Antiviral effects of ascorbic and dehydroascorbic acids in vitro.

In the present study, ascorbic acid weakly inhibited the multiplication of viruses of three different families: herpes simplex virus type 1 (HSV-1), influenza virus type A and poliovirus type 1. Dehydroascorbic acid, an oxidized form of ascorbic acid and hence without reducing ability, showed much stronger antiviral activity than ascorbic acid, indicating that the antiviral activity of ascorbic acid is due to factors other than an antioxidant mechanism. Moreover, addition of 1 mM Fe3+, which oxidizes ascorbic acid to dehydroascorbic acid and also enhances the formation of hydroxyl radicals by ascorbic acid in the culture media, strongly enhanced the antiviral activity of ascorbic acid to a level significantly stronger than that of dehydroascorbic acid. Although both ascorbic acid and dehydroascorbic acid showed some cytotoxicity, the degree of cytotoxicity of the former was 10-fold higher than the latter, suggesting that the observed antiviral activity of ascorbic acid with and without ferric ion is, at least in part, a secondary result of the cytotoxic effect of the reagent, most likely due to the free radicals. However, the possibility that oxidation of ascorbic acid also contributed to the antiviral effects of ascorbic acid exists, in particular in the presence of ferric ion, since dehydroascorbic acid exhibited a very strong antiviral activity. Characterization of the mode of antiviral action of dehydroascorbic acid revealed that the addition of the reagent even at 11 h post infection almost completely inhibited the formation of progeny infectious virus in the infected cells, indicating that the reagent inhibits HSV-1 multiplication probably at the assembly process of progeny virus particles after the completion of viral DNA replication.

Antiviral activities of coffee extracts in vitro.

Both hot water extracts of coffee grinds and instant coffee solutions inhibited the multiplication of herpes simplex virus type 1, a representative enveloped DNA virus, when they were added to the culture medium of the virus-infected cells at a dose of one fifth the concentration suitable for drinking. The antiherpetic activity was independent of the suppliers (companies) of the coffee grinds and of the locations where the coffee beans were produced. Further characterization revealed that there are two different mechanisms, by which the coffee extracts exert inhibitory activities on the virus infection; (1) a direct inactivation of the infectivity of virus particle (i.e., a virucidal activity) and (2) the inhibition of progeny infectious virus formation at the late stage of viral multiplication in the infected cells. Caffeine, but not quinic acid and chlorogenic acid, inhibited the virus multiplication to some extent, but none of them showed the virucidal activity, suggesting that other component(s) in the coffee extracts must play a role in the observed antiviral activity. In addition, the coffee extracts inhibited the multiplication of poliovirus, a non-enveloped RNA virus, but showed no virucidal effect on this virus.

Antiviral effect of octyl gallate against DNA and RNA viruses.

The effects of gallic acid (3,4,5-trihydroxybenzoic acid) and its alkyl esters on virus growth and virion infectivity were examined. All the compounds tested showed an inhibitory effect on the growth of herpes simplex virus type 1 (HSV-1) in HEp-2 or Vero cells. The antiviral activity of gallic acid alkyl esters was enhanced by increasing the number of carbon in the alkyl moieties of the compounds, reaching maximum at a carbon number of 12 (lauryl gallate), but both cytocidal activity and cytopathic effect of the compounds were also significantly increased simultaneously. Among these compounds, octyl gallate showed a marked antiviral effect with a relatively moderate cytotoxity. In addition, octyl gallate suppressed the multiplication of RNA viruses, such as vesicular stomatitis virus and poliovirus. Quantitative characterization of the HSV-1 infection in the presence of octyl gallate revealed that: (1) this reagent can directly inactivate HSV-1 (virucidal activity), (2) it suppresses both the intracellar multiplication and the release of the virus, (3) it selectively accelerates death of the virus-infected cells and (4) the addition of the reagent even at 6-h post infection completely abolishes the formation of progeny virus in the infected cells.

Antiviral effect of octyl gallate against influenza and other RNA viruses.

Octyl gallate inhibited the multiplication of several RNA viruses with widely different structure and replication strategies; i.e. vesicular stomatitis virus (VSV), influenza virus and poliovirus. In addition, octyl gallate showed virucidal activity against enveloped viruses at high concentrations. Characterization of the mode of action of octyl gallate against influenza virus infection revealed that the reagent delayed the onset of progeny virus formation. It did not, however, affect the rate of the formation, and the addition of the reagent at 2 h postinfection (p.i.), but not at 4 h p.i., almost completely abolished the formation of the progeny viruses, suggesting that the antiviral action of octyl gallate was not the result of a non-specific degeneration of the infected cells, but possibly the result of the inhibition at specific steps in the middle stage of the multiplication of influenza virus.

Inhibition by caffeic acid of the influenza A virus multiplication in vitro.

Caffeic acid has been shown to inhibit the multiplication of influenza A virus in vitro, whereas caffeine, quinic acid and chlorogenic acid do not. Caffeic acid has also been shown to have antiviral activity against herpes simplex virus (DNA virus) and polio virus (RNA virus). In the present study, a comparison of the one-step growth curve of the influenza virus in the presence of caffeic acid with that in the absence of the reagent showed that an eclipse period of the virus multiplication in the infected cells was not affected by the reagent, while the progeny virus yield was markedly decreased in the presence of caffeic acid. In additional experiments, it was found that the addition of caffeic acid at an early time point post-infection (within 3 h post-infection) was mandatory for extensive antiviral activity, suggesting that a major target of the reagent exists in the early stages of infection. Simultaneously with the decrease in the progeny virus yield, both the virus-induced cytopathic effects and apoptotic nuclear fragmentation were markedly suppressed by the reagent, suggesting that caffeic acid suppresses, at least temporally, the degeneration of the virus-infected cells and that the observed antiviral activity is likely not the secondary result of the cytotoxic effects of the reagent. These results suggest the potential pharmacological use of caffeic acid or its derivatives as an antiviral drug against influenza A virus.

Effects of electrolytes on virus inactivation by acidic solutions.

Acidic pH is frequently used to inactivate viruses. We have previously shown that arginine synergizes with low pH in enhancing virus inactivation. Considering a potential application of the acid inactivation of viruses for the prevention and treatment of superficial virus infection at body surfaces and fixtures, herein we have examined the effects of various electrolytes on the acid-induced inactivation of the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), the influenza A virus (IAV) and the poliovirus upon their incubation at 30˚C for 5 min. Eight electrolytes, i.e., phosphate, NaCl, glutamate, aspartate, pyrrolidone carboxylate, citrate, malate and acetate were tested. No detectable inactivation of the poliovirus was observed under the conditions examined, reflecting its acid-resistance. HSV-1 and HSV-2 responded similarly to the acid-treatment and electrolytes. Some electrolytes showed a stronger virus inactivation than others at a given pH and concentration. The effects of the electrolytes were virus-dependent, as IAV responded differently from HSV-1 and HSV-2 to these electrolytes, indicating that certain combinations of the electrolytes and a low pH can exert a more effective virus inactivation than other combinations and that their effects are virus-specific. These results should be useful in designing acidic solvents for the inactivation of viruses at various surfaces.

Inhibition of multiplication of herpes simplex virus by caffeic acid.

Hot water extracts of coffee grinds and commercial instant coffee solutions have been shown to exhibit marked antiviral and virucidal activities against herpes simplex virus type 1 (HSV-1). Specifically, it has been shown that caffeine and N-methyl-pyridinium formate inhibit the multiplication of HSV-1 in HEp-2 cells. The present study examined the virological properties and the antiviral activity of caffeic acid against HSV-1. Caffeic acid inhibited the multiplication of HSV-1 in vitro, while chlorogenic acid, a caffeic acid ester with quinic acid, did not. These reagents did not have a direct virucidal effect. The one-step growth curve of HSV-1 showed that the addition of caffeic acid at 8 h post infection (h p.i.) did not significantly affect the formation of progeny viruses. An analysis of the influence of the time of caffeic acid addition, revealed that addition at an early time post infection remarkably inhibited the formation of progeny infectious virus in the infected cells, but its addition after 6 h p.i. (i.e., the time of the completion of viral genome replication) did not efficiently inhibit this process. These results indicate that caffeic acid inhibits HSV-1 multiplication mainly before the completion of viral DNA replication, but not thereafter. Although caffeic acid showed some cytotoxicity by prolonged incubation, the observed antiviral activity is likely not the secondary result of the cytotoxic effect of the reagent, because the inhibition of the virus multiplication was observed before appearance of the notable cytotoxicity.

Modulation of small molecule solubility and protein binding by arginine.

We previously showed that arginine increases the aqueous solubility of the low molecular weight aromatic compounds octyl-gallate (OG) and coumarin. In this study, we focused on acycloguanosine (ACV), an antiherpic agent with moderate aqueous solubility, and examined the effects of arginine on its solubility at a neutral pH. The solubility of ACV increased by approximately 1.9- and 2.6-fold in the presence of 1 and 2 M arginine, respectively, while no solubility changes were observed at an arginine concentration below 0.1 M. These results were found to be consistent with observed changes in OG solubility in the presence of arginine. However, ACV and OG exhibited differential binding to bovine serum albumin (BSA): ACV showed no binding to BSA, while OG showed substantial BSA binding. In conclusion, Arginine partially blocked the protein binding of OG.

Antiviral effects of dehydroascorbic acid.

IN THE PRESENT STUDY, DEHYDROASCORBIC ACID INHIBITED THE MULTIPLICATION OF VIRUSES OF THREE DIFFERENT FAMILIES: herpes simplex virus type 1 (HSV-1), influenza virus type A and poliovirus type 1. Although dehydroascorbic acid showed some cytotoxicity at higher concentrations, the observed antiviral activity was not the secondary result of the cytotoxic effect of the reagent, as the inhibition of virus multiplication was observed at reagent concentrations significantly lower than those resulting in cytotoxicity. Characterization of the mode of the antiviral action of dehydroascorbic acid against HSV-1 revealed that the addition of reagent at any time post infection inhibited the formation of progeny infectious virus in the infected cells, and a one-step growth curve showed that the addition of reagent allowed formation for an additional 2 h, but then almost completely suppressed it. These results indicate that the reagent inhibits HSV-1 multiplication after the completion of viral DNA replication, probably at the step of the envelopment of viral nucleocapsids at the Golgi apparatus of infected cells.

Solvent-induced virus inactivation by acidic arginine solution.

Viral clearance is a primary concern for parenteral protein biopharmaceuticals. Low pH, detergent/solvent wash, or heating, called pasteurization, has been the main process for virus inactivation. Detergent/solvent wash is also used to treat superficial infectious diseases, including herpes simplex virus (HSV) infections. Herein we examined virus inactivation effects of acidic arginine on HSV type 2 (HSV-2) as a function of pH and temperature in an attempt to find solvent conditions that are effective for virus inactivation, yet are compatible with in vivo applications. Aqueous arginine at 0.7 M was highly effective on HSV-2, more so at lower pH and higher temperature. Its effects were stronger than 0.1 M citrate, 0.1 M citrate/0.6 M NaCl or 0.7 M citrate at any pH and temperature. This demonstrates that strong virus inactivation effects of arginine are not simply due to ionic strength or high concentration and arginine possesses a unique property that results in irreversible damage in virus particles. Such strong virus inactivation effects can be used in vivo for certain superficial infectious diseases, such as genital infections.

Antiviral effect of pyridinium formate, a novel component of coffee extracts.

N-methyl-pyridinium formate, a novel component of coffee extracts, inhibited the multiplication of both DNA and RNA viruses. In the presence of the compound, the progeny viral yields of both herpes simplex virus type 1 (HSV-1) and poliovirus in HEp-2 cells and those of influenza virus type A in MDCK cells decreased with increasing concentrations of the compound, although the degree of viral sensitivity to this compound differed. In addition, none of these viruses were directly inactivated by the compound at the concentrations tested. Characterization of the mode of action of this compound against HSV-1 multiplication revealed that it inhibits the viral growth primarily at the initial step of virus multiplication, i.e., within 2 h after the onset of multiplication, although the virus multiplication was affected by the compound throughout the multiplication cycle. In addition, this compound showed a significant cytotoxic effect, although the observed antiviral effect was unlikely to be attributed to the cytotoxic effect.

Effect of caffeine on the multiplication of DNA and RNA viruses.

The present study examined the effect of caffeine on RNA and DNA viruses, revealing that it inhibits the multiplication of both. In the presence of caffeine, the progeny virus yield of both herpes simplex virus type 1 (HSV-1) and poliovirus decreased with increasing concentrations of the reagent, although HSV-1 was much more sensitive than poliovirus. The influenza virus was not affected by caffeine at the same concentrations. None of the viruses were directly inactivated by caffeine at the tested concentrations. Characterization of the mode of action of caffeine against HSV-1 infection revealed that the addition of the reagent at 10 h post-infection significantly affected the formation of progeny virus, indicating that caffeine can inhibit the multiplication of HSV-1 during the step(s) following the completion of viral DNA replication and the formation of nucleocapsids. In addition, the reagent selectively enhanced the cytopathic effects and cell death of the infected cells over uninfected cells, suggesting that the antiviral action of caffeine against HSV-1 is, at least in part, the result of accelerated degeneration of the infected cells.