| Natural products are valuable resources for drug discovery,but the variety of ingredients and complex structures increase the difficulty of obtaining active ingredients.Therefore,it is urgent to use effective drug identification strategies to quickly discover potential target components and accelerate the process of obtaining active natural products.Traditional identification methods have various problems.The bioassay-guided fractionation methods are often tedious and time-consuming,and ligand fishing methods have high technical requirements,besides the enzymes are easy to deactivate.In order to solve the problems in the existing technology,this study has developed a 1H NMR-based biochemometric strategy to quickly and effectively identify and guide the separation of active natural products.Combined with high performance liquid chromatography(HPLC),the separation of active ingredients can be achieved,and the interference of inactive impurities can be eliminated.The specific research is as follows:Firstly,a 1H NMR-based biochemometric strategy was established to identify natural chemosensitizing compounds of colorectal cancer from Rauvolfia vomitoria.HPLC was used to obtain fractions of total alkaloids from Rauvolfia vomitoria.Fractions were subsequently analyzed by 1H NMR spectroscopy.In the meantime,the chemosensitization of those fractions were tested in the with HCT-8 and LoVo colorectal cancer cells using CCK-8(cell counting kit-8)assay.Biochemometrics was used to correlate the 1H NMR data with activity profiles of the fractions and to infer which signals(VIP>1)were important for the bioactivity.The next step was to identify the probable bioactive substances to which these signals belong using 2D NMR spectra.It was speculated that there are β-carboline-type and sarpagine-type alkaloids with potential chemosensitizing activity in Fr.7.Through systematic literature and database searches,the potential active ingredients of chemotherapy sensitization were identified as vellosimdinol,lochnerine,vinnajorine D,1-methyl-β-carboline and β-carboline.Among these five compounds,vellosimdinol,lochnerine,and vinnajorine D are sarpagine-type alkaloids,and 1-methyl-β-carboline and β-carboline are β-carboline-type alkaloids.Secondly,the chemical constituents of total alkaloids of Rauvolfia vomitoria were studied systematically with biochemometric strategy and the compounds were tested for their cytotoxic activity.A total of 21 compounds including 2 new and 19 known ones were isolated after column chromatography of Sephadex LH-20 and semi-preparative HPLC.The structures of the compounds were established on the basis of various spectroscopy techniques.The two new compounds were characterized as monoterpenoid indole alkaloids:rauvomtine A(6,2 mg)and rauvomtine B(7,2 mg).The 19 known compounds were identified as indole alkaloids,β-carboline(1,4 mg),1-methyl-β-carboline(2,10 mg),sarpagine(3,2 mg),lochnerine(4,15 mg),vellosiminol(5,5 mg),tetrahydroalstonine(8,14 mg),isoreserpiline(9,4 mg),10-methoxytetrahydroalstonine(10,2 mg),carapanaubine(11,5 mg),isocarapanaubine(12,4 mg),Na-methylrauflorine(13,5 mg),venoterpine(14,20 mg),mitoridine(15,2 mg),yohimbine(16,32 mg),ajmaline(17,35 mg),vinmajorine D/17-epi-vinmajorine D(18/19,16 mg),peraksine/17-epi-peraksine(20/21,5 mg).By using CCK-8 assay,the antiproliferation effect of some of the isolated compounds were evaluated,and the results showed that compound lochnerine and 1-methyl-β-carboline possessed moderate cytotoxic activities.Meanwhile,lochnerine showed significant inhibitory activity with an IC50 of 24.24 μM on LoVo cells.Further tests demonstrated that lochnerine could significantly increase active oxygen accumulation.The active compounds lochnerine and 1-methyl-β-carboline were isolated from the Fr.7,and those results gave strong evidence of effectiveness of such analytical strategy.Finally,after related literature retrieval,reserpine,yohimbine,ajmaline,ajmalicine,and serpentine were reported as the main bioactive components of R.vomitoria.An efficient and sensitive liquid chromatography tandem mass spectrometry(LC-MS/MS)method was developed and validated for the simultaneous determination of reserpine,yohimbine,ajmaline,ajmalicine and serpentine in rat plasma.The optimized conditions were as follows:50 μL of rat plasma samples were pretreated by a simple protein precipitation procedure.Adequate chromatographic retention was achieved on the Agilent Extend C18 column under gradient elution with acetonitrile and 0.1%formic acid aqueous solution.A triple-quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode.The validation results showed satisfactory accuracy(<10%)and precision(<15%)at linear dynamic range(0.2-200.0 ng/mL for reserpine and serpentine,0.2-100.0 ng/mL for ajmaline and ajmalicine,0.5-100.0 ng/mL for yohimbine).The validated LC-MS/MS method was applied for the determination of pharmacokinetic parameters of five analytes in rat plasma after a single oral administration of R.vomitoria extract(100 mg/kg).The contents of reserpine,yohimbine,ajmaline,ajmalicine and serpentine in the extracts were 4.7,27.04,26.03,26.51,and 25.33 mg/g,respectively.There are certain differences in the time to peak(Tmax)of the five alkaloids.Among them,ajmaline reached its peak quickly at 0.26 h,and the maximum of concentration(Cmax)was 130.67 ng/mL;Yohimbine has a slower Tmax(1.67 h),with Cmax of 299.17 ng/mL.For reserpine,ajmalicine and serpentine,the Tmax was between 0.79-0.96 h,and the Cmax was 3.70,414.67 and 34.94 ng/mL,respectively.For five alkaloids,MRT was 8.58,7.99,4.40,4.95 and 2.41 h,half-time(T1/2)was 5.01,6.41,4.94,5.43 and 1.16 h,respectively.The pharmacokinetic parameters indicate that ajmaline has the fastest absorption,ajmalicine has the highest absorption,and serpentine was eliminated faster than the other four components in vivo.These results indicate that both the absorption rate and elimination rate of alkaloid compounds were affected by structural changes.The aims of this thesis are to develop the screening methods to discover the chemosensitizing components of traditional Chinese medicine on colorectal cancer.A new 1H NMR combined with biochemistry strategy was developed for to screening the active chemosensitizing components of R.vomitoria.It could improve the efficiency in drug discovery using natural products.This work demonstrated the feasibility of NMR and biochemometric strategy in the screening of active ingredients of natural medicinal herbs. |
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