药学学报

Sample	Part	Producing area	Pith-decayed	Wild/Cultivate	Diameter/cm	Grade (1/2/3)
S1	Scutellaria baicalensis Georgi	Inner Mongolia	Yes	Wild	1.0-1.5	2
S2	Non-decayed part of S1	Inner Mongolia	Same as S1	Same as S1	Same as S1	Same as S1
S3	Decayed pith of S1	Inner Mongolia	Same as S1	Same as S1	-	-
S4	Scutellaria baicalensis Georgi	Inner Mongolia	Yes	Wild	1.0-1.5	2
S5	Scutellaria baicalensis Georgi	Inner Mongolia	Yes	Wild	1.0-1.5	2
S6	Scutellaria baicalensis Georgi	Hebei	Slightly	Cultivate	> 1.2	1
S7	Scutellaria baicalensis Georgi	Shanxi	No	Cultivate	0.7-1.2	2
S8	Scutellaria baicalensis Georgi	Shanxi	No	Cultivate	0.7-1.2	2
S9	Scutellaria baicalensis Georgi	Shaanxi	Yes	Cultivate	1.0-1.5	2
S10	Scutellaria baicalensis Georgi	Gansu	No	Cultivate	< 0.7	3
S11	Scutellaria baicalensis Georgi	Gansu	Yes	Cultivate	> 1.5	1
S12	Scutellaria baicalensis Georgi	Henan	No	Cultivate	0.7-1.2	2
S13	Scutellaria baicalensis Georgi	Shaanxi	No	Cultivate	0.7-1.2	2
S14	Scutellaria baicalensis Georgi stem-leaf	Heilongjiang	-	Cultivate	-	Substitute
S15	Scutellaria amoena C. H. Wright	Yunnan	No	Cultivate	-	Substitute

Sample	Part	Producing area	Pith-decayed	Wild/Cultivate	Diameter/cm	Grade (1/2/3)
S1	Scutellaria baicalensis Georgi	Inner Mongolia	Yes	Wild	1.0-1.5	2
S2	Non-decayed part of S1	Inner Mongolia	Same as S1	Same as S1	Same as S1	Same as S1
S3	Decayed pith of S1	Inner Mongolia	Same as S1	Same as S1	-	-
S4	Scutellaria baicalensis Georgi	Inner Mongolia	Yes	Wild	1.0-1.5	2
S5	Scutellaria baicalensis Georgi	Inner Mongolia	Yes	Wild	1.0-1.5	2
S6	Scutellaria baicalensis Georgi	Hebei	Slightly	Cultivate	> 1.2	1
S7	Scutellaria baicalensis Georgi	Shanxi	No	Cultivate	0.7-1.2	2
S8	Scutellaria baicalensis Georgi	Shanxi	No	Cultivate	0.7-1.2	2
S9	Scutellaria baicalensis Georgi	Shaanxi	Yes	Cultivate	1.0-1.5	2
S10	Scutellaria baicalensis Georgi	Gansu	No	Cultivate	< 0.7	3
S11	Scutellaria baicalensis Georgi	Gansu	Yes	Cultivate	> 1.5	1
S12	Scutellaria baicalensis Georgi	Henan	No	Cultivate	0.7-1.2	2
S13	Scutellaria baicalensis Georgi	Shaanxi	No	Cultivate	0.7-1.2	2
S14	Scutellaria baicalensis Georgi stem-leaf	Heilongjiang	-	Cultivate	-	Substitute
S15	Scutellaria amoena C. H. Wright	Yunnan	No	Cultivate	-	Substitute

Sample	Potency/U·g^-1	FL/%	Confidence interval/U·g^-1
S1	486.04	12.198 0	425.31-543.89
S2	459.68	3.223 6	444.26-473.89
S3	0.00	-	-
S4	518.91	3.358 0	500.58-535.43
S5	830.00	2.029 8	828.31-831.68
S6	605.10	2.281 3	591.21-618.82
S7	701.86	1.761 3	689.44-714.16
S8	901.15	3.766 8	885.25-917.09
S9	507.47	5.342 8	479.43-533.65
S10	297.60	6.257 5	278.29-315.53
S11	1 000.00	-	-
S12	389.85	4.999 8	369.95-408.93
S13	402.64	5.844 7	378.49-425.56
S14	438.45	1.486 8	431.47-444.51
S15	805.54	2.457 2	785.20-824.79

Sample	Potency/U·g^-1	FL/%	Confidence interval/U·g^-1
S1	486.04	12.198 0	425.31-543.89
S2	459.68	3.223 6	444.26-473.89
S3	0.00	-	-
S4	518.91	3.358 0	500.58-535.43
S5	830.00	2.029 8	828.31-831.68
S6	605.10	2.281 3	591.21-618.82
S7	701.86	1.761 3	689.44-714.16
S8	901.15	3.766 8	885.25-917.09
S9	507.47	5.342 8	479.43-533.65
S10	297.60	6.257 5	278.29-315.53
S11	1 000.00	-	-
S12	389.85	4.999 8	369.95-408.93
S13	402.64	5.844 7	378.49-425.56
S14	438.45	1.486 8	431.47-444.51
S15	805.54	2.457 2	785.20-824.79

Peak	Grey correlation degree	Grey relational order
P1	0.982 6	R22
P2	0.990 2	R15
P3	0.984 0	R20
P4	0.993 1	R10
P5	0.970 4	R28
P6	0.950 6	R33
P7	0.956 7	R31
P8	0.970 6	R27
P9	0.983 9	R21
P10	0.990 7	R14
P11	0.977 1	R24
P12	0.994 0	R8
P13	0.981 8	R23
P14	0.973 1	R26
P15	0.996 3	R2
P16	0.947 3	R34
P17	0.962 0	R29
P18	0.957 8	R30
P19	0.995 7	R4
P20	0.987 2	R18
P21	0.992 5	R11
P22	0.987 2	R19
P23	0.989 8	R16
P24	0.989 0	R17
P25	0.992 4	R12
P26	0.976 5	R25
P27	0.956 5	R32
P28	0.993 9	R9
P29	0.995 3	R5
P30	0.997 6	R1
P31	0.995 1	R6
P32	0.991 0	R13
P33	0.994 7	R7
P34	0.996 1	R3

Peak	Grey correlation degree	Grey relational order
P1	0.982 6	R22
P2	0.990 2	R15
P3	0.984 0	R20
P4	0.993 1	R10
P5	0.970 4	R28
P6	0.950 6	R33
P7	0.956 7	R31
P8	0.970 6	R27
P9	0.983 9	R21
P10	0.990 7	R14
P11	0.977 1	R24
P12	0.994 0	R8
P13	0.981 8	R23
P14	0.973 1	R26
P15	0.996 3	R2
P16	0.947 3	R34
P17	0.962 0	R29
P18	0.957 8	R30
P19	0.995 7	R4
P20	0.987 2	R18
P21	0.992 5	R11
P22	0.987 2	R19
P23	0.989 8	R16
P24	0.989 0	R17
P25	0.992 4	R12
P26	0.976 5	R25
P27	0.956 5	R32
P28	0.993 9	R9
P29	0.995 3	R5
P30	0.997 6	R1
P31	0.995 1	R6
P32	0.991 0	R13
P33	0.994 7	R7
P34	0.996 1	R3

基于抑菌效应成分指纹谱的黄芩质量评价研究

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任永申 ^#^,^* , 邓鑫 ^# , 雷蕾 , 梁帅 , 郑尧 , 艾娇 , 李燕 , 廖矛川 ^*

药学学报 | 专题报道：中药质量生物评价与精准控制 2019,54(12): 2155-2161

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药学学报 |专题报道：中药质量生物评价与精准控制 2019 , 54 (12) : 2155 -2161

基于抑菌效应成分指纹谱的黄芩质量评价研究

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任永申^#^,^*, 邓鑫^#, 雷蕾, 梁帅, 郑尧, 艾娇, 李燕, 廖矛川^*

作者信息

中南民族大学药学院, 湖北武汉 430070

通讯作者:

*任永申, E-mail:godreny@163.com;

廖矛川, E-mail:sippr1976@163.com

Establishment of an antibacterially effective components fingerprint for quality evaluation of Scutellariae Radix (Huangqin)

Yong-shen REN^*, Xin DENG, Lei LEI, Shuai LIANG, Yao ZHENG, Jiao AI, Yan LI, Mao-chuan LIAO^*

Affiliations

School of Pharmaceutical Science, South-central University For Nationalities, Wuhan 430070, China

出版时间: 2019-12-12 doi: 10.16438/j.0513-4870.2019-0655

文章导航

摘要

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化学指纹图谱是中药质控的重要手段，但常规指纹图谱存在冗余信息，影响了其准确性和可用性，需要进行改进和提升。本研究采用量反应平行线法原理检测黄芩抑菌活性生物效价；采用HPLC法检测不同黄芩样品的化学指纹图谱；采用灰色关联法（GRA）辨识主要抑菌效应成分。结果表明，15批黄芩样品抑菌效价在0~1 000 U·g^-1，平均抑菌效价为556.29 ±258.57 U·g^-1（1 U约相当于2.25 μg庆大霉素的抑菌活性）；15批黄芩样品的HPLC指纹图谱中共标定34个特征峰，样品间相似度为0.255~0.991；取灰色关联序前10位成分且与抑菌效价成正相关的P33、P30（黄芩素）、P19（黄芩苷）、P15、P29、P34、P31（汉黄芩素）、P28的8个成分并赋予权重，建立黄芩效应成分指纹谱，能够明确区分黄芩各规格等级样品，并能综合表征黄芩性状、成分、抑菌效价差异。综之，本研究构建了直接关切黄芩抑菌活性且信息简化的抑菌效应成分指纹谱，可综合评价黄芩质量，提高了中药指纹图谱的功效专属性和质量辨识能力，并可为其他中药质量综合评价提供有益参考。

关键词

黄芩 / 生物效价检测 / 灰色关联分析 / 抑菌效应成分指纹谱

Abstract

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A chemical fingerprint is an important mean for quality control of traditional Chinese medicine (TCM); however, there is much redundant information in a conventional fingerprint that can affect its availability and accuracy. In this work, the antibacterial biopotency of Scutellariae Radix (Huangqin, HQ) was determined according to the parallel line method of quantitative response. HPLC was adopted to detect the chemical fingerprint of HQ; Grey relational analysis (GRA) was used to identify the primary effective components. The results showed that the antibacterial biopotency of 15 batches of HQ ranged from 0 to 1 000 U·g^-1 and the average potency was 556.29 ±258.57 U·g^-1 (1 U is equivalent to the bacteriostatic activity of 2.25 μg gentamicin). There were 34 characteristic peaks in the fingerprints of the samples and their similarities were 0.255-0.991. Eight components (P33, P30/baicalein, P19/baicalin, P15, P29, P34, P31/wogonin and P28) are positively related to antibacterial biopotency and selected from the top ten components of the grey correlation sequence to define the antibacterially effective components fingerprint of HQ. This fingerprint can clearly distinguish the commodity specification and grade, and can also characterize the morphology, components and the bacteriostatic potency differences of HQ. In summary, we established an antibacterially effective components fingerprint which provides simplified information on the antibacterial activity of Scutellariae Radix and could significantly improve the efficacy, specificity, and discriminative ability of the fingerprint for HQ, and could be a useful reference for the comprehensive quality evaluation of other TCM.

Key words

Scutellariae Radix / antibacterial biopotency determination / grey relational analysis / antibacterial effective components fingerprint

引用本文

任永申, 邓鑫, 雷蕾, 梁帅, 郑尧, 艾娇, 李燕, 廖矛川. 基于抑菌效应成分指纹谱的黄芩质量评价研究. 药学学报, 2019 , 54 (12) : 2155 -2161 . DOI: 10.16438/j.0513-4870.2019-0655

Yong-shen REN, Xin DENG, Lei LEI, Shuai LIANG, Yao ZHENG, Jiao AI, Yan LI, Mao-chuan LIAO. Establishment of an antibacterially effective components fingerprint for quality evaluation of Scutellariae Radix (Huangqin)[J]. Acta Pharmaceutica Sinica, 2019 , 54 (12) : 2155 -2161 . DOI: 10.16438/j.0513-4870.2019-0655

正文

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中药化学成分指纹图谱具有整体性、指纹性、稳定性、关联性等特征, 能够较好表征中药多成分特征, 已成为中药质控的重要手段; 然而, 化学指纹图谱又具有一定的模糊性和不确定性, 各成分在指纹图谱中的作用地位不明确、各成分谱-效关联信息还需要进一步挖掘和指认, 需要建立明确的“成分/谱-效应关系”。本文拟将中药化学指纹图谱各成分信息与生物效价相关, 研究建立“效应成分指纹谱”, 使化学指纹谱成为能够直接关切中药药效的质控手段。

黄芩为唇形科植物黄芩(Scutellaria baicalensis Georgi)的干燥根, 主要为黄酮类成分^{[1, 2]}, 具有清热燥湿、泻火解毒等功效^[3], 具有抑菌、解热抗炎、抗氧化、抗肿瘤等药理作用^{[1, 4]}。黄芩是临床常用大宗药材, 具有多规格等级, 常规单指标成分含量测定方法不足以全面评价其质量, 需要建立质量综合评控方法。另外, 黄芩化学成分复杂、功效多样, 现有黄芩化学成分指纹图谱指纹信息复杂, 难以明确判定某一/类成分对某一功效/活性的具体贡献, 较难满足现今对中药质量评价的针对性、准确性、可用性的更高要求。

抑菌作用是黄芩的主要药理活性, 也是其清热燥湿解毒功效的重要基础^{[5, 6]}。为准确评价其抑菌作用效应成分(群), 本实验建立黄芩抑菌活性生物效价评价方法, 采用HPLC法建立不同黄芩样品特征峰图谱, 将黄芩化学成分指纹图谱与抑菌效价进行关联分析, 以准确辨识抑菌作用药效物质, 建立黄芩抑菌效应成分指纹谱, 为其质量评控提供依据。

根据邓聚龙教授在1982年创立的灰色系统理论, 信息完全明确的为白色系统, 完全未知的为黑色系统, 信息部分明确、部分不明确的称为灰色系统, 灰色系统适用于“小样本、贫信息”、“只知大概范围、不知确切内涵”的不确定性系统。随着中药基础研究的进步, 原来的黑色系统获得更多已知信息, 部分地转换为白色或灰色系统, 为更深入揭示中药物质基础与作用机制奠定了工作基础^{[7, 8]}。

根据信息差异论与灰性不灭原理, 灰色关联分析通过计算反应序列间发展趋势或曲线相对变化的接近程度以及发展过程或量级的相近性构建序列间的关联度, 并对关联度大小进行排序(关联序), 从而得到系统状态的正确描述和有效监控, 在医学、经济等众多领域得到大量应用^[9-13], 并特别适用于中药复杂系统研究。本研究在单味中药(黄芩)化学背景基本一致的前提下, 尽可能提供详尽的化学成分信息与药效信息, 采用谱-效灰色关联分析方法, 更准确地辨识各成分的作用, 获得直接关切黄芩抑菌活性的指纹图谱关键信息, 从而建立黄芩抑菌效应成分指纹图谱, 为中药质控提供新的技术手段和参考^[14-16]。

材料与方法

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仪器与试剂 洁净工作台(SW-CJ-1FD, 苏州安泰空气技术有限公司); 气浴恒温振荡器(THZ-92C, 上海博迅医疗生物仪器股份有限公司); 隔水式恒温培养箱(GRP-9050, 上海森信实验仪器有限公司); 紫外可见分光光度计[UV-1780, 岛津仪器(苏州)有限公司]; 高效液相色谱仪(UltiMate 3000, DAD检测器, 美国戴安公司)。

营养肉汤(批号:815B031)、营养琼脂培养基(批号: 621B031)均购自北京索莱宝科技有限公司; 黄芩素(批号: C20M8Y31962)、汉黄芩素(批号: P11M9F61014)、黄芩苷(批号: P20A9F59353)、汉黄芩苷(批号:R31M9F62605)、野黄芩苷(批号: Z17O7X22900)、白杨素(批号: X24O6C4947)、类叶升麻苷(批号: Y21A9H59554)对照品均购自上海源叶生物科技有限公司, 纯度≥98 %; 千层纸素A (批号:6180, 上海诗丹德标准技术服务有限公司)、木蝴蝶素A-7-葡萄糖醛酸苷(批号: P1443412, 上海泰坦科技股份有限公司); 硫酸庆大霉素(批号: 20180608, 华中药业股份有限公司); 乙腈为色谱纯; 甲醇(批号: 20170712, 分析纯)、磷酸(批号: 20170313, 分析纯)均购自国药集团化学试剂有限公司; 水为双蒸馏水。金葡菌Staphylococcus aureus (CMCC (B) 26003)由中国食品药品检定研究院提供。实验用15批黄芩经鉴定为唇形科植物黄芩Scutellaria baicalensis Georgi的干燥根/茎叶, 以及滇黄芩Scutellaria amoena C.H.Wright的干燥根。参照《中药材商品规格等级》标准^[17]将样品规格、等级进行划分。样品信息及性状图见表 1。

抑菌实验

供试品母液的制备精密称取各样品粉末(过4号筛) 10 g, 加8倍量水提取2 h, 过滤, 滤渣加5倍量水提取1 h, 合并滤液, 浓缩至0.8 g (原药材)·mL^-1溶液, 灭菌备用。

0.5麦氏浊度标准液的配制取0.5 mL BaCl₂溶液(0.048 mol·L^-1)于10 mL具塞试管, 加0.18 mol·L^-1 H₂SO₄溶液定容至10 mL, 摇匀, 于625 nm下测得吸光度为0.123, 符合要求(0.08~0.13), 4 ℃静置备用, 使用前混匀。

营养琼脂培养基的制备按照说明书指示制备营养琼脂培养基, 湿热灭菌法灭菌, 冷却至50~60 ℃, 无菌条件下, 每个培养皿加入25 mL培养基, 厚度约为4 mm, 水平放置冷却。检查培养基表面, 确保培养基表面平坦干燥。

药敏纸片的制备使用打孔器将滤纸制成直径为6 mm的滤纸片, 分装, 灭菌后烘干。无菌条件下, 将滤纸片置于无菌96孔板中, 每孔1片, 将“供试品母液的制备”项下供试品用无菌水分别配制成稀释比为1.25的系列浓度。每孔加入10 μL药液, 空白组滤纸片加入10 μL无菌水, 置于37 ℃烘干备用。

抑菌活性测定及量效关系考察将经MH培养活化后的金葡菌接种到MH培养基表面, 培养20 h, 用无菌的生理盐水将生长稳定、形态相似的菌落洗下, 目测法稀释至0.5麦氏浊度, 含菌量约为1.5×10⁸ cfu·mL^-1。使用无菌棉签蘸取校准的菌悬液, 将菌液均匀涂布于整个琼脂培养基表面, 确保条痕间无间隙。菌液接种的15 min内, 用无菌镊子将“药敏纸片的制备”项下药敏纸片取出, 置于已接种培养基表面。放置药敏纸片的15 min内, 将培养皿置于培养箱内培养20 h后测量抑菌圈直径, 平行测定3次。以黄芩(S11)载药量(mg)为横坐标X, 抑菌圈直径(mm)为纵坐标Y, 进行线性回归。

黄芩抑菌生物效价检测因目前尚无用于抑菌生物效价测定的黄芩标准对照药材, 本实验取最大载药量(8.00 mg)下抑菌圈直径最大的S11作为抑菌效价检测用对照药材并进行原始效价赋值, 定义黄芩对照药材效价为1 000 U·g^-1。将各样品提取物的抑菌圈直径作为测定结果, 参照《中国药典》2015版三部量反应平行线(3, 3)法原理, 采用中国药典生物检定统计程序BS 2000生物统计软件, 计算供试品的抑菌效价和效价的可信限率(FL%), 并进行可靠性检验。

研究同时以硫酸庆大霉素注射液(1 U·μg^-1)为参照, 检测5.24~20.00 μg (剂距1:0.8)的抑菌圈直径, 用以近似标定黄芩S11样品5.12~8.00 mg (剂距1:0.8)时对应的抑菌效力。

黄芩HPLC图谱的建立

液相用供试品溶液的制备精密称取各样品粉末(过4号筛) 10 g, 加8倍量水提取2 h, 过滤, 滤渣加5倍量水提取1 h, 合并滤液, 浓缩至每毫升1.0 g原药材溶液, 取2 mL药液浓缩挥干, 加10 mL甲醇超声溶解, 容量瓶补足至刻度, 摇匀, 用0.22 μm微孔滤膜过滤, 4 ℃保存备用。

液相用对照品溶液的制备精密称取黄芩素、汉黄芩素、黄芩苷、汉黄芩苷、野黄芩苷、白杨素、类叶升麻苷、千层纸素A、木蝴蝶素A-7-葡萄糖醛酸苷对照品适量, 置于5 mL量瓶内, 加甲醇至刻度, 制得质量浓度分别为1.02、1.00、1.00、1.04、0.98、1.00、0.98、1.02和1.01 mg·mL^-1的对照品溶液。分别精密量取各对照品溶液1 mL, 定容至10 mL, 制成含上述9种对照品的混合对照品溶液, 混合对照品色谱图见图3B。

色谱条件色谱柱: Thermo BDS HYPERSIL C18柱(4.6 mm×250 mm), 流动相:乙腈(A)-0.1%甲酸水溶液(B), 梯度洗脱程序: 0~30 min, 10%~25% (A); 30~60 min, 25%~45% (A), 60~65 min, 45%~90% (A), 流速: 1.0 mL·min^-1, 柱温: 30 ℃, 进样量: 15 μL, 检测波长: 274 nm。

化学特征图谱的建立将15批黄芩药材按“液相用供试品溶液的制备”项下方法制备供试品溶液, 按“色谱条件”项下色谱条件进行测定。将结果导入中国药典委员会相似度评价软件(2012版)进行处理, 设S11为参照图谱, 进行多点校正、自动匹配, 设定时间窗宽度为0.5, 按中位数法生成对照图谱R, 得到15批黄芩药材指纹图谱相似度。

灰色关联分析在15批黄芩样品的HPLC图谱中, 以黄芩苷(P19)为参照峰, 计算各样品特征峰相对峰面积。参照灰色关联分析方法, 以抑菌生物效价为母序列, 特征峰相对峰面积为子序列, 运用灰色建模软件V6.0进行灰色关联分析, 计算关联度, 根据关联度大小进行排序, 得到关联序。

效应成分指纹谱的建立及分析依据灰色关联序提取主要效应成分, 以主要效应成分的相对峰面积及各样品抑菌效价为指标, 采用SPSS进行Pearson相关分析并比较显著性, 取灰色关联序靠前且与抑菌活性成正相关的成分, 构建黄芩抑菌效应成分指纹谱, 以“原始峰面积/Pearson相关系数”对结果进行校正, 并以各效应成分峰面积为指标对样品进行PCA分析, 判别样品归属。

结果

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1 抑菌生物活性及量效关系考察

以金葡菌为受试菌, 对不同黄芩样品的抑菌活性及效价进行测定。黄芩(S11)的回归方程为: Y= 1.494 7 X + 5.191 2, r = 0.980 2, 在2.10~8.00 mg内抑菌圈直径满足测定要求。从结果可以看出, 不同黄芩样品抑菌圈直径差异明显, 呈良好的量效关系。

2 可靠性检验

可靠性结果表明, 回归项有非常显著意义(P < 0.01), 说明不同批次黄芩药敏片载药量与抑菌圈直径有较好的线性关系, 剂量间差异非常显著(P < 0.01), 说明15批黄芩样品剂间比安排合理; 偏离平行、二次曲线不显著(P > 0.05), 说明对照药材与供试药材呈平行直线关系, 可用(3, 3)法检测不同黄芩样品的抑菌生物效价。

3 抑菌生物效价计算

以金葡菌为受试菌, 硫酸庆大霉素为参照, 检测各剂量下硫酸庆大霉素与黄芩样品S11的抑菌圈直径。结果表明, 硫酸庆大霉素16.0 μg的抑菌圈直径为(15.13 ± 0.17) mm, 介于黄芩S11样品6.40 mg [(14.74 ± 0.35) mm]与8.00 mg [(16.62 ± 0.57) mm]之间, 由此可近似认定1.00 mg黄芩S11样品约相当于2.25 μg的硫酸庆大霉素对受试菌的抑菌效力。

以黄芩样品S11为抑菌生物效价检测用对照药材, 并标定其初始效价为1 000 U·g^-1, 对15批不同规格等级的黄芩药材进行效价计算并通过可靠性检验, 结果见表 2。结果表明, 15批黄芩样品抑菌效价在0~1 000 U·g^-1, 平均抑菌效价为556.29±258.57 U·g^-1, 提示不同黄芩药材品质存在差异, 其中以S11、S8、S5、S15抑菌生物效价值较高(> 800 U·g^-1)。

4 黄芩指纹图谱的建立及共有峰的标定

比对15批黄芩药材HPLC图谱与对照图谱(R), 共标定34个特征峰(P1~P34), 具有较好的分离度。共标定9个成分, 依次为野黄芩苷(P9)、类叶升麻苷(P11)、黄芩苷(P19)、木蝴蝶素A-7-葡萄糖醛酸苷(P24)、汉黄芩苷(P26)、黄芩素(P30)、汉黄芩素(P31)、白杨素(P32)、千层纸素A (P34)。黄芩对照图谱(R)及15批黄芩药材的指纹图谱见图 1、图 2A。9个成分的化学结构见图 2。15批黄芩指纹图谱相似度在0.255~0.991, 其中黄芩茎叶与滇黄芩相似度分别为0.255、0.679, 其余各批黄芩相似度均 > 0.800。

5 灰色关联分析

从灰色关联结果可以看出(表 3), 黄芩主要特征峰与抑菌生物活性具有较好的关联性(关联度均 > 0.9), 提示黄芩是通过多成分协调参与发挥抑菌作用; 关联度越大, 提示该成分对黄芩发挥抑菌(金葡菌)作用贡献越大; 根据关联度大小排序, 各成分对黄芩抑菌药效的贡献依次为: R1 (黄芩素/P30) > R2 (P15) > R3 (千层纸素A/P34) > R4 (黄芩苷/P19) > R5 (P29) > R6 (汉黄芩素/P31) > R7 (P33) > R8 (P12) > R9 (P28) > R10 (P4) > …… > R13 (白杨素/P32) > R17 (木蝴蝶素A-7-葡萄糖醛酸苷/P24) > R21 (野黄芩苷/P9) > R24 (类叶升麻苷/P11) > R25 (汉黄芩苷/P26)。从上述关联度结果可以看出, 黄芩主要成分的抑菌(金葡菌)作用以黄芩素/P30最优、次之为黄芩苷, 之后为汉黄芩素、再次之为汉黄芩苷。

6 黄芩抑菌效应指纹谱的建立

以灰色关联排序前十的黄芩素/P30、P15、千层纸素A/P34、黄芩苷/P19、P29、汉黄芩素/P31、P33、P12、P28、P4为指标, 计算其对应的Pearson相关系数。结果表明, P33 (r = 0.564 7)、P30 (r = 0.561 5)、P19 (r = 0.516 2)相关系数均大于0.5, 且有显著性差异(P < 0.05), 表明其是黄芩抑菌活性的主要效应成分; 其他5个成分P15 (r = 0.243 5)、P29 (r = 0.173 9)、P34 (r = 0.155 9)、P31 (r = 0.038 1)、P28 (r = 0.020 8)与抑菌效价成正相关, 但不具有显著意义, 可以作为次要效应成分。本文以此8个成分P33、P30 (黄芩素)、P19 (黄芩苷)、P15、P29、P34、P31 (汉黄芩素)、P28的效应成分校正峰面积(绝对峰面积除以其相关系数), 建立了黄芩抑菌效应成分指纹谱(图 2B); 结果表明, 与原始化学指纹图谱相比, 效应成分指纹图谱能够更清晰、准确地体现各等级样品的差异。

以效应成分指纹图谱进行主成分分析结果显示:黄芩一等品(S11、S6)与抑菌作用较强的S8 (抑菌效价901.15 U·g^-1)聚为一类; 黄芩二等品(S1、S4、S5、S7、S9、S12、S13, 抑菌效价389.85~830.00 U·g^-1)聚为一类; 4个特殊样品(S2、S3、S14、S15, 化学成分差异较大)与条芩三等品且抑菌效价值较低的S10 (抑菌效价297.60 U·g^-1)聚为一类(图 2D), PCA结果显示, 黄芩质量从优到劣存在一定的递减趋势, 且上述分类不仅可以体现性状分类的差异, 同时能够准确的体现黄芩抑菌效应的差异, 提示本研究建立的黄芩抑菌效应成分指纹谱具有较好的综合评价能力, 以此效应成分指纹谱为指标能够客观准确、关联药效地实现对黄芩样品质量的综合评价; 而以原始指纹图谱进行的主成分分析结果显示各类成分间均有交叉, 表明过多的冗余信息并不能更好地表征样品质量及药效信息(图 2C)。

讨论

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黄芩是临床常用中药, 具有多规格等级、多功效取向、多功效成分的特征, 其参与不同功效发挥的药效物质基础可能不同, 在药效成分识别与质量控制中理应区别对待; 而现今的质量控制均以黄芩苷等成分含量为依据, 并不能全面准确代表其各功效的药效成分(群)及作用地位; 另一方面, 现今建立的化学指纹图谱方法, 也不能明确关联药效和各成分的药效地位, 存在大量干扰冗余信息, 需要进行功效关联或校正, 并提取有效信息进而建立新的质量评控方法。

随着各种科学研究技术手段的进步, 中药研究也进入以各类“组学”为特征的大数据时代, 中药化学指纹图谱作为出现较早且较为成熟的中药组学技术, 也呈现出多手段、多维度、全息化发展的特征。海量数据的引入, 为中药质量的全面分析提供了可能, 但也不可避免的引入了大量“冗余信息”, 为分析工作带来较大难度; 特别是对于具有多功效属性的中药而言, 如何准确辨识关联具体功效/活性发挥的“有效成分/群”、剔除“冗余成分”或“类冗余成分”干扰, 进而建立直接关切药效、针对性、准确性、可用性较好的“效应相关-成分精简型专用指纹图谱”尚缺乏研究。

目前, 谱-效关联分析多采用灰色关联分析、相关分析、回归分析(普通多元回归分析、偏最小二乘回归分析)、主成分分析、典型相关分析、聚类分析等方法, 通过对谱-效相关程度分析、谱-效贡献程度或数据降维简化实现对主要药效成分的预测辨识; 本研究采用灰色关联分析与Pearson相关分析结合, 考察数列绝对变化与数据相对变化、满足对数据方向性、保序性和规范性的综合要求, 并实现对主要药效成分的权重赋值, 从而建立活性校正的化学指纹图谱^[18-21]。

因此, 本文选择黄芩清热解毒功效的基础活性抑菌作用建立生物效价评价方法, 并通过谱-效灰色关联分析, 辨识主要药效成分, 并采用各成分与抑菌活性呈正相关的Pearson相关系数的倒数作为权重, 重构“抑菌效应成分指纹谱”, 进而为黄芩质量控制提供依据^[22-24]。

研究结果显示, P33、P30 (黄芩素)、P19 (黄芩苷)、P15、P29、P34、P31 (汉黄芩素)、P28等在灰色关联序居于前列且与抑菌效价成正相关, 故本文以此8个成分建立了黄芩抑菌效应成分指纹谱, 能够较好地反映各样品规格等级、成分差异及抑菌效应差异, 为黄芩质量综合评价提供了客观依据。采用黄芩抑菌效应成分指纹图谱, 能够准确区分黄芩一等品、二等品、等外品, 能够较好地反映各等级间的质量传递状况; 且上述分类准确无交叉, 不仅可以体现性状分类的差异, 同时能够准确的体现黄芩抑菌效应的差异, 能够较好地改善传统性状鉴别在实际药效中存在的偏差、错判或失据, 提高了中药规格等级划分的准确性与可及性, 具有较好的综合评价能力。

综之, 本文以黄芩为例, 通过抑菌活性评价与主要效应成分辨识方法, 建立了黄芩抑菌效应成分指纹谱, 从而为黄芩及其他中药质量综合评价提供新的技术手段与科学依据。

基金

收起

国家自然科学基金资助项目(81202935)
国家自然科学基金资助项目(81773893)
国家“重大新药创制”科技重大专项资助项目(2017ZX09301060-001)
国家重点研发计划“中医药现代化研究”课题资助(2017YFC1701004)

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2019年第54卷第12期

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doi: 10.16438/j.0513-4870.2019-0655

接收时间：2019-08-20
首发时间：2026-01-26
出版时间：2019-12-12

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收稿日期：2019-08-20
修回日期：2019-09-25

基金

国家自然科学基金资助项目(81202935)

国家自然科学基金资助项目(81773893)

国家“重大新药创制”科技重大专项资助项目(2017ZX09301060-001)

国家重点研发计划“中医药现代化研究”课题资助(2017YFC1701004)

作者信息

中南民族大学药学院, 湖北武汉 430070

通讯作者:

*任永申, E-mail:godreny@163.com;

廖矛川, E-mail:sippr1976@163.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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Sample	Part	Producing area	Pith-decayed	Wild/Cultivate	Diameter/cm	Grade (1/2/3)
S1	Scutellaria baicalensis Georgi	Inner Mongolia	Yes	Wild	1.0-1.5	2
S2	Non-decayed part of S1	Inner Mongolia	Same as S1	Same as S1	Same as S1	Same as S1
S3	Decayed pith of S1	Inner Mongolia	Same as S1	Same as S1	-	-
S4	Scutellaria baicalensis Georgi	Inner Mongolia	Yes	Wild	1.0-1.5	2
S5	Scutellaria baicalensis Georgi	Inner Mongolia	Yes	Wild	1.0-1.5	2
S6	Scutellaria baicalensis Georgi	Hebei	Slightly	Cultivate	> 1.2	1
S7	Scutellaria baicalensis Georgi	Shanxi	No	Cultivate	0.7-1.2	2
S8	Scutellaria baicalensis Georgi	Shanxi	No	Cultivate	0.7-1.2	2
S9	Scutellaria baicalensis Georgi	Shaanxi	Yes	Cultivate	1.0-1.5	2
S10	Scutellaria baicalensis Georgi	Gansu	No	Cultivate	< 0.7	3
S11	Scutellaria baicalensis Georgi	Gansu	Yes	Cultivate	> 1.5	1
S12	Scutellaria baicalensis Georgi	Henan	No	Cultivate	0.7-1.2	2
S13	Scutellaria baicalensis Georgi	Shaanxi	No	Cultivate	0.7-1.2	2
S14	Scutellaria baicalensis Georgi stem-leaf	Heilongjiang	-	Cultivate	-	Substitute
S15	Scutellaria amoena C. H. Wright	Yunnan	No	Cultivate	-	Substitute

Sample

Part

Producing area

Pith-decayed

Wild/Cultivate

Diameter/cm

Grade (1/2/3)

Scutellaria baicalensis Georgi

Inner Mongolia

Yes

Wild

1.0-1.5

Non-decayed part of S1

Inner Mongolia

Same as S1

Decayed pith of S1

Inner Mongolia

Same as S1

Scutellaria baicalensis Georgi

Inner Mongolia

Yes

Wild

1.0-1.5

Scutellaria baicalensis Georgi

Inner Mongolia

Yes

Wild

1.0-1.5

Scutellaria baicalensis Georgi

Hebei

Slightly

Cultivate

> 1.2

Scutellaria baicalensis Georgi

Shanxi

Cultivate

0.7-1.2

Scutellaria baicalensis Georgi

Shanxi

Cultivate

0.7-1.2

Scutellaria baicalensis Georgi

Shaanxi

Yes

Cultivate

1.0-1.5

S10

Scutellaria baicalensis Georgi

Gansu

Cultivate

< 0.7

S11

Scutellaria baicalensis Georgi

Gansu

Yes

Cultivate

> 1.5

S12

Scutellaria baicalensis Georgi

Henan

Cultivate

0.7-1.2

S13

Scutellaria baicalensis Georgi

Shaanxi

Cultivate

0.7-1.2

S14

Scutellaria baicalensis Georgi stem-leaf

Heilongjiang

Cultivate

Substitute

S15

Scutellaria amoena C. H. Wright

Yunnan

Cultivate

Substitute

Sample	Potency/U·g^-1	FL/%	Confidence interval/U·g^-1
S1	486.04	12.198 0	425.31-543.89
S2	459.68	3.223 6	444.26-473.89
S3	0.00	-	-
S4	518.91	3.358 0	500.58-535.43
S5	830.00	2.029 8	828.31-831.68
S6	605.10	2.281 3	591.21-618.82
S7	701.86	1.761 3	689.44-714.16
S8	901.15	3.766 8	885.25-917.09
S9	507.47	5.342 8	479.43-533.65
S10	297.60	6.257 5	278.29-315.53
S11	1 000.00	-	-
S12	389.85	4.999 8	369.95-408.93
S13	402.64	5.844 7	378.49-425.56
S14	438.45	1.486 8	431.47-444.51
S15	805.54	2.457 2	785.20-824.79

Sample

Potency/U·g^-1

FL/%

Confidence interval/U·g^-1

486.04

12.198 0

425.31-543.89

459.68

3.223 6

444.26-473.89

0.00

518.91

3.358 0

500.58-535.43

830.00

2.029 8

828.31-831.68

605.10

2.281 3

591.21-618.82

701.86

1.761 3

689.44-714.16

901.15

3.766 8

885.25-917.09

507.47

5.342 8

479.43-533.65

S10

297.60

6.257 5

278.29-315.53

S11

1 000.00

S12

389.85

4.999 8

369.95-408.93

S13

402.64

5.844 7

378.49-425.56

S14

438.45

1.486 8

431.47-444.51

S15

805.54

2.457 2

785.20-824.79

Peak	Grey correlation degree	Grey relational order
P1	0.982 6	R22
P2	0.990 2	R15
P3	0.984 0	R20
P4	0.993 1	R10
P5	0.970 4	R28
P6	0.950 6	R33
P7	0.956 7	R31
P8	0.970 6	R27
P9	0.983 9	R21
P10	0.990 7	R14
P11	0.977 1	R24
P12	0.994 0	R8
P13	0.981 8	R23
P14	0.973 1	R26
P15	0.996 3	R2
P16	0.947 3	R34
P17	0.962 0	R29
P18	0.957 8	R30
P19	0.995 7	R4
P20	0.987 2	R18
P21	0.992 5	R11
P22	0.987 2	R19
P23	0.989 8	R16
P24	0.989 0	R17
P25	0.992 4	R12
P26	0.976 5	R25
P27	0.956 5	R32
P28	0.993 9	R9
P29	0.995 3	R5
P30	0.997 6	R1
P31	0.995 1	R6
P32	0.991 0	R13
P33	0.994 7	R7
P34	0.996 1	R3

Peak

Grey correlation degree

Grey
relational order

0.982 6

R22

0.990 2

R15

0.984 0

R20

0.993 1

R10

0.970 4

R28

0.950 6

R33

0.956 7

R31

0.970 6

R27

0.983 9

R21

P10

0.990 7

R14

P11

0.977 1

R24

P12

0.994 0

P13

0.981 8

R23

P14

0.973 1

R26

P15

0.996 3

P16

0.947 3

R34

P17

0.962 0

R29

P18

0.957 8

R30

P19

0.995 7

P20

0.987 2

R18

P21

0.992 5

R11

P22

0.987 2

R19

P23

0.989 8

R16

P24

0.989 0

R17

P25

0.992 4

R12

P26

0.976 5

R25

P27

0.956 5

R32

P28

0.993 9

P29

0.995 3

P30

0.997 6

P31

0.995 1

P32

0.991 0

R13

P33

0.994 7

P34

0.996 1