The Effect Of Lycium Ruthenicum Anthocyanins On Anxiety And Craving After Nicotine Withdrawal In Mice And Its Mechanism

BackgroundTobacco harm is one of the most serious public health problems in the world,and long-term smoking may cause many diseases.Smoking can be addictive which is called tobacco dependence.Many smokers have different degrees of tobacco dependence.Although some smokers have a strong desire to quit smoking,they usually relapse within a week.Studies have shown that the main substance in tobacco that causes tobacco dependence is nicotine.A series of withdrawal symptoms will occur after nicotine withdrawal,among them,anxiety and nicotine craving are very obvious,both of them could induce relapse of patients,so relieving the anxiety and craving after nicotine withdrawal would help to treat nicotine addiction and help patients quit smoking.At present,nicotine replacement therapy and non-nicotine therapy are commonly used in the treatment of nicotine dependence,but most of the drugs have many side effects or certain addictions.Therefore,it is of great significance to find effective drugs with little side effects for clinical treatment of nicotine addiction and helping quit smoking.The black Chinese wolfberry is the fruit of the Lycium ruthenicum Murray in the Solanaceae family and widely used in traditional ethnic medicine.Black Chinese wolfberry is rich in various components and the anthocyanins are considered to be one of the main active ingredients.Research shows that the anthocyanins of black Chinese wolfberry have many physiological activities such as antioxidation,free radical scavenging,lipid regulation and anti-aging.Our previous work found that the extract of anthocyanins in black Chinese wolfberry has a stable anti-anxiety effect,its mechanism includes reducing the content of corticotrophin releasing factor(CRF),regulating extracellular regulated protein kinase/mitogen-activated protein kinase(ERK/MAPK)pathway and so on,which are related to the mechanism of nicotine addiction.However,whether the antianxiety effect of black Chinese wolfberry could be applied to the treatment of nicotine addiction remains to be further studied.Clarifying this issue has good social significance and economic value for the development and application of anthocyanins in black Chinese wolfberry.Therefore,in this study,we extracted and purified the anthocyanins from black Chinese wolfberry,clarified its components,studied its influence on the anxiety and craving after nicotine withdrawal,and explored its mechanism.This study is supposed to provide theoretical and experimental basis for the application of anthocyanins in black Chinese wolfberry in the treatment of nicotine addiction,and provide reference for the treatment of nicotine addiction and smoking in clinical.ObjectiveThe purpose of this study is to optimize the purification process of anthocyanins in black Chinese wolfberry with macroporous resin and identify the structure of anthocyanins in the purified products;to explore the effect and mechanism of the anthocyanins in black Chinese wolfberry on anxiety and craving after nicotine withdrawal,so as to lay a foundation for the development of anthocyanins in black Chinese wolfberry into safe and effective smoking cessation products.Methods1 Optimization of purification process of anthocyanins in black Chinese wolfberryThe crude extract of anthocyanins in black Chinese wolfberry was extracted by water bath heating method,then the factors in the purification process of anthocyanins in black Chinese wolfberry with X-5 macroporous resin were optimized by single factor investigation and Box-Behnken response surface.The stable purification process parameters and high purity anthocyanins were obtained.2 Identification of anthocyanins in black Chinese wolfberryThe anthocyanins in black Chinese wolfberry were purified according to the optimal conditions,and the components of anthocyanins were identified by UPLC-Orbitrap MS.3 The effect of anthocyanins in black Chinese wolfberry on anxiety induced by nicotine withdrawal in miceIn order to study the effect of anthocyanins in black Chinese wolfberry at different doses(1 g/kg,2 g/kg,3 g/kg)on anxiety after nicotine withdrawal,Elevated cross maze and light-dark box experiments were used to evaluate their anti-anxiety effects in mice.4 The effect of anthocyanins in black Chinese wolfberry on the craving induced by nicotine withdrawal in miceWe established a mice model of conditioned place preference addiction induced by nicotine,and then used nicotine to induce the recovery of expression of conditioned place preference.Anthocyanins(3 g/kg)was administered during the regression period to evaluate its effect on the craving behavior in mice after withdrawal.5 The mechanism of anthocyanins in black Chinese wolfberry in the treatment of nicotine addictionNetwork pharmacology was used to analyze the action target of anthocyanins in black Chinese wolfberry.We constructed the "component-target-disease" network,and carried on the GO biological function analysis and KEGG pathway enrichment analysis to preliminarily explore the mechanism of its treatment of nicotine addiction.Results1 Optimization of purification process of anthocyanins in black Chinese wolfberryThe best process conditions for purification of anthocyanins in black Chinese wolfberry by X-5 macroporous resin were as follows:macroporous resin diameter to height ratio 1:15,loading solution concentration 32 g/L,loading solution pH=2.7,loading flow rate 1 mL/min;eluent concentration 92%,eluent pH=3.6,elution flow rate 1 mL/min,eluent volume 3.31 BV.Under this condition,the yield of anthocyanins of Lycium ruthenicum was 8.01%,and the color value was 33.8,which was 17.79 times higher than that of crude extract.2 Identification of anthocyanins in black Chinese wolfberryThere were 21 anthocyanins in the purified product,including 10 anthocyanins with petunidin as the mother nucleus,4 anthocyanins with delphinidin as the mother nucleus,2 anthocyanins with malvidin as the mother nucleus,1 anthocyanin with cyanidin as the mother nucleus and 4 proanthocyanidins.Petunidin-3-O-rutinoside(feruloyl)-5-O-glucoside and its methylated derivative were reported for the first time in black Chinese wolfberry.3 The relief effect of anthocyanins in black Chinese wolfberry on anxiety induced by nicotine withdrawal in miceIn the elevated cross maze experiment,the middle dose(2 g/kg)and high dose(3 g/kg)of anthocyanins in black Chinese wolfberry could significantly increase the percentage of time that nicotine withdrawal mice spend in the open arm,and the high dose could also significantly increase the percentage of entries to the open arm;in the light-dark box experiment,the low(1 g/kg),medium and high doses of anthocyanins in black Chinese wolfberry could significantly increase the time for nicotine withdrawal mice spend in the light box,and the high dose could also be significant increase the entries to the light box.4 The relief effect of anthocyanins in black Chinese wolfberry on the craving induced by nicotine withdrawal in miceIn the expression period,the conditioned place preference(CPP)score of model group was significantly higher than control group,and also significantly increased compared with the baseline value;after 3 days of extinction period,the CPP score of model group and treatment group was significantly lower than that of the expression period;after receiving nicotine again,the CPP score of model group was significantly higher than control group and significantly increased compared with the value in regression period,and the CPP score of treatment group was significantly lower than model group and there was no significant difference between the extinction period and reinstatement period.5 The mechanism of anthocyanins in black Chinese wolfberry in the treatment of nicotine addictionThe anthocyanins in black Chinese wolfberry may regulate signal transduction by acting on IL2,APP,SRC,MMP9,ESR1,PIK3R1,MCL1,EGFR,AKT1,MMP2,SIRT1,IGF1R,KDR,PTGS2 and TNF,and participate in the production and transmission of dopamine,choline and other nervous system functions,as well as steroid,estrogen,serotonin,prolactin,oxytocin and other biological molecules,so as to play a role in the treatment of nicotine addiction.ConclusionsIn this paper,the process conditions of purification of anthocyanins in black Chinese wolfberry by X-5 macroporous resin were optimized,and stable purification parameters were obtained.The process is simple and feasible,and the purification effect is good,which is suitable for the purification of anthocyanins in black Chinese wolfberry.21 anthocyanins were identified from the purified product,petunidin-3-O-rutinoside(feruloyl)-5-O-glucoside and its methylated derivative were reported for the first time in black Chinese wolfberry.This study clarified the types of anthocyanins in black Chinese wolfberry and provided the basis for later research.Anthocyanins in black Chinese wolfberry showed the therapeutic effect on the anxiety of nicotine addicted mice after withdrawal,inhibited the recovery of conditioned place preference induced by nicotine after withdrawal,and showed the therapeutic effect on the nicotine craving after withdrawal in mice.It is suggested that the mechanism is to regulate the signal transduction in dopamine,choline and other nervous systems,as well-as the production and transmission of steroids,estrogen,serotonin,prolactin,oxytocin and other biological molecules.The anthocyanins in black Chinese wolfberry have the therapeutic effect on nicotine withdrawal symptoms from the aspects of anti-anxiety and relieving nicotine craving,which is expected to be developed into a safe and effective smoking cessation product for the treatment of tobacco addiction and cessation.