| ObjectiveThe oligosaccharide from morinda officinalis how as the research object of the design.In order to improve the quality control system of Morinda Officinalis How.,the research group combined with previous research base to carry out the study on the quality control of the oligosaccharide from morinda officinalis how,also the study on extraction and purification in the same time.Use in situ and in vitro animal experimental methods to investigate oligosaccharide part and main active effective component-bajijiasu of Radix Morindae Officinalis’s mechanism of intestinal absorption in situ and their effect on metabolism of CYP3A4 enzyme in the liver,and to provide scientific basis on the research and development of oligosaccharide components being made into oral preparation thus lay foundation of its further research on pharmacokinetics and pharmacodynamics.The Alzheimer disease(AD)animal model is used for investgating the anti-AD efficacy and action mechanism of the oligosaccharides from Morinda officinalis(OMO)and the main active ingredient of Morinda A prime,which provided research-based for new anti-AD drug research.Methods1 Quality control of morinda officinalis how oligosaccharide’s effective fractionUsing hydrophilic interaction chromatography-evaporative light scattering detection to establish a determination method on five kinds of morinda officinalis how oligosaccharide,including scrose,kestose,nystose,1F-fructofuranosyl nystose and morinda A prime.The Orthogonal text is adopted to optimum choose the extraction technology of morinda officinalis how oligosaccharide.Multi-index comprehensive evaluation and analysis,which indexes are built up with the paste-forming rate of morinda officinalis how oligosaccharide’s extract,the content of oligosaccharide and nystose,morinda A prime,are used to optimum select the best extraction technology.Utilizing the solvent method combined with Activated carbon and the D-900 ion exchange resin column chromatography to pure morinda officinalis how oligosaccharide.And using HPLC-UV-ELSD、UV、LC-MS to detect the content and purity of scrose,kestose,nystose,1F-fructofuranosyl nystose and morinda A prime in morinda officinalis how.2 Pharmacokinetic research on oligosaccharide in Radix Morindae OfficinalisUse single-pass intestinal perfusion method to investigate oligosaccharide part and main active effective component-bajijiasu of Radix Morindae Officinalis’s absorption and transportation mechanism in rats’ intestine and investigated saccharose,kestose,nystose,1 F-fructofuranosyl nystose,bajijiasu these 5 kinds of oligosaccharide components’feature of rats intestinal absorption,then defined each component’s best absorption site and investigated the effect of P-gp on absorption of oligosaccharide part in Radix Morindae Officinalis.β-D-fructofuranosidase was used to hydrolyze bajijiasu and to make sure whether bajijiasu is the substrate of β-D-fructofuranosidase.And to confirm the enzymatic hydrolysates of bajijiasu then conduct preliminary investigation on its enzymatic reaction kinetics.Hepatic CYP3A4 enzyme’s probe substrate dapsone was used to investigate different doses of oligosaccharide extract in Radix Morindae Officinalis and main active effective component-bajijiasu’s effect on rats’ hepatic CYP3A4 enzyme and to predict the possibility of drug interactions when combining CYP3A4 subtypesenzyme metabolism drugs.3 The effects of oligosaccharides of Morinda officinalis(0M0)on AD model ratsThe AD model rat was established by injecting the D-semi lactose associate d with the long-term injection of AP25-35.After administrating with Morinda offi cinalis extract(BT),Morinda oligosaccharides parts(BD),and Morinda A prime(BJ)for 28 days,spatial learning and memory ability were evaluated by Morris wa ter maze,which used to investigate the effects of the pre and post administration o n level of body weight and organ coefficient in AD model rats.The Kits Enzyme s-standard instrument method was used to measure the indexes of super oxide dis mutase(SOD),malondialdehyde(MDA),catalase(CAT),Glutathione reductase(gsh-px)of the AD rat’s hippocampus and cortex,and other indexes,such as acetyl cholin e(ACH),Acetyl-cholinesterase(TchE),Na+/K+-ATPase,glutamic acid(Glu),γ-amino butyric acid(GABA),NO,NOS as well.HPLC-ECD was used to mesure the level s of Dopamine(DA),Norepinephrine(NE),Epinephrine(E),5-HT monoamine neurotra nsmitter and some relevant metabolites such as 5-hydroxy indole acetic acid(5-H IAA),Homovanillic acid(HVA),3,4-2 hydroxy benzene acetic acid(DOPAC),levo dopa(L-DOPA).Western blotting(Western blot)was used to assesse the BJ,BD,BT effect on AD model rat’s APP,Tau,Caspase-3,SYP protein expression in hi ppocampus.Results1 Quality control of morinda officinalis how oligosaccharide’s effective fractionThe five oligosaccharides of sucrose,kestose,nystose,1F-fructofuranosyl nystose,morinda A prime showed a good linear relationship at ranges of 2.128～21.28 μg(r=0.9993),1.864～18.64 μg(r=0.9996),1.92～19.2μg(r=0.9998),1.912～19.12 μg(r=0.9995),2.368～23.68 μg(r=0.9994),the recovery was at a range of 92.81%～102.8%(n=6).Results show the content of sucrose,kestose,nystose,1F-fructofuranosyl nystose,morinda A prime in morinda officinalis how from different places were at ranges of 1.25%～6.07%、0.57%～6.06%、1.61%～6.82%、2.41%～7.71%、3.84%～10.10%.In study on extraction process of morinda officinalis how oligosaccharide,the effect degree of all factors for paste-forming rate and extraction rate of oligosaccharide,morinda A prime,nystose were:volume fraction of ethanol in solvent>time of extraction>ratio of solid to liquid>times of extraction.All fators were no significant influence for results.For increasing production efficiency in the industrial production,the optimum condition was A2B1C2D2,namely ten times amount of 40%alcohol and extracting two times,1h each time.Purified morinda officinalis how oligosaccharide,the content of characteristic compounds were significantly increased,the purification process shows good reproducibility.2 Pharmacokinetic research on oligosaccharide in Radix Morindae OfficinalisThe single-pass intestinal perfusion of bajijiasu of rats in situ indicated that bajijiasu was a kind of medicine that could be absorbed in the whole intestinal tract and the absorption rate differed between each part of the whole gut,and the absorption rate descended successively in the order of duodenum,jejunum,ileum and colon.All the intestinal sections,drug absorption and accumulation amount ascended with linear approximation as the concentration of drugs increased,while the absorption rate constant scarcely changed,which indicated that the absorption method of bajijiasu in duodenum,jejunum,ileum and colon was a passive diffusion primarily and its absorption process accorded with first order kinetics.Results of investigation of P-gp inhibitor’s effect on the intestinal absorption of bajijiasu indicated that P-gp had a effect of intestinal excretion on bajijiasu and could reduce its intestinal absorption,which indicated that bajijiasu might be the substrate of P-gp.Research on enzymatic hydrolysis of bajijiasu defined bajijiasu as the substrate of P-D-fructofuranosidase,A single product β-D-fructofuranose was obtained after hydrolyzation.This preliminary enzymatic reaction’s Michaelis-Menten equation was achieved:rp=0.9565Cs/(12.8761+Cs),Km=12.8761mg·mL-1,Vm=0.9565mg·mL-1·h-1.The 5 kinds of oligosaccharide components in Radix Morindae Officinalis extract in rats’ whole intestinal tract showed no significant difference(P>0.05)in absorption rate,and drug absorption and accumulation amount ascended with linear approximation as the concentration of drugs increased,which illustrated that there was no drug inhibitory concentration phenomenon within itself and the absorption mechanism of these 5 kinds of oligosaccharide components in Radix Morindae Officinalis extract in rats’ whole intestinal tract was a passive diffusion primarily and its absorption process accorded with first order kinetics.The absorption rate of each component in Radix Morindae Officinalis extract was:bajijiasu>saccharose>kestose>nystose>1F-fructofuranosyl nystose.The absorption of each component in different intestinal sections was compared,population variance results showed there was a difference(P<0.05)between absorption of different components.Results of investigation of P-gp inhibitor’s effect on the intestinal absorption of Radix Morindae Officinalis extract indicated that verapamil hydrochloride could significantly increase the absorption amount of saccharose and bajijiasu,and this absorptive enhancer effect was achieved by inhibiting the excretion of P-gp,which indicated that saccharose and bajijiasu might be the substrates of P-gp.3 The effects of Morinda oligosaccharides on AD model ratsAfter intragastric administrating for 28 days,the Morris water maze tests showed that the latency and total route of place navigation of model group were significantly longer than blank group’s.While the other OMO treated groups were improved.Spatial probe tests showed that the swinging distance in the platform quadrant has been incresed compared with total distance,besides,the times through the position of the hiding platform was significantly incresed.The result of organ coefficient count showed that the organ Coefficient of the model group such as the brain,the spleen,the testis were lower,compared with normal control group.While all the organ Coefficient of BD,BT group had incresed,and BJ increased the organ Coefficient of the brain,the hepatic,and the testis.By injecting Aβ25-35 in the bilateral hippocampus in rats associated with the 1 ong-term injection of D-semilactose in the abdominal cavity,the contents of Neuro transmitter in rats’ hippocampus and cortex decreased obviously,while the contents of NE,DA,5-HT increased significantly after being treated with BJ,BD,BT,which e nhanced the ability of learning and memory.Meanwhile the ratio of DA/HVA,NE/E,5-HT/5-HIAA were improved in various degrees at different levels,showing that BJ,BD,BT can inhibit the relevant metabolic pathways.The level ofAPP,Tau,Caspase-3 protein expression in hippocampus of the rats were improved and the SYP expression markedly decreasedby via injecting with The D-semila ctose and the long term injection of Aβ25-35,while abnormal expression levels of each pr otein eased after being treated with BJ,BD,BT respectively,suggesting that oligosacchari des of Morinda officinali can improve the action mechanism of learning and memory diso rder,which may be relate to up-regulated expression of the SYP,do-wn-regulated expres sion of the APP,TAU,and Caspase-3 protein.Conclusion1 Quality control of morinda officinalis how oligosaccharide’s effective fractionEstablishing the determination method of morinda officinalis how oligosaccharide’s active ingredient content is simple,accurate and has a good reproducibility.It provides an effective analytical method for the morinda officinalis how.Combined with the results of this study in accordance with the requirements of the Chinese Pharmacopoeia,in this,the content limits of sucrose,kestose,nystose,1F-fructofuranosyl nystose,and morinda A prime from morinda officinalis how can be proposed preliminary recommendations:their contents are calculated according to morinda officinalis how’s dry product,separately,the nystose must not less than 2%,Bajijiasu must not less than 4%,and the total content of sucrose,kestose,nystose,1F-fructofuranosyl nystose,Bajijiasu must not less than 14%.The optimum morinda officinalis how’s extraction technology has a higher levels fo Each index components,and its process is stable,reasonable and feasible,which provides a reference for industrial production.Morinda officinalis how oligosaccharide’s purification process which has a good reproducibility,and production in a high purity that ingredient content is more than 50%,can be used to develop five new drugs of national regulations.2 Pharmacokinetic research on oligosaccharide in Radix Morindae OfficinalisBajijiasu was well absorbed in the whole intestinal tract and belonged to high permeability drugs,the absorption mechanisms of duodenum,jejunum,ileum and colon were passive diffusions primarily.This research will offer biological foundations for bajijiasu being designed into oral sustained/controlled-release preparations.The affinity between β-D-fructofuranosidase and bajijiasu was relatively strong and glucose had certain promoting effect for bajijiasu’s enzymolysis,which indicated that the glucose within body might have certain promoting effect for bajijiasu’s metabolism thus decreasing its bioavailability.This research will offer academic reference for bajijiasu’s pharmacokinetic research and it’s new drug development.As for the whole intestinal tract absorption of oligosaccharide in Radix Morin dae Officinalis,the absorption rate was:disaccharide>triose>tetrasaccharide>penta saccharide,it could be deduced that the absorption rate might have a relationship with each component’s molecular size.Under this experimental condition,each co mponent’s Papp in different intestinal sections was higher than 0.072 cm·h-1,which illustrated that all the oligosaccharide components were well absorbed in the who le intestinal tract and belonged to high permeability drugs,and this might have a relationship with each component’s relatively strong hydrophilicity making them ea sy to be diffused and absorbed into the body via the intestine.3 The effects of Morinda oligosaccharides on AD model ratsThe injection of Aβ25-35 in he bilateral hippocampus in rats associated with the Ion g-term injection of D-semilactose in the abdominal cavity were employed in this experim ent,inducing AD animal model.To observe effects of Morinda officinali oli-gosaccharid es on AD model rats from aspects of the behavior,Oxidative Stress,choli-nergic system,energy metabolism,gamma-aminobutyric acid(GABA),neurotransmitter and protein e xpression,The results show that Morinda oligosaccharides can im-prove the impairment of learning and memory ability of AD rats,whose mechanism of action may be relate to th e up regulation of neurotransmitter,up-regulated expression of the SYP and down-regulat ed expression of the APP,TAU,and Caspase-3 protein. |
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