Anti-influenza activity of dihydroquercetin from Larix sibirica (L.) on the model of lethal influenza virus infection. Zarubaev V.V. †, Garshinina A.V. †, Kalinina N.A. †, Anikin V.B. †, Babkin V.A. ‡, Ostroukhova L.A. ‡, Kiselev O.I. † † Influenza Research Institute, St. Petersburg, Russia ‡ Irkutsk Institute of Chemistry, Irkutsk, Russia Introduction. Influenza is a highly contagious acute respiratory infection in humans; it causes epidemic outbreaks or pandemics around the world annually and is associated with high morbidity and mortality worldwide. Since 2003, 440 cases with 262 deaths were identified of humans infected with the highly pathogenic avian influenza subtype H5N1. The April 2009 emergence of a swine-origin H1N1 virus is another example of a successful cross-species transmission of influenza A virus that led to another pandemic. Therefore, it is imperative that more anti- influenza chemotherapy drugs are identified. An influenza virus infection ranges in severity from an asymptomatic infection to a serious illness with systemic features. Severe influenza is manifested by virus-specific reactions with further development of reactive processes. These processes are induced by replicating virus in target cells and realized through host mechanisms. They include immune reactions, oxidative stress and other free radical processes, enhanced proteolytic activity, sharp elevation of the level of proinflammatory cytokines, etc. Plant flavonoids are known as compounds with a wide range of biological activities which include antioxidant activity, cell cycle – regulation (including apoptosis-induction) and anti-inflammatory activity. Based on the high anti-inflammatory and antioxidant activity of flavonoids, we supposed that these properties would play an important role in preventing reactive processes in lung tissue developed in the course of an influenza infection. Although not affecting virus replication, anti-inflammatory compounds and antioxidants are effective in suppression of tissue damage and prevention of complications. Here we describe the protective activity of dihydroquercetin (DHQ), the flavonoid from larch (Larix sibirica L.) wood, on the infection in white mice caused by pandemic isolate of influenza virus. Materials and Methods. DHQ (2,3-dihydro-3,5,7-trihydroxy-2-(3,4- dihydroxyphenyl)-4H-1-benzopyran-4-one, Fig.1) was extracted from larch heartwood as a microcrystalline light yellow powder and analyzed by HPLC. Fig.1. Structure of dihydroquercetin Mice were infected with an adapted influenza virus (0,5 or 5 LD 50 )and treated orally with DHQ. On day 3 p.i. level of virus’ replication in lung tissue and mean size of foci of inflammation were evaluated by titration in MDCK cells and lung morphology analysis, respectively. The mortality in each group of animals was calculated. Each group was checked daily for dead animals for two weeks post inoculation. Based on the data received, percent of mortality and index of protection (ratio of mortality in the control group over mortality in the experimental group) were calculated. Results. Infecting of animals with mice- adapted influenza virus resulted in typical influenza pneumonia leading to decreasing of food consumption, weight loss, tremor, ataxia, short breath and death of animals in severe cases. Microscopic findings showed a catarrhal tracheobronchitis with extensive degradation of tracheal epithelium and infiltration of sub- epithelial space with monocytes and macrophages. Virus-specific inclusions were detected in tracheal and bronchial epithelia. In lungs, diffuse alveolar damage was detected with hemorrhage, intra-alveolar serofibrinous exudate and monocytes and PMNLs infiltration. Mostly, cells lining the bronchi were absent or contained advanced signs of virus’ reproduction (condensation of chromatin, virus- specific inclusions, vacuoles, etc.). Beginning on day 5 p.i., bronchial and bronchiolar epithelium regenerated forming extensive cell nodules covering the denudated basal membrane. The chronic post- influenza lesions represented foci, or nodules, of overproliferation of low-differentiated cells of respiratory epithelium that filled the bronchial lumen and alveolar space. Table 1. Protective properties of DHQ against influenza virus infection in mice caused by A/Aichi/2/68 (H3N2) virus. * Values that differ from control with p<0.01 are indicated in bold ** Not determined Application of DHQ resulted in reduction of the pathological process of influenza pneumonia in mice. This was observed, in particular, as a dose-dependent decrease in mortality in the animals. The protective effect of DHQ was comparable with the protective activity of the reference drug rimantadine, to which the virus is highly susceptible. When applied, DHQ decreased mortality to 57.1 or 85.7 % with the 100 and 300 mg/kg doses, respectively, compared to 68.8% for rimantadine. The mean day of death in the DHQ- treated animals was later than in control animals without drug treatment (Table 1, Fig.2). Fig.2. Effect of DHQ on the dynamics of mortality of influenza virus- infected mice. Morphologically, DHQ has been shown to possess high protective activity against influenza virus infection in terms of preventing lung tissue damage. In particular, the foci of inflammation in the lungs of placebo- treated animals were extensive their relative size being approx % of total lung surface area. Treatment with DHQ resulted in reducing the signs of inflammation and dramatic decrease of this parameter to approx. 10% (Fig.2). a b Fig.2. Influenza pneumonia in mouse lungs of control (a) and DHQ-treated (b) mice on day 3 p.i. Conclusion. The results obtained suggest that DHQ can be considered as an affordable and prospective tool to be used in complex prophylaxis and/or treatment of severe influenza. Preparation Virus dose, LD 50 Mortality, % Index of protection, % Virus titer in the lungs (log 10 EID 50 /20 mg tissue) on day 3 p.i. (mean±SD) MDD (mean±S D) DHQ, 300 mg/kg ±0.3*14.8± N/D**13.8±2.2 DHQ 100 mg/kg ± ± N/D12.8±3.3 Rimantadine ± ± N/D13.7±2.4 Control (no drugs) ± ± N/D9.1±3.3