Study On The Active Components Of Dodder In Promoting The Proliferation Of Neural Stem Cells

Stroke,also known as infarction,is the disorder of consciousness after cerebral thrombosis.At the beginning of the onset of thrombolytic therapy,due to the short of thrombolytic time,most patients cannot acquire timely and effective treatment.When patients enter the sequelae of stroke,due to the damaged ischemic nerve,they will appear limb paralysis,aphasia and other disability symptoms of varying degrees.In modern clinical practice,stroke sequelae are mainly treated by modern rehabilitation methods(general language rehabilitation training,muscle spasm rehabilitation training).And there is a lack of related drugs that can be directly treated in clinic.So most patients can not achieve good recovery effect.In the final,these symptoms are the result of nerve death,and the key to nerve regeneration is neural stem cells.Neural stem cells(NSCs)exist in the central nervous system of adult animals.They are multifunctional potential cells that can self-replicate.In the state of brain injuried,it can proliferate,migrate and differentiate into neurons,astrocytes and oligodendrocytes.NSCs have two basic properties:self proliferation and multi differentiation;Self proliferation is the first step of NSCs.However,there is still a lack of related drugs to promote the proliferation of NSCs and repair nerve.According to the theory of traditional Chinese medicine,it is difficult to recover from limb paralysis due to the stagnation of brain collaterals,the failure of mental mechanism and the dysfunction of limbs' meridians.Our research group argue that tonifying the kidney,filling the essence and filling the sea of marrow should be the key points of the treatment.The result of related research showed that the extract of Cuscuta chinensis had strong proliferation effect on NSCs.Cuscuta chinensis is a traditional kidney tonifying Chinese medicine,which has the effect of tonifying liver and kidney,strengthening essence and reducing urine.We aim to find the active components of Cuscuta chinensis to promote the proliferation of NSCs by activity tracking method,so as to lay the foundation for obtaining potential drugs with clinical value.In this study,CCK-8 method was selected as the activity tracking method to detect the component of Cuscuta chinensis proliferation of NSCs.The ethanol extract of Cuscuta chinensis was separated by systematic solvent extraction,and the maximum cell proliferation rate of petroleum ether fraction was 24.32%,dichloromethane fraction was 51.87%(3.12 ?g/mL),ethyl acetate fraction was 53.31%(1.56?g/mL),nbutyl alcohol fraction was 20.30%(6.25?g/mL)and remaining water fraction was 21.05%(6.25 ?g/mL).The activity of ethyl acetate fraction(TSZE-EA)was the best;at a certain concentration(1.56?g/mL),the rate of cell proliferation was 53.31%.The ethyl acetate fraction was further separated by silica gel chromatography.Seven fractions(TSZE-EA-G1?G7)were isolated from TSZE-EA by silica gel chromatography.The maximum proliferation rates of each fraction were 18.82%(12.5?g/mL)for TSZE-EA-G2?30.76%for TSZE-EA-G3(6.25?g/mL)?24.07%for TSZEEA-G4(6.25?g/mL)?21.53%for TSZE-EA-G5(6.25?g/mL)?76.57%for TSZE-EAG6 76.57%(12.5?g/mL)?13.04%for TSZE-EA-G7(3.12?g/mL)and TSZE-EA-G1 has no proliferation activity.TSZE-EA-G6 was the main active component.Seven chemical constituents were identified from TSZE-EA-G6 by UPLC-QE-Orbitrap-MS,which were 4-O-pcoumaroylquinic acid,chlorogenic acid,5-O-p-coumaroylquinic acid,Hyperoside,astragaloside,isochlorogenic acid C,quercetin-3-O-galactose-7-O-glucoside.Six compounds(TSZE-EA-G6-A-F)were isolated by traditional chromatography.Five of them(TSZE-EA-G6-A-E)were identified as kaempferol,kaempferol-3-O-glucoside,quercetin-3-O-galactoside,chlorogenic acid and sucrose by NMR(1H?13C).The results showed that kaempferol and TSZE-EA-G6-F had the highest rate of cell proliferation:87.44%and 117.06%.The results of network pharmacology suggest that the target of flavonoids including kaempferol may be related to AKT1,the key kinase of PI3K/AKT signaling pathway,and flavonoid aglycone may promote the proliferation of NSCs better than flavonoid aglycone.The result of this research showed that the proliferation effect of kaempferol on NSCs was first discovered.It is also the first related active ingredient from Cuscuta chinensis.The further research of this project will focus on the following aspects:the structure identification of compound TSZE-EA-G6-F;search for active components in dichloromethane fraction;the study of the activity and structure relationship of flavonoids.The study of the drugability of the obtained components and their mechanism.