| Objective:Motherwort is a fresh or dry ground part of Leonurus japonicas Houtt.,which is mostly cultivated in most parts of China.This herb has functions of activating blood and removing stasis,nourishing blood and regulating menstruation,eliminating swelling and detoxifying and so on.It is a common clinical medicine in gynecology of traditional Chinese medicine.According to the literature research,motherwort contains alkaloids,flavonoids,diterpenoid,glycosides,fatty acids,volatile oils,cyclic peptides and other chemicals.At present,the research on quality control of motherwort is mostly focused on its preparations,such as injections,granules,fluid extracts,capsules and so on.The current quality standard of this herb is mainly used alkaloids as the index ingredient.The other chemicals are less concerned.The efficacy of Chinese herbal medicine is not from any single active ingredient,but the comprehensive results of various effective ingredients through the interaction with the human body on the body’s multipath and multiple targets.The fingerprint is used to evaluate the quality of traditional Chinese medicine,which can better adapt to the characteristics of the fuzziness and integrity of traditional Chinese medicine and control the quality of traditional Chinese medicine more comprehensively and scientifically.Modern pharmacological studies have shown that motherwort has many pharmacological effects,such as thrombolysis,anticoagulant,lipid lowering,blood viscosity reduction,reduction of erythrocyte aggregation,inhibition of platelet aggregation,improvement of microcirculation,oxygen free radicals,and reduction of intracellular calcium overload.It is commonly used in the treatment of thrombotic diseases such as irregular menstruation,hyperviscosity,myocardial ischemia,coronary heart disease,acute nephritis edema,and metabolic diseases caused by thrombotic diseases,such as hypertension and hyperglycemia.It has been proved that the zebrafish thrombus model induced by adrenaline hydrochloride is stable and reliable,which can be used as a screening model for antithrombotic drugs.Through the zebrafish thrombus model,the screening of antithrombotic activity in different extracts of motherwort and the establishment of the fingerprint of the antithrombotic active site of this herb will help to further improve the quality control standard of motherwort.Methods:(1)HPLC method and similarity evaluation,cluster analysis and principal component analysis were combined to establish HPLC fingerprint of motherwort.(2)The content of Leonurine and Rutin in motherwort was determined by HPLC.The quantitative analysis model of Leonurine was established by near infrared spectroscopy,which provided a reference for high-throughput determination and identification of motherwort.(3)Using the thrombus model induced by adrenaline hydrochloride in zebrafish,preliminary studies were conducted on the antithrombotic activity of different extracts from motherwort.(4)Using HPLC method and similarity evaluation,cluster analysis and principal component analysis to establish HPLC fingerprint of the antithrombotic activity of different extracts from motherwort.Results:(1)The HPLC fingerprint of motherwort was established,15 peaks were selected as the common peaks and the 2 common peaks were identified.The quality evaluation of 27 batches of motherwort was evaluated by similarity evaluation,cluster analysis and principal component analysis.The results of the three analysis methods were consistent and verified by each other,which was increase the reliability of results.The results of the analysis showed that the 27 species of motherwort were divided into 4 categories:3 batches in Sichuan Mianning;the 2 batches of Sichuan HuiLi and Hunan Guidong;and the 3 batches of Zhejiang,the 2 batches of Henan and Shandong,and Guangdong.In general,the quality difference between the cultivated samples from the Sichuan Mianning Motherwort Base and the market was obvious,suggesting that the cultivation method could have a great influence on the quality of motherwort.Zhejiang Sanmen had not been classified as one of the other Zhejiang origin,and Sichuan Mianning was also classified separately,suggesting that the quality of motherwort produced in the same province is different.Zhejiang,Henan,Shandong and Guangdong were classified into one category,suggesting that harvest time can have a great influence on the quality of motherwort.The quality of motherwort in the three Chinese herbal medicine market in Anhui,Hebei and Beijing,are close to the general quality of motherwort.(2)A HPLC method for the determination of Leonurine and Rutin was established.And the contents of two effective ingredients in motherwort from 27 different habitats were determined.It was found that the content of Leonurine in the northern origin was significantly higher than that in the southern origin,and the content of Leonurine in Zhejiang origin was lower than that of other habitats;the content of Rutin in Sichuan Mianning was significantly higher than that of other habitats,and the content of Leonurine and Rutin of Beijing Tongren Tang and Sichuan Mianning were more than the other productions.A near infrared quantitative correction model of Leonurine was established,and the results were compared with the results measured by HPLC method.The results showed that the predicted value of Leonurine content could be in accordance with the actual value.It was suggested that the quantitative analysis model established by NIR can be used to determine the content of Leonurine in motherwort from different habitats quickly and accurately.(3)The model of zebrafish thrombosis induced by adrenaline hydrochloride was verified,and the antithrombotic activity of the different extracts of motherwort was preliminarily studied by using this model.The results showed that AH induced thrombosis in the tail vein of zebrafish larvae,and the thrombus area was significantly greater than that in the blank group.The experimental results showed that the model was stable and could be used in the screening test of antithrombotic activity.The effect of low,medium and high dose treatment group can significantly inhibit the formation of thrombus.The effect of the water extract,ethyl acetate extract and the 70%ethanol extract were significantly.(4)The HPLC fingerprint of ethyl acetate extract of motherwort was established,15 peaks were selected as the common peaks and the 2 common peaks were identified.The quality evaluation of 27 batches of motherwort was evaluated by similarity evaluation,cluster analysis and principal component analysis.The results of the three analysis methods were consistent,and were verified by each other.The results showed that after the extraction of ethyl acetate,the general information of motherwort was retained.The results of the extraction clustering were slightly different from the results of the sample clustering,suggesting that the reasons for the extraction of some effective components were obtained.The ethyl acetate extract of motherwort from 27 regions were divided into 3 categories:Henan,Hunan,Guangdong and Zhejiang produced,and motherwort from Hebei,Beijing and Shandong.Southwest productions was different from these two types.It was suggested that the ethyl acetate extract of motherwort could be divided into two categories according to the Qinling-Huaihe Line.What’s more,the southwest region was different from these two types.Conclusion:This study established a HPLC fingerprint combined with two kinds of methods for the determination of active ingredient contents of motherwort,providing reference for improving quality standards of this herb.Established a near-infrared quantitative calibration model for motherwort,the model can quickly and accurately determine the content of Leonurine.The zebrafish thrombosis model induced by adrenaline hydrochloride was validated.Experimental studies showed that different extract fractions of motherwort had significant inhibitory effect on the formation of thrombosis induced by epinephrine hydrochloride in zebrafish.Among them,the water extract,ethyl acetate extract,and 70%ethanol extract have significant effects.The HPLC fingerprint of the anti-thrombotic active sites of motherwort,ethyl acetate of motherwort was established to provide reference for the comprehensive and effective quality control evaluation methods of motherwort. |
