Preparation And Evaluation Of Antitumor Effects In Vivo And In Vitro Of Baicalin Derivatives (BAD And BAL)

Objective:Baicalin(BA)is one of the major components of traditional Chinese medicine called radix scutellariae.it shows widely pharmacological effects such as antipyretic and detoxication,anti-oxidation,anti-inflamm-ation,and anti-tumor,etc.However,its poor water solubility,poor oral absorption and low bioavailability limited its clinical application.Therefore,BA derivatives(BAD and BAL)were synthesized by"green synthetic method"and evaluated their antitumor activities in vivo and in vitro.Methods:1.BAD and BAL were synthesized by using the idea of"green chemistry”.The structures of BAD and BAL were characterized by ultraviolet spectrophotometry(UV),high performance liquid chromatography(HPLC),thin layer chromatography(TLC),nuclear magnetic resonance spectroscopy(NMR)and mass spectrometry(MS).2.UV was used to measure the hydrolysis stabilities of BA,BAD and BAL in 1640 medium,1640 medium containing 10%fetal bovine serum and PBS solution(pH 7.2),respectively.3.MTT method was used to investigate the proliferation and inhibition activity of BA,BAD and BAL against three lung cancer cells(A549,H460 and Calu-1),three liver cancer cells(BEL-7402,SMMC7721 and HepG2)and three breast cancer cells(MDA-MB-435,MCF-7 and T74D)in 24 h and 48 h,respectively.4.The cell apoptosis of BA,BAD and BAL on A549 lung cancer was tested by flow cytometry instrument.5.In order to investigate respectively antitumor effects of BA,BAD and BAL,lung cancer cell line A549 subcutaneous transplantation tumor model in mice was established.Tumor growth,animal weight change,tumor inhibition rate and HE staining were observed to evaluate the effects of BA,BAD and BAL on the tumor.Results:1.The derivatives of BA(BAD and BAL)were synthesized successfu-lly for the first time.And their UV maximum absorption wavelength were278.4nm(BA),282.4nm(BAD)and 282.6nm(BAL),respectively.The contents of BAD and BAL were greater than 95%determined by HPLC method,Rf value of TLC was 0.45 and molecular weight which confirm-ed by ESI-MS m/z was 608.Besides,~1H NMR results were as followed:(400 MHz,DMSO)δ8.08(s,1H),7.98(s,1H),7.83(t,J=13.7 Hz,1H),7.54(s,1H),7.38(d,J=17.5 Hz,1H),7.12(d,J=6.4 Hz,1H),6.97(d,J=8.4 Hz,1H),6.88(d,J=6.6 Hz,1H),3.87(d,J=12.9 Hz,2H),3.80(s,4H),3.64(s,2H),2.15(s,3H).2.After 24 h in the three kinds of media,undegraded percentage of BA,BAD and BAL were followed by 84.73-92.75%,84.63-91.12%and85.59-92.70%,respectively.3.IC-50(μg/ml)on lung cancer cells A549,H460 and Calu-1 at 24h of BA,BAD and BAL,respectively,was as followed:BA:80.31±3.47,109.10±4.21 and 46.59±3.10;BAD:28.04±1.43,37.19±0.88 and37.83±2.94;BAL:34.71±1.78,49.57±2.49 and 31.93±2.91.IC-50(μg/ml)at 48 h was as followed:BA:63.55±2.51,51.96±1.46 and 26.37±0.75;BAD:37.30±1.49,55.20±1.69 and 37.61±1.09;BAL:27.96±1.68,38.86±1.57 and 26.13±1.32.For liver cancer cells SMMC7721,BEL-7402 and HepG2,IC-50(μg/ml)at 24 h was as followed:BA:56.93±3.67,87.62±4.13 and 52.18±2.72;BAD:41.25±2.83,-,-;BAL:33.50±2.07,39.89±3.12,-;IC-50(μg/ml)at 48 h was as followed:BA:40.71±1.09,53.68±2.19,-;BAD:32.28±1.02,44.45±1.53,-;BAL:33.50±2.07,39.89±3.12,-.For breast cancer cells MDA-MB-435,T74D and MCF-7,IC-50(μg/ml)at 24 h was as followed:BA:50.00±3.33,66.14±2.07 and 62.96±3.13;BAD:124.38±4.96,28.89±1.73 and47.34±3.47;BAL:83.53±4.14,25.10±1.07 and 43.58±3.56.IC-50(μg/ml)at 48 h was as followed:BA:18.61±0.56,29.08±0.72 and28.60±0.75;BAD:83.25±1.91,24.33±0.53 and 39.54±1.31;BAL:-,21.93±0.55 and 32.90±0.77.4.Different concentrations of BA(25,50,75μg/ml)on human lung adenocarcinoma cell line A549 early apoptosis percentage(%)were:6.46,11.28 and 12.87,respectively;BAD drugs with different concentra-tions(10,25,50μg/ml)were 5.93,949 and 14.39,respectively;BAL drugs with different concentrations(10,25,50μg/ml)were 11.92,13.99and 34.53,respectively.5.Compared with BA,both BAD and BAL could inhibit the growth of lung cancer cell A549 in nude mice,their tumor inhibition rates were 15.54%,44.68%and 65.27%,respectively.In addition,the nude mice body weight showed no significant difference between the treatment group.And HE staining of tumor results suggested that although tumor cells in the treatment group were no obvious morphological changes,the microvascular richness followed by:blank group≥BA group>BAD group>BAL group.Conclusion:1.BA derivatives(BAD and BAL)were synthesized successfully in this paper.To the best of our konwledge,there were no relevant reports about this novel structure at home and abroad.2.Compared with BA,no significant in the stabilities of BAD and BA-L in the three kinds of medium was observed(P>0.05).3.BAD and BAL had excellent inhibitory effects on proliferation of various tumor cells.Especially they were sensitive to lung cancer cell A549,Calu-1,liver cancer cell SMMC-7721,BEL-7402 and breast cancer cells T74D in comparison with BA.4.There was no significant difference between BAD and BA in promot-ing the apoptosis of A549 cells(P>0.05).Compared with BAD and BA,there was a significant difference(P<0.05)in BAL.Moreover,BAL can significantly improve the percentage of early apoptosis of lung cancer A-549 cells,and with a concentration-dependent manner.5.Compared with BA,BAD and BAL significantly improved their anti-tumor activity.