Preparation Of Arctigenin And Research Of Its Antiviral Activity Against PCV2 And PRRSV

The Chinese herbal medicines,with a long history in our country,have unique advantages in the prevention and treatment of viral diseases.Some medicines can not only share direct effects on virus replication,but also regulate the immune function with low side effects.Porcine circovirus type 2(PCV2)and porcine reproductive and respiratory syndrome virus(PRRSV)are important pathogens that endanger the global pig industry.They can cause immunosuppression in pigs,leading to the secondary infection of other pathogens,and result in huge economic losses.Vaccination alone cannot achieve the desired effect of prevention and control.Therefore,it is necessary to develop novel prevention and control measures.In this study,we extracted and purified the arctigenin(ACT)from the herbal medicine fructus arctii and analyzed its antiviral activity for PCV2 and PRRSV in vitro and vivo,in order to provide scientific evidence for the development and clinical use of ACT.A mixture of degreased powder of fructus arctii and 50%alcohol solution containing 5%hydrochloric acid was refluxed for 5 h.After filtration,the solid residue was re-suspended in 50%alcohol solution,and refluxed for 1 h again.The pH value of the filtrate was adjusted to neutral by 5mol/L NaOH,and then extracted twice with chloroform.Then,the combined organic phase was evaporated using a rotary evaporator under reduced pressure.The crude extract was purified by silica gel column chromatography twice,and finally the ACT was obtained.The average ACT concentration in the three batches was 98.2%,compared with reference substance.And the average ratio of extraction reached 1.62%.The effect of ACT on the proliferation of PCV2 in PK-15 cells and mice was analyzed.The results showed that 15.6-62.5?g/mL of ACT could significantly inhibit the replication of PCV2 in PK-15 cells(p<0.01).In vivo,the ACT could significantly inhibit the replication of PCV2 in the lung,spleen,and inguinal lymph nodes of mice at the dose of 0.2mg/kg by intraperitoneal injection(p<0.01).The effects of ACT on PCV2 replication and histopathology induced by PCV2 infection were analyzed in piglets.Compared to the control group,the viral loads in drug-treated groups(20?g/kg and 50?g/kg)decreased significantly on day 7,14,and 21 post-challenge(p<0.05,p<0.01).The piglets were sacrificed on day 21 post-challenge and related tissues were collected for pathological,virological and immunohistochemical examinations.In group 1(20?g/kg),the proliferation of PCV2 significantly lowered in the inguinal lymph nodes(p<0.05)and mesenteric lymph nodes(p<0.05),and in group 2(50?g/kg),the proliferation of PCV2 significantly decreased in mesenterium lymph nodes(p<0.001),inguinal lymph nodes(p<0.01),tonsilla(p<0.05)and lungs(p<0.05).The results of histopathological section and scores showed that both doses of the ACT could reduce the pathological damage of the aforementioned organs caused by PCV2 infection.The effects of ACT on the proliferation of PRRSV were analyzed.The results showed that the ACT had direct inactivation on PRRSV in 100TCID50 and 10TCID50,at dosage of 12.5-100?g/mL(p<0.01),and significantly inhibited the proliferation of PRRSV with 100TCID50 and 1OTCID50 in MARC-145 cells at dosage of 0.39-100?g/mL(p<0.01).However,the proliferation of PRRSV was not inhibited when MARC-145 cells was pretreated with the ACT at the dosage of 0.19-100?g/mL.The mechanism of ACT inhibition on PCV2 proliferation was analyzed from the perspective of cell cycle regulation.The results showed that 62.5 ?g/mL ACT could inhibit PK-15 cells S-phase arrest(p<0.01)caused by PCV2 infection.Meanwhile,the increase of CyclinE2,CDK2 and p53 mRNA caused by PCV2 infection were significantly inhibited,and further Western Blot results were consistent with qPCR.It is suggested that the ACT may inhibit the cell cycle distribution by inhibiting the expression of CyclinE2,CDK2 and p53 proteins,thus inhibiting the proliferation of PCV2 in PK-15 cells.Taken as a whole,we established a simple and stable method for ACT extraction.Moreover,the antiviral activity of ACT against PCV2 and PRRSV were analyzed in vitro and vivo.The results showed that the ACT can effectively inhibit the proliferation of PCV2 in vitro,mice and piglets.ACT was also shown to inhibit the growth of PRRSV in vitro.Further research found that ACT could inhibited PCV2 proliferation in PK-15 cells by regulating of cell cycle.Our findings provided novel ideas and experimental data for further understanding the antiviral mechanism of ACT and the clinical control of PCVAD and PRRS.