Center for Medical Research and Innovation, First Hospital of Hunan University of Chinese Medicine, Changsha 410007.
Key Laboratory of Research on Prevention and Treatment of Major Diseases in Chinese Internal Medicine and Transformation of Achievements of Ministry of Education, Changsha 410007.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2024 Mar 28;49(3):435-446. doi: 10.11817/j.issn.1672-7347.2024.230344.
Farfarae Flos has the effect of cough suppression and phlegm elimination, with cough suppression as the main function. Studies have revealed that certain components of Farfarae Flos may be related to its cough suppressant effect, and some components have been confirmed to have cough suppressant activity. However, the antitussive material basis of Farfarae Flos has not been systematically elucidated. This study aims to elucidate the group of active ingredients in Farfarae Flos with cough suppressant activity by correlating the high performance liquid chromatography (HPLC) fingerprint of Farfarae Flos extract with its cough suppressant activity.
HPLC was used to establish the fingerprint profiles of 10 batches of Farfarae Flos extract and obtain their chemical composition data. Guinea pigs were selected as experimental animals and the citric acid-induced cough model was used to evaluate the antitussive efficacy data of 10 batches of Farfarae Flos extract. SPF-grade healthy male Hartley guinea pigs were randomly divided into the S1 to S10 groups, a positive control group, and a blank control group (12 groups in total), with 10 guinea pigs in each group. The S1 to S10 groups were respectively administered Farfarae Flos extract S1 to S10 (4 g/kg), the positive control group was administered pentoverine citrate (10 mg/kg), and the blank control group was administered purified water. Each group received continuous oral administration for 5 days. The guinea pigs were placed in 5 L closed wide-mouth bottles, and 17.5% citric acid was sprayed into the bottle with an ultrasonic atomizer at the maximum spray intensity for 0.5 minutes. The cough latency period and cough frequency in 5 minutes were recorded for each guinea pig. Grey relational analysis (GRA) and partial least squares regression (PLSR) were used to conduct spectral-effect correlation analysis of the chemical composition data of Farfarae Flos extract and the antitussive efficacy data, and predict the group of active ingredients in Farfarae Flos with antitussive activity. The bioequivalence verification was conducted to verify the predicted group of active ingredients in Farfarae Flos with antitussive activity: SPF-grade healthy male Hartley guinea pigs were randomly divided into a S9 group, an active ingredient group, a positive control group, and a blank control group (4 groups in total), with 10 guinea pigs in each group. The S9 group was administered Farfarae Flos extract S9 (4 g/kg), the active ingredient group was administered the predicted combination of antitussive active ingredients (dose equivalent to 4 g/kg of Farfarae Flos extract S9), the positive control group was administered pentoverine citrate (10 mg/kg), and the blank control group was administered purified water. Each group received continuous oral administration for 5 days, and animal modeling and observation of efficacy indicators were the same as above.
The HPLC fingerprint of 10 batches of Farfarae Flos extract was established, and the peak area data of 14 main common peaks were obtained. The antitussive effect data of 10 batches of Farfarae Flos extract were obtained. Compared with the blank control group, the cough latence in the positive control group and S1, S2, S3, S4, S6, S7, S8, S9, S10 groups was prolonged (all <0.01), while the cough frequency in 5 minutes in the positive control group and S1, S2, S4, S6, S8, S9, S10 groups was decreased (all <0.05). The analysis of spectrum-effect relationship revealed that isochlorogenic acid C, isochlorogenic acid A, chlorogenic acid, isochlorogenic acid B, isoquercitrin, and rutin had high contribution to the antitussive effect of Farfarae Flos, and the 6 components were predicted to be the antitussive component group of Farfarae Flos. The verification of bioequivalence showed that there were no statistically significant differences in the antitussive effect between the S9 group and the antitussive component composition group(all 0.05), which confirmed that isochlorogenic acid C, isochlorogenic acid A, chlorogenic acid, isochlorogenic acid B, isoquercetin, and rutin were the antitussive component group of Farfarae Flos.
The analysis of spectrum-effect relationship combined with the verification of bioequivalence could be used to study the antitussive material basis of Farfarae Flos. The antitussive effect of Farfarae Flos is the result of the joint action of many components.
款冬花具有镇咳和祛痰作用，以镇咳作用为主。研究表明，款冬花的某些成分可能与其镇咳作用有关，一些成分已被证实具有镇咳活性。然而，款冬花的镇咳物质基础尚未系统阐明。本研究旨在通过关联款冬花提取物的高效液相色谱（HPLC）指纹图谱与其镇咳活性，阐明具有镇咳活性的款冬花活性成分组。
采用 HPLC 建立 10 批款冬花提取物的指纹图谱，获得其化学成分数据。选择豚鼠作为实验动物，采用柠檬酸诱导咳嗽模型评价 10 批款冬花提取物的镇咳功效数据。SPF 级健康雄性 Hartley 豚鼠随机分为 S1 至 S10 组、阳性对照组和空白对照组（共 12 组），每组 10 只。S1 至 S10 组分别给予款冬花提取物 S1 至 S10（4 g/kg），阳性对照组给予戊乙奎醚柠檬酸酯（10 mg/kg），空白对照组给予纯化水。每组连续口服给药 5 天。将豚鼠放入 5 L 密闭大口瓶中，用超声雾化器以最大喷雾强度将 17.5%柠檬酸喷雾入瓶中 0.5 分钟。记录每只豚鼠的咳嗽潜伏期和 5 分钟内的咳嗽次数。采用灰色关联分析（GRA）和偏最小二乘回归（PLSR）对款冬花提取物的化学成分数据和镇咳功效数据进行谱效关联分析，并预测具有镇咳活性的款冬花活性成分组。进行生物等效性验证，以验证具有镇咳活性的款冬花活性成分组：SPF 级健康雄性 Hartley 豚鼠随机分为 S9 组、活性成分组、阳性对照组和空白对照组（共 4 组），每组 10 只。S9 组给予款冬花提取物 S9（4 g/kg），活性成分组给予预测的具有镇咳活性的 6 种活性成分组合（剂量相当于 4 g/kg 的款冬花提取物 S9），阳性对照组给予戊乙奎醚柠檬酸酯（10 mg/kg），空白对照组给予纯化水。每组连续口服给药 5 天，动物造模和观察疗效指标同前。
建立了 10 批款冬花提取物的 HPLC 指纹图谱，得到了 14 个主要共有峰的峰面积数据。获得了 10 批款冬花提取物的镇咳功效数据。与空白对照组相比，阳性对照组和 S1、S2、S3、S4、S6、S7、S8、S9、S10 组的咳嗽潜伏期延长（均<0.01），而阳性对照组和 S1、S2、S4、S6、S8、S9、S10 组的 5 分钟内咳嗽次数减少（均<0.05）。谱效关系分析表明，异绿原酸 C、异绿原酸 A、绿原酸、异绿原酸 B、异槲皮苷和芦丁对款冬花的镇咳作用有较高的贡献，这 6 个成分被预测为款冬花的镇咳成分组。生物等效性验证显示，S9 组与镇咳成分组成组之间的镇咳效果无统计学差异（均 0.05），证实异绿原酸 C、异绿原酸 A、绿原酸、异绿原酸 B、异槲皮苷和芦丁是款冬花的镇咳成分组。
谱效关系分析结合生物等效性验证可用于研究款冬花的镇咳物质基础。款冬花的镇咳作用是多种成分共同作用的结果。
