| In recent year, the outbreaks of SARS, MERS, avian flu, Ebola, and other emerging infectious diseases have become more and more frequent. As viral infections are posing increasingly serious threats, existing or potential, to human society, people now face more pressure to prevent and control the outbreaks of infectious diseases. But more and more evidences indicate that the existing medications with some specificity are normally less effective or even ineffective in preventing and controlling infectious diseases that are induced by virus variants or new viruses. Moreover, researchers are unable to develop new medicines with remarkable efficacy and specificity for disease prevention and treatment in a short period of time, due to the limits of our level of cognition and scientific research cycle. This can easily lead to rapid spread and regional catastrophic outbreaks of epidemics and thus cause great damage to local public health service, politics and economy. Therefore, new types of broad-spectrum viral infection inhibitors with lasting efficacy should be researched and developed according to the basic biological properties of viruses and their common targets in host cells. This is significant for coping with the ongoing challenges posed by viral infections.Phytochemicals, also known as the Seventh Nutrients, are chemical common compounds in our daily foods. They are a type of non-nutritional chemicals that have special effects on human health. Related research has discovered that some phytochemicals have biological activities that can inhibit viral infections. These phytochemicals, such as(-)-epigallocatechin gallate(EGCG), Tannins, and Astragalus Polysacharin(APS), are gradually becoming a hot topic in antivirals research. Preliminary research results indicated that phytochemicals’ biological activities are mainly reflected in their effects on the biological characteristics of cell membrane and the balance of local microenvironment. By embedding themselves in the lipid bilayer structure of cell membrane, some phytochemicals can change the normal functional flow of cell membrane and then perform their biological activities. Virus is a type of intracellular parasitic organism, and only the viruses that enter host cells through cell membrane can complete their infection and replication cycles. Therefore, cell membrane is the first, common, and unavoidable barrier for most viruses to enter cells and induce infection. From the perspective of host cells, the invasion of viruses is a pathologic process that will inevitably result in some changes in the physiological properties of cell membrane to some degree, such as changes in the fluidity of the lipid bilayer structure and in the electric potential of cell membrane. Previous research on the mechanism of these phytochemicals’ inhibition of viral infections mostly focused on disturbing the specific infection target of one virus, while the phytochemicals’ broad-spectrum inhibition of viral infections has seldom been studied. In particular, no research has been conducted on if the phytochemicals’ biological activities can resist or disrupt viruses’ pathologic effects on the infected host cells and then obstruct the invasion, pinocytosis and endocytosis of the virusesIn this study, a preliminary assessment of the viral infection-inhibiting effects of EGCG, Tannins, and APS was made based on the cell infection models of influenza virus and adenovirus. The patterns and efficacy of the three phytochemicals’ viral infectioninhibiting effects in different conditions were investigated. Then using the techniques of fluorescence polarization and fluorescence labeling for membrane potential detection respectively, the influences of these phytochemicals and viruses on cell membrane’s fluidity and potential as well as the patterns of resulting changes in different conditions were compared. Through the preliminary exploration of the mechanisms of phytochemicals’ broad-spectrum inhibition of viral infections, the research is expected to provide a technical support for the research and development of broad-spectrum viral infection inhibitors and help improve the whole society’s capability of preventing and controlling viral infections. 1. Analysis of inhibitory effect on EGCG, tannins and APS in vitro viral infectionThe adenovirus-5 cell infection model and A H1N1 cell infection model were used in combination with neutral erythrocyte phagocytosis technology for the analysis of toxicity of three plant compounds EGCG, tannin and APS for 293 A cells and MDCK cells. The results indicated that toxic concentration 0(TC0) of EGCG, tannins and APS for 293 A cells was respectively 640μg/m L, 1,280μg/m L and 1,280μg/m L, and that of the control sample ribavirin was 640μg/m L; TC0 of EGCG, tannins and APS for MDCK cells was respectively 1,280μg/m L, 640μg/m L and 1,280μg/m L, and that of the control sample ribavirin was 640μg/m L. Apparently, the cytotoxicity of these three plant compounds is not higher than that of the control sample ribavirin that has obtained the safety permit.To further clarify the inhibitory effect of these three plant compounds on in vitro viral infection, adenovirus-5 and A H1N1 of 100TCID50 were used. Before, during and after viral infection, three plant compounds were respectively added. The cytopathic effect(CPE) was observed to analyze the effective concentration of these three plant compounds to inhibit CPE. The results showed that with adenovirus infection, inhibitory concentration 50(IC50) of EGCG under the above three conditions was respectively 162±11μg/m L, 607±29μg/m L and 325±26μg/m L; IC50 of tannins was respectively 303±47μg/m L, 197±19μg/m L and 400±40μg/m L; APS had certain inhibitory effect only added before viral adsorption, whose IC50 was 449±50μg/m L; IC50 of the control sample ribavirin was respectively 604±25μg/m L, 561±52μg/m L and 177±27μg/m L. With A H1N1 infection, inhibitory concentration 50(IC50) of EGCG under the above three conditions was respectively 465±15μg/m L, 543±37μg/m L and 519±55μg/m L; when tannins was added at the same time before and during viral adsorption, significant inhibitory effect could be detected, whose IC50 was respectively 411±54μg/m L and 234±36μg/m L; the inhibitory effect of APS on CPE was not detected, while the control sample ribavirin had inhibitory effect on CPE only added after viral adsorption, whose IC50 was 330±24μg/m L.Obviously, with the safe working concentration of cellular level, EGCG and tannins were first added and incubated for 60 minutes before viral infection and they had significant inhibitory effect on CPE, indicating that they had a high application value for the preventive inhibition of adenovirus and influenza virus infection.EGCG and other plant compounds have a significant biological effect to increase cell membrane potential and reduce cell membrane mobility. Such effect can effectively antagonize the pathological changes such as the reduced cell membrane potential and increased cell membrane fluidity caused by viral infection, and this antagonistic effect is closely related to its inhibitory effect of a variety of viral infections, indicating that the functional impact of such plant compounds on the lipid bilayer membrane structure of host cells is one of the important mechanisms for them to have broad-spectrum inhibitory effect on viral infection.2. Preliminary study of inhibitory mechanism of EGCG, tannins and APS on viral infection 8As is known to all, Influenza A H1N1 is a segmented RNA virus with envelope, while adenovirus is a single double-stranded DNA virus with envelop. They are significantly different in shape, structure and protein composition and completely different in intracellular proliferation strategy, but both are required to overcome the cell membrane barrier with the same structural and functional characteristics in order to complete productive infection and proliferation, therefore, the host cell membrane should be one of the ideal targets for EGCG, tannins, APS and other plant compounds play their broadspectrum effects to interfere viral infection. For the in-depth exploration of the effect mechanism of such plant compounds, relevant testing and comparative analysis were carried out with a series of data and indicators such as the pathological changes in cell membrane caused by viral infection were studied, the impact of EGCG, tannins and APS on the structure and function of cell membrane and the interference/antagonism of biological effects of plant compounds on virus infection.1) Based on the cell membrane potential specificity probe fluorescent Di BAC4(3) labeling, flow cytometry fluorescence detection technology was used for analyzing the characteristics of cell membrane potential changes. The results indicated that the cell membrane potential of the host infected with E adenovirus and A H1N1 of 100 TCID50 took on a decline trend and the depolarization rate of cell membrane increased by 38.12% and 83.91% respectively. Three plant compounds had different impacts on in the cell membrane potential, especially EGCG, which could significantly increase the membrane potential of 293 A and MDCK cells, and with the effective concentration of 640ug/ml, could decrease the membrane potential depolarization rate of 293 A and MDCK cells by 29.38% and 43.76% respectively. EGCG could significantly reduce the cell membrane potential increase caused by viral infection. Before E adenovirus infection, the 10-640μg/m L EGCG added to interfere viral infection could reduce host cell membrane depolarization rate by 1.31-31.86%. It was also indicated by Spearman’s bivariate rank correlation analysis that only EGCG was used for 293 a cells pretreatment before E adenovirus infection, its interference effect on the host cell membrane potential was correlated to its inhibitory on viral infection(R=0.931, r>0.05).2) Based on DPH(1,6-Diphenyl-1,3,5-hexatriene) fluorescent probe labeling, the fluorescence polarization technology was used for detecting the change characteristics of fluorescence anisotropy(r) of cell membrane lipid bilayer structure(note: cell membrane fluorescence anisotropy r was inversely proportional to fluidity). The results indicated that after the host cells were infected with E adenovirus and A H1N1 of 100 TCID50, the membrane fluidity significantly increased, and the membrane fluorescence anisotropy of sensitive cells reduced 13.57% and 15.76% respectively. EGCG, tannins and APS could reduce the membrane fluidity of 293 a and MDCK cells in varying degrees. With the concentration 640ug/ml could increase the membrane fluorescence anisotropy of 293 A cells by 32.61%, 25.75% and 5.22% respectively and that of MDCK cells by 36.56%, 74.60% and 3.37%. After three plant compounds were added before, during and after viral infection, the pathological change of increased host cell membrane fluidity caused by viral infection could be reduced in varying degrees. All the three phytochemicals with concentration of 640 μ g/ml were added respectively before, during and after virus infection, and the pathological changes of increased host cell membrane fluidity resulted from virus infection were reduced to different degrees. With addition of Tannins, the fluorescence anisotropy of 293 A cell membrane increased by 5.68%, 5.08% and 21.17% respectively, and that of MDCK cells increased by 78.45%, 75.50% and 70.92% respectively; with addition of EGCG, the fluorescence anisotropy of 293 A cell membrane increased by 19.71%, 2.26% and 15.56% respectively; with addition of EGCG before and after virus infection, the fluorescence anisotropy of MDCK cell membrane increased by 32.69% and 34.82% respectively; with addition of APS(astragalus polysaccharide) before virus infection, the fluorescence anisotropy of 293 A cell membrane increased by 4.33%; with addition of APS before and during virus infection, the fluorescence anisotropy of MDCK cell membrane increased by 1.00% and 7.12% respectively. The result of Spearman bivariate correlation test also indicated that under three different conditions, the biological effects of EGCG and Tannins on the host cell membrane fluidity presented obvious correlation with their inhibitory effects on virus infection.In summary, with safe working concentration, the preventive use of EGCG, tannins and other plant compounds can effectively inhibit adenovirus-5 and A H1N1 virus infection and have enormous potential for further research and development of broadspectrum virus infection inhibitors. Their biological effects on host cell membrane potential and liquidity as well as the antagonistic/interference mechanism of such unique effects on viral infection lay a solid theoretical foundation and technical support for the in-depth development of their biological characteristics of broad-spectrum inhibition of viral infection. |
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