Studies On The Inhibitory Activity On The HIV Gp41 Six-helix Bundle Formation Of Extractions From Psidium Guajava Leaves

BackgroundAIDS(acquired immunodeficiency syndrome) are communicable diseases caused by human immunodeficiency virus(HIV).Since the initial report of clinical cases of AIDS at 80's the 20th century,the diseases have spread rapidly allover the world, which turned out to be a global outbreak trend recently.It has been put a dramatic impact upon the average life expectancy of the population and the world's economic development,and become the most major public health problem in the 21st century. AIDS has become the most destructive and unprecedented disease.So far,developed countries have invested substantial funds and human resources in the research of HIV and anti-HIV drugs.At present,drugs for the clinical treatment of AIDS are reverse transriptase inhibitor and proteinase inhibitor.By binding on the enzyme or competitive binding on the enzyme with the substrate,these drugs make enzyme denatured and inhibit activity of enzyme,and thus destroy HIV's life cycle and inhibit viral replication.To some extent,these drugs can delay the deterioration of patients,but it can not completely wipe out the virus in patients.A majority of patients have been living at a low level virus replication status.Patients with long-term use these drugs will result in resistance,and if the medication stopped, virus levels in patients restore soon,even higher than the level before treatment. In the course of HIV infection,the first procedure is the fusion of HIV envelope membrane and target cells.This fusion is mainly mediated by gp160.Gp 120 combine CD4 on target cells with supporting elements receptor(chemokine receptor CCR5 or CXCR4,etc.) by turns.Then it resulted in a change in the structure of gp41,and the formation of 6 shares the coreα-helix bundle structure.The 6 shares the coreα-helix bundle structure narrowed the virus envelope with the target cell membrane,and induces fusion.Therefore,inhibiting the process of integration of any one link,you can suppress HIV to enter target cells and thus the prevention and treatment of HIV infection.Gp120,gp41,CD4,chemokine receptor(CXCR4 or CCR5,etc.) can serve as HIV entry inhibitors target the drug.Chemical drugs for AIDS are not efficient enough and have lots of side effects. So many medicine practitioners seek treatments from Chinese traditional medicine for AIDS,so as to promote the research of plants,which used to screen,prevent and treat AIDS.In the analysis of the active ingredients of Chinese herbal medicine,it finds that some polysaccharides,saponins,alkaloids,flavonoids and protein in some Chinese herbs can inhibit HIV replication material,and show good activity against HIV virus.Flavonoids are a large class of compounds which show anti-virus activity.Some studies find that flavonoids show good inhibitory activity against HIV.Saponins are a essential class of natural products in the chemical composition.Saponins have diverse and important pharmacological activity and biological activity,such as anti-tumor, anti-fungal,prevention and treatment of cardiovascular disease,lowering blood sugar and immune regulation and thus show a broad application prospects.Volatile oil,also known as essential oil,are a class of general term of volatile oil-like liquid,which can be distilled from the plants.Distribution of volatile oil in a wide range of plants, hundreds of plants used as traditional Chinese medicine contain volatile oil, particularly in the Compositae,Rutaceae,Umbelliferae,Zingiberaceae.Volatile oil show anti-tumor,anti-bacterial,anti-allergy and anti-viral effect,and may have enzyme inhibitory activity.In the experiments,we extracted flavonoids,saponins and volatile oil from Psidium Guajava Leaves,and studied their inhibitory activity on the HIV gp41 six-helix bundle formation.ObjectivesFlavonoids,saponins and volatile oil isolated from the Psidium Guajava Leaves, were used as a compound library to screen and identify compounds which can effectively inhibit the formation of HIV gp41 core structure,the six-helix bundle structure.So as to provide experimental basis for searching a new efficacious drug for treatment of AIDS.Method1.HeLa Cells were transferred of culture into the 96-well culture plate by the density of 1×10~5/ml,100μl per well.After cells are cultivated 24 hours,the different groups treated with different drugs which were disposed by RPMI-1640 medium: control group and other group of different concentration(flavonoids and saponins are divided into 1mg/ml,0.5mg/ml,250μg/ml,125μg/ml,62.5μg/ml,31.25μg/ml, volatile oil are divided into 50μ/ml,25μg/ml,10μg/ml,1μg/ml,0.1μg/ml).Each group included 4 wells,and 100μl different drugs were added into each well.Being incubated for 24 hours,the cells were detected the optical density which reacts the quantity of living cells by technology of MTT,and get the median toxic concentration (TC50).2.The inhibitory activity of those compounds(saponins,flavonoids and volatile oil) on HIV gp41 six-helix bundle formation was screened by enzyme linked immunosorbent assay(ELISA).Peptide C34 derived from HIV gp41 CHR region and N36 from NHR region,can form a similar helix bundle in vitro like the six-helix bundle formed by CHR and NHR of gp41.Therefore,the inhibitory activity of HIV gp41 six-helix bundle can be determined by inhibiting the interaction of C34 with N36.3.Statistical Analysis:All data used SPSS 13.0 statistical analysis software. Measurement data are expressed as mean±standard deviation((?)±S).Differences among groups were analyzed by One-way ANOVA.LSD(Least Significant Difference)method should be used in the case of homogeneity of variance;otherwise Dunnett's T3 methods should be used in the case of variance nonhomogeneity. P＜0.05 was considered statistically significant.Result1.MTT method measured the cell survival rate,and the relationship between the concentration of the samples is calculated by the PHARM/PCS-VERSION4 software. By calculated we find the TC50 of saponin,flavonoids and volatile oil on HeLa cells is137.6μg/ml,71.62μg/ml,23.63μg/ml respectively.When saponins and flavonoids are at the greatest concentration(1mg/ml) the cell survival rate was 17.28%and 14.53%,at the lowest concentration(0.03125mg/ml) the cell survival rate was 93.32%and 85.64%.When the concentration of volatile oil is 50μg/ml and 0.1μg/ml the cell survival rate was 25.39%and 82.21%,respectively.2.Based on the antigen-antibody reaction of the sandwich ELISA method, saponins,flavonoids and volatile oil were screened at final concentration of 25μg/ml. It showed that saponin and flavone had obvious inhibitory activity on the formation of HIV gp41 six helix bundle structure,and their inhibition rate were more than 50%.While the volatile oil in the final concentration of 25μg/ml showed lower inhibition rate than saponins and flavonoids,which was lower than 50%.Saponins and flavonoids inhibit group was significantly higher than that of volatile oil group. The differences among volatile oil group and the group of saponins,flavonoids group were significant(P＜0.001).However,saponin and flavonoid group compared with no significant difference(P = 0.507).So,after the initial screening volatile oil was discarded,left saponins and flavonoids to do further calculation of the IC50.Saponins, flavonoids diluted into five concentration,25μg/ml,15μg/ml,2.5μg/ml,1μg/ml, 0.25μg/ml gradient.At the concentration of 25μg/ml both of them inhibited most of the formation of gp41 six helix bundle structure.Saponins in the concentration of 2.5μg/ml and flavonoids in the concentration of 1μg/ml at some time be able to suppress six helical bundle structure.Compared to the inhibitory activity on the formation of HIV gp41 six helix bundle structure,flavonoids slightly stronger than the saponin.Through calculated the IC50 of saponins and flavonoids inhibitory concentration were 5.65μg/ml,0.87μg/ml.It showed that saponins and flavonoids could target HIV gp41 and inhibit the formation of the core six-helix bundle structure, might also play a role in blocking HIV fusion with target cells.ConclusionWe had screened saponins,flavonoids and volatile oil isolated from the Psidium Guajava Leaves on the effects of inhibiting the formation of HIV gp41 six-helix bundle.Saponins and flavonoids showed favourable inhibitory activities.Thy study shows a light on how to find HIV fusion inhibitors from Chinese herbs and other natural resourses.