Oligomeric procyanidins combined with Parabacteroides distasonis ameliorate high-fat diet-induced atherosclerosis by regulating lipid metabolism,inflammation reaction and bile acid metabolism in ApoE^(-/-)mice

Atherosclerosis(AS)is the main pathological basis of cardiovascular diseases.Hence,the prevention and treatment strategies of AS have attracted great research attention.As a potential probiotic,Pararabacteroides distasonis has a positive regulatory effect on lipid metabolism and bile acids(BAs)profile.Oligomeric procyanidins have been confirmed to be conducive to the prevention and treatment of AS,whose antiatherosclerotic effect may be associated with the promotion of gut probiotics.However,it remains unclear whether and how oligomeric procyanidins and P.distasonis combined(PPC)treatment can effectively alleviate high-fat diet(HFD)-induced AS.In this study,PPC treatment was found to significantly decrease atherosclerotic lesion,as well as alleviate the lipid metabolism disorder,inflammation and oxidative stress injury in ApoE^(-/-)mice.Surprisingly,targeted metabolomics demonstrated that PPC intervention altered the BA profile in mice by regulating the ratio of secondary BAs to primary BAs,and increased fecal BAs excretion.Further,quantitative polymerase chain reaction(qPCR)analysis showed that PPC intervention facilitated reverse cholesterol transport by upregulating Srb1 expression;In addition,PPC intervention promoted BA synthesis from cholesterol in liver by upregulating Cyp7a1 expression via suppression of the farnesoid X receptor(FXR)pathway,thus exhibiting a significant serum cholesterol-lowering effect.In summary,PPC attenuated HFD-induced AS in ApoE^(-/-)mice,which provides new insights into the design of novel and efficient anti-atherosclerotic strategies to prevent AS based on probiotics and prebiotics.

Diet with high content of advanced glycation end products induces oxidative stress damage and systemic inflammation in experimental mice: protective effect of peanut skin procyanidins

Non-enzymatic glycation reaction in food can produce diet-derived advanced glycation end products(dAGEs),which have potential health risks.Thus,it is of great significance to find efficient substances to improve the negative effects induced by dAGEs on human health.This study investigated the intervening effects of peanut skin procyanidins(PSP)on the dAGEs-induced oxidative stress and systemic inflammation in experimental mice model.Results showed that the accumulation of AGEs in serum,liver,and kidney was significantly increased after mice were fed dAGEs(P<0.05).The expression of advanced glycation product receptor(RAGE)was also significantly increased in liver and kidney(P<0.05).PSP could not only effectively reduce the accumulation of AGEs in serum,liver and kidney of mice,but also reduce the expression of RAGE in liver and kidney of mice.And the levels of pro-inflammatory cytokines interleukin-6(IL-6),tumor necrosis factor(TNF-α),and IL-1βin serum of mice were significantly decreased(P<0.05),while the levels of antiinflammatory factor IL-10 were increased,and the inflammatory injury in mice was improved.In addition,the levels of superoxide dismutase(SOD),glutathione(GSH),catalase(CAT)in liver and kidney of mice were increased(P<0.05),and the level of malondialdehyde(MDA)was decreased(P<0.05),which enhanced the antioxidant capacity of mice in vivo,and improved the oxidative damage of liver and kidney.Molecular docking technique was used to confirm that the parent compound of procyanidins and its main metabolites,such as 3-hydroxyphenylacetic acid,could interact with RAGE,which might inhibit the activation of nuclear transcription factor(NF-κB),and ultimately reduce oxidative stress and inflammation in mice.

Effect of dual targeting procyanidins nanoparticles on metabolomics of lipopolysaccharide-stimulated inflammatory macrophages

Inflammation plays an important role in the occurrence and development of many inflammatory diseases.The purpose of this study was to evaluate the anti-inflammatory effect and metabolic behavior of the dual targeting procyanidins(PC)nanoparticles on lipopolysaccharide(LPS)-stimulated inflammatory macrophages by metabolomics method.The double-targeting PC nanoparticles could specifi cally target both the CD44 receptor and mitochondria,while the single targeting PC-loaded nanoparticles that could target the CD44 receptor on the surface of macrophages.The double-targeting PC nanoparticles had better inhibitory effect than single-targeting PC nanoparticles on the leakage of lactate dehydrogenase and reactive oxygen species overexpression induced by LPS.Amino acid metabolism,energy metabolism and purine metabolism were disordered in LPS-treated group,and metabolic pathway analysis indicated that the double-targeting PC nanoparticles reversed some of LPS impacts.The changes of these potential biomarkers and their corresponding pathways are helpful to further understand the mechanism of PC nanoparticles in alleviating inflammation,and promote their application in nutrition intervention.

Procyanidins Inhibit Tumor Angiogenesis by Crosslinking Extracellular Matrix

Objective： Procyanidins （PC） are widely available natural polyphenols. The present study is designed to investigate if PC can inhibit angiogenesis in lung adenocarcinoma xenografts through crosslinking vascular extracellular matrix （ECM） and preventing proteolysis by matrix metalloproteinases （MMPs）. Methods： Using the in vitro MMP-2 proteolysis and in vivo subcutaneous implantation models, we investigated if PC crosslinking inhibits MMP-mediated proteolysis. Using a cultured cell detachment assay, an in vitro angiogenesis assay, and a cell proliferation assay, we investigated if PC inhibits MMP-2-mediated endothelial cell detachment, angiogenesis, and cell proliferation, respectively. Using tumor xenografts, we evaluated if PC can inhibit growth of lung adenocarcinoma. Results： PC crosslink vascular ECM proteins, protecting them against proteolysis by MMPs in vitro and in vivo, protecting cultured human umbilical vein endothelial cells from detachment by MMP-2, and inhibiting in vitro angiogenesis. However, PC （0.75-100 μg/mL） did not inhibit vascular and tumor cells proliferation. PC injections （30 mg PC/kg bodyweight） in situ had anticancer effects on xenografts of lung adenocarcinoma, most likely by inhibiting angiogenesis during ECM proteolysis by MMPs. Conclusion： The results suggest that PC may be important MMP inhibitors that can be used as therapeutic anticancer agents.

The Protective Role of Procyanidins and Lycopene Against Mercuric Chloride Renal Damage in Rats

Objective This study aims to investigate the protection of procyanidins and lycopene from the renal damage induced by mercuric chloride.Methods Rats were treated with either procyanidins or lycopene 2h before HgCl 2 subcutaneously injection,once daily treatment for 2 successive days.Results In comparison with HgCl 2 group,markers of renal function such as blood urea nitrogen in serum and urinary protein were decreased to （18.45±11.63） mmol/L and （15.93±9.36） mmol/L,（4.54±0.78） g/（g Cr） and （4.40±1.12） g/（g Cr）.N‐acetyl‐beta‐D‐glucosaminidase,lactate dehydrogenase,alkaline phosphatase in urine were depressed to （125.49±11.68） U/（g Cr）,（103.73±21.79） U/（g Cr）,（101.99±12.28） U/（g Cr）,and （113.19±23.74） U/（g Cr）,（71.14±21.80） U/（g Cr）,（73.64±21.51） U/（g Cr） in procyanidins and lycopene groups.Indicators of oxidative stress,for example,Glutathion was reduced to （45.58±9.89） μmol/（g pro） and （45.33±5.90） μmol/（g pro）,and antioxidant enzymes such as superoxide dismutase,glutathione‐peroxidase were enhanced to （43.07±10.97） U/（mg pro） and （39.94±6.04） U/（mg pro）,（83.85±18.48） U/（mg pro）,and （85.62±12.68） U/（mg pro）.Malondialdehyde was lowered to （0.95±0.12） （μmol/g pro） and （1.03±0.12） μmol/（g pro） in procyanidins and lycopene groups.ROS generation was decreased by 27.63% and 16.40% and apoptosis was also decreased in procyanidins and lycopene groups respectively.Pathological changes were much better as well.Conclusion Procyanidins and Lycopene play some protective role against mercury kidney damage.

Technical Study on Extraction of Procyanidins in Lycium ruthenicum Murr. Using Sub-critical Fluid 1,1,1,2-Tetrafluoroethane( R134a)and Content Determination from Different Producing Areas

[Objectives] To study the optimal conditions for extracting procyanidins fromLycium ruthenicum Murr. with sub-critical fluid R134 a( 1,1,1,2-tetrafluoroethane) in 1 L extraction kettle. [Methods]Taking the extraction rate of procyanidins as an indicator,the influence of pressure,temperature,and extraction time on extraction rate of procyanidins fromL. Ruthenicum Murr. was studied by single factor experimental methods and orthogonal array design. [Results]The order of factors affecting extraction rate of procyanidins was extraction temperature > extraction pressure > extraction time. The optimum extraction conditions were as follows: the extraction rate of procyanidins fromL. ruthenicum Murr. was the highest with extraction pressure of 1. 2 MPa,extraction temperature of 50℃ and extraction time of 90 min. The content of procyanidins in L. ruthenicum Murr. from different producing areas was determined by vanillin-HCl method under the optimal conditions. [Conclusions] The method has the advantages of easy operation,good selectivity,low extraction temperature and high extraction efficiency,which is suitable for extraction of procyanidins in L. ruthenicum Murr.

Efficient semisynthetic approach for large preparation of procyanidin C1 through degradation of grape seed polymeric procyanidins

An efficient method for producing trimeric procyanidin C1(PCC1)was developed through degradation of grape seed polymeric procyanidins(PPCs),using epicatechin(EC)as nucleophile and hydrochloric acid as catalyst.With the yield of PCC1 as the evaluation index,the degradation conditions were optimized by Box-Behnken Design(BBD)based on the results of single-factor experiments.The results showed that the optimal conditions were reaction temperature of 40℃,the ratio of EC and PPCs 2:1,acidity of 0.10 mol/L,and reaction time of 20 min.The yield of PCC1 reached up to 17.7 mg by only one-step degradation of 3 g PPCs.This work proposed a new method for large preparation of PCC1 from waste grape seed polymeric procyanidins.

Crosslinking of Bovine Pericardia Using Procyanidins

The aim of this study was to evaluate the crosslinking effect of a naturally crosslinking reagent-procyanidins （PA）-on the materials of bioprosthetic heart valves. After fixing bovine pericardial tissues by procyanidins, crosslikng characteristics, mechanical properties, in vitro enzymatic degradation resistance, the hydrophilicity and hemolysis tests were examined. The results showed that the fixation of biological tissue with glutaraldehyde （GA） or procyanidins increased its denaturation temperature, the surface hydrophilieity and mechanical properties as well as in vitro enzymatic degradation resistance. There were no significant differences in denaturation temperature, mechanical properties, the hydrophilicity and the in vitro enzymatic degradation between the glutaraldehyde and procyanidins fixed tissues. However, the ultimate tensile strength of the procyanidins fixed tissues was significantly superior to the glutaraldehyde fixed tissues. The hemolysis tests showed that hemolysis rate of the proeyanidins fixed tissues was lower than that of the glutaraldehyde fixed tissues. This study shows that procyanidins can crosslink which bovine pericardiaa effectively without toxicity. Our results suggest that this method might be a useful approach for the preparation of bioprosthetic heart valve.

Inhibitory effects of grape procyanidins on free radical-induced cell damage in rat hepatocytes in vitro

AIM： To study the protective effect of grape procyanidins on oxidative injury induced by ethanol and carbon tetrachloride in rat hepatocytes. METHODS： Normal rat hepatocytes as well as cells damaged by ethanol or carbon tetrachloride were incubated with different doses of grape procyanidins for 24 h. Cell proliferation, apoptosis and TNFα mRNA expression were subsequently determined using MTT assay, cell death ELISA and in situ hybridization. RESULTS： Proliferative levels of the control cells from ethanol and CCh injury groups significantly decreased while apoptosis and TNFα mRNA expression significantly increased compared to the normal control and grape procyanidins co-treatment groups （0.455 ± 0.051 vs 0.318 ±0.045, P 〈 0.05）. In comparison with the normal control, 50 and 100 mg/L grape procyanidins significantly stimulated cell growth, with a better effect observed with 100 mg/L grape procyanidins. CONCLUSION： Grape procyanidins inhibit the hepatocyte damage induced by ethanol and carbon tetrachloride, and stimulate normal hepatocyte proliferation.

Optimization of Enzyme-assisted Extraction Technology for Tartary Buckwheat Shell Procyanidins with Response Surface Methodology

This study was conducted to investigate the effects of cellulase dosage, enzymolysis time, pH and enzymolysis temperature on procyanidin extraction rate by single factor experiment, with tartary buckwheat shell as an experimental material.Main process parameters were optimized to obtain a regression model by response surface methodology. The results of variance analysis indicated that the regression model reflected the relationship between buckwheat shell procyanidin extraction rate with enzyme dosage, enzymolysis time, pH and enzymolysis temperature; and the optimal process parameters were enzyme dosage of 6.5 mg/g, enzymolysis time of 1.5 h, pH at 4.7 and enzymolysis temperature at 46 ℃. Three parallel experiments were conducted under these process parameters. In practice, the highest procyanidin extraction rate was 6.78 g/100 g. The relative error between the predicted value of regression model and the actual value was 1.3%. The regression equation fitted the real situation better.

Effect of enzymatic extraction conditions on the yield and the polymerization degree of procyanidins

Grape seeds are rich sources of procyanidin(PCs)known for potential health benefi ts.In this study,PCs were extracted from defatted grape seeds by enzymatic method in which pectinase and cellulase were used.The enzyme extraction process was further optimized by single factor experiment and response surface methodology.The optimal conditions were as follows:ethanol concentration of 70%,extraction time of 70 min,extraction temperature of 35℃,liquid/solid ratio of 103:1(mL/g),pectinase/cellulase ratio of 1:1,enzyme/solid ratio of 1:314 w/w.Under the above conditions,the extraction yields and mean degree of polymerisation(mDP)of PCs reached 47.18 mg/g dry material weight and 11.2,respectively.Compared with other extraction methods,enzyme extraction can obtain PCs with higher yield and lower mDP.According to the antioxidant activity test,PCs extracts with lower mDP showed better ability to clear 1,1-Diphenyl-2-picrylhydrazyl radical(DPPH).Enzymatic extraction was an effi cient method to obtain oligomeric procyanidin which has stronger antioxidant activity.

The mixture of procyanidins extracted from the lotus seed pod and bilobalide ameliorates scopolamine-induced memory impairment in mice

Objective To study the co-effect of procyanidins extracted from the lotus seed pod （LSPC） and bilobalide （BIL） on ameliorating scopolamine-induced learning and memory impairment in young mice. Methods Fifty male Kunming mice with similar learning and memory capabilities were selected by Morris water maze test and were randomized into 5 groups （n=10 in each group）： control group, scopolamine group, L-（LSPC＋BIL） group （50 mg/kg LSPC＋10 mg/kg BIL）, M-（LSPC＋BIL） group （100 mg/kg LSPC＋20 mg/kg BIL）, H-（LSPC＋BIL） group （150 mg/kg LSPC＋30 mg/kg BIL）. Scopolamine model with impaired learning and memory was established by scopolamine treatment （1 mg/kg）, and after 10 min mice were tested. In L-, M-, and H- （LSPC＋BIL） groups, mice were treated with LSPC and BIL ig. for 30 days, while mice in the other 2 groups were treated with normal saline ig. instead. After the 30-day＇s treatment, the co-effect of LSPC and BIL on learning and memory was tested by Morris water maze and the step-down avoidance tests. Results The memory impairment caused by scopolamine in young mice could be ameliorated by co-treatment of LSPC and BIL, as indicated by significantly shorter escape latency and swimming distance in the Morris water maze test, when compared with those in the scopolamine group. In the step-down avoidance test, mice in all the 3 dose groups showed significantly smaller number of errors and longer latency than mice in the scopolamine group did. Conclusion Co-treatment of LSPE and BIL can ameliorate scopolamine-induced learning and memory impairment in young mice.

Structural Elucidation of the Polymeric Condensed Tannins of Acacia nilotica Subspecies by ^(13)C NMR, MALDI-TOF and TMA as Sources of Bioadhesives

Tannin was extracted from different subspecies of Acacia nilotica,Acacia nilotica nilotica(Ann),Acacia nilotica tomentosa(Ant)and Acacia nilotica adansonii(Ana).The aim was to elucidate their structure and evaluate their reactivity as bioadhesives in the wood industry.The extracts were prepared by hot water extraction(90°C tem-perature).Their gel time with paraformaldehyde was used atfirst to compare their reactivity.The tannin contents and the percentage of total polyphenolic materials in different solutions of the extracts spray dried powder were determined by the hide powder method.Concentrated solutions(47%)were tested by both MALDI ToF,13CNMR.The thermomechanical analysis(TMA)was performed to evaluate their modulus of elasticity(MOE)at different pHs.The gel times of all the three tannin extracts showed that their reactivity and it was com-parable to other known procyanidin/prodelphinidin tannin extract types.Ana,Ann and Ant showed highest per-cent of total polyphenolic materials at 70%,64%,and 57%,respectively.The 13CNMR spectra showed that the three subspecies of condensed tannins were mainly constituted of procyanidins(PC)and prodelphinidins(PD)in slightly different ratios.Ann(56.5%PC and 43.4%PD),Ant(57%PC and 43%PD)and Ana(58%PC and 42%PD).MALDI–TOF spectra showed the presence offlavonoid monomers,and oligomers some of which linked to short carbohydrates monomers or dimers.TMA revealed that the three types of tannins had high MOE at their initial pH(5).

Procyanidins-crosslinked small intestine submucosa: A bladder patch promotes smooth muscle regeneration and bladder function restoration in a rabbit model

Currently the standard surgical treatment for bladder defects is augmentation cystoplasty with autologous tissues,which has many side effects.Biomaterials such as small intestine submucosa(SIS)can provide an alternative scaffold for the repair as bladder patches.Previous studies have shown that SIS could enhance the capacity and compliance of the bladder,but its application is hindered by issues like limited smooth muscle regeneration and stone formation since the fast degradation and poor mechanical properties of the SIS.Procyanidins(PC),a natural bio-crosslinking agent,has shown anti-calcification,anti-inflammatory and anti-oxidation properties.More importantly,PC and SIS can crosslink through hydrogen bonds,which may endow the material with enhanced mechanical property and stabilized functionalities.In this study,various concentrations of PC-crosslinked SIS(PC-SIS)were prepared to repair the full-thickness bladder defects,with an aim to reduce complications and enhance bladder functions.In vitro assays showed that the crosslinking has conferred the biomaterial with superior mechanical property and anti-calcification property,ability to promote smooth muscle cell adhesion and upregulate functional genes expression.Using a rabbit model with bladder defects,we demonstrated that the PC-SIS scaffold can rapidly promote in situ tissue regrowth and regeneration,in particular smooth muscle remodeling and improvement of urinary functions.The PC-SIS scaffold has therefore provided a promising material for the reconstruction of a functional bladder.

Oligomeric Procyanidins Induce Generation of Reactive Oxygen Species and Collapse of Mitochchondrial Membrane Potential in Glioblastoma Cell Lines

Objective The aim of the present study was to clarify the mechanism underlying glioma cell death upon oligomeric procyanidins (F2) exposure. Methods The cytotoxicity of F2 on U87 (human malignant glioblastoma cell line) and C6 (rat glioma cell line) cancer cells was evaluated, and changes of mitochondrial membrane potential (MMP) and production of reactive oxygen species (ROS) in drug-treated cells were monitored. Moreover, morphological changes associated with F2-induced cells death were examined. Results F2 induced a concentration-dependent increase in ROS production and decrease in MMP. Furthermore, pre-incubation with N-acetylcysteine (NAC) and rotenone (Rt), resulted in partial inhibition of F2-induced ROS generation and marked attenuation of cell death and the cytoplasmic vacuolization induced by F2. In addition, pretreatment with Rt markedly attenuated the MMP loss in F2-treated cells. However, pretreatment with NAC only markedly attenuated the MMP loss in F2-treated C6 cells. Conclusion The increase in ROS level is at least one of mechanisms associated with F2-induced glioma cell death as well as the cytoplasmic vacuolization formation that contribute to the cytotoxicity of F2 in glioma cells.

Procyanidin A_1 and its digestive products alleviate acrylamide-induced IPEC-J2 cell damage through regulating Keap1/Nrf2 pathway

Our previous study has revealed that procyanidin A_(1)(A_(1))and its simulated digestive product(D-A,)can alleviate acrylamide(ACR)-induced intestine cell damage.However,the underlying mechanism remains unknown.In this study,we elucidated the molecular mechanism for and D-A_(1) to alleviate ACR-stimulated IPEC-J2 cell damage.ACR slightly activated nuclear factor erythroid 2-related factor 2(Nrf2)signaling and its target genes,but this activation could not reduce intestine cell damage.A_(1) and D-A_(1) could alleviate ACR-induced cell damage,but the effect was abrogated in cells transiently transfected with Nrf2 small interfering RNA(siRNA).Further investigation confirmed that A_(1) and D-A_(1) interacted with Ketch-like ECH-associated protein 1(Keapl),which boosted the stabilization of Nrf2,subsequently promoted the translocation of Nrf2 into the nucleus,and further increased the expression of antioxidant proteins,thereby inhibiting glutathione(GSH)consumption,maintaining redox balance and eventually alleviating ACR-induced cell damage.Importantly,there was no difference between A_(1) and D-A_(1) treated groups,indicating that A_(1) can tolerate gastrointestinal digestion and may be a potential compound to limit the toxicity of ACR.

Antioxidant procyanidin B2 protects oocytes against cryoinjuries via mitochondria regulated cortical tension

Background:Irreversible cryodamage caused by oocyte vitrification limited its wild application in female fertility preservation.Antioxidants were always used to antagonist the oxidative stress caused by vitrification.However,the comprehensive mechanism underlying the protective role of antioxidants has not been studied.Procyanidin B2(PCB2)is a potent natural antioxidant and its functions in response to vitrification are still unknown.In this study,the effects of PCB2 on vitrified-thawed oocytes and subsequent embryo development were explored,and the mechanisms underlying the protective role of PCB2 were systematically elucidated.Results:Vitrification induced a marked decline in oocyte quality,while PCB2 could improve oocyte viability and further development after parthenogenetic activation.A subsequent study indicated that PCB2 effectively attenuated vitrification-induced oxidative stress,rescued mitochondrial dysfunction,and improved cell viability.Moreover,PCB2 also acts as a cortical tension regulator apart from strong antioxidant properties.Increased cortical tension caused by PCB2 would maintain normal spindle morphology and promote migration,ensure correct meiosis progression and finally reduce the aneuploidy rate in vitrified oocytes.Further study reveals that ATP biosynthesis plays a crucial role in cortical tension regulation,and PCB2 effectively increased the cortical tension through the electron transfer chain pathway.Additionally,PCB2 would elevate the cortical tension in embryo cells at morula and blastocyst stages and further improve blastocyst quality.What's more,targeted metabolomics shows that PCB2 has a beneficial effect on blastocyst formation by mediating saccharides and amino acids metabolism.Conclusions:Antioxidant PCB2 exhibits multi-protective roles in response to vitrification stimuli through mitochondria-mediated cortical tension regulation.

超声提取葡萄籽原花青素工艺的优化及其抗氧化活性研究

研究利用响应曲面法优化超声提取葡萄籽原花青素的工艺。在单因素实验基础上,采用中心组合设计响应面实验,考察了提取温度、液料比、乙醇浓度以及超声时间对原花青素提取率的影响,并建立回归模型。优化后的工艺:提取温度55℃,液料比20(mL/g),乙醇浓度65%,超声时间10min;在此条件下葡萄籽原花青素的提取率为2.482%,与回归模型预测值的相对偏差为0.36%。同时进行了提取物的抗氧化性检验,结果表明葡萄籽原花青素对超氧阴离子自由基具有较好的清除能力,且呈剂量相关性。

荔枝核化学成分的研究

利用硅胶柱色谱、聚酰胺柱色谱、制备薄层色谱等方法，从荔枝（Litchi chinensis Sonn．）核乙醇提取物中分离出9个化合物，根据理化性质和波谱分析鉴定了7个化合物，分别为胡萝卜苷（daucosterol，1）、原儿茶醛（protocatechuic aldehyde，2）、原儿茶酸（protoeatechuic acid，3）、表儿茶素（（-）-epicafichin，4）、procyanidin A2（5）、procyanidin A1（6）、芦丁（rulin，7）。化合物2,4-7为首次从荔枝核中分得，化合物2．4、5为首次从该属植物中分离得到。

Effects of Pinus massoniana bark extract on cell proliferation and apoptosis of human hepatoma BEL-7402 cells

AIM: To study the effects of Pinus massoniana bark extract (PMBE) on cell proliferation and apoptosis of human hepatoma BEL-7402 cells and to elucidate its molecular mechanism.METHODS: BEL-7402 cells were incubated with various concentrations (20-200 μg/mL) of PMBE for different periods of time. After 48 h, cell proliferation was determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was evaluated by morphological observation, agarose gel electrophoresis,and flow cytometry analysis. Possible molecular mechanisms were primarily explored through immunohistochemical staining.RESULTS: PMBE (20-200 μg/mL) significantly suppressed BEL-7402 cell proliferation in a time- and dose-dependent manner. After treatment of BEL-7402 cells with 160 μg/mL PMBE for 24, 48, or 72 h, a typical apoptotic 'DNA ladder'was observed using agarose gel electrophoresis. Nuclear condensation and boundary aggregation or split, apoptotic bodies were seen by fluorescence and electron microscopy.Sub-G1 curves were displayed by flow cytometry analysis.PMBE decreased the expression levels of Bcl-2 protein in a time-dependent manner after treatment of cells with 160 μg/mL PMBE.CONCLUSION: PMBE suppresses proliferation of BEL-7402 cells in a time- and dose-dependent manner and induces cell apoptosis by possibly downregulating the expression of the bcl-2 gene.