The Study Of Anti-influenza Virus And Anti-inflammatory Pharmacodynamics Of Flavonoids And Aetherolea In Laggera Pterodonta(DC.) Benth

Laggera pterodonta(DC.) Benth has been used as folk medicine to treat upper respiratory tract infection in Yunnan province. Its curative effect has been preliminarily verified by clinical trials. As a traditional Chinese herb, Laggera pterodonta (DC.) Benth, is cool-nature, pungent and bitter in flavor. From TCM point of view, influenza falls under the category of warm disease, the distinctive nature and flavor of Laggera pterodonta(DC.) Benth is consistent with the basic rules of treatment of warm disease. Total flavonoids can be Served as typical functional composition of’bitter flavor’of Laggera pterodonta (DC.) Benth, and aetherolea as of’pungent flavor’. Based on the TCM theory of’flavor-effect’, a series of studies of multi-target anti influenza pharmacology mechanism is to be carried out furtherly.ObjectiveThis study is to verify the anti-influenza effect of total flavonoids and aetherolea in Laggera pterodonta(DC.) Benth, and analyze the multi-target anti influenza pharmacology mechanism of them, including the influence of Laggera pterodonta(DC.) Benth total flavonoids to the host signaling pathway and cytokine, chemokine induced by influenza virus, and anti-inflammation and analgesia effect of Laggera pterodonta(DC.) Benth aetherolea, also to analysis the component of total flavonoids and aetherolea of Laggera pterodonta (DC.) Benth.Meanwhile, the new train of thought about TCM anti-influenza research will be validated.MethodsThe antiviral effection and spectrum were testing with cytopathic effect assay (CPE asasy) in vitro. The effect of Laggera pterodonta (DC.) Benth total flavonoids to the host signaling pathway is studied by western-blot. The cytokines and chemokines mRNA expression induced by influenza virus are observe by QRT-PCR assay. Apoptosis rate was measured by flow cytometry (FCM). The mice model of influenza virus infection was applieded to evaluate the potency in vivo. Conventional models including pinna swelling and capillary permeability model in mice, and cotton pelletgranuloma in rats are used to observe the anti-inflammation effect of Laggera pterodonta (DC.) Benth aetherolea. The analgesic effects of aetherolea were studied by using of ache model caused by acetic acid, formaldehyde and hot plate test in mice. The component analysis was measuring with GC-MS assay.Results1. Laggera pterodonta(DC.) Benth total flavonoids showed effective to influenza viruses (H1N1, H3N2 (Aichi), the 2009 new H1N1, FluB (lee), H9N2, H6N2 and H7N3) infection in the model of giving drug after infection, the 50% inhibiting concentration (IC50) were 25mg/mL,19.84 mg/mL, 45.53mg/mL,25mg/mL,50mg/mL,50mg/mL and 42.4mg/mL respectively.2. P38/MAPK Phosphorylation were inhibited by Laggera pterodonta(DC.) Benth total flavonoids at the concentration 15μg/mL、30μg/mL、60μ g/mL.3. The mRNA expression of COX-2, CCL-5, MCP-1, IP-10, MIP-1α, CXCL9, TNF-α, IL-6 were inhibited by Laggera pterodonta(DC.) Benth total flavonoids at the concentration 15 μg/mL,30μg/mL,60μg/mL (P<0.05, 0.01 or 0.005). The mRNA expression of IL-8 is inhibited by total flavonoids at the concentration 30μg/mL,60μg/mL (P<0.01 or 0.005)4. Apoptosis rate was inhibited by Laggera pterodonta(DC.) Benth total flavonoids, apoptosis rates of influenza group is 10.10%, total flavonoids 30 μg/mL group 8.84%, total flavonoids 60 μg/mL group 8.00%.5. Method for Laggera pterodonta(DC.) Benth total flavonoids preparation were established.4 flavonoid monomer were isolated, including a new obtained compound.6. Laggera pterodonta(DC.) Benth aetherolea can suppress inflammation in mice with pneumonia induced by influenza, but has no inhibitory effect in flu cells.7. Laggera pterodonta(DC.) Benth aetherolea (4%, v/v) suppressed mouse ear edema (P<0.01), resistant rate is 59.58%. Aetherolea (1%, v/v and 2%, v/v)suppressed peritoneal capillarypermeability(P<0.01). Aetherolea (v/v:1%,2% and 4%)inhibit the cotton granuloma of rats(P<0.01 or 0.05).8. Laggera pterodonta(DC.) Benth aetherolea (4%, v/v) reduced time of writhing reaction causing by glacial acetic acid in mice (P<0.05), also decreased the licking rear feet times of mice induced by formaldehyde (P<0.05). There is no positive result in hot plate test.9. The component analysis of Laggera pterodonta(DC.) Benth aetherolea shows,there are 64 components, mainly including terpenoids, sesquiterpenoids, phenolic compounds, alcohols and ketones,23 chemical composition were testified(64.041%).GonclusionLaggera pterodonta(DC.) Benth total flavonoids has broad-spectrum antiviral efficacy and anti-inflammatory effects. The anti-influenza pharmacology mechanism is multi-target, includes suppressing the virus directly, inhibiting p38/MAPK Phosphorylation, inhibiting mRNA expression of COX-2, CCL-5, MCP-1, IP-10, MIP-1α, CXCL9, TNF-α, IL-6 and IL-8, and inhibiting apoptosis caused by influenza viruses.Laggera pterodonta(DC.) Benth aetherolea can not inhibit influenza virus replication directly. Its has analgesic and anti-inflammatory effects so as to relieve flu symptoms, such as sore throats, muscle pain and soreness, and fever.To some degree, efficacies of total flavonoids and aetherolea in Laggera pterodonta(DC.) Benth are consistent with TCM theory of’flavor-effect’. TCM theory, which was supposed to be guiding principle of Chinese medicine research, has guiding significance for TCM-anti-influenza study, and should be given more attention in future work.