ZNs And Its Components,Inhibition Effect,and Mechanism On Chinese Sacbrood Disease

As a native bee species in China,Apis cerana cerana plays an important role in agricultural production.In recent years,due to the epidemic of diseases,climate change,species invasion and other reasons,the number of bees has been decreasing year by year,and the reduction of bee population caused by bee diseases has been widely concerned.Chinese sacbrood disease is one of the main reasons for the decrease of honeybee population.The main reason is that the Chinese sacbrood virus(CSBV)poses a serious threat to the health and population growth of Apis cerana cerana.However,little is known about the anti-CSBV drugs,especially the research of Chinese herbal medicine anti-CSBV.The main problems are as follows:(1)there are more studies in vitro,but less studies on culture conditions.(2)There are few studies on chemical constituents,and the studies on antiviral constituents are not comprehensive enough.(3)There are more studies on antiviral alone,but less on the overall regulatory effect.In order to provide a feasible basis for the development of green and pollution-free alternative chemical drugs of Chinese herbal medicine,it is of great significance to protect the health of Apis cerana cerana,prevent the disease and produce safe and pollution-free products.In this paper,we focus on the above three issues,and lay the foundation for the discovery of the effective substance basis of Chinese herbal medicine and the clarification of the mechanism of anti CSBV pharmacological action.(1)Selection of culture conditions of larvae in vitro.Six kinds of common bee viruses were not detected in the Chinese bee colony as samples,and the second instar larvae of the colony were cultured in vitro.The results showed that when the temperature was 32℃,the relative humidity was 75%,the growth of the larva was the best,and the relative humidity had a greater impact on the larvae,while the temperature and sugar concentration had no significant impact on the larvae.(2)Inhibition effect of ZNs.ZN was mainly composed of root and leaves of Radix Isatidis(Isatis indingtica Fort.)and propolis(Apis mellifera)on CSBV.First,the appropriate CSBV concentration and ZNs were selected to feed the larvae,and the death of the larvae was observed.Then,the virus copy number in the larvae was detected by fluorescence quantitative PCR.The results showed that when the concentration of ZN8(root: leaves: propolis=1: 1: 1.5)was 0.32 mg/m L,the survival rate of the healthy larvae was the highest,and there was no difference between the size of the larvae and that of the CK group.Moreover,it could reduce the mortality of the infected CSBV larvae,promote their growth and development,and significantly inhibit the number of virus copies in the infected larvae.(3)Inhibitory effect of ZN8 active ingredients on CSBV.Three compounds were identified from ZN8 by UPLC-Q-Exactive quadrupole electrostatic field orbital well high-resolution mass spectrometry was used for full scan,and adenosine,L-proline and caffeic acid were identified.The results showed that adenosine(7.5 μg/m L)and L-prolin(75 μg/m L)could inhibit the disease and the larvae could pupate into bees.(4)The mechanism of the inhibition of CSBV by the active components of ZN8 was discussed.The results showed that adenosine and L-proline could inhibit the proliferation of CSBV,and adenosine could induce the overexpression of hymenoptaecin and defensin genes,while L-proline could induce the overexpression of hymenoptaecin.(5)To sum up,this study explored the control effect of ZNs and ingredients on Chinese sacbrood disease,and preliminarily discussed the mechanism,obtained the Chinese medicine preparation ZN8 with inhibition effect,and successfully obtained the effective ingredients in ZN8.Through inhibiting the proliferation of CBSV and inducing the high expression of hymenoptaecin and defensin genes,it provided the theoretical basis for Chinese medicine antiviral.