Mechanism Of Action Of Total Barley Malt Alkaloids In The Treatment Of Hyperprolactinemia And Optimization Of Granule Formulation

Objective: Malt is traditionally used to treat abnormal milk secretion.It has also been clinically confirmed that malt can improve abnormal lactation and is mainly used in the clinical treatment of hyperprolactinemia,but the mechanism of malt’s anti-hyperprolactinemia is still unclear.Based on the previous experiments,this study explored the target and mechanism of the active ingredient in malt in inhibiting prolactin,so as to provide reference and theoretical basis for the traditional use of malt in the treatment of hyperprolactinemia.Methods:(Ⅰ)CCK-8 method was used to screen the optimal culture time of MMQ cells and GH3 cells;PRL levels were detected by enzyme linked immunosorbent assay(ELISA),Real-time PCR and Western blot,and the effective concentrations of total malt alkaloid,malt alkaloid and arrowine were screened,respectively.(Ⅱ)MMQ cells were pretreated with the dopamine D2 receptor antagonist haloperidol,and then the total malt alkaloid and malt alkaloid were added for further culture.PRL levels were detected by ELISA,Real-time PCR and Western blot.DRD2 levels were detected by Real-time PCR and Western blot.The cAMP content was detected by ELISA,and the protein expression levels of PKA-C,CREB and p-CREB were detected by Western blot.(Ⅲ)Rat pheochromocytoma PC12 cells were cultured with total malt alkaloid and malt alkaloid,respectively.Real-time PCR was used to detect the mRNA expression levels of DRD2 and DAT,and Western blot was used to detect the protein expression levels of DRD2 and DAT.(Ⅳ)MMQ cells were cultured with total malt alkaloid,malt base and adenylate cyclase agonist respectively.PRL levels were detected by ELISA,Real-time PCR and Western blot.The cAMP content was detected by ELISA,and the protein expression levels of PKA-C,CREB and p-CREB were detected by Western blot.MMQ cells were cultured with total malt alkaloid and malt alkali combined with protein kinase A agonist,respectively.PRL levels were detected by ELISA,Real-time PCR and Western blot.The cAMP content was detected by ELISA,and the protein expression levels of PKA-C,CREB and p-CREB were detected by Western blot.(Ⅴ)Orthogonal experimental design was designed to optimize the formulation of total alkaloid granules of malt,and score was made according to molding rate,pile density,Angle of recline and hygroscopicity.The solubility,drying weight loss and particle size were tested to control the quality of the optimal formula.The content of malt in total alkaloid granules was determined by high performance liquid chromatography.Results:(Ⅰ)The PRL level of MMQ cells in 35.2 and 70.4 μg/mL total malt alkaloid groups and 3.25 and 6.5μg/mL large malt alkaloid groups was significantly decreased(P < 0.05),but there was no significant change in PRL level of GH3 cells.(Ⅱ)Compared with the total malt alkaloid group,the PRL of MMQ cells in the total malt alkaloid + haloperidol group was significantly increased(P <0.05);Compared with the BMA group,PRL of MMQ cells in BMA +Haloperidol group was significantly increased(P < 0.05).The level of DRD2 and the content of cAMP in MMQ cells were significantly increased(P <0.05),and the activities of PKA and CREB were significantly decreased(P <0.05).Compared with the total malt alkaloid group,the expression of DRD2 was significantly decreased,the secretion of PRL was significantly increased,and the expression of cAMP/PKA/CREB was significantly increased in the total malt alkaloid + haloperyl alcohol group(P < 0.05).(Ⅲ)The expression levels of DRD2 and DAT in PC12 cells of malt total alkaloid group and malt base group were significantly increased(P < 0.05).(Ⅳ)Compared with Forskolin group,PRL secretion and cAMP/PKA/CREB expression in MMQ cells of malt total alkaloid +Forskolin group were not significantly changed.Compared with 8-Bromo-cAMP group,PRL secretion and cAMP/PKA/CREB expression in MMQ cells in the malt total alkaloid+8-Bromo-cAMP group were significantly decreased(P < 0.05).Compared with Forskolin group,PRL secretion and cAMP/PKA/CREB expression of MMQ cells in malt +Forskolin group were not significantly changed.Compared with 8-Bromo-cAMP group,PRL secretion and cAMP/PKA/CREB expression in MMQ cells of malt +8-Bromo-cAMP group were not significantly changed.(Ⅴ)The orthogonal test results showed that when dextrin:lactose :MCC=1:2:1,the formability of granules was the best;The ratio of extract powder and excipients is 1,which is beneficial to control the dosage.The solubility,particle size and dry weight loss of the granules prepared according to the optimal formulation were all in line with the regulations of Chinese Pharmacopoeia 2020 edition.The content of maltine in the total malt alkaloids was 4.953 mg/g.Conclusion:(1)Malt exerts an anti-hyperprolactinemia effect through a single target pathway dependent on dopamine D2 receptor: Malt stimulates DRD2,which in turn activates the cAMP/PKA/CREB signaling pathway to reduce PRL.(2)Malt total alkaloids can exert anti-hyperprolactinemia effects through multi-target pathways: 1.Malt total alkaloids can stimulate DRD2,activate cAMP/PKA/CREB signaling pathway,and inhibit PRL secretion.2.Malt total alkaloids can directly act on PKA and reduce PRL level by inhibiting PKA activity.(3)Both malt alkaloids and malt alkaloids can up-regulate DRD2 and DAT,suggesting that both malt alkaloids and malt alkaloids act on at least two components of the dopamine pathway,not only on DRD2.(4)The granules of malt total alkaloid were successfully prepared in accordance with the provisions of Chinese Pharmacopoeia 2020 edition.