Effects of ginsenoside Rh2 on growth and migration of pancreatic cancer cells

AIM:To investigate the effects of ginsenoside Rh2 on the human pancreatic cancer cell line Bxpc-3.METHODS:The human pancreatic cancer cell line Bxpc-3 was cultured in vitro and treated with or without ginsenoside Rh2.Growth rates for Bxpc-3 cells were assessed by methyl thiazolyl tetrazolium(MTT) and colony formation assays.Cell cycle changes were analyzed by flow cytometry.Apoptosis was measured by flow cytometry and Hoechst 33258 fluorescence staining.A scratch assay and a Matrigel invasion assay were used to detect cell migration and invasion.Expression of Bax,Bcl-2,survivin,cyclin D1,matrix metalloproteinase(MMP)-2,MMP-9,cleaved caspase-3,caspase-8,and caspase-9 mRNA were determined by reverse transcriptase-polymerase chain reaction(RT-PCR).Bax,Bcl-2,survivin,cyclin D1,cleaved caspase-3,caspase-8 and caspase-9 protein levels were examined by western blotting.Expression of MMP-2 and MMP-9 proteins in culture supernatants were determined by enzymelinked immunosorbent assay(ELISA).RESULTS:Rh2 significantly inhibited Bxpc-3 cell proliferation in a dose-and time-dependent manner,as evaluated by the MTT(P < 0.05) and colony formation assays(P < 0.05).Compared to the control group,Rh2 significantly increased the percentage of Bxpc-3 cells in the G 0 /G 1 phase from 43.32% ± 2.17% to 71.32% ± 1.16%,which was accompanied by a decrease in S phase(from 50.86% ± 1.29% to 28.48% ± 1.18%) and G 2 /M phase(from 5.81% ± 1.19% to 0.20% ± 0.05%) in a dose-dependent manner(P < 0.05),suggesting that Rh2 arrested cell cycle progression at the G 0 /G 1 phase,as measured by flow cytometry.Compared to the control group,cells treated with Rh2 showed significantly higher apoptosis ratios in a dosedependent manner(percentage of early apoptotic cells:from 5.29% ± 2.28% to 38.90% ± 3.42%(F = 56.20,P < 0.05);percentage of late apoptotic cells:from 4.58% ± 1.42% to 36.32% ± 2.73%(F = 86.70,P < 0.05).Rh2 inhibited Bxpc-3 cell migration and invasion,as detected by scratch wound healing assay and Matrigel invasion assay [percentages of scratch wound healing for 12 h,24 h and 48 h(control vs experimental group):37.3% ± 4.8%vs 18.30% ± 1.65%,58.7% ± 3.5% vs 38.00% ± 4.09% and 93.83% ± 4.65% vs 65.50% ± 4.09%,respectively;t = 6.489,t = 6.656 and t = 7.926,respectively,P < 0.05;the number of cells invading at various concentrations(0 μmol/L,35 μmol/L,45 μmol/L and 55 μmol/L):81.10 ± 9.55,46.40 ± 6.95,24.70 ± 6.88 and 8.70 ± 3.34,respectively(F = 502.713,P < 0.05)].RT-PCR,western blotting or ELISA showed that mRNA and protein expression of Bax,cleaved caspase-3 and caspase-9 were upregulated(P < 0.05),while mRNA and protein expression of Bcl-2,survivin,cyclin D1,MMP-2 and MMP-9 were downregulated(P < 0.05).CONCLUSION:Ginsenoside Rh2 inhibits proliferation,migration and invasion and induces apoptosis of the human pancreatic cancer cell line Bxpc-3.

One Stone Four Birds:A Novel Liposomal Delivery System Multi-functionalized with Ginsenoside Rh2 for Tumor Targeting Therapy

Liposomes hold great potential in anti-cancer drug delivery and the targeting treatment of tumors.However,the clinical therapeutic efficacy of liposomes is still limited by the complexity of tumor microenvironment(TME)and the insufficient accumulation in tumor sites.Meanwhile,the application of cholesterol and polyethylene glycol(PEG),which are usually used to prolong the blood circulation and stabilize the structure of liposomes respectively,has been questioned due to various disadvantages.Herein,we developed a ginsenoside Rh2-based multifunctional liposome system(Rh2-lipo)to effectively address these challenges once for all.Different with the conventional’wooden’liposomes,Rh2-lipo is a much more brilliant carrier with multiple functions.In Rh2-lipo,both cholesterol and PEG were substituted by Rh2,which works as membrane stabilizer,long-circulating stealther,active targeting ligand,and chemotherapy adjuvant at the same time.Firstly,Rh2 could keep the stability of liposomes and avoid the shortcomings caused by cholesterol.Secondly,Rh2-lipo showed a specifically prolonged circulation behavior in the blood.Thirdly,the accumulation of the liposomes in the tumor was significantly enhanced by the interaction of glucose transporter of tumor cells with Rh2.Fourth,Rh2-lipo could remodel the structure and reverse the immunosuppressive environment in TME.When tested in a 4T1 breast carcinoma xenograft model,the paclitaxel-loaded Rh2-lipo realized high efficient tumor growth suppression.Therefore,Rh2-lipo not only innovatively challenges the position of cholesterol as a liposome component,but also provides another innovative potential system with multiple functions for anti-cancer drug delivery.

20(S)-原人参二醇组皂苷的制备及其转化制取人参皂苷Rh2

目的：制取２０（Ｓ）原人参二醇组皂苷并将其转化制备抗癌活性单体２０（Ｓ）人参皂苷Ｒｈ２。方法：以国产西洋参茎叶总皂苷为原料，通过萃取法制备２０（Ｓ）原人参二醇组皂苷；将２０（Ｓ）原人参二醇组皂苷碱催化水解后，经柱层析分离纯化制备２０（Ｓ）人参皂苷Ｒｈ２。结果：２０（Ｓ）原人参二醇组皂苷收率为４１．５％，其转化成２０（Ｓ）人参皂苷Ｒｈ２的转化率为９．６４％。结论：制取方法简便，收率较高。

Ginsenoside Rh_2 Showing Ability to Induce Apoptosis in HeLa Cells

This paper deals with the inhibitory mechanisms of ginsenoside \{G Rh 2\} on the growth of tumor cells. \{G Rh 2\} significantly inhibited the proliferation of human cervical adenocarcinoma HeLa cells in a time and dose dependent manner. G Rh 2 induced apoptotic manifestations in HeLa cells as evidenced by the changes in the cell morphology, the DNA fragmentation and the activation of caspases. Caspase inhibitors, caspase family inhibitor, z Val Ala Asp fmk(z VAD fmk); caspase 1 inhibitor, Ac Tyr Val Ala Asp chloromethyl ketone(Ac YVAD cmk); caspase 3 inhibitor, z Asp Glu Val Asp fmk(z DEVE fmk) and caspase 8 inhibitor, \{z Ile \}Glu Asp fmk(z IETD fmk) effectively attenuated G Rh 2 induced cell death. The activities of caspase 1 and caspase 3 were increased in the G Rh 2 induced apoptotic process. However, caspase inhibitors can not inhibit G Rh - 2 induced cell death completely. These results suggest that G Rh 2 induced cell death is mediated by the activation of caspase cascade, but there might be some other pathways for induction of this apoptosis.

Preparation and Inhibitory Effects of 20(S)-Ginsenoside Rh_2 on Hep-A-22 Cells

A modified method of preparing 20（S）-ginsenoside Rh2（G-Rh2） and the inhibitory effect of 20（S）-ginsenoside Rh2 on Hep-A-22 cells were investigated. The total saponins and strong alkali were dissolved in glycerol at the atmospheric pressure, and the degradation was performed at a high temperature. After G-Rh2 had been isolated and purified, MTT（methyl thiazolyl tetrazolium） assay was applied to evaluating the effect of 20（S）-ginsenoside Rh2 on the cells viability and morphological changes were observed. It was shown that 20（S）-ginsenoside Rh2 can reduce Hep-A-22 cells viability in dose-dependent manner and the cells took on cell shrinkage, membrane blebbing, chromosomal condensations, especially under the higher concentrations of it. In conclusion, 20（S）-ginsenoside Rh2 can be prepared effectively that not only decreases viability but also induces the apoptosis of Hep-A-22 cells.

20(S)-人参皂苷Rh_2对人结肠癌细胞增殖和周期的影响

目的研究20(S)-人参皂苷Rh2对人结肠癌Caco-2和HT-29细胞增殖及周期的影响。方法采用荧光微孔法测定20(S)-人参皂苷Rh2对人结肠癌Caco-2、HT-29细胞增殖的影响;利用显微镜观察给药后HT-29细胞形态变化;流式细胞术分析20(S)-人参皂苷Rh2对HT-29细胞周期分布的影响。结果 20(S)-人参皂苷Rh2对Caco-2、HT-29细胞生长有抑制作用,并呈剂量依赖性;20(S)-人参皂苷Rh2作用48 h后,对HT-29、Caco-2细胞的半数抑制浓度(IC50)分别为19.68、26.79μg/mL;流式细胞分析结果显示,与对照组细胞相比,20(S)-人参皂苷Rh2能显著减少HT-29细胞的G0/G1期和G2/M期细胞比例,增加S期细胞比例。结论 20(S)-人参皂苷Rh2可抑制人结肠癌细胞增殖,主要原因是阻滞细胞处于S期。

西洋参叶中20(s)-人参皂苷-Rh_1,-Rh_2和人参皂苷Rh_3的分离与鉴定

目的:从西洋参叶中分离、鉴定20(s)人参皂苷Rh1,Rh2和人参皂苷Rh3,为了深入研究它们的生理活性。方法:以水提取,用多孔树脂吸附,再以体积分数为95%的乙醇洗脱,最后以硅胶柱层析分离。通过理化性质及IR,NMR光谱等方法测定,鉴定其结构。结果:共分得3个化合物,经鉴定后分别证明为20(s)人参皂苷Rh1,Rh2和人参皂苷Rh3。结论:人参皂苷Rh3为首次从西洋参叶中分离获得。

人参皂苷Rh2对人红白血病K562细胞HDAC1/2活性和周期蛋白的影响

目的 探讨人参皂苷单体Rh2[20（S）-ginsenoside Rh2,Rh2（S）]对人红白血病K562细胞增殖及对组蛋白去乙酰化酶1（HDAC1）、HDAC2活性和周期蛋白的影响。方法 以10-80μmol/L的Rh2（S）作用于体外培养的K562细胞,采用CCK-8法检测Rh2（S）对K562细胞增殖活性的影响;流式细胞术（FCM）检测细胞周期、细胞凋亡的变化;化学比色法检测细胞中HDAC活性;Western blot法检测（10、20、40、60）μmol/L Rh2（S）诱导48 h后HDAC1、HDAC2、细胞周期蛋白D1（cyclin D1）、细胞周期蛋白依赖激酶4（CDK4）、p16INK4A和p21蛋白的表达。结果 CCK-8结果显示,Rh2（S）在20-80μmol/L浓度范围内能有效抑制K562细胞生长,并呈时间剂量依赖性;FCM结果显示,60μmol/L Rh2（S）将K562细胞周期阻滞在G0/G1期;（20、40、60）μmol/L Rh2（S）诱导K562早期凋亡,其凋亡率分别为（8.09±0.86）%、（9.44±0.53）%、（22.80±2.16）%,与空白对照组（2.63±0.14）%相比,差异有统计学意义（P〈0.05）;40~60μmol/L Rh2（S）能降低K562细胞中HDAC的活性;Western blot结果显示,Rh2（S）能够下调HDAC1/2和cyclin D1、CDK4,激活p16INK4A和p21的表达。结论 Rh2（S）可能是通过抑制HDAC1、HDAC2的活性,下调cyclin D1激活p16INK4A和p21的表达,从而抑制白血病细胞增殖,诱导周期阻滞和细胞凋亡。

应用流式细胞光度计研究人参单体Rh_1和Rh_2对小鼠S180肉瘤细胞周期移行的影响

人参皂甙单体Rh_1和Rh_2给予接种S180肉瘤细胞3天后的载瘤小鼠腹腔注射,50mg/kg.d,连续7天。应用FACS 420荧光细胞激活器对肿瘤细胞进行周期分析发现,在短期内Rh_1对肿瘤细胞周期影响不大;Rh_2可阻断S_(180)肉瘤细胞从S期向G_2期过度(P<0.005),抑制肿瘤细胞生长。

20(s)-原人参二醇、人参皂苷Rh_2及人参皂苷Rg_3抗肿瘤作用的对比

目的对比20(s)-原人参二醇(Ppd)与人参皂苷Rh2(Rh2)及人参皂苷Rg3(Rg3)的抗肿瘤作用。方法建立小鼠肝癌H22、Lewis肺癌及黑色素瘤B16三种移植瘤动物模型,将小鼠随机分成11组,对照组给予0.5%羧甲基纤维素钠,阳性药组给予环磷酰胺(CTX)30 mg/kg,Ppd、Rh2及Rg3三个剂量组均给予相应受试药25、50、100 mg/kg。阳性药组隔日腹腔注射给药1次,其余各组均每日灌胃给药1次,给药容积为20 ml/kg,连续10 d。每天记录小鼠体重,末次药后称取小鼠体重及肿瘤重量并计算肿瘤抑瘤率。结果 Ppd、Rh2及Rg3在25、50、100 mg/kg剂量下,对肝癌H22、Lewis肺癌及黑色素瘤B16均有一定的抑瘤作用,仅Ppd在50、100 mg/kg剂量下对肝癌H22、Lewis肺癌及黑色素瘤B16抑制效果明显,其抑瘤率超过40%,Rh2与Rg3的抑瘤率无明显差别。结论 Ppd、Rh2及Rg3对小鼠三种移植瘤均具有抗肿瘤活性,以Ppd的抗肿瘤作用最明显,Rh2与Rg3之间无明显差别。

RP-HPLC测定西洋参叶皂苷碱降解物中20(S)-人参皂苷Rh_2的含量

目的：对西洋参叶皂苷碱降解物中20（S）-人参皂苷Rh2进行含量测定。方法：采用RP-HPLC，ZORBAX EXTEND C18柱（4．6mm×250mm，5μm），流动相甲醇-水（85：15），流速1．2mL·min^-1，检测波长203nm，柱温25℃。结果：20（S）-人参皂苷Rh2的进样量在0．5～25μg有良好的线性关系，r=0．9999；平均加样回收率为99．7％，RSD1．0％。结论：该方法测定20（S）-人参皂苷Rh2简便快捷，准确度高，测定结果证实该降解方法20（S），人参皂苷Rh2的转化率高。

黑曲霉Aspergillus niger对人参皂苷Re的微生物转化

目的:筛选长白山人参土壤中的活性微生物,转化单体人参皂苷产生稀有人参皂苷成份。方法:从长白山人参根际土壤中分离各类菌株,对单体人参皂苷Re进行微生物转化,通过硅胶柱层析等方法对转化产物进行分离纯化,采用波谱解析及理化常数对其进行结构鉴定。结果:从长白山人参根际土壤中分离各类真菌菌株68株,3株真菌对三醇组人参皂苷Re具有转化作用,其中黑曲霉Aspergillusniger的转化活性较强,转化产物为人参皂苷Rg1、Rg2和Rh1。结论:首次报道黑曲霉能将人参皂苷Re转化为人参皂苷Rg1、Rg2和Rh1这一转化过程。

微生物酶催化制备人参皂苷20(S)-Rg2,20(S)-Rh1和20(S)-PPT

以微生物Microbacterium esteraromaticum GS514的培养液中分离的粗酶为催化剂水解人参皂苷Re和Rg1,并通过1HNMR和13C NMR谱对水解产物的结构进行了表征.实验结果表明,反应体系中无机盐NaCl的存在与否直接影响人参皂苷Re,Rg1与粗酶液的反应结果.人参皂苷与粗酶液直接反应时,人参皂苷Re不发生反应,人参皂苷Rg1通过C6所连β-D-吡喃葡萄糖的选择性水解转化成人参皂苷F1.而该反应在无机盐NaCl存在下进行时,人参皂苷Re通过对C20所连β-D-吡喃葡萄糖的选择性水解定向转化为20(S)-人参皂苷Rg2;人参皂苷Rg1定向转化成20(S)-人参皂苷Rh1以及20(S)-原人参三醇(PPT).表明NaCl的加入激活了C20β-D-吡喃葡萄糖苷酶的活性,这对定向合成不同次级人参皂苷具有重要意义.

人参皂苷Rh2和Rg3抗肿瘤作用研究进展

人参皂苷是人参抗肿瘤的有效成分,其中人参皂苷Rh2（ginsenoside Rh2,GRh2）和人参皂苷Rg3（ginsenoside Rg3,GRg3）是人参抗肿瘤的主要活性成分。研究表明,二者可阻滞细胞周期,诱导分化,抑制转移,调节免疫功能,逆转多药耐药性,诱导凋亡等（[1]）,综术如下。1人参皂苷Rh2和Rg3药理作用抑制肿瘤细胞的增殖。洪新雨等（[2]）用聚合酶链反应检测到GRh2可使人脑胶质瘤SHG44中PCNA表达与转录产物明显下降。游智梅等（[3]）研究发现。

20(R)和20(S)-人参皂甙Rg_2碳氢NMR信号全指定

20 (R)和 2 0 (S) 人参皂甙 Rg2 属于达玛烷型四环三萜类化合物 .应用 2DNMR技术 :1 H 1 HCOSY、HMQC和HMBC全归属 2 0 (R)和 2 0 (S) 人参皂甙 Rg2 碳和氢质子信号 ,为该类型化合物的结构鉴定提供波谱学依据 .

人参皂苷Rh2两种差向异构体的HPLC含量测定

目的：建立2种人参皂苷Rh2异构体的高效液相色谱测定方法。方法：采用Merck Lichro-spherRP-18e C18色谱柱（250mm×4．6mm，5μm），流动相为CH3CN—CH3OH（65：35），检测波长为203nm，流速为1．0mL·min^-1。结果：Rh2线性范围为0．06～1．20g·L^-1，以峰面积（y）对浓度（x，g·L^-1）求得线性方程，S构型线性方程为y=2260．9x-12．533，r=0．9999；R构型线性方程为Y=2596．1x-7．9654，r=0．9999。结论：本方法简便、准确、可靠，可用于Rh2中的2种差向异构体化合物的鉴别和含量测定。

Liquid Chromatography-Electrospray Ionization Mass Spectrometry Method for Determination of Protopanaxadiol in Rat Plasma

A simple, rapid and sensitive method for the determination of protopanaxadiol in rat plasma with ginsenoside Rh2 as internal standard was developed and validated. The analyte and internal standard were extracted from plasma with ether-dichloromethane（3：2, volume ratio） and then were analyzed by reversed-phase HPLC on a short Zorbax Extend C18 column（50 mm×2.1 mm, 3.5 μm i. d.） eluted with a mobile phase consisting of acetonitrile/methanol 0.10 mmol/L ammonium acetate（45：45：10, volume ratio） at 0.4 mL/min. Detection was performed on an Applied Biosystems Sciex API 4000 mass spectrometer set at unit resolution in the multiple reaction monitoring mode. Electrospray ionization was used for ion production. The assay method shows linear over a range of 5-2000 ng/mL and intra- and inter-day precisions over this range were 〈10.0% with accuracy ranged from 86.3% to 114.1%. The limit of detection was 500 pg/mL in the plasma. The method was successfully applied to a preclinical pharmacokinetic study of protopanaxadiol（17.5 mg/kg） administered as a single oral dose.

Mechanism of Shenfu Decoction in the treatment of critically ill patients with Coronavirus Disease 2019 (COVID-19) based on network pharmacology

Objective:The purpose of this thesis is to explore the mechanism of ShenFu Decoction in the treatment of critically ill patients with COVID-19 based on network pharmacology.Methods:The primary active ingredients and potential targets of ShenFu Decoction were searched from the TCMSP database.The targets of COVID-19 were obtained by searching the GeneCards and OMIM databases.A ShenFu Decoction-compound-target-COVID19 network and a protein-protein interaction(PPI)network were respectively constructed through the Cytoscape 3.5.1 software and the STRING database.Gene Ontology(GO)function enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were performed via Bioconductor bioinformatics software package and R programming language.Results:ShenFu Decoction contains 255 compounds and 94 potential targets.43 primary active ingredients were searched from the TCMSP database with oral bioavailability(OB)≥30%and drug-likeness(DL)≥0.18 as the retrieval condition.Numbers of targets of COVID-19 were 352 by searching the GeneCards and the OMIM databases.16 key targets were acquired by intersecting the targets of drug with the targets of disease.There were 49 GO terms and 102 pathways after analyzing GO and KEGG.Conclusion:Kaempferol,ginsenoside rh2,beta-sitosterol,Stigmasterol and Deoxy andrographolide might be the main active ingredients which may cause the inhibition of the SARS-CoV-23CL hydrolase activity and regulate ACE2.As a result,the antiviral effect,immunoregulation,targeting cytokine storm of SFD may play an important role in the treatment of critically ill patients with COVID-19 through regulating multiple signaling pathways such as AGE-RAGE signaling pathway in diabetic complications,IL-17 signaling pathway,C-type lectin receptor signaling pathway,HIF-1 signaling pathway.

Enhancement of oral bioavailability and immune response of Ginsenoside Rh2 by co-administration with piperine

Ginsenoside Rh2(Rh2)is one of the major bioactive ginsenosides in Panax ginseng.However,the oral bioavailability of Rh2 is low,with P-glycoprotein(P-gp)and CYP3A4 being reported to be the main factors.The purpose of the present study was to determine the enhancing effect of piperine on the oral bioavailability as well as bioactivity of Rh2.The inhibitory effect of piperine on P-gp and CYP3A4 was determined using a Caco-2 monolayer model and a recombinant CYP3A4 metabolic system,respectively.The pharmacokinetics of oral Rh2(10 mg·kg^(-1))administered alone or in combination with piperine(10 and 20 mg·kg^(-1))was performed in rats.The immune boosting effect of Rh2 was assessed in rats by measuring IL-12 level after treated by Rh2 alone or co-administered with piperine.The results indicated that piperine significantly increased the permeability of Rh2 and inhibited the metabolism of Rh2.The pharmacokinetic study results showed that the AUC of Rh2 was significantly increased in combination with piperine at high dose(20 mg·kg^(-1))when compared to the control group,with relative bioavailability of 196.8%.The increase of Rh2 exposure led to increased serum levels of IL-12.In conclusion,piperine may be used as a bioenhancer to improve pharmacological effect of Rh2 when given orally.