Research On Screen Neuroprotective Ingredients Of Buyanghuanwutang Using Neuron-Like PC 12 Cell Binding Assay

Buyanghuanwutang(BYHWT)is a popular Traditional Chinese Medicine formula consisting of seven herbal medicines which include i)Radix Astragali(huangqi),the dried roots of Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao;ii)the carda part of Radix Angelicae Sinensis root(guiwei),the dried lateral roots of Angelica sinensis(Oliv.)Diels;iii)Radix Paeoniae Rubra(chishao),the dried roots of Paeonia lactiflora Pall.;iv)Rhizoma Chuanxiong(chuanxiong),the dried rhizomes of Ligusticum chuanxiong Hort;v)Flos Carthami(honghua),the dried flowers of Carthamus tinctorius L.;vi)Semen Persicae(taoren),the dried seeds of Amygdalus persica L.;and vii)Pheretima(dilong),the dried bodies of Pheretima aspergillum(E.Perrier),and is widely for prevention of ischemic cerebral vascular diseases and stroke-induced disability.BYHWT can invigorat Qi,activate blood flow and dredge collaterals.This research will screen neuroprotective components of BYHWT by HPLC fingerprint,neuron-like PC 12 Cell bingding assay,UPLC-MS/MS and further to verify the efficacy of specific binding components using oxygen glucose deprivation reperfusion model(OGD/R)of cortical and hippocampal neurons.This research will lay the foundation for the research modern prescription and bioactive compounds of BYHWT.Objective:A HPLC method for BYHWT fingerprint and simultaneous determination of four components were established.At the same time,a method to analysis neuroprotective components in BYHWT by neuron-like PC 12 Cell bingding assay and UPLC-MS/MS was established.A method to verify the efficacy of specific binding components using OGD/R cortical and hippocampal neurons was established.Methods:1.Establishment of fingerprint and content determination method of BYHWTThe BYHWT was separated on a Hypersil ODS2 column(4.6mm×250mm,5?m)by gradient elution using acetonitrile-0.1%formic acid and detected at 250nm.The common mode of HPLC fingerprint for 10 batches of BYHWT was established by"Similarity evaluation system of chromatographic fingerpirnt for TCM revision 2012.130723".Astragaloside,ferulic acid,paeoniflorin and Chuanxiongzine in BYHWT were separated on a Hypersil ODS2 column by gradient elution using acetonitrile-0.1%phosphate.2.Culture and identification of neuron-like PC 12 cellsInduction of undifferentiated PC 12 cells into neuron-like PC 12 cells by co-culture of nerve growth factor(NGF)and identification of neuron-like PC 12 cells by immunofluorescence technique,which providing sufficient cell sources for neuron-like PC 12 Cell bingding assay.3.Establishment of neuron-like PC 12 cell binding assayScreening the specific binding ingredients of BYHWT by using neuron-like PC 12 Cell binding assay.Enrichment and concentration of the specific binding components by solid phase microextraction.4.Establishment of UPLC-MS/MS method to screening the specific binding ingredients of BYHWTStudy on the specific binding components of neuron-like PC 12 cells by UPLC-MS/MS method.5.Preliminary validation of the specific binding component of neuron-like PC 12 cell binding assayTo verify the efficacy of specific binding components by CCK8 using OGD/R cortical and hippocampal neurons.Results:1.Establishment of fingerprint and content determination method of BYHWTUnder the selected spectrum condition,the BYHWT fingerprint of 10 batches were obtained.15 peaks were found in BYHWT's fingerprint.The similarity of 10 batches BYHWT fingerprint chromatograms were over 0.9.The motheds were reproducible,stable and accurate.The content of astragaloside and ferulic acid,paeoniflorin and ligustrazine in BYHWT were 2493.423?2693.621,2.540?4.392,1428.322?1564.047,2.763?3.73?g/mL in 10 batches.2.Culture and identification of neuron-like PC 12 cellsThe undifferentiated PC 12 differentiated into neuron-like PC 12 cells in 4d by co-culture of NGF in the concentration of 100ng/mL.3.Establishment of neuron-like PC 12 cell binding assay9 chromatographic peaks were obtained using established neuron-like PC 12 Cell binding assay and this method has good reproducibility(RSD<3.00%).4.Establishment of UPLC-MS/MS method to screening the specific binding ingredients of BYHWTThe specific binding composition of neuron-like PC 12 Cell binding assay was investigated by UPLC-MS/MS technology.Six kinds of compounds were found as follows:6-Hydroxykaempferol-tri-O-glucoside,6-Hydroxykaempferol-di-O-glucoside,Calycosin-7-O-?-D-glucoside,Galloyl-paeoniflorin,Formononetin-7-O-?-D-glucoside,(3R)-7,2'-Hydroxyl-3',4'-dimethoxy-isoflavan.5.Preliminary validation of the specific binding component of neuron-like PC 12 cell binding assayUsing the OGD/R model of primary cortical neurons and hippocampal neurons,the CCK8 results showed absorbance values of specificity binding drug group were higher than OGD/R model group.The CCK8 results showed that six kinds of compounds:6-Hydroxykaempferol-tri-O-glucoside,6-Hydroxykaempferol-di-O-glucoside,Calycosin-7-O-?-D-glucoside,Galloyl-paeoniflorin,Formononetin-7-O-?-D-glucoside,(3R)-7,2'-hydroxy-3 ',4'-dimethoxy-isoflavan had neuroprotective effects.Conclusion:The establishment of HPLC fingerprint of BYHWT and the determination of Astragaloside,ferulic acid,paeoniflorin and Chuanxiongzine provide more reference for the quality control of BYHWT.Six kinds of compounds:6-Hydroxykaempferol-tri-O-glucoside,6-Hydroxykaempferol-di-O-glucoside,Calycosin-7-O-?-D-glucoside,Galloyl-paeoniflorin,Formononetin-7-O-?-D-glucoside,(3R)-7,2'-hydroxy-3',4'-dimethoxy-isoflavan had neuroprotective effects.