Events associated with apoptotic effect of p-Coumaric acid in HCT-15 colon cancer cells

AIM:To investigate the events associated with the apoptotic effect of p-Coumaric acid,one of the phenolic components of honey,in human colorectal carcinoma(HCT-15)cells.METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertazolium-bromide assay was performed to determine the antiproliferative effect of p-Coumaric acid against colon cancer cells.Colony forming assay was conducted to quantify the colony inhibition in HCT15 and HT 29 colon cancer cells after p-Coumaric acid treatment.Propidium Iodide staining of the HCT15 cells using flow cytometry was done to study the changes in the cell cycle of treated cells.Identification of apoptosis was done using scanning electron microscope and photomicrograph evaluation of HCT 15cells after exposing to p-Coumaric acid.Levels of reactive oxygen species(ROS)of HCT 15 cells exposed to p-Coumaric acid was evaluated using 2’,7’-dichlorfluorescein-diacetate.Mitochondrial membrane potential of HCT-15 was assessed using rhodamine-123 with the help of flow cytometry.Lipid layer breaks associated with p-Coumaric acid treatment was quantified using the dye merocyanine 540.Apoptosis was confirmed and quantified using flow cytometric analysis of HCT15 cells subjected to p-Coumaric acid treatment after staining with YO-PRO-1.RESULTS:Antiproliferative test showed p-Coumaric acid has an inhibitory effect on HCT 15 and HT 29 cells with an IC50(concentration for 50%inhibition)value of 1400 and 1600μmol/L respectively.Colony forming assay revealed the time-dependent inhibition of HCT 15 and HT 29 cells subjected to p-Coumaric acid treatment.Propidium iodide staining of treated HCT 15cells showed increasing accumulation of apoptotic cells(37.45±1.98 vs 1.07±1.01)at sub-G1phase of the cell cycle after p-Coumaric acid treatment.HCT-15 cells observed with photomicrograph and scanning electron microscope showed the signs of apoptosis like blebbing and shrinkage after p-Coumaric acid exposure.Evaluation of the lipid layer showed increasing lipid layer breaks was associated with the growth inhibition of p-Coumaric acid.A fall in mitochondrial membrane potential and increasing ROS generation was observed in the p-Coumaric acid treated cells.Further apoptosis evaluated by YO-PRO-1 staining also showed the timedependent increase of apoptotic cells after treatment.CONCLUSION:These results depicted that p-Coumaric acid inhibited the growth of colon cancer cells by inducing apoptosis through ROS-mitochondrial pathway.

p-Coumaric acid ameliorates ethanol-induced kidney injury by inhibiting inflammatory cytokine production and NF-κB signaling in rats

Objective: To examine the effects of p-coumaric acid on ethanol-induced kidney injury in Swiss Wistar rats.Methods: Ethanol(25% v/v) was used to induce nephrotoxicity in rats. p-Coumaric acid was orally administered at 50, 100, or 200 mg/kg body weight. The levels of oxidative parameters were determined; pro-inflammatory biomarkers were analyzed by Western blotting and apoptotic protein was analyzed by immunohistochemistry. Results: Ethanol treated rats showed decreased levels of antioxidants and aberrant production of pro-inflammatory cytokines(IL-6, IL1β, TNF-α), NF-κB activation and imbalance of proand anti-apoptotic proteins(Bcl-2, Bax, caspase 3). Meanwhile, p-coumaric acid restored antioxidant levels and decreased the levels of inflammatory cytokines, NF-κB, and proapoptotic proteins and increased Bcl-2 expression. Conclusions: p-Coumaric acid ameliorates ethanol-induced kidney injury by restoring antioxidant production and suppressing cellular apoptosis and inhibiting NF-κB expression.p-Coumaric acid should be further investigated as a promising candidate for ethanol-induced kidney toxicity.

MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.

18β-glycyrrhetinic acid inhibits proliferation of gastric cancer cells through regulating the miR-345-5p/TGM2 signaling pathway

BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the molecular mechanism of 18β-glycyrrhetinic acid(18β-GRA)regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells.METHODS CCK-8 assay was used to determine the effect of 18β-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells.Cell cycle and apoptosis were detected by flow cytometry,cell migration was detected by a wound healing assay,the effect of 18β-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated,and the cell autophagy level was determined by MDC staining.TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18β-GRA intervention,and then the protein-protein interaction was predicted using STRING(https://string-db.org/).MicroRNAs(miRNAs)transcriptome analysis was used to detect the miRNA differential expression profile,and use miRBase(https://www.mirbase/)and TargetScan(https://www.targetscan.org/)to predict the miRNA and complementary binding sites.Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18β-GRA treated cells,and western blot was used to detect the expression of autophagy related proteins.Finally,the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression.RESULTS 18β-GRA could inhibit GC cells viability,promote cell apoptosis,block cell cycle,reduce cell wound healing ability,and inhibit the GC cells growth in vivo.MDC staining results showed that 18β-GRA could promote autophagy in GC cells.By TMT proteomic analysis and miRNAs transcriptome analysis,it was concluded that 18β-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells.Subsequently,we verified that TGM2 is the target of miR-345-5p,and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2.Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced,and LC3II,ULK1 and AMPK expression was significantly increased in GC cells treated with 18β-GRA.Overexpression of miR-345-5p not only inhibited the expression of TGM2,but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle.CONCLUSION 18β-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.

18β-glycyrrhetinic acid promotes gastric cancer cell autophagy and inhibits proliferation by regulating miR-328-3p/signal transducer and activator of transcription 3

BACKGROUND Gastric cancer(GC)is one of the most common cancer types worldwide,and its prevention and treatment methods have garnered much attention.As the active ingredient of licorice,18β-glycyrrhetinic acid(18β-GRA)has a variety of pharmacological effects.The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC,in order to provide effective ideas for the clinical prevention and treatment of GC.AIM To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells.METHODS Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs(miRNAs)in GC cells after 18β-GRA intervention.Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability,cell colony formation ability was detected by the clone formation assay,and flow cytometry was used to detect the cell cycle and apoptosis.A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells.Tumor tissue morphology was observed by hematoxylin and eosin staining,and microtubule-associated protein light chain 3(LC3)expression was detected by immunohistochemistry.TransmiR,STRING,and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3(STAT3)-related information.Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction(qPCR)and the expression levels of STAT3,phosphorylated STAT3(p-STAT3),and LC3 were detected by western blot analysis.The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system.AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label.LC3 was labeled and autophagy flow was observed under a confocal laser microscope.RESULTS The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells(P=4.51E-06).Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability,arrested the cell cycle in the G0/G1 phase,promoted cell apoptosis,and inhibited the growth of subcutaneous tumors in BALB/c nude mice(P<0.01).No obvious necrosis was observed in the tumor tissue in the negative control group(no drug intervention or lentivirus transfection)and vector group(the blank vector for lentivirus transfection),and more cells were loose and necrotic in the miR-328-3p group.Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3,and STAT3 was closely related to autophagy markers such as p62.After overexpressing miR-328-3p,the expression level of STAT3 mRNA was significantly decreased(P<0.01)and p-STAT3 was downregulated(P<0.05).The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT33’untranslated regions of the wild-type reporter vector group was significantly decreased(P<0.001).Overexpressed miR-328-3p combined with bafilomycin A1(Baf A1)was used to detect the expression of LC3 II.Compared with the vector group,the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated(P<0.05),and compared with the Baf A1 group,the expression level of LC3 II in the overexpressed miR-328-3p+Baf A1 group was upregulated(P<0.01).The expression of LC3 II was detected after intervention of 18β-GRA in GC cells,and the results were consistent with the results of miR-328-3p overexpression(P<0.05).Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis(P<0.001).qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention(P<0.05).Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention(P<0.05).CONCLUSION 18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.

栀子厚朴汤中p-香豆酸在正常及抑郁大鼠尿液中排泄动力学差异研究

目的建立一种基于HPLC-UV的快速稳定方法,测定栀子厚朴汤灌胃给药后大鼠尿液中p-香豆酸的含量,并应用于正常及抑郁大鼠的尿液排泄动力学研究。方法采用慢性温和不可预知应激进行大鼠抑郁造模,甲醇沉淀蛋白进行尿液样本前处理。色谱柱:Phenomenex C_(18)(250 mm×4.6 mm,5μm);流动相:乙腈∶0.1%甲酸水溶液为21∶79,等度洗脱;柱温:35℃;检测波长:310 nm;进样量:20μL;流速:1.0 mL/min。结果方法学结果表明回收率在98%~104%,RSD<3%,在0.2~64μg/mL范围内呈良好的线性关系(r=1);尿液排泄动力学结果表明,单次灌胃给予栀子厚朴汤后,正常及抑郁大鼠体内的p-香豆酸在24 h内均基本排泄完全;p-香豆酸在抑郁组大鼠体内以原型形式经肾脏排泄率较正常组大鼠高。结论该研究所建立的p-香豆酸测定方法灵敏、快速、可靠,可用于正常及抑郁大鼠p-香豆酸尿液排泄动力学的研究。抑郁组大鼠的尿液总排泄率较空白组大鼠高,提示可能与抑郁大鼠肠道菌群及肝脏代谢酶功能变化有关。

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis

AIM To determine the effects of ω-3 fatty acids(ω-3FA) on the toll-like receptor 4(TLR4)/nuclear factor κB p56(NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis(SAP).METHODS A total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay. RESULTS The expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group(P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h(P < 0.05).CONCLUSION During the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

Changes of chlorogenic acid content and its synthesis-associated genes expression in Xuehua pear fruit during development

According to synthetic pathway of plant chlorogenic acid （CGA）, the expression patterns of genes encoding enzymes that are associated with CGA synthesis were studied in normally developed Xuehua pear fruit. The study demonstrated that CGA content in peel and flesh of Xuehua pear decreased as fruit development progressed, with a higher level in peel. The expression levels of PbPAL 1, PbPAL2, PbC3H, PbC4H, Pb4CL 1, Pb4CL2, Pb4CL6, PbHC T1 and PbHC T3 genes decreased in fruit, which was consistent with the pattern of variation in CGA content. That indicated that these genes might be key genes for influencing fruit CGA synthesis in Xuehua pear. However, Pb4CL7 gene expression profile is not consistent with variation of CGA content, hence, it may not be a key gene involved in CGA synthesis.

Cytochrome P450 monooxygenase-mediated eicosanoid pathway:A potential mechanistic linkage between dietary fatty acid consumption and colon cancer risk

Human consumption of linoleic acid(LA,18:2ω-6,abundant in vegetable oils)is very high.Animal experiments showed that excessive LA intake increased azoxymethane-induced colon tumorigenesis,however,the impact of excessive LA on colon cancer in human is not conclusive,making it difficult to make dietary recommendations for optimal intake of LA.Understanding the molecular mechanisms of LA on colon tumorigenesis could help to clarify its health effect,and facilitate development of mechanismbased strategies for preventing colon cancer.Recent studies show that the previously unappreciated cytochrome P450 monooxygenase-mediated eicosanoid pathway is upregulated in colon cancer and plays critical roles in its pathogenesis,and could contribute to the effects of dietary LA,as well asω-3 fatty acids,on colon tumorigenesis.In this review,we will discuss recent studies about the roles of cytochrome P450 monooxygenases in fatty acid metabolism and its roles in colonic inflammation and colon cancer,and how this information could help us to clarify the health impacts of dietary fatty acids.

p-香豆酸对西洋参的化感作用及生理机制

西洋参(Panax quinquefolium L.)栽培中存在严重的连作障碍现象,前期发现p-香豆酸在以滤纸片为基质的条件下,能够显著抑制西洋参胚根的生长。为了明确p-香豆酸在土壤基质中对种胚的化感活性以及对成株西洋参生长的作用及生理机制,以自然土壤为基质,观察p-香豆酸作用后种胚的生长情况;采用室内水培试验,观察p-香豆酸作用下2年生西洋参种根从出苗至结果期的生长及部分生理指标的变化。种胚生长实验在土壤中分别添加0.0024、0.012、0.06、0.3、1.5、7.5 mg/g的p-香豆酸,处理7 d后测定西洋参种胚的胚根长和胚芽长。水培试验中全营养液中分别添加0.012 mg/m L、0.06 mg/m L、0.3 mg/m L p-香豆酸,处理后每隔5 d测定植株叶片展开情况、株高、冠幅等生长指标;于展叶期(10 d)、现蕾期(20 d)、结果期(30 d)测定地上部分及新生须根的生物量,同时测定新生须根苯丙氨酸解氨酶(PAL)活力;叶片完全展开后测定植株净光合速率(Pn)、表观电子传递速率(ETR)和最大光化学效率(Fv/Fm)等光合特性参数。结果表明,土壤中添加0.0024—7.5 mg/g p-香豆酸西洋参胚根长度降低28.52%—100%,胚芽长度降低1.09%—100%,并呈现一定的剂量抑制效应。实验浓度内的p-香豆酸可显著抑制西洋参植株地上部分生长,推迟展叶期;结果期地上部生物量比对照降低17.17%—54.55%(P<0.05,Dunnett-t test);同时,叶片的Pn、ETR受到抑制(P<0.05),但Fv/Fm不变;对须根的影响主要表现为0.06 mg/m L p-香豆酸处理组在展叶期PAL酶活力提高69.05%,之后PAL活力和生物量均比对照下降,浓度增加至0.3 mg/m L时整个培养期内PAL酶活力和生物量均低于对照。由此推论,根系环境中的p-香豆酸在自然土壤中对西洋参种胚具有显著抑制其生长的化感作用;对成株西洋参的作用主要为抑制地上部分生长,并通过降低成株西洋参叶片光合能力,从而表现出明显的化感作用,0.06 mg/m L p-香豆酸诱导须根PAL酶活力先升高再降低并最终降低生物量的结果也表明p-香豆酸是西洋参根系生长的胁迫因素。结果证实p-香豆酸对西洋参种胚和成株的生长均具有自毒作用,其抑制生长的生理机制在于抑制叶片的光合作用。

HPLC法同时测定不同配伍比例三棱-莪术药对中P-香豆酸、阿魏酸的含量

目的:建立同时测定三棱-莪术药对中P-香豆酸、阿魏酸含量的方法,探讨不同比例药对中二者含量的变化。方法:采用高效液相色谱法。色谱柱为Agilent 20RBAX XDB-C_(18),流动相为乙腈-0.1%冰醋酸溶液(梯度洗脱),流速为1.0 m L/min,检测波长为266 nm,柱温为30℃,进样量为10μL。结果:P-香豆酸、阿魏酸检测质量浓度线性范围分别为4.218 6~21.093μg/m L(r=0.999 8)、1.836 0~9.180μg/m L(r=0.999 9);精密度、稳定性、重复性试验的RSD<2.0%;加样回收率分别为98.72%~100.30%(RSD=0.18%,n=9)、99.11%~100.45%(RSD=0.46%,n=9)。三棱-莪术药对4个配伍比例(1∶1、2∶1、1∶2、1∶0,m/m)中,三棱-莪术比例(m/m)为2∶1时P-香豆酸、阿魏酸含量最高。结论:该方法操作简便,精密度、稳定性、重复性好,可用于三棱-莪术药对中P-香豆酸、阿魏酸含量的同时测定;该药对中三棱-莪术比例(m/m)为2∶1时主要有效成分含量最高。

分散液相微萃取-HPLC法用于大鼠血浆中对香豆酸测定

目的建立了分散液相微萃取与高效液相色谱联用技术测定大鼠血浆中对香豆酸浓度。方法微萃取条件为:20μL 1-己基-3-甲基咪唑六氟磷酸盐离子液体(1-hextyl-3-methylimidazoliumhexafluorophosphate,[C6MIM][PF6])作萃取剂,80μL乙腈作分散剂,0.5 mol.L-1硫酸作酸化剂,萃取时间为1 min。结果在优化的萃取条件下,血浆中对香豆酸质量浓度在0.012～2.400 mg.L-1内,线性关系良好(相关系数r=0.997 5),日内和日间精密度(RSD%)<9%(n=6),相对回收率为96.7%～101.6%,提取回收率为59.0%～70.4%。结论该方法适于血浆中对香豆酸浓度的测定。

Induced Effects of Exogenous Phenolic Acids on Allelopathy of a Wild Rice Accession(Oryza longistaminata,S37)

Four exogenous phenolic acids, including salicylic acid, fumalic acid, p-coumaric acid and p-hydroxybenzonic acid, were used to investigate the regulatory effects on allelopathy of a wild rice accession of S37 （Oryza Iongistaminata）, which is a known allelopathic rice. The four exogenous phenolic acids induced the enhancement of the allelopathic potential of wild rice S37 in target weeds though the weed-suppressive activities were low, and the inducible effects were dependent on the specific phenolic acid, concentration and treatment time. After foliar application of exogenous phenolic acids, the inhibition rates for plant height, root length and fresh weight of barnyard grass （Echinochioa crus-galli） were significantly higher than those of the control. Especially at the concentration of 100 mg/L, the inhibition rates for plant height and fresh weight of barnyard grass by fumalic acid were 38.12% and 26.31% higher than those of the control, showing that fumalic acid was more effective compared with other phenolic acids in inhibiting monocotyledon weed growth. Furthermore, the weedsuppressive activity of aqueous extract from the leaves of wild rice S37 treated with exogenous phenolic acids was increased, and it peaked at 48 h after the treatment with the aqueous extract, and then gradually declined.

Uncoupling tumor necrosis factor-αand interleukin-10 at tumor immune microenvironment of breast cancer through miR-17-5p/MALAT-1/H19 circuit

Triple Negative Breast Cancer(TNBC)immunotherapy has recently shown promising approach.However,some TNBC patients presented with resistance.One of the reasons was attributed to the excessive release of cytokines at the tumor microenvironment(TME)such as Tumor necrosis factor alpha(TNF-α)and Interleukin-10(IL-10).Fine regulation of these cytokines’levels via non-coding RNAs(ncRNAs)might alleviate the immune quiescent nature of TME at TNBC tumors.However,the extrapolation of ncRNAs as therapeutic tools is highly challenging.Therefore,disentanglement the nature for the isolation of natural compounds that could modulate the ncRNAs and their respective targets is an applicable translational therapeutic approach.Hence,this study aimed to targeting the chief immune suppressive cytokines at the TME(TNF-αand IL-10)via ncRNAs and to examine the effects of Rosemary aerial parts extract on the expression levels of these ncRNAs in TNBC.Results revealed miR-17-5p as a dual regulator of TNF-αand IL-10.Moreover,an intricate interaction has been shown between miR-17-5p and the oncogenic lncRNAs:MALAT1 and H19.Knocking down of MALAT1 and/or H19 caused an induction in miR-17-5p and reduction in TNF-αand IL-10 expression levels.miR-17-5p was found to be down-regulated,while TNF-α,IL-10,MALAT1 and H19 were up-regulated in BC patients.Forced expression of miR-17-5p in MDA-MB-231 cells reduced TNF-α,IL-10,MALAT1 and H19 expression levels,as well as several BC hallmarks.In a translational approach,ursolic acid(UA)isolated from rosemary induced the expression of miR-17-5p,MALAT1 and decreased H19 expression levels.In conclusion,this study suggests miR-17-5p as a tumor suppressor and an immune-activator miRNA in BC through tuning up the immunological targets TNF-α,IL-10 at the TME and the oncological mediators MALAT1 and H19 lncRNAs.

HPLC法同时测定芦根中对香豆酸和阿魏酸含量

采用HPLC法同时测定对香豆酸和阿魏酸的含量以评价芦根质量。芦根经碱性乙醇水溶液回流提取对香豆酸和阿魏酸,Diamonsil C18柱(4.6 mm×250 mm,5μm),乙腈-0.3%醋酸水溶液流动相梯度洗脱分离,紫外310 nm波长检测。对香豆酸和阿魏酸分别在0.2~200μg/m L和0.1~120μg/m L范围有良好线性,平均回收率分别为(96.9±2.91)%和(98.7±1.78)%。测得15批市售芦根饮片中对香豆酸和阿魏酸的含量(x珋±s,max^min)分别为(0.88±0.16,1.21~0.70)%和(0.41±0.035,0.49~0.36)%。所建立的对香豆酸和阿魏酸含量的同时测定方法可用于芦根质量的评价。

HPLC测定百合中对香豆酸、没食子酸含量

目的建立药食两用植物百合中2种有效成分对香豆酸、没食子酸的HPLC含量测定方法。方法采用Hypersil BDC-C18色谱柱(200 mm×4.6 mm,5μm),对香豆酸的测定以甲醇-0.1%甲酸水溶液(30:70)为流动相等度洗脱,没食子酸的测定以甲醇-0.1%磷酸水溶液(15∶85)为流动相等度洗脱,检测波长分别为310、270 nm,流速1.0 mL/min,柱温25℃,进样量10μL。结果在上述色谱条件下,对香豆酸、没食子酸与相邻色谱峰之间分离度良好。对香豆酸线性回归方程为Y=1×10~8X+57 335(r=0.998 7),线性范围为10.00~160.03 ng;没食子酸线性回归方程为Y=3×10~7X-21 414(r=0.998 2),线性范围为9.99~159.95 ng。对香豆酸、没食子酸的平均回收率分别为95.63%、96.62%。结论本研究建立的方法操作简单、方便、可靠,可为百合饮片、百合药膳产品开发及相关药材的质量控制提供依据。

HPLC法同时测定3种红景天药材中5种化学成分的含量

目的:建立同时测定大花红景天、狭叶红景天和长鞭红景天药材中没食子酸、红景天苷、酪醇、咖啡酸、对香豆酸含量的方法,并比较3种红景天药材中5种化学成分的含量差异。方法:采用高效液相色谱法测定含量,色谱柱为Wonda Sil C18,流动相为乙腈-0.3%磷酸溶液(梯度洗脱),检测波长为275 nm,柱温为25℃,流速为1.0 m L/min,进样量为10μL;采用Graph Pad Prism 5.0软件对含量测定结果进行单因素方差分析和Tukey’s多重比较。结果:没食子酸、红景天苷、酪醇、咖啡酸、对香豆酸的检测进样量线性范围分别为0.020 48~0.614 4μg(r=0.999 7)、0.118~3.54μg(r=0.999 5)、0.010 84~0.325 2μg(r=0.999 7)、0.008 48~0.254 4μg(r=0.999 5)、0.004 1~0.123μg(r=0.999 5);精密度、稳定性、重复性试验的RSD均小于2%;加样回收率分别为99.62%~104.92%(RSD=1.68%,n=9)、96.55%~100.69%(RSD=1.67%,n=9)、98.91%~103.39%(RSD=1.48%,n=9)、100.93%~104.98%(RSD=1.37%,n=9)、97.71%~103.89%(RSD=1.99%,n=9)。大花红景天中没食子酸、红景天苷、酪醇和咖啡酸含量均显著高于长鞭红景天,大花红景天中的红景天苷、咖啡酸含量均显著高于狭叶红景天,差异均有统计学意义(P<0.05,P<0.01或P<0.001)。结论:该方法简便可行、结果准确,可用于同时测定大花红景天、狭叶红景天和长鞭红景天药材中没食子酸、红景天苷、酪醇、咖啡酸、对香豆酸的含量。3种常见红景天药材中的化学成分含量存在较大差异,大花红景天中红景天苷的含量最高。

基于改进AHP-熵权-关联度及化学计量学的经典名方一贯煎提取工艺研究

目的 筛选经典名方一贯煎的最优提取工艺,为其制剂研究提供实验依据。方法 对HPLC法测定的甜菜碱、地黄苷D、对香豆酸、阿魏酸、毛蕊花糖苷、洋川芎内酯Ⅰ进行化学计量学分析;以筛选出的关键成分含量、出膏率及指纹图谱相似度为评价指标,采用改进AHP-熵权法确定各个指标的组合权重系数,以基准样品关联度评价样品与基准样品的质量一致性,在单因素试验的基础上,采用响应面法优选一贯煎的提取工艺参数。结果 Box-Behnken设计建立的数学模型具有统计学意义,一贯煎最佳提取工艺参数为加12倍量水,提取时间80 min,提取2次。此条件下设计空间的综合评分为0.963。结论 采用改进AHP-熵权-基准样品关联度法优选一贯煎提取工艺参数,所拟合的模型显著,与基准样品质量基本一致,为进一步研究经典名方一贯煎复方制剂究提供了参考。

中药菟丝子中生物活性成分的毛细管电泳-电化学检测

采用毛细管电泳-电化学检测法(CE-ECD)同时测定了菟丝子中芦丁、金丝桃甙、山柰酚、对香豆酸和槲皮素等5种主要生物活性成分的含量,考察了运行缓冲液酸度和浓度、分离电压、氧化电位和进样时间等实验参数对分离检测的影响。在最佳实验条件下,以直径300μm的碳圆盘电极为工作电极,检测电位为+950m V(vs.参比电极),以50mm o l/L的硼砂缓冲溶液(pH9.0)为运行缓冲液,上述各组分在19m in内能完全分离。芦丁、金丝桃甙、山柰酚、对香豆酸和槲皮素在两个数量级的范围内呈良好线性关系,检测下限(按S/N=3计)分别为1.93×10-5,3.55×10-4,3.65×10-5,1.73×10-5和1.46×10-4g/L。该法已成功地应用于菟丝子中活性成分的分离检测,结果令人满意。

对-香豆酸和绿原酸对美拉德反应体系中5-羟甲基糠醛形成的影响

以谷氨酸、赖氨酸、甘氨酸、半胱氨酸分别与葡萄糖反应构建美拉德反应体系,研究5-羟甲基糠醛(5-HMF)的形成量随时间的变化规律及对-香豆酸和绿原酸对5-HMF形成量的影响。结果表明:在相同反应时间内,模拟体系中5-HMF形成量由高到低排列依次是:谷氨酸-葡萄糖模拟体系>赖氨酸-葡萄糖模拟体系>甘氨酸-葡萄糖模拟体系>半胱氨酸-葡萄糖模拟体系;对-香豆酸、绿原酸对美拉德反应中5-HMF形成均表现出抑制和促进的"双重作用";不同质量浓度的对-香豆酸、绿原酸对5-HMF的影响趋势不同,相同质量浓度的同种酚酸对不同模拟反应体系中5-HMF的影响也有差异。