保加利亚乳杆菌对绞股蓝皂苷Gypenoside XLⅥ的微生物转化

目的:分析绞股蓝皂苷GypenosideⅩLⅥ的微生物转化产物。方法:利用益生菌德氏乳杆菌保加利亚亚种的脱脂牛奶培养基对GypenosideⅩLⅥ进行微生物转化,并利用液相色谱离子阱飞行时间串联质谱方法鉴定GypenosideⅩLⅥ的微生物转化产物。结果:GypenosideⅩLⅥ的微生物转化产物为绞股蓝皂苷Gypenoside L、Gypenoside LI、Damulin B和Damulin A。结论:通过微生物转化方法可以获得更多天然产物活性成分。

Mechanism of action of gypenosides on type 2 diabetes and non-alcoholic fatty liver disease in rats

AIM:To explore the mechanism of action of gypenosides(GPs)on type 2 diabetes mellitus and non-alcoholic fatty liver disease(T2DM-NAFLD)in rats.METHODS:Sixty rats were randomly divided into a healthy group,an untreated disease model group andGP-treatment groups.The study involved the evaluation of biochemical parameters,including serum aspartate transaminase(AST),alanine transferase(ALT),blood glucose(BG),triglycerides(TG)and total cholesterol(TC).Additionally,the protective effect of the treatments were confirmed histopathologically and the expression of TNF-αand NF-κB in the rat liver was analyzed using immunohistochemistry.The expression of proliferatoractivated receptor gamma(PPARγ)and cytochrome P450(CYP450)1A1 m RNA was determined by quantitative RTPCR.RESULTS:GP treatments at oral doses of 200,400,and800 mg/kg per day significantly decreased the levels of serum AST and ALT(P<0.05,P<0.01),especially at the dose of 800 mg/kg per day.To a similar extent,GP at800 mg/kg per day reduced the levels of BG(4.19±0.47,P<0.01),TG(80.08±10.05,P<0.01),TC(134.38±16.39,P<0.01)and serum insulin(42.01±5.04,P<0.01).The expression of TNF-αand NF-κB measured by immunohistochemistry was significantly reduced by GPs in a dose-dependent manner,and the expression of PPARγand CYP4501A1 m RNA,as measured using quantitative real-time PCR,were significantly down-regulated by GPs.Moreover,GPs decreased the infiltration of liver fats and reversed the histopathological changes in a dosedependent manner.CONCLUSION:This study suggests that GPs have a protective effect against T2DM-NAFLD by down-regulating the expression of TNF-αand NF-κB proteins,and PPARγand CYP4501A1 m RNAs.

Evaluating effects of gypenosides and soyasaponins on differentiation of neural stem cells as an in vitro model

Neural stem cell has a potential to differentiate into neurons, astrocytes and oligodendrocytes. It provides an in vitro model to screen herbal medicines on the cellular differentiation and development level. In this work, active component from gypenosides and soyasaponins was prepared to investigate their effects on the differentiation of neural stem cells.. Both gypenosides and soyasaponins promote the differentiation of neural stem cells. This method provides speed and practicality for screening effective herbal medicine. It is well suited for studying the mechanism of cell differentiation and development.

Neuroprotective effects of gypenosides in experimental autoimmune optic neuritis

AIM：To determine whether gypenosides have protective effects in experimental autoimmune optic neuritis（EAON）.METHODS：Mice were randomly divided into seven groups：control group,model group,three different density gypenosides monotherapy,methylprednisolone monotherapy,combination of gypenosides and methylprednisolone group.The control group was subcutaneously injected with oil emulsion adjuvant and all other groups were subcutaneously immunized with an emulsified mixture of myelin oligodendrocyte glycoprotein（MOG） 35-55 peptide to induce EAON.Mice in the gypenosides groups were administered injections daily with three concentrations（15 mg/kg,30 mg/kg,45 mg/kg） of gypenosides respectively.Mice in the methylprednisolone group and the combination treatment group were injected daily with methylprednisolone（20 mg/kg） or methylprednisolone（20 mg/kg） ＋ gypenosides（30 mg/kg）,respectively.After MOG immunization,visual evoked potential（VEP）,optical coherence tomography（OCT）,and histopathologic examination were performed at 14,20,30,and 40 d post-inoculation（p.i.）.All results were expressed as mean±SEM.The data were evaluated by oneway ANOVA followed by Tukey or Games-Howell test.RESULTS：Compared with the control group,p2 latency was prolonged in the model group（P=0.041）.Combination treatment can alleviated the change in VEP at 20 d p.i.（P=0.012）.Average peripapillary retinal nerve fiber layer（RNFL） thickness was reduced in the model group（P= 0.000,30d;P=0.000,40d） and gypenosides treatment remarkably diminished the degree of RNFL degenerationat 30 d and 40 d p.i（P=0.000,30d;P=0.000,40d）.The pathomorphological results showed a decrease in demyelination（P=0.020） and inflammatory reactions in the combination group compared with the model group（20d p.i.）.Gypenosides treatment also alleviated the degree of axonal loss（40d p.i.）（P=0.003）.CONCLUSION：Treatment with gypenosides exerts protective effects on retinal nerve fibers and axons in EAON.When combined with gypenosides,methylprednisolone reduces demyelination in the acute stage of EAON.

Gypenoside ameliorates insulin resistance and hyperglycemia via the AMPK-mediated signaling pathways in the liver of type 2 diabetes mellitus mice

Gynostemma pentaphyllum,also called"Southern Ginseng"in China,is a traditional Asian folk medicinal plant.Gypenosides(Gps)are the biologically active constituents of G.pentaphyllum,which have been reported with hypoglycemic activity.However,the underlying mechanisms are unclear.The effects of two Gps(Gp-Ⅰand Gp-Ⅱ)on type 2 diabetic mellitus(T2DM)mice,induced by high-fat and high-sugar diet and streptozotocin,were evaluated to explore the mechanism of their hypoglycemic actions.Gps reduced fasting blood glucose and serum lipids,as well as significantly improved T2DM mice glucose tolerance and insulin resistance(IR).After Gps treatment,the severity of liver injury was reduced and liver glycogen content increased.In addition,Gps promoted the phosphorylation of adenosine monophosphate-activated protein kinase(AMPK),and downregulated the key proteins phosphoenolpyruvate carboxy kinase and glucose-6 phosphatase,in the AMPK signaling pathway.Thus,our study suggests that Gps mediate hepatic gluconeogenesis and improve IR via activating AMPK signaling pathway in T2DM mice.

DAMAGES OF GYPENOSIDES ON THE ULTRASTRUCTURES OF S-180 SARCOMA IN MOUSE

The significant inhibition action of Gypenosides (GP) to many kinds or tumor strain wesreported before. The erfects of GP on the ultrastructures, without any findiys in present litrature,of S-180 sarcoma in mouse were studied through electron microscope in this article. The results indicated that the ultrastructures of.S-180 sarcoma cells were destructed obviously by the administratiouof GP to the tumor-bearing mouse with a Pattern or dose-depeudant, especially to the cellular nuclear. We saw both apoptosis and necrosis in morphologic alterations in tumor cells, suck as a reductionin cellular volume. an increase in cytoplasm, uucleoplasm electron density and condensation of nuclear chromatiu either to periphery or the nuclear membrane or inclumps within the cell, lots of inthe cytoplasm and apoptotic body in some turner cells or some cytoplast breaking into small frag- ments etc.

Gypenosides ameliorate morphine-induced immunosuppression with an increased proportion of thymic T lymphocyte subsets and are involved in the regulation of the cAMP-CREM/CREB-IL-2 pathway

Opioid abuse can suppress the lymphatic system function,and produce severe immunosuppression that poses a significant risk of opportunistic infections such as methicillinresistant Staphylococcus aureus(MRSA)pneumonia.^(1,2)Gypenosides(Gps)are the most important immunomodulator components in the Chinese herbal medicine Gynostemma pentaphyllum.

Gypenoside, the Main Active Compound of Gynostemma pentaphyllum , Mitigates the Diabetic Nephropathy through Down-regulating mTOR

Background:Diabetic nephropathy(DN),as a complication of diabetes,is featured with hypertension,hyper-glycemia,proteinuria and edema.Gypenoside(GP),the main active compound of Gynostemma pentaphyllum,is proved to be effective for DN.In our previous research,we found that GP could protect the glomerulus and re-duce proteinuria by up-regulating the expression of nestin and down-regulating TGFB1.However,the panoramic mechanism of GP against DN is still unclear.Objective:This research is designed to reveal the mechanism of GP on DN through network pharmacology and in vivo and in vitro experimental verification.Methods:In this study,active compounds and targets of Gynostemma pentaphyllum were collected from TCMSP.DisGeNET was used for obtaining the targets of DN.The protein-protein interaction network was acquired from the STRING database and analyzed by the MCODE plugin.GO and KEGG enrichment analysis were constructed to explore further information.In vivo and in vitro experiments were also carried out to evaluate the reliability of this study.Western blotting and RT-PCR were used to detect mTOR,4E-BP1,p70s6k protein expression and Mtor mRNA expression in DN rats,respectively.AKT1,TP53,ESR1 and PTEN protein expression in MPC-5 cells were detected by Western blotting.Results:Twenty-four compounds and 217 targets were selected from Gynostemma pentaphyllum,of which 36 targets overlapped with DN were taken for the potential targets.The results showed that Quercetin,Rhamnazin,Isofucosterol and 3′-methyleriodictyol corresponded to more targets,AKT1,TP53,MYC,ESR1,PTEN were more active.36 potential targets were mainly involved in autoimmunity,inflammatory response,metabolism and autophagy.In vivo and in vitro experiments showed that GP might protect the podocytes of DN rats by decreasing the protein expression of mTOR,4EBP1,p70s6k,as well as the mRNA expression of Mtor,and it had the function in regulating the potential targets through decreasing the protein expression of AKT1,TP53 and ESR1 and increasing the expression of PTEN.Conclusion:This research demonstrates that various compounds of Gynostemma pentaphyllum may intervenes in DN through targets of multiple signaling pathways,which involves a large number of biological processes.It can provide novel insights for further research of the mechanism of GP in the treatment of DN.

Analysis of the Inhibitory Effect of Gypenoside on Na^+, K^+-ATPase in Rats' Heart and Brain and Its Kinetics

Objective： To study the effects of gypenoside （Gyp） on the activity of microsomalNa^＋, K^＋-ATPase in rat＇s heart and brain in vitro. Methods： The microsomal Na^＋, K^＋-ATPase was prepared from rat＇s heart and brain by differential centrifugation. The activity of microsomal Na^＋, K^＋-ATPase was assayed by colorimetric technique. Enzyme kinetic analysis method was used to analyze the effect of Gyp on the microsomal Na^＋, K^＋-ATPase of rats. Results： Gyp reversibly inhibited the brain and heart＇s microsomal Na^＋, K^＋-ATPase in a concentration-dependent manner, and showed a more potent effect on enzyme in the brain. The IC50 of Gyp for the heart and brain were 58.79± 8.05 mg/L and 52.07± 6.25 mg/L, respectively. The inhibition was enhanced by lowering the Na^＋, or K^＋-concentrations or increasing the ATP concentration. Enzyme kinetic studies indicated that the inhibitory effect of Gyp on the enzyme is like that of competitive antagonist of Na^＋, the counter-competitive inhibitor for the substrate ATP, and the mixed-type inhibitor for K^＋. Cenclusien： Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain＇s microsomal Na^＋, K^＋-ATPase activities in rats.

Effect of light quality on total gypenosides accumulation and related key enzyme gene expression in Gynostemma pentaphyllum

Objective： Light quality has effect on the accumulation of gypenosides in the medicinal plant Gynosternma pentaphyllum in the family Cucurbitaceae, while the squalene synthase （SS） and squalene epoxidase （SE） are the key enzymes for gypenoside biosynthesis, The objective of this study was to elucidate the rela- tionship between light quality and biosynthesis key enzyme involving the regulation of gypenoside accu- mulation. Methods： The content of total gypenosides was measured by colorimetric method and the expression of SS and SE gene was determined by quantitative Real-time PCR in the seedlings of G. pentaphyllum which were grown with different light quality. Results： Light quality showed remarkable impacts on the accumulation of total gypenosides. The highest content of total gypenosides in the plant under red light condition was determined, followed by blue light and white light, while the lowest content was recorded under dark condition, qRT-PCR analysis proved that the expression levels of SS and SE genes were also affected by light quality. The high-level gene expressions of SS and SE were found in the plant under red light condition, followed by blue light, with the least content in darkness. The statistical analysis revealed that the total gypenosides were significantly different in different light treatment and the content of total gypenosides was positively related to the expression of SS and SE genes. Conclusions： Light quality regulates gypenoside accumulation via altering the expression of SS and SE in G. pentaphyllum.

SYNTHESIS OF THE POLYSTYRENE-TYPE ADSORBENTS CONTAINING POLAR GROUPS AND THEIR CAPACITIES IN THE ADSORPTION OF STEVIOSIDE AND GYPENOSIDES

<正> Seven polystyrene-type adsorbents (Ⅰ—Ⅶ) were synthesized by the modification of styrene-divinylbenzene copolymer respectively with hydroxymethyl, aminomethyl, methoxymethyl, phenoxymethyl, (4-hydroxy phenoxy)methyl, (4-acetylphenoxy)methyl, and acetyl groups. Their structures were characterized with chemical methods and physical techniques. The adsorption capacities of the adsorbents to stevioside and gypenosides were determined via batch test in which the HPLC method was used for the analysis of Saponin concentration of solution. The results showed that all the adsorbents could adsorb these saponins to a certain extent, but Ⅵ and Ⅶ had large capacities owing to their moderate polarities.

绞股蓝皂苷成分的分离与鉴定

目的提取、分离、鉴定绞股蓝皂苷主要成分。方法利用硅胶柱色谱及反相半制备柱色谱等分离手段,分离、纯化绞股蓝皂苷成分,并用核磁共振波谱(1H-NMR、13C-NMR)、液相色谱离子阱飞行时间质谱(LCMS-IT-TOF)等方法鉴定化合物结构,并对相关成分的抑癌作用进行了研究。结果绞股蓝总提物的主要成分经分离得到2种达玛烷类皂苷,分别为绞股蓝皂苷gypenoside XLVI和gypenoside LVI,而且它们具有一定的抑制癌细胞增殖作用。结论利用硅胶柱色谱和反相半制备色谱方法从绞股蓝总提物中分离得到主要的皂苷成分,为进一步研究其抑癌有效成分提供了参考。

绞股蓝热处理产物中活性成分的纯化与鉴定及其对A549细胞的抑制活性

目的:提取、分离、鉴定绞股蓝热处理产物中抑制肺癌A549细胞的活性成分。方法:绞股蓝叶在温度125℃、压力0.24MPa条件下,加热处理3h,用80%乙醇加热回流提取3h,利用活性跟踪方法,通过大孔树脂HP-20及反相柱等分离手段,分离抑制肺癌A549细胞的活性成分,并用核磁共振波普(1H-NMR、13C-NMR)、电喷雾质谱(ESI-MS)等数据鉴定有效成分。结果:绞股蓝热处理产物中分离得到2个达玛烷类皂苷,分别为绞股蓝皂苷Gypenoside L和Gypenoside LI,而且它们对肺癌A549细胞具有质量浓度依赖性的抑制活性(P<0.05),其IC50值分别为(48.43±1.33)μg/mL和(34.35±0.88)μg/mL。结论:热处理绞股蓝产物可提高对肺癌A549细胞的抑制作用,其中有效成分为达玛烷类皂苷Gypenoside L和Gypenoside LI。

绞股蓝皂苷对2型糖尿病合并非酒精性脂肪性肝病大鼠葡萄糖和脂质代谢的影响

目的研究绞股蓝皂苷(gypenosides,GPS)对2型糖尿病并非酒精性脂肪性肝病(type2 diabetes mellitus and nonalcoholic fatty liver disease,T2DM-NAFLD)大鼠糖和血脂代谢的影响。方法60只SD大鼠被分为正常对照组(Ⅰ组,n=8)和T2DM-NAFLD模型组。Ⅰ组予普通饵料持续饲养;余大鼠通过高脂高糖饵料(HFSD)加小剂量链脲佐菌素注射经8周饲养建立T2DM-NAFLD模型。将T2DM-NAFLD模型大鼠分为模型组(Ⅱ组)、T2DM-NAFLD模型GPS小剂量干预组(Ⅲ组)、T2DM-NAFLD模型GPS中剂量干预组(Ⅳ组)、T2DM-NAFLD模型GPS大剂量干预组(Ⅴ组);继续HFSD饲养;Ⅲ组、Ⅳ组、Ⅴ组分别予GPS 200、400、800 mg/(kg·d)干预,时间6周;实验周期14周。检测血天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、血糖(BS),甘油三酯(TG)、总胆固醇(TC)。进行大鼠口服葡萄糖耐量(OGTT)实验。观察肝脏病理学变化。结果 (1)ALT、AST水平:与Ⅱ组比较,Ⅲ组、Ⅳ组、Ⅴ组显著降低AST、ALT水平(P<0.05,P<0.01),呈剂量依赖性。(2)TG、TC水平:与Ⅱ组比较,Ⅲ组、Ⅳ组、Ⅴ组显著降低TG,TC水平(P<0.05,P<0.01),呈剂量依赖性。(3)BS水平和OGTT曲线:与Ⅱ组比较,Ⅲ组、Ⅳ组、Ⅴ组显著降低BS(P<0.01),呈剂量依赖性;与Ⅱ组比较,Ⅲ组、Ⅳ组、Ⅴ组显著改善OGTT曲线。(4)血胰岛素水平:与Ⅱ组比较,Ⅲ组、Ⅳ组、Ⅴ组显著降低血胰岛素水平(P<0.01),呈剂量依赖性。⑸肝脏病理学:干预组能减轻治疗组大鼠肝脏脂肪浸润程度,呈剂量依赖性。结论 GPS对T2DM-NAFLD大鼠糖代谢和脂质代谢异常具有调整作用,呈剂量依赖性。

Ethanol extract from Gynostemma pentaphyllum ameliorates dopaminergic neuronal cell death in transgenic mice expressing mutant A53T human alpha-synuclein

Gynostemma(G.) pentaphyllum(Cucurbitaceae) contains various bioactive gypenosides. Ethanol extract from G. pentaphyllum(GP-EX) has been shown to have ameliorative effects on the death of dopaminergic neurons in animal models of Parkinson’s disease(PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-and 6-hydroxydopamine. PD patients exhibit multiple symptoms, so PD-related research should combine neurotoxin models with genetic models. In the present study, we investigated the ameliorative effects of GP-EX, including gypenosides, on the cell death of dopaminergic neurons in the midbrain of A53 T α-synuclein transgenic mouse models of PD(A53 T). Both GP-EX and gypenosides at 50 mg/kg per day were orally administered to the A53 T mice for 20 weeks.α-Synuclein-immunopositive cells and α-synuclein phosphorylation were increased in the midbrain of A53 T mice, which was reduced following treatment with GP-EX. Treatment with GP-EX modulated the reduced phosphorylation of tyrosine hydroxylase, extracellular signal-regulated kinase(ERK1/2), Bcl-2-associated death promoter(Bad) at Ser112, and c-Jun N-terminal kinase(JNK1/2) due to α-synuclein overexpression. In the A53 T group, GP-EX treatment prolonged the latency of the step-through passive avoidance test and shortened the transfer latency of the elevated plus maze test. Gypenosides treatment exhibited the effects and efficacy similar to those of GP-EX. Taken together, GP-EX, including gypenosides, has ameliorative effects on dopaminergic neuronal cell death due to the overexpression of α-synuclein by modulating ERK1/2, Bad at Ser112, and JNK1/2 signaling in the midbrain of A53 T mouse model of PD. Further studies are needed to investigate GP-EX as a treatment for neurodegenerative synucleinopathies, including PD. This study was approved by the Animal Ethics Committee of Chungbuk National University(approval No. CBNUA-956-16-01) on September 21, 2016.

Multidimensional autophagy nano-regulator boosts Alzheimer's disease treatment by improving both extra/intraneuronal homeostasis

Intraneuronal dysproteostasis and extraneuronal microenvironmental abnormalities in Alzheimer’s disease(AD)collectively culminate in neuronal deterioration.In the context of AD,autophagy dysfunction,a multi-link obstacle involving autophagy downregulation and lysosome defects in neurons/microglia is highly implicated in intra/extraneuronal pathological processes.Therefore,multidimensional autophagy regulation strategies co-manipulating“autophagy induction”and“lysosome degradation”in dual targets(neuron and microglia)are more reliable for AD treatment.Accordingly,we designed an RP-1 peptide-modified reactive oxygen species(ROS)-responsive micelles(RT-NM)loading rapamycin or gypenoside XVII.Guided by RP-1 peptide,the ligand of receptor for advanced glycation end products(RAGE),RT-NM efficiently targeted neurons and microglia in AD-affected region.This nanocombination therapy activated the whole autophagy-lysosome pathway by autophagy induction(rapamycin)and lysosome improvement(gypenoside XVII),thus enhancing autophagic degradation of neurotoxic aggregates and inflammasomes,and promoting Aβ phagocytosis.Resultantly,it decreased aberrant protein burden,alleviated neuroinflammation,and eventually ameliorated memory defects in 3×Tg-AD transgenic mice.Our research developed a multidimensional autophagy nano-regulator to boost the efficacy of autophagy-centered AD therapy.

Dammarane-type triterpenoid saponins isolated from Gynostemma pentaphyllum ameliorate liver fibrosis via agonizing PP2Cαand inhibiting deposition of extracellular matrix

Gypenosides,structurally analogous to ginsenosides and derived from a sustainable source,are recognized as the principal active compounds found in Gynostemma pentaphyllum,a Chinese medicinal plant used in the treatment of the metabolic syndrome.By bioactive tracking isolation of the plants collected from different regions across China,we obtained four new gypenosides(1−4),together with nine known gypenosides(5−13),from the methanol extract of the plant.The structures of new gypenosides were elucidated by one-dimensional(1D)and two-dimensional(2D)nuclear magnetic resonance(NMR)spectra,complemented by chemical degradation experiments.Through comprehensive evaluation involving COL1A1 promoter assays and PP2Cαactivity assays,we established a definitive structure-activity relationship for these dammarane-type triterpenoids,affirming the indispensability of the C-3 saccharide chain and C-17 lactone ring in effectively impeding extracellular matrix(ECM)deposition within hepatic stellate cells.Further in vivo study on the CCl4-induced liver damage mouse model corroborated that compound 5 significantly ameliorated the process of hepatic fibrosis by oral administration.These results underscore the potential of dammarane-type triterpenoids as prospective antifibrotic leads and highlight their prevalence as key molecular frameworks in the therapeutic intervention of chronic hepatic disorders.

A New Triterpene Saponin from Gynostemma pentaphyllum

Objective To study the triterpene saponins from Gynostemma pentaphyllum with antitumor activities. Methods The 75% EtOH extract of G. pentaphyllum was used for isolation by silica gel column chromatography and preparative HPLC. The structures of pure compounds isolated were identified by the spectral analysis and chemical evidence. Results Two compounds were isolated and identified as 23(S)-3β,20ξ,21ξ-trihydroxy-19-oxo-21,23- epoxydammar-24-ene 3-O-α-L-rhamnopyranosyl (1→2)-[β-D-xylopyranosyl (1→3)]-β-D-arabinopyranoside (1) and 23(S)-21(R)-O-n-butyl-3β,20ξ-dihydroxy-21,23-epoxydammar-24-ene 3-O-α-L-rhamnopyranosyl (1→2)-[β-D-xylo- pyranosyl (1→3)]-β-D-arabinopyranoside (2). Conclusion Compound 2 is a new triterpene saponin with moderate antitumor activities against the HL-60, Colon205, and Du145 cell lines.

绞股蓝中皂苷类与黄酮类化合物的分离与鉴定

目的:研究绞股蓝中皂苷类与黄酮类化合物的分离与鉴定。方法:采用硅胶、大孔树脂、中压柱色谱、半制备型HPLC及分析型HPLC等现代分离技术对绞股蓝的化学成分进行分离纯化,并根据其理化性质、UV、MS、NMR等方法进行结构鉴定。结果:从绞股蓝70%乙醇提取物中分离得到6个化合物,分别鉴定为gypenoside S5(1)、gypenoside XLIX(2)、gypenoside VN2(3)、ombuine(4)、yixingensin(5)、ombuoside(6)。结论:从绞股蓝中分离得到3个皂苷类和3个黄酮类化合物,其中化合物1为新化合物,命名为gypenoside S5。