Study On The Quality Evaluation Of Dracocephalum Tanguticum Maxim Based On The Pharmacodynamic Components And Biological Effect Index Of Clearing Liver Heat

Dracocephalum tanguticum Maxim,is one of the most commonly used Tibetan medicinal materials in Tibetan medicine.At present,the research on Dracocephalum tanguticum Maximmainly focuses on the chemical constituents and pharmacological effects,but also a small amount of herbal research,resource distribution.The chemical composition of Dracocephalum tanguticum is complex.Most of the studies are based on traditional phytochemistry methods for extraction,isolation,identification and structural analysis.The pharmacological studies reported in the literature mainly focused on the effects of the crude extract of Dracocephalum tanguticum on anti-hypoxia and ischemia of heart and brain,bacteriostasis and anti-toxin,liver protection and enzyme reduction.However,there is no genuine producing area of Dracocephalum tanguticum medicinal materials,only through literature review found that the study subjects produced in Qinghai as the most.The quality control of the Dracocephalum tanguticum is still based on the standards promulgated by the ministry.The quality control level is low,and it is difficult to ensure the effectiveness and safety of the application.Therefore,this study aimed at the current practical problems of quality control and evaluation,based on its traditional efficacy of clearing liver-heat,to validate the efficacy evaluation model,definite effective components,screen biological effect evaluation indicators,construct and validate the quality evaluation method.The main points are as follows:1 Confirmed the efficacy evaluation model of clearing liver-heatThe efficacy evaluation model of clearing liver-heat was defined by consulting literature.The results showed that the etiology and pathogenesis of alcoholic liver injury(ALD)in Tibetan medicine were related to“Liver-heat”,and its therapeutic principle was clearing liver-heat which corresponded to the efficacy of Dracocephalum tanguticum and could be used as a disease model for evaluating its efficacy.2 Screened pharmacodynamic components in Dracocephalum tanguticum Maximin based on its clinical efficacy of clearing liver-heatThe medicinal materials were extracted by water,the extracts were filtered and the liquid was extracted by chloroform,ethyl acetate and n-butanol respectively.After concentration using rotary evaporator,the DrT-C,DrT-B,and DrT-E were obtained.The mice model of acute alcoholic liver injury(ALD)was established by intragastric administration of 40%EtOH.The activities of ADH,SOD,MDA,CAT,GSH-Px in liver tissueand the levels of AST,ALT,ADH,TC,TG,HDL-C,LDL-C in serum were determined following the manufacturer’s instructions for the respective kits.HE staining,oil red O staining and Masson staining were used to observe the pathological morphology,steatosis and fibrosis degree of liver tissue respectively.According to the results of pharmacological experiments,the active part of Dracocephalum tanguticum Maximin based on its clinical efficacy of clearing liver heat was screened out.HPLC was used to establish the fingerprint of the effective part,analyzed its relation with the liver function indexes,so as to screen out the active components.Compared with the control group,the levels of AST,ALT,TG,TC,LDL-C in serum and MDA in liver tissue were significantly increased,while the levels of HDL-C in serum and GSH-Px,SOD,CAT,ADH in liver tissue were significantly decreased in the model group(P<0.05).He staining showed that the structure of the hepatic lobules was unclear,the sinusoids were narrow,the hepatocytes were swollen and showed sheet-like balloon degeneration,a few nuclei of the hepatocytes were pyknosis,the infiltration of lymphocyte in the hepatic lobules was easy to be seen.Large amounts of fat droplets were seen in oil red o staining.Blue collagen fibers were seen in Masson staining.The above biochemical and pathological indexes indicated that the acute ALD mouse model was successfully induced.The all Dracocephalum tanguticum Maximin extracts could improve the liver injury induced by EtOH,and DrT-E had the best effect.After the intervention of DrT-E,the levels of ALT,AST,TC,TG,HDL-C and LDL-C in serum were increased,the levels of Lipid peroxidation related enzymes(MDA,SOD,CAT,GSH-Px)and alcohol metabolizing enzymes(ADH)in liver tissues tended to be normal(P<0.05).In addition,the liver tissues of ALD mice also showed significant improvement in pathology.Therefore,DrT-E was selected as the active part of Dracocephalum tanguticum Maximin.Using18 common peaks of DrT-E fingerprint as independent variables,and liver-protective activity(AST,ALT)as dependent variables,the spectral-effect relationship was analyzed,the chromatographic peaks 1,2,4,8,11,16 were positively correlated with AST and Alt,and VIP>1.Therefore,the six components were finally identified as the pharmacodynamic components of Dracocephalum tanguticum Maximin.3 Screened biological effect evaluation index of Dracocephalum tanguticum Maximin.The LO2 cell model was induced by EtOH,the effects of Dracocephalum tanguticum Maximin extracts on the activity of normal and injured LO2 cells were investigated.q PCR was used to detect the mRNA expression of Nrf2,NF-κB,STAT3,AP-1,CREB and HIF-1α.q PCR and Western blot were used to detect the mRNA and protein expression levels of KEAP1 and its downstream key enzyme genes(HO-1,NQO1,GSTA1).At the same time,the antioxidant activity of Dracocephalum tanguticum Maximin extracts were studied by DPPH free radical scavenging method.When the concentration of EtOH was 200μm,the activity of LO2 cells was significantly decreased with the activity of(70.77±5.53)%.The LO2 cell model induced by EtOH was successfully established.The extracts of Dracocephalum tanguticum Maximin had no effect on the activity of normal LO2 cells,while all of them could increase the activity of injured cells to some extent,especially when the concentration of DrT-E was 40μg/m L.These results suggest that DrT-E has protective effect on EtOH-induced hepatocyte injury,which is dose-dependent.Compared with the model group,DrT-E activated transcription factor NRF2 and up-regulated the mRNA and protein expressions of HO-1,NQO1 and GSTA1,and down-regulated the mRNA and protein expressions of KEAP1,suggesting that the liver protective effect of DrT-E is related to oxidative stress.DrT-C,DrT-B and DrT-E all had some antioxidant activity in vitro with the sequence of Vit C>DrT-E>BHT>DrT-B>DrT-C.At the concentration of 1.00 mg/m L,the antioxidant activity of DrT-E was(89.51±1.00)%,which was between the positive drug VITC and BHT.The results showed that the liver protective effect of DrT-E was significantly correlated with its antioxidant activity,so the antioxidant activity was selected as biological effect evaluation index of Dracocephalum tanguticum Maximin.4 Construction and validation of quality evaluation method for Dracocephalum tanguticum Maximin based on its pharmacodynamic components and biological effect indexThe fingerprints of 40 batches of DrT-E were established by RP-HPLC and the similarity was calculated.Ward method is applied to cluster analysis with square Euclidean distance as measure.PLS-DA model was established by SMICA software.The TOPSIS analysis model was established by using the six characteristic peaks of DrT-E and the free radical scavenging activity of DPPH as the evaluation indexes.The Dracocephalum tanguticum Maximin from different regions were sorted according to the C_i,the higher the C_ivalue,the better the overall quality.8 samples were randomly selected as test set,6 characteristic peaks and DPPH free radical scavenging activity were used as evaluation indexes to calculate Ci value,and their effects on serum indexes of ALD model were detected,to verify the feasibility of the method.There were 17 common peaks in the fingerprint of DrT-E,and the similarity was between 0.045 and 1.000.When the distance is 1,the Dracocephalum tanguticum Maximin in Qinghai,Gansu and Sichuan were clustered into one group.The clustering of Dracocephalum tanguticum Maximin in Tibet was more dispersed and cross-clustering with that of Gansu and Sichuan,the results were consistent with the similarity evaluation.The PLS-DA model was effective in distinguishing Dracocephalum tanguticum Maximin from four regions,and the contribution components X17,X14,X15,X10,X16,X6,X7,X9 were selected according to the VIP values.The Euclidean approximation of samples from different regions ranged from 0.107 to 0.711.The samples in Tibetan(S11-S22)has the best quality,with the Euclidean approximation range of 0.383-0.576,the Euclidean approximation range of samples in Gansu(S23-S32)was 0.239-0.711,the Euclidean approximation range of samples in Qinghai(S1-S10)was 0.162-0.231,the Euclidean approximation range of samples in Sichuan(S33-S40)was 0.107-0.449.The results can provide reference for the quality evaluation and control of Dracocephalum tanguticum Maximin.The validation tests results showed that the quality evaluation order based on TOPSIS model was the same as that based on liver function index,which shows that the model is stable and can be used to evaluate the quality of DrT.To sum up,this study based on DrT’s traditional efficacy of clearing liver-heat,screened active ingredients and biological activity evaluation indicators through experiments in vitro and in vivo,established the quality evaluation model of Topsis,it can be used for quality control and evaluation of DRT.Although the active ingredients have been screened out in this paper,only some of them have been identified,and the unknown active ingredients need to be clarified.The reasons for the quality difference of DRT also need to be further explored.