Pharmacological Activity And Mechanism Z. Bungeanum Attenuated Scopolamine-induced Cognition Impairments In Mice

Zanthoxylum bungeanum is the dried pericarp of ripe fruit of the Zanthoxylum bungeanum Maxim plant of family Rutaceae,Zanthoxylum genus.As part of our continuing study of Z.bungeanum pericarp,we found that Z.bungeanum pericarp can significantly activate the spontaneous activities of the certain brain regions,such as the hippocampus(HIPP)and paraHIPP,using the functional magnetic resonance imaging(fMRI),suggesting that Z.bungeanum pericarp might enhance the formation of learning and memory ability.However,its effective active fraction against learning and memory impairment and its related mechanisms are still unclear so far.Objective:The Functional magnetic resonance(fMRI)was used to study the central response mechanisms of Z.bungeanum percarp under amplitude of low-frequency fluctuation(0.1 ~ 0.8 Hz).The learning and memory impairments model induced by scopolamine(1.0 mg/kg,i.p.)was used to systematically evaluate the effect of Z.bungeanum pericarps against the cognitive impairment,and the effective active fraction was further screened.Furthermore,the cognition-improving effect of HAS,as the most representative compound isolated from Z.bungeanum percarp,was studied and its related mechanisms of action were preliminarily explored.The stability of HAS in water extraction of Z.bungeanum percarp were investigated.Methods:(1)Study on the central brain response mechanism of Z.bungeanum pericarps.To explore the central response mechanism of ZBP,the data was collected before and after sublingual ZBP powder(0.1 g)stimulation using the functional magnetic resonance(fMRI)technique,and the ampitude of low frequency amplitude(ALFF)and fractional ALFF(fALFF)were used to analyze data.Furthermore,the functional connectivity(FC)of the bilateral hippocampus as region of interest was also investigated.(2)Effective fraction of ZBP against the learning and memory impairments: The Morris water maze(latency time,crossing times of platform,and residence time in the platform quadrant),passive avoidance test(latency time)and HE staining were performed to screen the effective fraction of ZBP(petroleum ether,ethyl acetate,n-butyl alcohol and water)on learning and memory impairment based on learning and memory defict induced by scopolamine.(3)The pharmacological activity and its mechanism of HAS to improving the learning and memory impairments: The pharmacological activity of HAS on improving learning and memory impairment(Morris maze,passive test and HE staining)was further investigated.Furthermore,its mechanism was explored by evaluating the activity of acetylcholinesterase(AChE),mRNA and protein expression level of the CREB and BDNF in hippocampus.(4)Stability study of HAS in aqueous extract of Z.bungeanum pericarps: The stability of HAS was explored by focusing on the effects of temperature,air oxygen,ultraviolet light,pH value,artificial gastrointestinal fluid and other factors.This study could improve the retention rate and clinical efficacy of HAS in the aqueous extract of Z.bungeanum.Results:(1)We found that significant ALFF and fALFF signal increases in response to ZBP stimulation were mainly focused in the regions of the bilateral HIPP,paraHIPP,and medial prefrontal cortex(mPFC),superior parietal,precuneus,and especially in the HIPP,paraHIPP and mPFC regions(P < 0.001,uncorrected).Additionally,the functional connectivity between the bilateral HIPP and the contralateral HIPP,paraHIPP and some other brain regions were obviously enhanced.The results suggested that ZBP might enhance the learning and memory ability.(2)Petroleum ether and ethyl acetate fraction(1.0 g/kg,i.g.)both significantly reduced the escape latency time during the acquisition phase,increased the number of platform crossing in the probe trial and the platform quadrant residence time of Morris water maze,prolonged the escape latency in the passive test,and ameliorated scopolamine-induced hippocampal neuron damage of mice;however,the n-butanol and water fraction were not significantly different from the model group.(3)HAS(1.25,2.5 and 5.0 mg/kg,i.g.)reduced the escape latency during the acquisition phase,increased the number of platform crossing and the platform quadrant residence time in the probe trial in Morris water maze,prolonged the escape latency of the passive test,and dose dependently prevented changes in hippocampal neuron morphology and apoptosis.HAS inhibited the acetylcholinesterase(AChE)activity,increased the mRNA and protein expression of the brain-derived neurotrophic factor(BDNF)and phosphor cAMP response element-binding(p-CREB)compared with the model group by western blotting,quantitative real-time polymerase chain reaction(qRT-PCR)and immunohistochemical staining.(4)The results of stability experiments showed that UV light,high temperature(80 °C),strong acid(pH = 2)and strong alkali(pH = 12)have a great influence on the stability of HAS,and the content degradation is as high as 58.0%.The stability of HAS is better in artificial intestinal fluid than in the gastric juice;it is unstable in artificial gastric juice,and the remaining percentage is only 17.8% within 8 hours.Conclusion:In this study,HAS isolated from the Z.bungeanum was found to improving the learning and memory impairment in the pathological model of cognitive deficit induced by scopolamine in mice;furthermore,the mechanism of action is related to the activation of cholinergic system and CREB/BDNF signaling pathways;HAS is an compound that is extremely sensitive to acid and alkali environments,therefore it can be prepared into microcapsules,cyclodextrin inclusion complexes,etc.to ensure its biological activity.