| 1 ObjectiveSinapis Semen and Raphani Semen are representative Chinese medicines containing glucosinolates in cruciferous plants.Raw and processed slices have different focuses on their pharmacological activities,and they are used in clinical practice and have similar changes in efficacy after being processed,which is longer than cough and phlegm,but what is the material basis of the medicinal effect after processed,how to exert the medicinal effect and how the metabolic process is,so far it is not very clear.Based on the study of the changes in the chemical composition of the raw and processed slices,this study carried out a comparative study on the antitussive and expectorant biological activities of 6 kinds of processed slices,including before and after processing of Sinapis Semen and Raphani Semen,and selected the most effective processed slices.In order to investigate the change of plasma concentration at different time points after a single oral administration of rats by UPLC-MS/MS,further observe the correlation between plasma concentration and pharmacological effects of plasma samples taken at different time points under pathological conditions.Combined with the results of the preliminary analysis of the in vitro pharmacodynamic substances,and correlate with the results of pharmacodynamic studies,it revealed the correlation between in vitro chemical constituents,metabolic changes in the body,and pharmacodynamic effects before and after processing Sinapis Semen and Raphani Semen.The interrelationships should be searched for its anti-tussive,expectorant,and asthmatic medicinal substance basis,and provide scientific basis for clinical rational and effective application.2 Contents2.1 Comparative study on antitussive and expectorant effects before and after processing of Sinapis Semen and Raphani Semen2.1.1 Study on the Ammonia Induced Cough in Mice by Before and After Processing of Sinapis Semen and Raphani SemenMethods:112 sensitive mice with a cough latency of less than 90 s and more than 5 coughs within 2 minutes were pre-screened first,and the sensitive mice were randomly divided into control group,Tween group,high and low dose group of raw and stir-fired decoction pieces of Sinapis alba Semen;high and low dose group of raw and stir-fired decoction pieces of Brassica juncea Semen;high and low dose group of raw and stir-fired decoction pieces of Raphani Semen according to their weight,8 mice in each group,and were given orally for 5 days.30 minutes after the last administration,and stimulated with ammonia spray.The number of coughs and the latency of cough within 2 min of each group of mice were recorded.Results:Compared with the control group,the 6 kinds of decoction of stir-fried Raphani Semen and stir-fried Brassica juncea Semen both increased the cough latency and reduced the number of coughs in mice(P<0.05,P<0.01).The results of stir-fried semen Raphani were significantly different from those of their own raw slices(P<0.05,P<0.01).2.1.2 Comparative study on phenol red excretion in mice by Before and After Processing of Sinapis Semen and Raphani SemenMethods:Mice were randomly divided into control group,Tween group,high and low dose group of raw and stir-fired decoction pieces of Sinapis alba Semen;high and low dose group of raw and stir-fired decoction pieces of Brassica juncea Semen;high and low dose group of raw and stir-fired decoction pieces of Raphani Semen,continuous oral administration for 5 days,the last dose for 30min,mice in each group were injected with 0.5%phenol red solution 0.5 mL,The phenol red excretion in the trachea was determined.Results:Compared with the control group,the 6 kinds of decoction of stir-fired Raphani Semen high-dose group,raw Sinapis alba Semen high,low-dose group,and stir-fried Brassica juncea Semen low-dose group could significantly increase the tracheal phenol red excretion in mice(P<0.05,P<0.01);and the results of the high-dose stir-fired Raphani Semen group were significantly different from those of their raw slices(P<0.05).In summary,the antitussive and expectorant activities of stir-fired Raphani Semen are better than five kinds of raw and stir-fired decoction pieces,which will be used as the test drug for the subsequent pharmacokinetic experiment.2.2 Study on pharmacokinetics of stir-fried Raphani Semen in normal rats2.2.1 Development of UPLC-MS/MS simultaneous determination of sinapic acid and sinigrin in rat plasmaMethods:The chromatographic column was HSS T3 column(2.1 mm×100 mm,1.8 ?m),0.1%formic acid aqueous solution-acetonitrile as mobile phase gradient elution,the flow rate was 0.3 mL·min-1 and the sample size was 5 ?L for chromatographic separation,electrospray ion source,negative ion detection mode,ion pair selection erucic acid m/z 223.1?164.1,sinigrin m/z 358.0?97.0,nipagin butyl ester m/z 193.0?121,0,Multiple reaction monitoring for mass scan.Results:The linear relationship between sinapic acid and sinigrin components was good(R>0.999),the intra-day and inter-day precision RSD were less than 4.1%,the recovery RSD was 0.8%?3.1%and there was no significant matrix effect on the tested components and internal standards,and the stability was good.The method is simple,sensitive and meets the analysis requirements of biological samples.2.2.2 Pharmacokinetics of different doses of stir-fired Raphani Semen in a single administration in normal ratsMethods:According to the clinical design of low,medium and high dose groups(4.5,9,18 g·kg-1),normal animals were used as experimental subjects to establish a pharmacokinetic model.The concentration of sinapic acid and sinigrin in plasma of rats in each group were measured 0 hours before administration and 0.083,0.167,0.333,0.5,1,1.5,2,2.5,3,6,9 hours after oral administration of stir-fired Raphani Semen alcohol extracts,the relevant pharmacokinetic parameters were calculated of non-compartmental model by DAS3.2.8 software.Results:After normal rats were given orally with 4.5,9,and 18 g·kg-1 doses of Stir-fried Raphani Semen alcohol extracts,the peak concentrations(Cmax)of sinapic acid in plasma were(29.35±10.32),(62.70±27.47),(137.33±40.95)?g·L-1;The area under the curve(AUC0-1)at the time of drug is(92.83±27.16),(240.74±75.09),(633.95±195.88)?g·L-1·h;the peak time(Tmax)is(2.58±0.80),(3.00±0),(5.50±1.23)h;Compared with the low-dose group of stir-fired Raphani Semen alcohol extracts,the AUC0-1 and MRT0-1 of the medium and high-dose group increased significantly(P<0.05,P<0.01);AUC0-t in sinapic acid had a good linear dependence at 4.5?18 g·kg-1 and no sinigrin component was detected this time.2.3 Correlation between PK-PD of stir-fried Raphani Semen in asthmatic rats2.3.1 Establishment and evaluation of rat asthma modelMethods:An allergic asthma rat model was established by intraperitoneal injection of ovalbumin with aluminum hydroxideon on day 1 and 8 and atomization with 1%OVA physiological saline solution for 2 weeks.Model evaluation was performed by using behavioral observation,white blood cell count and classification,and Elisa method to detect the inflammatory factor content.Results:Compared with the control group,the rats in the model group showed symptoms such as scratching the nose,sneezing,coughing,and dull hair,and the total number of eosinophils,neutrophils and white blood cells in the whole blood increased significantly(P<0.05,P<0.01).In addition,the levels of IL-5,IgE,and TNF-? in serum increased significantly(P<0.05,P<0.01),indicating that the model was successful.2.3.2 Fitting analysis of PK-PD model in asthmatic ratsMethods:Starting from the day of atomization,oral administration started early 0.5 h before atomization for 3 weeks,UPLC-MS/MS method was used to determine 0.083,0.167,0.5,1,1.5,3,4.5,6,and 7.5,9 h sinapic acid and sinigrin concentrations.At the same time,the Elisa method was used to determine the levels of IL-5,IgE and TNF-? in the serum at corresponding time points to obtain pharmacodynamic indexes for evaluating allergic asthma.DAS software fits the PK-PD model.Results:Compared with the model group,asthma rats which were administrated with 4.5 and 9 g·kg-1 of Stir-fried Raphani Semen alcohol extracts respectively in 0.083 and 0.167 h,the levels of IL-5,IgE,TNF-? in serum were reduced to varying degrees(P<0.05,P<0.01);Cmax of sinapic acid in plasma was(58.43±27.94),(61.16±18.79)?g·L-1:AUC0-t were(188.75±37.07),(247.90±36.89)?g·L-1·h;AUC0-t,apparent volume of distribution(Vz/F)and clearance(CLZ/F)all increased with increasing doses.Cmax in the plasma of the sinigrin was(6.38±2.94),(7.03±3.83)?g·L-1;AUC0-t were(23.28±11.60)and(22.24±10.83)?g·L-1·h;both Vz/F and CLz/F increased with increasing dose.The analysis of data from DAS3.2.8 software showed that the optimal pharmacokinetic models of sinapic acid and sinigrin were fitted to one and three-compartment models for extravascular administration,respectively,and Emax connected by effect chamber was established Model,PK-PD model may be suitable for indirect connection model.3 ConclusionsAfter being stir-fried,Raphani Semen has good antitussive,expectorant and antiasthmatic effects than five kinds of raw and stir-fried decoction pieces.There are a certain correlation between the blood concentration of sinapic acid and sinigrin in the asthmatic rats and the levels of IL-5,IgE,TNF-? in the serum,the dosage and functional status(Pathological or physiological status)will affect the pharmacokinetic behavior of sinapic acid and sinigrin in Stir-fried Raphani Semen in rats,and it is related to its antiasthmatic effect. |
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