Total Saponins of Panax notoginseng Activate Akt/mTOR Pathway and Exhibit Neuroprotection in vitro and in vivo against Ischemic Damage

Objective:To reveal the neuroprotective effect and the underlying mechanisms of a mixture of the main components of Panax notoginseng saponins(TSPN)on cerebral ischemia-reperfusion injury and oxygenglucose deprivation/reoxygenation(OGD/R)of cultured cortical neurons.Methods:The neuroprotective effect of TSPN was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay,flow cytometry and live/dead cell assays.The morphology of dendrites was detected by immunofluorescence.Middle cerebral artery occlusion(MCAO)was developed in rats as a model of cerebral ischemia-reperfusion.The neuroprotective effect of TSPN was evaluated by neurological scoring,tail suspension test,2,3,5-triphenyltetrazolium chloride(TTC)and Nissl stainings.Western blot analysis,immunohistochemistry and immunofluorescence were used to measure the changes in the Akt/mammalian target of rapamycin(mTOR)signaling pathway.Results:MTT showed that TSPN(50,25 and 12.5μg/m L)protected cortical neurons after OGD/R treatment(P<0.01 or P<0.05).Flow cytometry and live/dead cell assays indicated that 25μg/m L TSPN decreased neuronal apoptosis(P<0.05),and immunofluorescence showed that 25μg/m L TSPN restored the dendritic morphology of damaged neurons(P<0.05).Moreover,12.5μg/m L TSPN downregulated the expression of Beclin-1,Cleaved-caspase 3 and LC3 B-Ⅱ/LC3 B-Ⅰ,and upregulated the levels of phosphorylated(p)-Akt and p-mTOR(P<0.01 or P<0.05).In the MCAO model,50μg/m L TSPN improved defective neurological behavior and reduced infarct volume(P<0.05).Moreover,the expression of Beclin-1 and LC3 B in cerebral ischemic penumbra was downregulated after 50μg/m L TSPN treatment,whereas the p-mTOR level was upregulated(P<0.05 or P<0.01).Conclusions:TSPN promoted neuronal survival and protected dendrite integrity after OGD/R and had a potential therapeutic effect by alleviating neurological deficits and reversing neuronal loss.TSPN promoted p-mTOR and inhibited Beclin-1 to alleviate ischemic damage,which may be the mechanism that underlies the neuroprotective activity of TSPN.

Chemical constituents from acid hydrolyzates of Panax quinquefolius total saponins and their inhibition activity to α-glycosidase and protein tyrosine phosphatase 1B

Objective:To investigate the hypoglycemic components from the acid hydrolyzates of Panax quinquefolius total saponins,and screen the active compounds by in vitro inhibitory activities toα-glycosidase enzymes and protein tyrosine phosphatase-1 B(PTP1 B).Methods:The hydrolyzates were chromatographed repeatedly over silica gel column,and the structures of the compounds were determined by means of NMR.The in vitro bioassay was performed through the inhibitory effects onα-glucosidase or/and PTP1 B.Results:Eight compounds were isolated,which identified as 20(S)-panaxadiol(1),(20 S,24 R)-dammarane-20,24-epoxy-3β,6α,12β,25-tetraol(2),20(R)-dammarane-3β,12β,20,25-tetraol(3),20(S)-dammarane-3β,6α,12β,20,25-pentol(4),20(R)-dammarane-3β,12β,20,25-tetrahydroxy-3β-O-β-D-glucopyranoside(5),β-sitosterol(6),oleanolic acid(7)and 20(S)-protopanaxadiol(8).Compound 5 was ginseng triterpenoid isolated from the acid hydrolysates of total saponins from P.quinquefolius for the first time.In this paper,the possible in vitro inhibitory activities were investigated.Compound 5 exhibited significantly inhibitory activity againstα-glucosidase,and the IC50 value[(0.22±0.21)μmol/L]was about 43-fold lower than positive control.For the PTP1 B inhibition assay,compound 5 indicated the strongest inhibitory effect with IC50 of(5.91±0.38)μmol/L,followed by compound 4 with IC50 of(6.21±0.21)μmol/L,which were all showed competitive inhibitory pattern by using a Lineweaver-Burk plot.Conclusion:These results supported the potential application of dammaranes from acid hydrolyzates of P.quinquefolius total saponins can be used as ingredients of ancillary anti-diabetic agent or functional factor.

A mathematical model for quality evaluation of total saponins of Panax japonicas based on hypolipidemic activity

Objective: The chemical finger printing-based methods for evaluating TCMs quality can report partial of TCMs quality without linking to effective constituents. In this study, a mathematical model was established for the quality evaluation of total saponins of Panax japonicus(TSPJ), a folk medicine in China and Japan for treating diseases, through coupling the dynamic changes of chemical constitutions with corresponding activities.Methods: High-performance liquid chromatography(HPLC) fingerprints were applied to establish the chromatographic database of TSPJ. The associated hypolipidemic activity database was determined by TG assay using Hep G2 cell model. Correlation analyses of two databases were performed by partial least squares(PLS) for calculating regression coefficients, and the interval value of YZL value(the ratio of positive and negative peak-to-peak area coefficient) closely related to hypolipidemic activity was refined by the formula of Norminv function to value the quality of TSPJ.Results: In this study, the chromatographic data of 16 common peaks were obtained from 20 batches of TSPJ. After the estimate by this mathematical evaluation model, seven peaks were positively correlated with hypolipidemic activity, and nine peaks were negatively correlated with hypolipidemic activity.When the YZL value was less than 0.7861, the quality of sample was inferior, while YZL value was more than 6.6992, and the quality of samples was superior. The quality of another ten batches of TSPJ was further assessed to verify this method.Conclusion: These results indicated that the established model could be usefully applied to evaluate the quality of TSPJ in the hypolipidemic activity.

Neuroprotection of total steroid saponins from Dioscorea zingiberensis C.H.Wright against transient focal cerebral ischemia-reperfusion injury in rats via its anti-inflammatory and anti-apoptotic effects

OBJECTIVE The total steroid saponins(TSSN)isolated from Dioscorea zingiberensis C.H.Wright(D.zingiberensis)has shown a variety of beneficial bioactivities.However,there are no reports about the neuroprotective effects of the TSSN until now.Therefore,we explored the neuroprotective effects of TSSN on rats against transient focal cerebral ischemia-reperfusion(I/R)and the underlying mechanisms.METHODS The healthy adult Sprague-Dawley rats were randomly assigned into six groups.After pre-treatment with the TSSN intragastrically for six days,the rats were subjected to the ischemia injury by the surgery of middle cerebral artery occlusion(MCAO)for 90 min.Some indexes were evaluated and detected.RESULTS As compared to the I/R group,TSSN group of rats,especially given the 30 mg·kg^-1 of TSSN,not only marked reduction in the neurological deficit scores,cerebral infarct volume,and brain edema,but also an increase in neuron survival(Nissl bodies)in the hippocampal cornuammons 1(CA1)and cortex hemisphere of the ipsilateral ischemia.At the same time,the inflammatory cytokines in serum induced by MCAO were significantly alleviated by the TSSN pre-administration.What′s more,the increase of caspase-3 was evidently reduced in the CA1 and cortex of the hemisphere injured brain.Finally,the down-regulating anti-apoptotic Bcl-2 and up-regulating pro-apoptotic Bax proteins were obviously suppressed.CONCLUSION TSSN plays a potential neuroprotective role against a severe injury induced by transient focal cerebral ischemic reperfusion in a rat experimental model,and this role may be mediated by its antiinflammatory and anti-apoptotic actions.

Mechanism of TSCS promote anti-Fas antibody-induced FLS apoptosis via blocking MC-tryptase-PAR-2-Rho-FLS signaling pathway

OBJECTIVE It has been supposed that mast cells have important participation in the physiopathology of RA,however,the role of mast cells in the pathogenesis of RA remains unclear.In this study,we observed the antiapoptotic effects of tryptase released by mast cell on RA synovial fibroblasts.METHODS Mast cells and fibroblasts synovial were obtained from mouse.Chemical mediator release was assessed by measuringβ-hexosa-minidase release.TSCS and bone marrow-derived mast cells were co-cultured;the toxic effects of TSCS on mast cell was measured by MTT and CCK-8 method;the releasing amount of tryptase in cell supernatants was measured by Elisa assay;the expression of FLS cell membrane receptor PAR-2 was detected by flow cytometry;the apoptosis of FLS cell was detected through flow cytometry and Western blotting;the level of activated Rho-GTP was detected by the pull-down method and Western blotting.RESULTS In this study,we observed the antiapoptotic effects of tryptase released by mast cell on RA synovial fibroblasts,and found that tryptase significantly increased the expression of PAR-2 on the surface of fibroblast-like synovial cell,significantly activated Rho kinase,significantly inhibited apoptosis of fibroblast-like synovial cell induced by CH11.The release rates ofβ-hexosaminidase and the level of tryptase from bone marrow-derived mast cells after stimulation with different antigen and co-cultured with TSCS significantly decreased compared to the control group.In the co-culture system of mast cells and fibroblast-like synovial cells,TSCS treatment significantly inhibited Rho kinase(P<0.05),significantly promoted apoptosis of fibroblast-like synovial cell induced by CH11(P<0.05).CONCLUSION These results demonstrate that tryptase may play a key role in the physiopathology of RA.TSCS can inhibit mast cells activation and promote FLS cells apoptosis.This provide theoretical and experimental basis for the study of mast cells as targets for new anti-RA drugs.

Changes in Active Components before and after Microorganism Fermentation of Rhizoma Paridis(Chonglou)

[Objectives] To study the changes in active components before and after microorganism fermentation of Rhizoma Paridis( Chonglou). [Methods]Analytical methods using total saponins and total flavonoids as indicators were established. UV spectrophotometry was used to determine the changes in active components before and after fermentation of Rhizoma Paridis. [Results] The content of total saponins decreased after fermentation,while the content of total flavonoids increased. [Conclusions]The content of total saponins decreased after fermentation and the content of total flavonoids increased. The fermentation process of Monascus purpureus Went. needs further optimization.

Application of Multivariate Analysis Method in Evaluation of Quality and Traits of Sichuan Ophiopogon japonicus

[Objectives] To study the relationship between the yield and total saponin content of Sichuan Ophiopogon japonicus. [Methods]The multi-indicator function model of Sichuan O. japonicus was established by using the multivariate analysis method and taking the yield of and total saponin content of Sichuan O. japonicus as indicators. [Results] In the multivariate non-linear fitting,the Pearson correlation test was used to reduce the dimension of the model,and the significant correlated variables were rejected,leaving three independent variables: the total fresh weight of the plant( x_1),the fresh weight of the aboveground part(x_2) and the fresh weight of fibrous roots(x_3),established the total saponin( y) function model for Sichuan O. japonicus: y = a_1+a_2x_2+ a_3x_3+ a_4x_1x_2+ a_5x_1x_3+ a_6x_2x_3+ a_7x_1~2+ a_8x_2~2+ a_9x_3~2.[Conclusions]When the total fresh weight of plant,fresh weight of aboveground part and fresh weight of fibrous roots were known in Sichuan O. japonicus root tuber,the total saponin content could be estimated by polynomial of these three variables. The establishment of this functional model system is expected to provide a scientific basis for the scientific prediction of the yield and total saponin content of Sichuan O. japonicus.

Effects of Organic and Medium and Trace Element Fertilizers on Yield and Quality of Panax notoginseng

[Objectives]This study aimed to investigate the effects of organic and medium and trace element fertilizers(Ca,Zn,B)on yield and quality of Panax notoginseng to provide theoretical support for rational fertilization in cultivation of P.notoginseng.[Methods]Five fertilization treatments,control(CK),organic fertilizer(OM),zinc fertilizer(ZF),boron fertilizer(BF)and lime(LF),were designed.A two-consecutive-year field plot trail was conducted.The biological traits,yield and saponin content of P.notoginseng were determined.[Results]The application of organic fertilizer had no significant effect on the biological traits of P.notoginseng.Trace element fertilizers significantly increased the scape length of P.notoginseng.Among the treatments,ZF significantly increased the single flower weight but reduced the inflorescence diameter,while the effects of BF were opposite to those of ZF;LF significantly increased the stem thickness and reduced the plant height.All treatments significantly increased the seedling rate of three-year-old P.notoginseng,and the increase in the LF group(20.49%)was the largest,followed by those in the ZF(16.80%)and OM(16.40%)groups,and the increase in the BF group(13.08%)was the smallest.Although OM,ZF and BF treatments caused the root weight of individual plants to decrease,the final yield of each treatment was higher than that of the control group,and the increases in the BF and LF groups exceeded 17%(P<0.05).The total saponin outputs of all the treatments except OM were significantly higher than that of the control group.[Conclusions]Under the conditions of this test,the supplementation of organic and medium and trace element fertilizers on the basis of conventional fertilization will help to increase the yield of P.notoginseng.However,the reduction of the total saponin output of P.notoginseng caused by organic fertilizer cannot be ignored.

Attenuation of Brain Inflammatory Response after Focal Cerebral Ischemia/Reperfusion with Xuesaitong Injection (血塞通注射液) in Rats

Objective: To investigate the neuro-protective effect of Xuesaitong Injection (血塞通注射液, XST) on brain inflammatory response after transient focal cerebral ischemia/reperfusion in rats. Methods: Focal cerebral ischemia/reperfusion models of male rats were induced by transient occlusion for 2 h of middle cerebral artery (MCA) which was followed by 24 h reperfusion. XST was administered through intraperitoneal injection of 25 mg/kg or 50 mg/kg at 4 h after the onset of ischemia. After reperfusion for 24 h, the neurological function score was evaluated, the brain edema was detected with dry-wet weight method, the myeloperoxidase (MPO) activity and the expression of intercellular adhesion molecule-1(ICAM-1) of ischemic cerebral cortex and caudate putamen was determined by spectrophotometry and immunohistochemistry respectively. Results: XST not only lowered neurological function score at the dose of 50 mg/kg, but reduced brain edema and inhibited MPO activity and ICAM-1 expression as compared with the ischemia/reperfusion model group (P<0.01).Conclusion: XST has a definite effect on inhibiting the expression of ICAM-1 and neutrophil infiltration in rats with cerebral ischemia/reperfusion when treatment started at 4 h after ischemia onset, and also attenuates inflammation in the infarcted cerebral area.