Preliminary Exploration Of Inhibitory Effect Of Kandelia Candel Fruit Tannins On 3T3-L1 Adipocytes

Obesity often leads to adverse pathological conditions such as type 2 diabetes,cardiovascular disease,and cancers.Current anti-obesity drugs are expensive and limited applicability,accompanied with serious side effects.The research shows that natural plant polyphenols have significant anti-obesity effects and have promising application.Therefore,this study explores the extracts of mangrove plant Kandelia candel fruit in order to replace drugs or make drinks for the prevention and treatment of obesity in the future.Tannins were extracted from Kandelia candel fruits by ultrasonic extraction combined with dextran gel,and the inhibitory effect of this tannin on Mucor oryzae lipase(MML)was studied using MML and p-nitrophenol palmitate p-PNC16 as enzymes and substrates in vitro.In vitro enzymatic experiments showed that the ICso of Kandelia candel fruit tannin on MML-catalyzed p-PNC16 was 288.639 ug/mL,and the enzymatic reaction showed reversible inhibition mechanism and non-competitive inhibition type.The KSV value greater than 100 L/mol was determined by fluorescence quenching experiment as static quenching with slight blue shift.Then 3T3-L1 preadipocyte was selected as the cell model,and the effects of tannin on cell proliferation,cycle,apoptosis and ROS were detected by MTT and flow cytometry.The results showed that the tannin could inhibit cell proliferation,block cell cycle in G2/M phase,and promote cell apoptosis,but had the ability to scavenge intracellular free radicals.Finally,3T3-L1 preadipocytes were induced to differentiate into mature adipocytes by MDI method.Oil red O staining was used to detect whether the differentiation was successful.Flow cytometry was used to detect the effect of this tannin on ROS of mature adipocytes.The effects of this tannin on genes and proteins related to ROS?lipolysis and lipogenesis of mature adipocytes were detected by qRT-PCR and Western blotting.Flow cytometry results showed that ROS production in mature adipocytes could be inhibited;qRT-PCR results showed that it could inhibit transcription levels of gene of TNF-alpha,IL-6,PPARgamma,C/EBPalpha,ACC and FASN,and promote transcription levels of gene of ATGL and HSL;Western blotting results showed that it could upregulate HO-1,p-ACC/total-ACC,p-HSL/total-HSL,p-AMPK/total-AMPK proteins,and down-regulate IKKbeta,FASN,C/EBPalpha and PPARgamma proteins.In this study,it was confirmed that Kandelia candel fruit tannins could inhibit the catalytic activity of MML,scavenge free radicals on 3T3-L1 precursor adipocytes,and also have anti-ROS and inflammation effects on mature adipocytes,which will promote the decomposition of lipids and inhibit the synthesis of lipids.It laid a theoretical foundation for the application of these compounds in weight-loss products.