Mianma Guanzhong Phloroglucinol Compounds Anti-H5N1 Influenza Virus Activity Screening And Metabolism Study

Male Fern Rhizome is the dried rhizome and frond bases of Dryopteris crassirhizoma Nakai(DC),with the properties of bitter,slightly cold and toxicity.It mainly enters into liver and stomach channels.It is active in clearing heat,removing toxin and expelling parasites,DC been traditionally used for treating worm accumulation,abdominal pain and sore.In the present,it is usually used for treating various inflammatory and infectious diseases.Studies have been demonstrated that fraction rich in phloroglucinols has the inhibiton effect on influenza virus.However,the anti-influenza virus activity of phloroglucinols compounds have never been reported.H5N1 influenza virus characterized with high pathogenicity,infectivity,highmortality.Therefore,for screening the most active phloroglucinol on influenza virus(H5N1)from DC,twenty-three phloroglucinols that had been isolated from DC were screened their inhibitory effect on the neuraminidase(NA)of influenza virus H5N1 by molecular docking.Meanwhile,the rhizomes of DC were subjected to isolation and purification processes to obtain the inhibitor candidates.Then anti-influenza virus activity in vitro and vivo of candidates were performed to find the most active phloroglucinol compound.Moreover,the immune mechanism of the most active compound was investigated by assaying various cytokines and the metabolites of the active phloroglucinols compounds in mice were also explored.The main research items in this dissertation are as follows:Part one Literature reviewThe chemical constituents and pharmacological activities of D.crassirhizoma were summarized.Many kinds of chemical compounds such as phloroglucinols,flavonoid,terpenoid,steroid,aliphatic hydrocatbons,aromatic hydrocarbons,carbohydrate,et al.Many studies demonstrated that phloroglucinols from DC had a wide range of pharmacological effects,such as anti-virus(influenza virus,HIV,hepatitis B virus,coxsackie virus),antibacterial,anti-malarial,anti-inflammatory,expelling parasite,anti-tumor,antioxidant,anti-aging,analgesia,uteri excitation,hemostasis,estrogen-like effects.The effects of anti-virus,expelling parasite,antibacterial awalys being pay attention.Phloroglucinols compounds are the active constituents of anti-influenza virus.Therefore,the effects of phloroglucinols compounds on influenza virus H5N1 were investigated in this paper.Phenols compounds have been drawn attention for their antivirus activity recently.Phloroglucinols compounds from DC are the active constituents of anti-influenza virus.Therefore,activities of phenols were summarized as follows:phenols constituents that could inhibit many kinds of influenza A virus with low toxicity and no drug resistance.In addition,when combination with chemical drug,phenols constituents could inhibit the development of drug resistance.The anti-influenza mechanism is related to the inhibition of virus absorption,penetration,the expression and synthesis of hemagglutinin and neuraminidase,the transcription of RNA,the exportation of nucleogluco protein;the reduction of oxidative stress damage caused by viruses.In addition,phenols constituents could enhance the functions of cellular immunity and humoral immune.Therefore,lead compounds may be screened from phenols constituents by structural modification.The great developments of computing science become the driving forces of life sciences.Nowadays,computing technology becomes one of the most important approaches in new drug research and development.Through the molecular docking operation of the computer simulation,the interaction between drug and target can be rapidly and accurately described.Molecular docking was used in this paper to screen the inhibitory effect of phloroglucinols on the neuraminidase(NA)of influenza virus H5N1.Therefore,progress on molecule docking and the application of it in drug design were summarized to lay foundation for virtual screening the inhibitory effect of phloroglucinols on the neuraminidase(NA)of influenza virus H5N1 and structure-activity relationship.Part two Experimentation2.1 The screening on phloroglucinols of anti-influenza H5N1 virus from DC and the structure-activity relationship analyzingFor screening the active phloroglucinols on influenza virus(H5N1)from Dryopteris crassirhizoma NaKai,a database was established including twenty-three phloroglucinols from the literature that had been isolated from Dryopteris crassirhizoma.The five kinds neuraminidase(NA)were the target protein that downloaded from PDB database.Autodock Vina was first used in this papter for molecular docking of twenty-three phloroglucinols and five target protein.The results showed that binding energies were ranged from-6.8 to-8.3 kal/moL among phloroglucinols and NA.Compounds that could interaction with the important amino acid residues of the binding pocket and embed into both binding pocket and 150-cavity were selected as candidate compounds.As a result,dryocrassin ABBA(GZ-A),filixic acid ABA,filixic acid ABB,filixic acid ABP,flavaspidic acid AB and albaspidin AA were selected as the candidate compounds.The structure-activity relationship was analyzed and the results showed that the antiinfluenza activity of phloroglucinols compounds were list in order as follows:tetrameric>trimerie>dimeric>pentameric>monomeric.Carbonyl and hydroxyl groups were essential.The activity was higher when phloroglucinols with more carbonyl and hydroxyl groups.However,the structures of phloroglucinols compounds should not be more than four rings,otherwise,compounds could not dock into the active site pocket.2.2 Isolation and characterization of phloroglucinols compoundsIn order to obtain the six candidate compounds,column chromatography was performed on 200-300 mesh silica gel and all purifications were monitored both by TLC(Thin-Layer Chromatography)and pHPLC(preparative High performance Liquid Chromatography).The structures of all compounds were determined by combined spectroscopic methods(UV,IR,1HNMR,13C-NMR,HMBC,HSQC,HR-ESI-MS).Thirteen phloroglucinols compounds were characterized including two tetrameric compounds:GZ-A(1)and dryocrassin ABBP(2),four trimeric compounds:filixic acid ABA(3),filixic acid ABP(4),filixic acid ABB(5)and nortrisflavaspidic acid ABB(6),seven dimeric compounds:albaspidin AA(7),albaspidin AP(8),albaspidin AB(9),albaspidin PP(10),albaspidin PB(11),norflavaspidi acid AB(12)and flavaspidic AB(13).Compounds 2 and 6 are new compounds;Compounds 1,3,4,5,7,13 are the candidates.2.3 Anti-Influenza virus H5N1 activity in vitro of the candidatesSix candidates on NA was assayed by quantifying the fluorescent product resulting from the cleavage of the substrate 4-MUNANA.The two new compounds and GZP fraction that rich in GZ-A were also evaluated their activity.The results showed that GZ-A,filixic acid ABA and GZP fraction exhibited inhibitory activity against NA with IC50 values of 18.59 ± 4.53,29.57 ±2.48 ?M and 22.07 ± 2.73 ?g/mL.The other compounds showed moderate activity in NA inhibition assay.GZ-A and filixic acid ABA were evaluated for their antiviral activity against H5N1(A/Vietnam/1203/2004)strain on MDCK cells via CCK8 and CPE assay in a biosafety BSL-3 laboratory,and oseltamivir was used as a positive control.The antiviral assays demonstrated that GZ-A exhibited anti-influenza virus activity with EC50 value of 18.45±2.69?M.It was found that the EC50 value of anti-influenza virus activity was equal to the IC50 value of NA inhibition activity.The results suggested that GZ-A has the strongest activity on influznea H5N1 virus and the target was NA.2.4 The anti-influenza H5N1 virus activity and immune mechanism of GZ-AThe aim of our current study is to evaluate the antiviral activity of GZ-A against influenza virus H5N1 in a mouse model.In the present study,120 female BALB/c mice(SPF level,18?20 g)were randomly divided into six groups with 20 mice per group:control group,untreated mouse model group,three dosages of GZ-A groups high,middle,low dosage,amantadine hydrochloride group(20 mg/kg).On day 2 post inoculation mice received of GZ-A or amantadine hydrochloride by oral gavages once daily for 7 days.Body weight,activity,mortality rate,and survival time were monitored daily for 14 days.Post inoculation on day 7 and on day 14,5 mice were euthanized to collect the lungs and the lung index was determined as previously described:the dry lung-to-body weight ratio(%)=weight of the whole dry lung/body weight × 100%.The cytokine contents in bronchial alveolar lavage fluid were used to assess the protective efficacy of drug treatment against AIV H5N1-derived pneumonia.The result showed that mice administered with high dosage of GZ-A with an 87%survival rate against influenza H5N1 virus.Moreover,body weight gain increased significantly while lung lesions and virus loads were reduced compared to those of the untreated mice,which suggested that GZ-A could alleviate pulmonary inflammation of mice infected with H5N1 virus.Moreover,the three groups treated with GZ-A were found to be a dose-dependent manner in the mouse model and better than amantadine-treated group.The mechanism of GZ-A on influenza virus H5N1 was also be investagted.The alleviation of lung inflammation after administrated with GZ-A was probably mediated by decreased pro-inflammatory cytokines IL6,IL-12,IFN-?,TNF-? and increased anti-inflammatory cytokines IL-10 and MCP-1.2.5 Toxicity studies in vivo and vitro of GZ-ATo investigate the safety of GZ-A,MDCK cells and ICR mice were used to observe the cytotoxicity,acute toxic symptoms.The results showed that the inhibition rate of GZ-A(400?M)was only 23.90 ± 0.59%,which suggested that GZ-A has not obvious cytotoxicity;when administrated GZ-A(25 mg/mL,0.4 mL/10g)with mice,no death and obvious toxicity and pathologic changes of the viscera were observed.The dosage 1 g/kg was equivalent to 5 7?222 times of clinical dose.Therefore,GZ-A was considered safety under dose of common usage.2.6 Quantitative determination of 3 phloroglucinols compounds in active fraction of anti-influenza virus and preparation of GZ-AThe active fraction(GZP fraction)exhibited inhibitory activity against NA with IC50 value of 22.07±2.73 ?g/mL,which suggested that it may has the potential to anti-influenza virus H5N1.Therefore,UPLC was used to perform the quantitative determination of phloroglucinols compounds in the fraction.The result showed that the content of GZ-A,dryocrassin ABBP and filixic acid ABB were 74.71%,6.51%and 4.95%,respectively.2.7 Metabolites studies of active fraction in vivoThis study is the first to investigate the metabolism of phloroglucinols compounds in vivo by UPLC-LTQ-Orbitrap mass spectrometry.A total of 34 metabolites were identified based on accurate mass measurements,fragementation patterns and chromatographic retention times.Four parent durg and 12 metabolites were found in plasma;14 metabolites in urine;1 parent durg and 23 metabolites in fece.The metabolic pathways of phloroglucinols compounds were also proposed for the first time.The results demonstrated that phloroglucinols compounds underwent extensive metabolic reaction including demethylation(-CH2),oxidation(+O-H2),dihydroxylation(-OH)and ammonification(-O+NH),reduction(+H2),methylation(+CH2),desacylation(-C2H2O,-C3H4O),cysteine conjugation(+C3H5ONS),sulfoconjugation(+SO3).In the present study,GZ-A were screened as the most active phloroglucinols compound that anti-influenza virus H5N1 from Dryopteris crassirhizoma,two new compounds were obtained at the same time.The immune mechanism and target of GZ-A were also explored.The experiments in vitro and vivo were demonstrated the safety of GZ-A.This study is the first to investigate the metabolism of phloroglucinols compounds in vivo,34 metabolites were identified and metabolic pathways were also proposed.