半枝莲标准汤剂的指纹图谱建立与抗氧化活性成分的化学模式识别
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篇名: 半枝莲标准汤剂的指纹图谱建立与抗氧化活性成分的化学模式识别
TITLE: Fingerprint establishment of Scutellaria barbata standard decoction and chemical pattern recognition of anti- oxidant components
摘要: 目的 测定半枝莲标准汤剂中总黄酮的含量,评价其体外抗氧化活性,建立其指纹图谱,并进行化学模式识别分析。方法采用紫外-可见分光光度法测定半枝莲标准汤剂中总黄酮含量(以野黄芩素计);采用1,1-二苯基-2-三硝基苯肼(DPPH)自由基、2,2′-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)自由基清除实验考察半枝莲标准汤剂的体外抗氧化活性;采用高效液相色谱(HPLC)法,以野黄芩苷峰为参照峰,采用《中药色谱指纹图谱相似度评价系统(2004A版)》绘制16批半枝莲标准汤剂的指纹图谱并进行相似度评价,确定共有峰;采用SPSS24.0软件进行Pearson相关性分析,筛选具有体外抗氧化活性的潜在物质,并以其为变量,采用SPSS24.0、SIMCA14.1软件进行聚类分析和主成分分析。结果总黄酮检测质量浓度的线性范围为2.106~21.06μg/mL(R2=0.9993);精密度、重复性、稳定性(120min)试验的RSD均小于2%;加样回收率为100.62%(RSD=0.55%,n=6);总黄酮含量为0.634~1.053mg/mL。DPPH自由基清除实验的半数抑制浓度(IC50)为1.120~3.602mg/mL,ABTS自由基清除实验的IC50为0.684~1.327mg/mL;相关性分析结果显示,半枝莲标准汤剂中总黄酮含量与DPPH自由基、ABTS自由基清除实验的IC50均呈负相关,相关系数分别为-0.976、-0.940(P<0.01)。16批半枝莲标准汤剂共有18个共有峰,相似度为0.964~0.997;共指认出其中4个共有峰,分别为野黄芩苷(8号峰)、野黄芩素(14号峰)、木犀草素(15号峰)、芹菜素(17号峰)。半枝莲标准汤剂HPLC指纹图谱中3~4、8~9、12~15、17号峰峰面积与DPPH自由基、ABTS自由基清除实验的IC50均呈显著负相关(P<0.05或P<0.01)。聚类分析结果显示,16批半枝莲标准汤剂可聚为两类,其中S2、S7~S8、S14~S16为一类,S1、S3~S6、S9~S13为一类;主成分分析将16批半枝莲标准汤剂分为两类,其中S2、S4、S7、S14~S16为一类,S1、S3、S5~S6、S8~S13为一类,S4、S13、S15样品的综合评分较高。结论所建HPLC指纹图谱及化学模式识别分析方法可用于评价半枝莲标准汤剂的质量;3~4、8~9、12~15、17号峰及总黄酮是半枝莲标准汤剂清除DPPH自由基和ABTS自由基的潜在物质基础。
ABSTRACT: OBJECTIVE To determi ne the contents of total fla vonoids i n Scutellaria barbata standard decoction ,evaluate in vitro antioxidant activity ,establish the fingerprint and conduct chemical pattern recognition analysis. METHODS The contents of total flavonoids in S. barbata standard decoction (calculated by scutellarein )were determined by ultraviet-visible spectrophotometry. In vitro antioxidant activity of S. barbata standard decoction was investigated by free radical scavenging tests of 1,1-diphenyl- 2-trinitrophenylhydrazine(DPPH)and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid )ammonium salt (ABTS);HPLC method was adopted. Using scutellarin as reference ,the fingerprints of 16 batches of S. barbata standard decoction were drawn and evaluated by Similarity Evaluation System of TCM Chromatogram Fingerprint (2004 A edition ),and the common peaks were determined;Pearson correlation analysis was carried out by using SPSS 24.0 software to screen substances with in vitro antioxidant activity. Taking them as variables ,cluster analysis and principal component analysis were carried out by using SPSS 24.0 and SIMCA 14.1 software. RESULTS The linear range of total flavonoids were 2.106-21.06 μg/mL(R2=0.999 3);RSDs of precision , reproducibility and stability tests (120 min)were all lower than 2%;the recovery was 100.62%(RSD=0.55%,n=6);the contents of total flavonoids were 0.634-1.053 mg/mL. Median inhibitory concentration (IC50) of DPPH radical scavenging experiment ranged 1.120-3.602 mg/mL,and IC 50 of ABTS radical scavenging e xperiment range d 0.684-1.327 mg/mL. The results of correlation analysis showed that the content of total flavonoids Δ 基金项目 :河北省高校省级重点学科建设项目 (No.冀教 in S. barbata standard decoction was negatively correlated 高〔2013〕4号);承德医学院自然科学研究计划项目(No.201824) *讲师,硕士。研究方向:中药质量控制 。电话:0314-2291186。 with the IC 50 of DPPH free radical and ABTS free radical E-mail:duyilongww@sina.com scavenging experiment ,and the correlation coefficients were # 通信作者 :教授,硕士。研究方向 :中药质量控制 。电话: -0.976 and -0.940 respectively(P<0.01). There were 18 0314-2291186。E-mail:phf2301@163.com common peaks in the fin gerprints of 16 batches of S. barbata 中国药房 2022年第33卷第4期 China Pharmacy 2022Vol. 33 No. 4 ·425· standard decoction ;the s imilarities were 0.964-0.997. A total of 4 common peaks were identified ,such as scutellarin (peak 8), scutellarein(peak 14),luteolin(peak 15),apigenin(peak 17).In the HPLC fingerprints of S. barbata standard decoction ,the peak areas of peak 3-4,8-9,12-15 and 17 were significantly negatively correlated with the IC 50 of DPPH free radical and ABTS free radical scavenging experiment (P<0.05). The results of cluster analysis showed that 16 batches of S. barbata standard decoction could be clustered into two categories ,of which S 2,S7-S8 and S 14-S16 were clustered into one category ,S1,S3-S6 and S 9-S13 were clustered into one category. By principal component analysis ,16 batches of S. barbata standard decoction were divided into two categories ,of which S 2,S4,S7 and S 14-S16 were clustered into one category ,and S 1,S3,S5-S6 and S 8-S13 were clustered into one. The comprehensive scores were high in the samples of S 4,S13,S15. CONCLUSIONS Established HPLC fingerprint and chemical pattern recognition analysis method can be used to evaluate the quality of S. barbata standard decoction ; peak 3-4,8-9,12-15 and 17 and total flavonoids are the potential material basis for S. barbata standard decoction to scavenge DPPH free radical and ABTS free radical.
期刊: 2022年第33卷第04期
作者: 杜义龙,李赛,李艳荣,潘海峰
AUTHORS: DU Yilong ,LI Sai,LI Yanrong ,PAN Haifeng
关键字: 半枝莲;标准汤剂;总黄酮;体外抗氧化作用;指纹图谱;主成分分析;聚类分析
KEYWORDS: Scutellaria barbata ;standard decoction ;total flavonoids ;in vitro antioxidant effect ;fingerprint;principal
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