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Influenza infection is a common respiratory disease, causing high morbidity in the general population, a high mortality in at-risk groups, as well as consi-derable cost of hospitalization and treatment and loss in productivity (38). According to WHO data, 10%-20% of the world population fall victims to influenza yearly. Although influenza usually proceeds as a 'self-limited' infection, it can cause severe extra-pneumonic complications (20). The possibility of both pneumonic and extra-pneumonic complications increases dramatically in less healthy and senior patients. An efficient protec-tion against influenza is vaccination which, in patients above 65 years of age, could reduce the probability of death by up to 80% (26). This is particularly true in situations in which vaccines are unavailable or ineffective due to viral antigenic changes or poor host immune responses. In this regard, the development of antiviral drugs provides another important way to combat influenza.
Despite recent success in the development of new antiviral agents, the need for effective therapies for influenza virus infections continues to exist. The M2channel inhibitors are effective against influenza strains, but their use is limited because of central nervous system and gastrointestinal side effects, emergence of viral resistance and lack of effectiveness against influenza B (6). Meanwhile the broad-spectrum antiviral, ribavirin, is a recognized inhibitor of influenza virus infections in vitro and in animal models (30, 12, 13), but ribavirin has a relatively small therapeutic index, causes hemolytic anemia induced at high doses and produces potential teratogenic effects (8, 31).The search for viral inhibitors with plant origins is a promising approach in the development of new therapeutic agents. In this respect a large number of extracts and pure substances have been tested and some of them have been proved to have a selective antiviral effect (5, 39).
The plant Hypericum perforatum L belongs to the genus Guttiferae which contains about 400 species, and is found in Europe, West Asia, North Africa and North America. It is used in herbal medicine exter-nally for the treatment of skin wounds, eczema and burns and internally for several applications such as diseases of the alimentary tract (21, 9). Many studies have shown that Hypericum perforatum L. extract (HPE) has antiviral activity (1, 29) and can also have considerable effect against cytomegalovirus (CMV) and human immunodeficiency virus (HIV) (2, 10, 11, 40, 27, 28). The antiviral action of HPE has been studied on lipid enveloped and non-enveloped DNA and RNA viruses (14, 15, 16, 36) and also, clinical studies have been conducted in HIV infected patients (4, 37).
Besides the above-mentioned activities, HPE has been reported to possess marked antimicrobial (17) and antiretroviral activity (15). However, scientific information is rarely available for the mechanism of antiviral activity of HPE at cellular and molecular levels. Therefore, the present study was designed to study the effect of HPE at cellular level using the culture of MDCK cells infected with IAV in vitro, and the animal model of influenza A virus infection by comparing its therapeutic effects with those of Ri-bavirin, whose mechanisms of antiviral activities are well known. The results of these experiments are reported here.
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Hypericum perforatum L. extract was provided by Lanzhou Institute of Animal and Veterinary Phar-maceutics Sciences, Chinese Academy of Agricultural Sciences and the ribavirin (H19993462) was purchased from Tianjin Pharmaceutical Group Xinzheng Co., Ltd, Henan, China.
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The human influenza A virus (IAV) strain (H1N1) (A/Gansu/1771/2006, Gansu Center for Disease Control and Prevention, Lanzhou, China) was used in this study. The virus used in cell culture experiments was passaged through MDCK cells at least once to prepare pools. The pools were then titrated in MDCK cells before use and were then propagated in the allantoic cavities of 9-day-old chicken eggs for 72 h at 35 ℃. Then the virus was passaged five times through mice, after which the recovered virus from the lungs was used to prepare a pool in MDCK cells, which were provided by the Gansu Center for Disease Control and Prevention. MDCK cells were grown in DMEM (Dulbecco's Modified Eagle Medium) (from Sigma Company) supplemented with 0.55% w/v NaHCO3 (Jianghai Biological Engineering Co., Ltd, Beijing, China), 100 units/mL penicillin, 100 mg/mL strep-tomycin and 10% fetal calf serum (FCS) (Sijiqing Company, Hangzhou, China). FCS was reduced to 2% for the viral infection. The cell suspension containing 100 000 cells per mL was obtained after enzymatic dissociation with trypsin (1 g/L, 1:250, Lvshengyuan Biotechnology). 0.1 mL of this cell suspension was added to each well of a 96-well cell culture plate (Corning, USA, NY. 14831). The cells were incubated (at 37 ℃ in a 5%CO2 incubator) for 3 d and after forming a continuous layer, they were inoculated with the virus.
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Female specific pathogen-free BALB/c mice weig-hing 18-21 g were purchased from The Cancer Hospital of Gansu Province, China.
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SaO2 was determined using the Ohmeda Biox 3800 pulse oximeter (Medi Tech Group Co., Ltd, Qingdao, China). The ear probe attachment was used with the probe placed on a thigh of the mice. Readings were made after a 30 s stabilization time on each animal (32). All SaO2 determinations were made on days 3 through 11 after virus exposure.
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Each mouse lung was homogenized and varying dilutions assayed in triplicate for infectious virus in MDCK cells grown in 96-well flat-bottomed micro-plates (34). Each lung homogenate was centrifuged at 2500 rpm for 30 min and the supernatants were used in these assays.
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Three methods were used to assay antiviral activity in vitro: inhibition of virus-induced cytopathic effect (CPE) determined by visual (microscopic) exami-nation of the cells, increase of neutral red (NR) dye uptake into cells and virus yield reduction (35). Eight concentrations of the test agents, each concentration varying in 2X steps, were evaluated in MDCK cells. Standard virus controls, toxicity controls and normal medium controls were included in all assays. CPE inhibition data were expressed as 50% effective (viral CPE-inhibitory) concentration (EC50), 50% cytotoxic (cell-inhibitory) concentration (CC50) and selectivity index (SI), determined as the CC50/EC50. Virus yield reduction data were expressed as the concentration inhibiting virus yield by one log10 (EC90); the SI for virus yield results was calculated as CC50/EC90.
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HPE and ribavirin were each evaluated for the dose considered lethally toxic to mice, which was done by treating ten mice per dose with HPE and ribavirin. The ribavirin doses studied were 320, 160, 80, 40, and 20 mg/kg/day and HPE doses were 2000, 1000, 500, 250 and 125 mg/kg/day. The mice were treated by oral gavage (po) twice daily for 5 days and their weights were determined prior to the first treatment and again 18 h after the final treatment. Animals were checked daily for 14 d to check whether any had died.
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The general procedure of initial evaluation for the relative efficacies of HPE and ribavirin was to infect groups of mice intranasally with the IAV. This was done by anesthetizing them with aether and instilling 50 μL of 25 LD50concentration of virus on the nares. The human influenza A virus (IAV) strain was studied in vivo. The mice were treated with varying dosages (as described in Table 3) of either drug po twice daily for 5 d beginning 4 h prior to virus ex-posure. Parameters for determining the effects of treatment included prevention of death through 14 d, slowing of SaO2decline, inhibition of lung consolidation [scores ranging from 0 (normal) to 4 (maximal plum coloration) and lung weight], and lessening of lung virus titers. The lung parameters were assayed on d 3, 6 and 9 of the infection, with 3-4 mice being killed at each time point. Ten mice were used per treated group to assay for death and SaO2. Virus control was used in each experiment with the same numbers of animals used for each disease parameter. Toxicity control was also run in parallel for each dose; three mice were used per dose, with their weights taken before the start of the treatment and again 18 h after the last treatment and the animals were observed for overt signs of toxicity and death for 14 d. A normal control was run in parallel; these animals were weighed along with the toxicity controls and SaO2 levels were ascertained on the same days as the animals were infected. The normal control was sacrificed along with the infected mice to provide background lung data.
Table 3. Inhibition of influenza A virus infections in mice treated po with HPE or ribavirin
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To determine the relative efficacy of ribavirin and HPE against the IAV infection when initiation of therapy was delayed, groups of 10 mice infected with the IAV were treated po twice daily for 5 d with either agent beginning 4, 12 or 20 h post-virus exposure. Effects on mortality and slowing of SaO2 decline were determined, with the numbers of animals per group described in Table 4. The dose of ribavirin was equivalently non-toxic (1/3 LD50 dose), which was 70 mg/kg/day, and the dose of HPE was equiva-lently nontoxic, 500 mg/kg/day. The animals were held for 14 d. When it was determined that all periods of treat-ment were highly effective, the experiment was repeated with treatments delayed until 28, 36 or 48 h post-virus exposure.
Table 4. Effect of delay of p.o. HPE or ribavirin treatment on an influenza A virus infection in mice
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Increases in number of survivors were evaluated using χ2 analyses. Differences in the mean days to death, mean SaO2 values, lung scores and mean lung virus titers were analyzed by t-test.
Agents
Virus and cell
Animals
Arterial oxygen saturation (SaO2) determination
Lung virus titer determination
In vitro antiviral evaluation
In vivo toxicity determination
Initial in vivo antiviral studies
In vivo antiviral effects of delay in therapy initiation
Statistical evaluations
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Both HPE and ribavirin were inhibitory to the in-fluenza A virus evaluated, the EC50 values for ribavirin being 5.0 μg/mL, whereas HPE was approximately five-fold less potent, its EC50 being 24 μg/mL (Table 1). Multiple cytotoxicity assays using neutral red uptake indicated the mean CC50 values for ribavirin and HPE was 520 μg/mL and 1.5 mg/mL respectively.
Table 1. In vitro inhibition of influenza A virus by HPE and ribavirin
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Oral gavage treatment with ribavirin and HPE using therapies twice daily for 5 d indicated that the ap-proximate LD50 dose of ribavirin was 200 mg/kg/day, whereas HPE was better tolerated since below a dosage of 2 000 mg/kg/day, almost all treated mice survived. It should also be noted that some weight gain was seen at dosages below 2000 mg/kg/day. No attempt was made to determine the cause of death in the mice in this range finding study (Table 2).
Table 2. Comparison of toxicity of po administered HPE and ribavirin in mice
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Multiple dosages of ribavirin and HPE were utilized and both were evaluated against the IAV in young adult mice. The results of each treatment against the IAV infections are summarized in Table 3. Due to the large amount of data available, only the last day of SaO2 determination, day 11, is shown in the tables; it was on this day that the values usually reached their lowest levels in virus control animals and thus differences resulting from therapy were the most pronounced. The day 6 lung consolidation data are shown in the tables, since it was at this time that lung scores and weights were maximal; day 3 lung virus titers are indicated because these titer differences are usually best seen early in the infection. According to these parameters each agent was significantly inhibitory to the virus.
Against the influenza A virus infection, HPE was effective in inhibiting death and SaO2 decline at doses down to 31.25 mg/kg/day. Ribavirin's inhibition of this virus infection was seen down to 40 mg/kg/day.
Each agent has approximately the same therapeutic index. But HPE represented the minimum effective dose and had the least toxicity in the mice, as described in Table 3.
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The results of delaying the initiation of therapy with ribavirin and HPE on the influenza A virus infection are presented in Table 4. In this experiment, 500 mg/kg/day of HPE and 70 mg/kg/day of ribavirin were used, including a nontoxic dose of HPE and an approximately one-third of the LD50 dose of ribavirin. Both agents remained highly protective of the mice despite the delay in starting the treatment, and a highly significant difference in reduction of mortality was seen as late as the 20 h post-virus exposure initiation time, which was the longest delay evaluated in the first experiment. One infected animal treated with ribavirin and HPE respectively died at this latest therapy initiation time. All mice survived in the other infected and treated groups. In the second experiment, with the therapy initiation being further delayed, both agents were highly protective of the mice even when the treatment was begun 48 h after virus exposure, which was the latest time evaluated.