (B) in the blood tonic effect

下载: 全尺寸图片幻灯片

图 2 Hemin(A)和当归饮片(B)补血活性线性考察

Figure 2. Linear graph of Hemin (A) and angelica sinensis decoction pieces (B) in the blood tonic effect

下载: 全尺寸图片幻灯片

图 3 不同批次(A)、炮制方法(B)、入药部位(C)当归饮片的补血效价

Figure 3. Different batches(A), processing methods(B) and drug parts(C) of Angelica sinensis pieces in the blood tonic effect

下载: 全尺寸图片幻灯片

表 1 当归饮片样品采集情况表

Table 1. Collection of Angelica sinensis pieces

编号	饮片名称	饮片批号	产地	编号	饮片名称	饮片批号	产地
1	生当归	20170205-1	四川	16	酒当归	1804002	甘肃
2	生当归	20170306	河北	17	酒当归	18010261	甘肃
3	生当归	20170205-2	云南	18	酒当归	20180101	甘肃
4	生当归	M-1-1	甘肃禾驮乡	19	土炒当归	1804002	甘肃
5	生当归	W-1-1	甘肃渭源	20	土炒当归	18010261	甘肃
6	生当归	M-3-1	甘肃岷县	21	土炒当归	20180101	甘肃
7	生当归	1705051	甘肃	22	当归头	2018-03-20	甘肃
8	生当归	1705052	甘肃	23	当归头	2018-03-08	甘肃
9	生当归	1705053	甘肃	24	当归头	2018-03-09	甘肃
10	生当归	20170202	甘肃	25	当归身	2018-03-20	甘肃
11	生当归	1709040571	甘肃	26	当归身	2018-03-08	甘肃
12	生当归	20170212	甘肃	27	当归身	2018-03-09	甘肃
13	生当归	1804002	甘肃	28	当归须	2018-03-20	甘肃
14	生当归	18010261	甘肃	29	当归须	2018-03-08	甘肃
15	生当归	20180101	甘肃	30	当归须	2018-03-09	甘肃

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表 2 加样方法与加样量

Table 2. Sample addition method and dosage

组别	加样方法与加样量
无细胞组	2 000 μL完全培养基+1 000 μL空白培养基
空白组	2 000 μL细胞悬液+1 000 μL空白培养基
标准品组	2 000 μL细胞悬液+1 000 μL的Hemin溶液
供试品组	2 000 μL细胞悬液+500 μL的Hemin溶液(40 μmol·L^-1)+500 μL当归饮片提取物溶液

下载: 导出CSV

表 3 检测结果表

Table 3. Table of test results

	标准品剂量组			供试品剂量组			行总和
	d_s1	d_s2	d_s3	d_t1	d_t2	d_t3	行总和
	y(s1, 1)	y(s2, 1)	y(s3, 1)	y(t1, 1)	y(t2, 1)	y(t3, 1)	∑y1
平行组	y(s1, 2)	y(s2, 2)	y(s3, 2)	y(t1, 2)	y(t2, 2)	y(t3, 2)	∑y2
	y(s1, 3)	y(s2, 3)	y(s3, 3)	y(t1, 3)	y(t2, 3)	y(t3, 3)	∑y3
列总和	S₁	S₂	S₃	T₁	T₂	T₃	∑y
注: 标准品和供试品剂量组d₁、d₂、d₃, 分别对应低、中、高剂量, 且两两剂间比均为1 ∶ 0.5;y表示各组血红蛋白含量(相对值), ∑y表示y值总和。

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表 4 当归样品生物效价(3·3)法可靠性测验结果

Table 4. Results of reliability test of Angelica sinensis by biological potency method

变异来源	f	差方和	方差	F	F查表值		P	显著性
变异来源	f	差方和	方差	F	0.05	0.01	P	显著性
试品间	1	0.00	0.00	1.32×10^-6			＞0.05	不显著
回归项	1	0.93	0.93	1.53×10³			＜0.01	非常显著
偏离平行	1	0.00	0.00	2.84×10^-3	4.75	9.33	＞0.05	不显著
二次曲线	1	0.00	0.00	4.12			＞0.05	不显著
反向二次曲线	1	0.00	0.00	4.15×10^-2			＞0.05	不显著

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