Study On Active Substance Group Of Hawthorn Leaf Extract On Promoting Blood Circulation And Removing Blood Stasis Based On Multidimensional Spectrum-Effect Relationship

Traditional Chinese medicine?TCM?is a complex system composed of many ingredients and factors,the curative effects are principally based on the synergic effect of their multi-targeting and multi-ingredient preparation.The fingerprint of TCM,a widely accepted quality control mode,which can reflect the kinds and contents of chemical components to the maximum extent,and evaluate the quality of TCM as a whole.However,the present fingerprint technique of TCM can only explain the existence of chemical composition group,but not explain the correlation between it and the efficacy of TCM.Therefore,to study the relationship between the changes of chemical components and the efficacy,further to find out effective substance group related to the pharmacological activity by means of mathematical statistics,that is,the study of"spectrum-effect relationship",has become the main means to explore material basis of specific efficacy of TCM.In recent years,the research model of spectrum-effect relationship explored and established by scholars at home and abroad,which includes the establishment of a suitable fingerprint of TCM,the establishment of a suitable evaluation model of the drug effect,and correlation analysis of fingerprint and pharmacological data with statistical software.Among them,the key is to establish the appropriate fingerprint,the point is to evaluate the pharmacodynamics,and the application of mathematical statistical method is the core.In order to establish a meaningful spectrum-effect relationship,it is necessary to think over the professional knowledge of TCM.This paper aimed to study the spectrum-effect relationship of Hawthorn leaf extract in promoting blood circulation and removing blood stasis from these three aspects.Hawthorn leaves,with the functions of promoting blood circulation and removing blood stasis,regulating qi and dredging veins,turbid and lipid-lowering,is a member of the Rosaceae family.Hawthorn leaves comes from Crataegus pinnatifida Bge.Var.major N.E.Br.or Crataegus pinnatifida Bge.,flavonoids are main active components,such as vitexin-rhamnoside,quercetin,vitexin and hyperoside etc.Nowadays,the prevention and treatment of cardiovascular disease has become a hot research topic for many scholars.Hawthorn leaves are being developed and used to make different preparations because of its rich resources,low cost,multi-target comprehensive treatment and low toxic side effects.For example,Shan Mei capsule and Xin an capsule used to treat coronary heart disease and improve myocardial blood supply,are made of 50%ethanol extract.Yi xin ketone tablet used to treat chest obstruction caused by blood stasis and angina pectoris of coronary heart disease,are made of alcohol extract from macroporous resin recorded in the Pharmacopoeia of People's Republic of China?2010 edition?.In this paper,four different extracts from Hawthorn leaves,50%ethanol extract?A?,alcohol extract from macroporous resin?B?,ethylacetate extract?C?,n-butyl alcohol extract?D?were selected to study the spectrum-effect relationship of hawthorn leaf extract in promoting blood circulation and removing blood stasis.At present,there are many reports about promoting blood circulation and removing blood stasis of hawthorn leaves,which mainly focus on gene expression,antioxidant effect and the effect on myocardial mitochondria,etc.However,the effects of hawthorn leaf extract on hemorheology,whole blood viscosity?WBV?and plasma Viscosity?PV?have not been reported.Therefore,on the basis of the previous study of the research group,four hawthorn leaf extracts were taken as the research objects.Under the same chromatographic conditions,the fingerprints of four hawthorn leaf extracts were established respectively.The rat model of acute blood stasis was used and the drug was administered by gavage at low,middle and high doses.Principal component analysis?PCA?and one-way ANOVA were used to screen out the indexes related to promoting blood circulation and removing blood stasis of four extracts from 27 biochemical indexes.Then a multivariate linear regression equation between pharmacodynamic values of each biochemical index Y and the area of common peak X was established by Matlab software with backward stepwise.Further explore on the active substance group of hawthorn leaf extract would provide basis for establishment of the internal quality standard between hawthorn leaves with promoting blood circulation and removing blood stasis.Part one Establishment of chemical fingerprints and comparison of total flavonoids contents of four hawthorn leaf extractsObjective:Under the same amount and chromatographic conditions,the fingerprints of four different extracts from Hawthorn leaves,50%ethanol extract?A?,alcohol extract from macroporous resin?B?,ethylacetate extract?C?,n-butyl alcohol extract?D?were established respectively.The total flavonoids content in four extracts was determined by ultraviolet spectrophotometry.To compare the similarities and differences in chemical composition and the total flavonoids content.Methods:10 batches of A and B were purchased.The hawthorn leaves were collected to prepare 10 batches of C and D respectively.Chromatographic separation was conducted by High Performance Liquid Chromatography?HPLC?,the mobile phase consisted of acetonitrile?A?,0.1%glacial acetic acid?B?and tetrahydrofuran?C?in gradient elution mode,and the gradient of A,B and C were changed as follows,T?0⁵¹5³0⁵0⁶0⁷0⁸0¹20min?,A?10%¹2%¹2%¹2%¹2%²0%²0%³0%⁸0%?,B?90%⁸8%⁸8%⁸5%⁸5%⁷6%⁷3%⁶6%¹6%?,C?0%⁰%⁰%³%³%⁴%⁷%⁴%⁴%?.The detection wavelength was 320nm,the flow rate was 0.9ml/min,the column temperature was 30?,the injection amount was 10?l.The fingerprints of four hawthorn leaf extracts were established by Similarity Evaluation System for chromatographic fingerprinting of traditional Chinese medicine?2004A,Pharmacopoeia Commission of China?.Peaks existing in all chromatograms of the samples were as signed as“common peaks”.According to the method,recorded in the Pharmacopoeia of People's Republic of China?2015 edition?,the total flavonoids content of four hawthorn leaf extracts were determined,and the average value was calculated.Results:Under the same conditions,18,18,25,20 common peaks were found in the hawthorn leaf extracts A,B,C,and D respectively.Five common peaks were unambiguously identified as chlorogenic acid?S1?,vitexin-glucoside?S2?,vitexin-rhamnoside?S3?,vitexin?S4?,rutin?S5?and hyperoside?S6?.From the overall fingerprint information,the common peaks with a medium-area in C were distributed evenly in polar,middle polar and non-polar parts,and the number of common peaks was the most.Compared with the C,the common peaks in A were mainly distributed in the polar and middle polar parts,fewer peaks in the non-polar parts and the area were small.The common peaks with a large-area in B were concentrated in the polar and middle polar parts.The distribution of common peaks in D was similar to that in A.From the results of the total flavonoids content,the content of total flavonoids in B was the highest,with an average value of 83.56%,and C followed,with an average of 58.61%.The contents of A and D were close,the average values were 33.81%and 39.80%,respectively.Conclusion:The chemical constituents and total flavonoids contents in four hawthorn leaf extracts were obviously different,which may lead to different in efficacy.This part would provide a theoretical basis for further study on the spectrum-effect relationship.Part two Screening of related biochemical indexes of hawthorn leaf extract on promoting blood circulation and removing blood stasisObjective:Four extracts,10 batches uniformly mixed of each,were made into high,middle and low 3 doses.The rat model of acute blood stasis was used and the drug was administered by gavage.PCA and one-way ANOVA were used to screen out the indexes related to promoting blood circulation and removing blood stasis of four extracts from 27 biochemical indexes.Methods:The wistar rat model of blood stasis was induced by ice water and adrenaline.Four extracts?10 batches uniformly mixed of each,were made into high,middle and low 3 doses to ensure the homogeneity of drug delivery matrix?were given respectively by gavage.A total of 27 indexes were measured.Among them,the prothrombin time?PT?,activated partial thromboplastin time?APTT?,thrombin time?TT?,fibrinogen?FIB?were determined by XN06-IV coagulation instrument.WBV?3 tests included low,medium and high shear rate?was determined by whole blood viscosity tester.Blood routine test?19 tests included total white blood cells,lymphocyte percentage and total number of red blood cells,etc.?were determined by automatic purging blood rheometer.PV was determined by microplasma measuring instrument.Dimensionless and dimensionality reduction processing of data was completed by PCA,and the new variables were then analyzed by one-way ANOVA.The analysis results were combined the contribution rate of each principal component with the correlation coefficient of each index,and the index group and single index in each group are comprehensively analyzed.To screen some biochemical indexes related to promoting blood circulation and removing blood stasis by comparing the effects of four extracts on the biochemical indexes of rats with acute blood stasis syndrome.Results:Compared with the blank group,the FIB,total number of red blood cells,total number of thrombocytes,mean platelet volume?MPV?,thrombocytocrit,the low,median,high shear rates,and PV in the model group were significantly higher than those in the blank group?P<0.01?.The results showed that the rat model of blood stasis was successful.?2?Compared with the model group,for extract A,total number of thrombocytes,MPV,thrombocytocrit,low,median,high shear rates,and PV were significantly decreased in a dose-dependent manner at low,medium and high doses?P<0.01?.Low FIB?P<0.05?and total number of red blood cells decreased significantly?P<0.01?at high dose.?3?Compared with the model group,for extract B,total number of red blood cells,total number of thrombocytes,MPV,thrombocytocrit,low,median,high shear rates,and PV were significantly decreased in a dose-dependent manner at low,medium and high doses?P<0.01?.Hemoglobin?HGB?,total number of red blood cells and FIB decreased significantly?P<0.01?at high dose.?4?Compared with the model group,for extract C,the changes of indexes at low,medium and high dose were consistent with that of extract B?P<0.01?.?5?Compared with the model group,for extract D,the changes of indexes at low,medium and high dose were consistent with that of extract A?P<0.01?,low hematocrit?HCT?and total number of red blood cells at high dose?P<0.05?,low FIB at medium and high dose.Conclusion:The four extracts had different effects on FIB,total number of red blood cells,total number of thrombocytes,MPV,thrombocytocrit,the low,median,high shear rates,and PV.Therefore,the above nine biochemical indexes have correlation with promoting blood circulation and removing blood stasis,and can be used as the"effect"in the third part of the study on the spectrum-effect relationship.Part three Study on the spectrum-effect relationship of hawthorn leaf extract on promoting blood circulation and removing blood stasisObjective:The fingerprint was re-established with four extracts,10 batches uniformly mixed of each.At the same time,the area of common peaks were marked as X,and the biochemical indexes selected in the second part were marked as Y.Then the equation of spectrum-effect relation was calculated by Matlab software.To explore the active substance group of hawthorn leaf extract on promoting blood circulation and removing blood stasis.Methods:In accordance with the chromatographic conditions in first part,the chromatograms of 12 samples of four extracts were collected by HPLC method.The standard fingerprint of four extracts was established by Similarity Evaluation System for chromatographic fingerprinting of traditional Chinese medicine?2004A,Pharmacopoeia Commission of China?.The area of common peaks were marked as X,and the biochemical indexes selected in the second part were marked as Y.Then the equation of spectrum-effect relation was calculated by Matlab software with backward stepwise.To obtain the active substance group of hawthorn leaf extract on promoting blood circulation and removing blood stasis.Results:32 common peaks were found in the fingerprint re-established by four extracts.The area of each common peak was taken as independent variable X,and the data of 9 biochemical indexes related to promoting blood circulation and removing blood stasis were taken as dependent variable Y,FIB?Y₁?,total number of red blood cells?Y₂?,total number of thrombocytes?Y₃?,MPV?Y₄?,thrombocytocrit?Y₅?,the low?Y₆?,median?Y₇?,high?Y₈?shear rates and PV?Y₉?.Then the equatios of spectrum-effect relation were calculated by Matlab software with backward stepwise respectively.Y₁=3.684+32.919X₆-137.424X₉+5.917X₁₀+12.739X₁₃-21.789X₁₅-8.911X₁₇+55.929X₁₉+14.380X₂₁+40.266X₂₃-65.414X₂₇,r²=0.9999,P=0.0001<0.01Y₂=100.35+732.23X₁-2329.6X₅+43.44X₁₄+2433X₁₉+194.06X₂₁+885.61X₂₃+3617.6X₂₅+766.71X₂₆-13.119X₃₀-11672X₃₂,r²=0.9999,P=0.0002<0.01Y₃=155.241-38382X₁+5228.5X₈-11320X₁₅+23536X₁₈-41184X₁₉-17866X₂₃-54465X₂₇+74870X₂₈+48244X₃₁+733000X₃₂,r²=0.9999,P=0.0031<0.01Y₄=9.0806-262.25X₁+386.12X₈-158.30X₁₀-188.03X₁₁+3.2921X₁₂+69.553X₁₅-370.63X₁₉-96.547X₂₇+333.62X₃₁+4883X₃₂,r²=0.9999,P=0.0019<0.01Y₅=0.3323-7.3505X₁-46.676X₂-10.1838X₈-34.954X₉+13.7663X₁₀+23.7524X₁₁+21.272X₂₂-23.1355X₂₇-14.986X₂₉+797.285X₃₂,r²=0.9999,P=0.0027<0.01Y₆=7.9105-129.44X₃-594.09X₆+243.2993X₁₀-22.3509X₁₃+125.849X₁₉+62.4919X₂₁-59.7529X₂₃-584.1149X₂₇+141.7714X₃₁+11059X_32,r²=0.9999,P=0.0042<0.01Y₇=8.5820-6.1227X₃+60.0425X₉-21.4660X₁₀+0.5350X₁₄+33.8677X₁₅-36.8762X₁₉-5.7435X₂₁+30.2422X₂₃-45.0794X₂₈+593.8178X₃₂,r²=0.9999,P=0.0009<0.01Y₈=5.2432-25.7911X₄+48.0672X₅+37.3757X₁₀-19.1838X₁₃-0.3789X₁₅-70.3253X₁₉+43.1946X₂₂-91.9206X₂₃+8.7604X₃₀+892.2150X₃₂,r²=0.9999,P=0.0001<0.01Y₉=1.4767-4.5919X₉-1.9720X₁₀+3.5956X₁₁-0.5178X₁₃-0.7469X₁₄+1.7472X₁₅-0.4215X₁₇+1.5535X₁₉+6.8144X₂₇-13.8635X₃₁,r²=0.9999,P=0.0045<0.01Among them,FIB can be affected by X₁₉?rutin?,X_23,X_6,X_21,X_13,X_10,X₁₇?vitexin?,X₁₅,X₂₇ and X₉.The total number of red blood cells can be affected by X₂₅,X₁₉?rutin?,X₂₃,X₂₆,X₁,X₂₁,X₁₄?vitexin-glucoside?,X₃₀,X₃₂2 and X_5.Thrombocytes can be affected by X₃₂,X₂₈,X₃₁,X₁₈,X₈,X₁₅,X₂₃,X₁,X₁₉?rutin?and X_27.MPV can be affected by X₃₂,X₈,X₃₁,X₁₅,X₁₂,X₂₇,X₁₀,X₁₁,X₁ and X₁₉?rutin?.Then the components X_32,X₁₁,X₂₂,X₁₀,X₁,X₈,X₂₉,X₂₇,X₉and X₂ can affect thrombocytocrit.The components X₃₂,X₁₀,X₃₁,X₁₉?rutin?,X₂₁,X₁₃,X₂₃,X₃?chlorogenic acid?,X₂₇ and X₆ can affect low shear rate.Median shear rate can be affected by X₃₂,X₉,X₁₅,X₂₃,X₁₄?vitexin-glucoside?,X₂₁,X₃?chlorogenic acid?,X₁₀,X₁₉?rutin?and X_28.High shear rate can be affected by X₃₂,X₅,X₂₂,X₁₀,X₃₀,X₁₅,X₁₃,X₄,X₁₉?rutin?and X_23.PV can be affected by the components X₂₇,X₁₁,X₁₅,X₁₉?rutin?,X₁₇?vitexin?,X₁₃,X₁₄?vitexin-glucoside?,X₁₀,X₉ and X_31.Conclusion:In this part,32 common peaks were found in the fingerprint re-established by four extracts.The area of each common peak was taken as independent variable X,and the data of 9 biochemical indexes related to promoting blood circulation and removing blood stasis were taken as dependent variable Y,then the equation of spectrum-effect relation was calculated by Matlab software with backward stepwise.Finally,the active substance group of hawthorn leaf extract on promoting blood circulation and removing blood stasis were screened out successfully.This paper would provide basis for establishment of the internal quality standard between hawthorn leaves with promoting blood circulation and removing blood stasis.