| Curcumin,[1,7-bis(4-hydroxy-5-methoxy)phenyl-1,6-heptane-3,5-dione]is phenolic compound isolated from the rhizome of Curcuma longa L.and been used as traditional medicines in India and China.Pharmacological studies have shown that curcumin has anti-tumor,anti-liver fibrosis,anti-inflammatory,anti-microbial,anti-oxidant etc.Curcumin is currently being tested in clinical trials to treat various types of cancer,including liver cancer,colon cancer,multiple myeloma,pancreatic cancer etc.However,its relatively poor solubility and instability at physiological condition limited its absorption and bioavailability to a great extent,which restricts the therapeutic usage of curcumin.Oligopeptide transporter 1(PepT1),is a low-affinity,high transporter protein,responsible for the uptake of dipeptides and tripeptides,which belongs to proton-dependent oligopeptide transporter(POT)family.PepT1 as been widely explored as a target for increasing the membrane permeability and oral bioavailability of poorly absorbed drugsIn this work,curcumin was used as the research object,and 10 new curcumin dipeptide derivatives were designed and synthesized based on PepT1.Ten compounds were identified and the solubility of curcumin and its dipeptide derivatives was determined by HPLC.The fluorescence method was used to assessed the uptake of curcumin and its dipeptide derivatives.The LC-MS/MS method was applied to determined cellular uptake.The Caco-2 cell model was established to evaluate drug transmembrane transport.The metabolism was further researched by in vitro metabolic model(rat liver microsomal enzyme incubation).Human liver stellate cells LX-2 and liver cancer cells HepG-2 and SMMC-7721 were used to evaluate their anti-proliferation activity on hepatic stellate cells and anti-tumor activity.The research content is as follows:1.Synthesis and Solubility Determination of Curcumin Dipeptide DerivativesIn this paper,ten curcumin dipeptide derivatives(5a-5j)were designed and synthesized by glycine,valine,alanine,phenylalanine and isoleucine,and ten dipeptides were obtained by DCC condensation.The N-Boc protection of amino group in dipeptides were deprotected with Trifluoroacetate(TFA)(20%)in dry dichloromethane,and and curcumin and dipeptide were linked by chloroacetyl chloride.then the methyl ester group is removed under alkaline conditions.The structure was confirmed by HRMS-ESI,1H NMR and 13C NMR.The HPLC was utilizated to determine the purity,solubility and chemical stability of compounds.The results showed that the solubility and stability of the ten new compounds were improved compared with curcumin.2.Absorption Evaluation of Curcumin Dipeptide DerivativesThe Caco-2 cell model was used to evaluate the absorption of curcumin and 10 compounds in vitro.1)The fluorescence intensity of drugs in cells was measured by full-wavelength scanning multifunctional microplate reader instrument,and optimal uptake time was 60 min,optimal concentration was 100 μM.The uptake of curcumin and compounds 5a-5j in Caco-2 cells was determined by microplate reader instrument.The results showed that compounds 5d and 5e showed stronger fluorescence intensity than curcumin,and the others were worse than curcumin.By adding the PepT1 classical substrate Gly-Sar as an inhibitor,curcumin uptake was not related to Gly-Sar,and the fluorescence intensity of 5d and 5e could be significantly decreased by Gly-Sar.2)The LC-MS/MS method was established to determine curcumin and its dipeptide derivatives in cell lysate and determined the uptake of curcumin and its compounds 5d and 5e in Caco-2 cells and in the presence or absence of inhibitor Gly-Sar.The results showed that compared with curcumin,compounds 5d and 5e had higher cellular uptake in Caco-2 cells.The celluar uptake of curcumin was not related to Gly-Sar,but celluar uptake of 5d and 5e in Caco-2 cells was inhibited in a dose-dependent manner by adding Gly-Sar.The enhanced cellular uptake may be attributed to PepTl-mediated uptake and their differential affinity toward PepTl.3)The Transwell chamber model of Caco-2 cells was established to investigate the transmembrane transport of curcumin,compounds 5d and 5e.According to the measured apparent permeability coefficient(Papp),compounds 5d and 5e were 1.5 times and 2.8 times than that of curcumin,and 5e permeation absorption was significantly better than curcumin.The accumulation of 5d and 5e was significantly higher than that of curcumin,and the transport was significantly reduced with the participation of Gly-Sar,further demonstrating that the increased absorption of compounds 5d and 5e was due to PepT1.4)In order to investigate whether compounds 5d and 5e may be prodrugs of curcumin,a rat liver microsome incubation model in vitro was constructed,and phase I metabolism was studied by NADPH hydrogen supply,and glucuronic acid binding reaction was carried out with UDPGA as a glycosyl donor.The prototype drug and its metabolic metabolites were analyzed by liquid chromatography-high resolution mass spectrometry(UFLC-QTOF-MS).The results showed that curcumin was detected in the metabolites of compounds 5d and 5e;compounds 5d and 5e detected tetrahydrogen,hexahydrogen and other reduction products and glucuronic acid binding products in liver microsomes,suggesting that compounds 5d and 5e may be the prodrug of curcumin,but this need further study.3.Biological Activity Evaluation of Curcumin Dipeptide DerivativesThe anti-proliferation activity on hepatic stellate cells of curcumin and its dipeptide derivatives were evaluated against human hepatic stellate cell LX-2 in vitro by MTT assay.Most of the derivatives showed effective activities,especially the IC50 value of compound 5e was 13.08μM.which showed higher efficacy than curcumin(IC50＝22.62 μM).In order to explore the antitumor effects of curcumin and its dipeptide derivatives,the antitumor activities of curcumin against human hepatocellular carcinoma cell lines HepG2 and SMMCC-7721 in vitro were evaluated by MTT assay.Most of the derivatives showed effective antitumor activities,especially the IC50 value of compound 5e were 23.35μM and 19.67 δμM in HepG2 and SMMC-7721 respectively,which showed higher potencies than curcumin(HepG2:IC50=35.81μM,SMMC-7721:IC50=22.89μM).Compound 5d(HepG2:IC50=40.25μM,SMMC-7721:IC50=35.40μM)showed similar activity to curcumin. |
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