Alleviatory effect of isoquercetin on benign prostatic hyperplasia via IGF-1/PI3K/Akt/mTOR pathway

We evaluated the effect of isoquercetin(quercetin-O-3-glucoside-quercetin,IQ)as a functional component of Abeliophyllum disistichum Nakai ethanol extract(ADLE)on prostate cell proliferation and apoptosis and its effects on the IGF-1/PI3K/Akt/mTOR pathway in benign prostatic hyperplasia(BPH).Metabolites in ADLE were analyzed using UHPLC-qTOF-MS and HPLC.IQ was orally administered(1 or 10 mg/kg)to a testosterone propionate-induced BPH rat model,and its effects on the prostate weight were evaluated.The effect of IQ on androgen receptor(AR)signaling was analyzed in LNCaP cells.Whether IGF-1 and IQ affect the IGF-1/PI3K/Akt/mTOR pathway in BPH-1 cells was also examined.The metabolites in ADLE were identified and quantified,which confirmed that ADLE contained abundant IQ(20.88 mg/g).IQ significantly reduced the prostate size in a concentration-dependent manner in a BPH rat model,and significantly decreased the expression of AR signaling factors in the rat prostate tissue and LNCaP cells in a concentration-dependent manner.IQ also inhibited the PI3K/AKT/mTOR pathway activated by IGF-1 treatment in BPH-1 cells.In BPH-1 cells,IQ led to G0/G1 arrest and suppressed the expression of proliferation factors while inducing apoptosis.Thus,IQ shows potential for use as a pharmaceutical and nutraceutical for BPH.

Transformation of trollioside and isoquercetin by human intestinal flora in vitro

The present study was designed to determine the intestinal bacterial metabolites of trollioside and isoquercetin and their antibacterial activities. A systematic in vitro biotransformation investigation on trollioside and isoquercetin, including metabolite identification, metabolic pathway deduction, and time course, was accomplished using a human intestinal bacterial model. The metabolites were analyzed and identified by HPLC and HPLC-MS. The antibacterial activities of trollioside, isoquercetin, and their metabolites were evaluated using the broth microdilution method with berberine as a positive control, and their potency was measured as minimal inhibitory concentration(MIC). Our results indicated that trollioside and isoquercetin were metabolized by human intestinal flora through O-deglycosylation, yielding aglycones proglobeflowery acid and quercetin, respectively The antibacterial activities of both metabolites were more potent than that of their parent compounds. In conclusion, trollioside and isoquercetin are totally and rapidly transformed by human intestinal bacteria in vitro and the transformation favors the improvement of the antibacterial activities of the parent compounds.

Evaluation of the wound healing potential of isoquercetin-based cream on scald burn injury in rats

Background:The present study was designed to evaluate the potential of isoquercetin-based cream formulation on scald burn wound injury in rats.Methods:Four isoquercetin-based cream formulations viz.0.01,0.02,0.04,and 0.06%w/w were prepared.Cream base and standard anti-burn cream containing silver sulfadiazine were also used for comparison.Scald burn was given to rats by pouring water at 90℃on a shaved dorsal area of 20 mm2.Deep second-degree burn injury was produced which was evaluated for the next 21 days for the percentage of wound contraction and period of epithelialization.On day 21,the rats were sacrificed and histopathological slides were prepared using hematoxylin-eosin staining.Burned tissue was also screened for levels of oxidative stress using thiobarbituric acid reactive species(TBARS)and reduced glutathione(GSH)estimation.Results:There was a significant increase in the percentage of wound contraction and a significant decrease in the period of epithelialization in isoquercetin-based cream-treated groups as compared with the control group.However,most significant results were obtained with isoquercetin 0.06%w/w cream.Histological y,isoquercetin 0.06%w/w cream treatment resulted in almost complete re-epithelialization and re-structuring of the wound tissue.There was a significant rise in TBARS and a decrease in GSH levels in the burn injury group which was reversed to a major extent by the application of isoquercetin-based cream.Conclusions:The results indicate the wound healing potential of isoquercetin-based cream.Tissue biochemical studies indicate towards a possible role of free radical scavenging in the observed effects of isoquercetin in wound healing.

极耐热α-L-鼠李糖苷酶的性质及其在异槲皮素制备中的应用研究

L-鼠李糖苷酶可特异性切除糖苷类化合物上连接的α-L-鼠李糖基,已被广泛用于食品、医药等工业领域。作者旨在获得耐受高温的α-L-鼠李糖苷酶,提高工业生产的效率和降低使用成本。首先对嗜热菌Sulfolobus islandicus来源的α-L-鼠李糖苷酶基因进行克隆及大肠杆菌的异源表达,随后测定了系列酶学性质,接着优化了该酶制备异槲皮素的酶法制备工艺。结果表明:重组酶SisRha在90℃下酶活力最佳,具有极好的热稳定性,其在80℃下孵育120 min后酶活力几乎没有损失;最适反应pH为5.5,在pH 4.5~7.0时,pH稳定性良好;重组酶SisRha对人工底物对硝基苯酚鼠李糖糖苷(p NPR)的Km值为(0.15±0.03) mmol/L,Vmax值为(6.26±0.51)U/mg,k_(cat)值为(10.48±0.86) s^(-1);对底物p NPR、芦丁、朝藿定C、柚皮苷、橘皮苷和淫羊藿苷的比活力分别为25.06、11.89、6.74、3.14、2.77、0.42 U/mg。重组酶SisRha转化芦丁生成异槲皮素的适宜工艺为:用酶量0.40 U/mL,在85℃、pH 5.0的条件下反应1 h,可将2 mmol/L芦丁几乎全部转化,摩尔转化率高达98.56%。本研究丰富了现有的α-L-鼠李糖苷酶资源,为嗜热菌来源的α-L-鼠李糖苷酶的研究奠定了基础,同时提供了一种高温转化芦丁制备异槲皮素的方法。

槲皮素及异槲皮素、芦丁抗自由基活性的比较研究

目的评价3种黄酮类同系化合物槲皮素及其单糖苷异槲皮素和双糖苷芦丁的体外抗自由基作用,进一步研究其构效关系。方法采用H2O2/Fe2+体系,通过Fenton反应生成羟自由基(·OH);采用邻苯三酚自氧化法产生超氧阴离子(O2-)。利用·OH造成肝细胞脂质过氧化以及红细胞膜破裂,观察受试化合物的保护作用,并计算IC50。结果在以上4种体外模型中,3种药物均表现出极强的清除自由基能力。在·OH以及O2-清除实验中,抗氧化活性顺序为芦丁>异槲皮素>槲皮素;在抑制肝匀浆脂质过氧化及红细胞膜破裂实验中,抗氧化活性顺序为槲皮素>异槲皮素>芦丁。结论3种受试黄酮类化合物均具有十分强大的自由基清除作用,且呈现明显的剂量依赖性和糖基结构依赖性,在不同的实验体系中其抗自由基活性强弱顺序不同。

锁阳化学成分研究Ⅱ

目的研究常用补肾助阳中药锁阳(Herba Cynomorii)的化学成分。方法采用大孔吸附树脂、聚酰胺、硅胶、SephadexLH-20和ODS柱等色谱手段进行化学成分的分离纯化;依据理化常数、光谱数据、对照品和相关文献进行结构鉴定。结果分离得到并鉴定了11个化合物,分别为柑桔素(naringenin,1)、柑桔素-4′-O-β-吡喃葡萄糖苷(naringenin-4′-O-β-pyranoglucose;choerospondin,2)、异槲皮苷(isoquercetin,3)、儿茶素[(+)catechin,4]、表儿茶素[(-)epicatechin,5)]、乙酰熊果酸(acetyeursolic acid,6)、葡萄糖(glucose,7)、熊果酸(ursolic acid,8)、β-谷甾醇(β-sitosterol,9)、棕榈酸(palmitic acid,10)和胡萝卜苷(daucosterol,11)。结论化合物1,3,5,7为首次从锁阳中得到。对其中5种成分1,2,4,6,8首次建立了定性鉴别和含量测定方法,为锁阳药材的质量控制标准提供了科学依据。

黄蜀葵花黄酮成分的研究

目的 :对黄蜀葵花 (AbelmoschusmanihotL Medic)总黄酮进行化学成分的研究。方法 :利用色谱和分部结晶方法进行成分分离 ,根据物理化学性质和波谱学手段对化合物进行结构鉴定。结果 :分离到 4个黄酮醇单糖苷 ,分别鉴定为棉皮素 3′ O β 葡萄糖苷 (Gossypetin 3′ O β glucoside ,1) ,异槲皮苷 (Isoquercetin ,2 ) ,金丝桃苷 (Hyperoside ,3)和槲皮素 3′ 葡萄糖苷 (Quercetin 3′ glucoside ,4 )。结论 :其中 1为新化合物 ,2为从该植物中首次分得。

苦荞异槲皮苷对人胃癌细胞SGC-7901增殖及凋亡的影响

从苦荞中提取制备异槲皮苷,研究其对人胃癌细胞SGC-7901增殖、凋亡、迁移和细胞周期的影响。将异槲皮苷作用于人胃癌SGC-7901细胞和人肾上皮细胞系293T,通过噻唑蓝法检测异槲皮苷对其增殖的影响;4’,6-二脒基-2-苯基吲哚(4’,6-diamino-2-phenyl indole,DAPI)荧光染色法观察细胞核的形态学变化;划痕擦伤迁移实验检测异槲皮苷对SGC-7901细胞迁移能力的影响;流式细胞术检测异槲皮苷对SGC-7901细胞凋亡及细胞周期的影响。结果表明:苦荞异槲皮苷可以抑制SGC-7901细胞的增殖,并呈时间和剂量依赖性,当用100μmol/L异槲皮苷作用细胞48 h后,对SGC-7901细胞的增殖抑制率达到35.92%,而对人肾上皮细胞系293T的增殖抑制率仅为3.15%;DAPI荧光染色法观察异槲皮苷处理细胞后,染色体凝聚,有凋亡小体产生;划痕擦伤实验显示,异槲皮苷能抑制SGC-7901细胞的迁移;流式细胞术检测结果表明,异槲皮苷可使G1和S期细胞减少,G2/M期细胞增多,且细胞凋亡率明显增加。综上所述,苦荞麦异槲皮苷能够诱导SGC-7901细胞发生凋亡,阻断细胞周期并抑制细胞增殖和迁移。

RP-HPLC法测定桑叶中的芦丁和异槲皮苷含量

目的:建立HPLC法测定桑叶中芦丁、异槲皮苷含量的定量分析方法。方法:HPLC外标法。采用色谱柱:Agilent TC-C18柱(250mm×4.6mm,5μm);流动相:乙腈-甲醇-水-磷酸(100:10:340:0.3,V/V);检测波长365nm;流速1.0ml/min;进样量20μl;柱温:室温。结果:在上述色谱条件下,芦丁、异槲皮苷获良好分离,浓度分别在7.5～150μg/ml(R2=0.9995)、7.1～142μg/ml(R2=0.9998)范围内呈良好的线性关系,平均加样回收率为98.79%、99.01%,RSD分别为3.37%、2.43%,重复性实验RSD分别为0.0133%和0.0214%。结论:该方法分离度好,简便快速,为桑叶生药及其制剂的质量控制提供可借鉴的分析方法。

HPLC法同时测定桑叶中芦丁、异槲皮苷、紫云英苷的含量

目的建立晾晒、烘干及鲜桑叶中芦丁、异槲皮苷、紫云英苷的含量测定方法。方法采用HPLC法,Shim pack ODS色谱柱(250 mm×4.6 mm,5μm),柱温35℃,流动相为0.4%磷酸乙腈溶液(A)和0.4%磷酸水溶液(B),梯度洗脱,检测波长为360 nm,流速设为1.0 ml.min-1。结果三种黄酮类成分在35 min内达到良好分离,芦丁在0.076～1.920 mg.L-1,异槲皮苷在0.086～2.148 mg.L-1,紫云英苷在0.042～2.108 mg.L-1范围内线性关系良好(r>0.999 3,n=6),三种成分平均加样回收率分别为99.7%(RSD=2.5%)9、8.8%(RSD=2.4%)和99.5%(RSD=4.1%)。结论该方法操作简单,结果准确,具有较好的重复性和稳定性,为评价桑叶药材炮制方法提供了一个很好的依据,同时也可用于桑叶药材的质量控制。

木贼中三种黄酮苷的含量测定

目的测定木贼中三种黄酮苷的含量.方法采用双波长薄层扫描法测定木贼中三种黄酮苷含量.结果异槲皮素,山柰酚-3-芸香糖-7-葡萄糖苷,山柰酚-3,7-双葡萄糖苷分别在0.5～2.5μg,0.2～1.0μg,0.2～1.0μg范围内呈良好的线性关系,平均回收率分别为97.96%,98.06%,97.43%,RSD分别为2.08%,2.31%,2.46%.结论用薄层扫描法测定木贼中三种黄酮苷的含量是可行的,为木贼的合理利用和质量控制提供了一定的科学依据.

青钱柳叶中黄酮类化合物的地理变异

以青钱柳Cyclocarya paliurus11个种源1年生苗的叶片为材料,研究青钱柳叶片中黄酮类化合物的地理变异,初步筛选出黄酮类化合物质量分数高的优良药用种源。黄酮类化合物各成分用液相色谱测定,数据采用方差分析、多重比较和相关分析等进行统计分析。结果表明:青钱柳叶中3种黄酮类化合物质量分数紧密相关,其中山奈酚质量分数高于槲皮素和异槲皮苷。研究的各种源中,安徽歙县和广西资源种源的黄酮类化合物的质量分数较高,分别为6.770和6.494mg·g-1,安徽舒城种源的黄酮类化合物质量分数最低,仅为1.230mg·g-1;以经纬度为自变量,各成分的质量分数为因变量进行趋势面分析,青钱柳叶中山奈酚质量分数呈经纬度双向变异,但其变化幅度随经度变化幅度不如纬度变化大,其表现出以108°E和27°N交叉区域为中心向东西递增、向南北递减的趋势,而槲皮素和异槲皮苷质量分数的变化没有明显的规律性。青钱柳黄酮类化合物较高的优良药用种源为安徽歙县和广西资源种源。

荞麦黄酮和荞麦糖醇对胰脂肪酶的抑制作用

目的:了解荞麦麸皮中活性成分抑制胰脂肪酶的活性。方法:以苦荞麸皮醇提物(主要成分为芦丁)、醇提残渣水解物(主要成分为芦丁、槲皮素和异槲皮素的混合物)和甜荞麸皮水提物(主要成分为荞麦糖醇)为实验材料,测定3个样品对胰脂肪酶的抑制作用,以市售胰脂肪酶抑制剂——奥利司他为参照;通过研究底物、抑制剂和酶的加入顺序对抑制率的影响,初步确定醇提水解物的抑制机理。结果:实验材料对胰脂肪酶的抑制作用依次为奥利司他>醇提水解物>醇提物>水提物,半数抑制浓度(IC50)分别为0.47、1.94、2.53、17.6 mg/m L;醇提水解物的抑制作用机理主要是通过苦荞黄酮与底物相互作用,从而阻碍了底物与酶的作用,属于竞争性抑制。结论:荞麦黄酮(即醇提物和醇提水解物)对胰脂肪酶有抑制活性;醇提残渣经高压水解后更有利于其对胰脂肪酶的抑制作用;荞麦糖醇(即水提物)在低质量浓度下抑制效果不明显,在较高的质量浓度下则表现出抑制活性。

人肠内菌转化模型的稳定性研究

目的:以异槲皮素为转化对象对肠内菌体外转化模型的转化稳定性进行考察。方法:按照先前建立的《人肠内细菌生物转化模型和标准操作规程》,采用人源不同及人源相同但培养代数不同的肠内菌建立体外转化模型,对异槲皮素进行生物转化。结果:在24 h内,采用不同人源的肠内菌建立的转化模型均可将异槲皮素转化为槲皮素,但是转化所需的时间存在差异;而随着培养代数的增加,肠内菌表现出转化效率增强的趋势。结论:本研究证明,按照《标准操作规程》建立的肠内菌体外转化模型比较稳定,所得实验结果具有重现性,可用于中药化学成分的肠内菌转化研究。

基于荧光光谱技术的异槲皮素抑制晚期糖基化产物形成的机制研究

荧光光谱学法具有灵敏度高、选择性强、样品用量少、操作简便的优点,同时又可提供多种分子间相互作用的物理参数,所以被广泛用于研究小分子和蛋白质的相互作用。因此,通过内源荧光、同步荧光以及三维荧光光谱技术,研究异槲皮素抑制α-乳白蛋白(α-lactalbumin,α-La)-果糖体系糖基化过程中晚期糖基化产物(AGEs)形成的能力,并从分子间相互作用的角度阐述其作用机制,最终阐明异槲皮素抑制食品热加工过程中AGEs形成的能力。结果表明,异槲皮素可显著降低糖基化反应诱导的α-La构象变化,抑制AGEs的形成,样品浓度为36.58μmol·L^-1时,其抑制率达74.66%,高于阳性对照品氨基胍盐酸盐。同步荧光和三维荧光光谱分析结果表明,异槲皮素会改变α-La的构象,使色氨暴露于更亲水的环境中,增加其微环境的亲水性。抑制动力学和热力学分析结果显示,异槲皮素通过静态方式猝灭α-La的内源荧光,且与α-La按照1∶1的摩尔比例结合形成了稳定的复合物,该结合是一个自发放热的过程,范德华力和氢键是异槲皮素-α-La复合物形成的主要作用力。

罗布麻花中异槲皮素的蜗牛酶提取工艺研究

目的研究罗布麻花中异槲皮素的提取工艺。方法以蜗牛酶提取作为提取方法,高效液相色谱法测定异槲皮素含量为指标,通过正交实验,考察蜗牛酶用量、酶解温度、酶解时间和酶解pH值等对提取效果的影响。结果罗布麻花中异槲皮素的最佳提取工艺为酶解温度为60℃,酶用量为0.2 mg/ml,酶解时间为1 h,酶解pH值为5.0,测得异槲皮素含量为4.058 5%。结论酶法提取具有反应温和。设备简单的特点,提取过程节能环保,并且减少对提取物的氧化,适合中药材的大量提取。

金盏银盘指纹图谱的建立及其活性成分的研究

目的:建立不同产地金盏银盘药材的高效液相色谱指纹图谱,并同时测定金丝桃苷、异槲皮苷、鬼针聚炔苷和紫云英苷4种主要成分的含量。方法:采用反相高效液相色谱法,选用Hypersil ODS C18(250 mm×4.6 mm,5μm)色谱柱,乙腈-3%冰醋酸为流动相梯度洗脱,采用药典委员会颁布的中药色谱指纹图谱相似度评价系统(2004 A)评价图谱的相似性,同时测定药材中4种主要成分的含量。结果:建立了不同产地金盏银盘药材的HPLC指纹图谱,方法学符合指纹图谱要求,不同产地药材相似度差异不大,但4种主要成分含量差异较大。结论:该方法稳定、准确、可靠,指纹图谱结合含量测定为金盏银盘药材质控标准的制定提供了科学依据。

Anti-free radical and cytoprotective effects of quercetin and its sugar-containing natural congeners in cultured HEK293 cells injured by anoxia/hypoglycemia and the structure-effect relationship thereto

Objective To comparatively study anti-free radical and cytoprotective effects of quercetin(Q)and its monoglucoside isoquercetin(I),diglucoside rutin(R),which differs only in glycosyl-substitution at C-3 position of the molecules,using anoxia/hypoglycemia-induced cell injury model and thereby to explore the structure-effect relationship thereto.Methods The cell injury model was established by HEK293 cells cultured in vitro with Na2S2O3 plus sugar-free Earle's fluid as incubation medium.Cell survival rate(CSR),total antioxidant capacity(TAC),SOD and LDH levels were determined.The effect intensity of the 3 flavonoids was compared by means of IC50,the concentration required to achieve 50% inhibition of the changes in the above indices in injured cells.Results Q,I and R all concentration-dependently elevated CSR,TAC and SOD and reduced LDH level.The all of IC50s for the above indices were ranked in order of IC50,Q<IC50,I<IC50,R,namely,the effect intensity should be Q>I >R.Conclusions The 3 structurally similar flavoloids all have significant and concentration-dependent anti-free radical and cyto-protective effects with the intensity being in order of aglycone>monoglucoside>diglucoside;the substitution of-OH by sugar group at C-3 position of flavoloids and increase in the sugar-substituent number are associated with the effect intensity reduced;namely,the intensity of these effects of flavonoids is negatively related the substutution by sugar group at C-3 position.

Cytoprotective effects of quercetin and its sugar-containing natural congeners in cultured HEK293 cells injured by anoxia/hypoglycemia and the structure-effect relationship thereto

Objective To comparatively study anti-free radical and cytoprotective effects of quercetin(Q)and its monoglucoside isoquercetin(I),diglucoside rutin(R),which differs only in glycosyl-substitution at C-3 position of the molecules,using anoxia/hypoglycemia-induced cell injury model and thereby to explore the structure-effect relationship thereto.Methods The cell injury model was established by HEK293 cells cultured in vitro with Na2S2O3 plus sugar-free Earle's fluid as incubation medium;Cell survival rate(CSR),total antioxidant capacity(TAC),SOD and LDH levels were determined;The effect intensity of the 3 flavonoids were compared by means of IC50,the concentration required to achieve 50% inhibition of the changes in the above indices in injured cells.Results Q,I and R all concentration-dependently elevated CSR,TAC and SOD,and reduced LDH level;the all of IC50s for the above indices were ranked in order of IC50,Q<IC50,I<IC50,R,namely,the effect intensity should be Q>I>R.Conclusions The 3 structurally similar flavoloids all have significant and concentration-dependent anti-free radical and cyto-protective effects with the intensity being in order of aglycone>monoglucoside>diglucoside;the substitution of-OH by sugar group at C-3 position of flavoloids and increase in the sugar-substituent number are associated with the effect intensity reduced;namely,the intensity of these effects of flavonoids is negatively related the substutution by sugar group at C-3 position.

槲皮素和异槲皮素对猕猴桃糖蛋白影响的研究

采用荧光光谱、紫外光谱和傅里叶红外光谱法研究不同温度下槲皮素和异槲皮素对猕猴桃糖蛋白GP2的影响。确定了GP2与槲皮素和异槲皮素发生相互作用生成新的复合物,在300 K和310 K温度下的荧光猝灭机制主要是静态猝灭;结合常数分别为107.66 L/moL和63.07 L/moL,对应的结合位点数为0.65和0.57;氢键和范德华力是二者之间主要的相互作用力;通过同步荧光光谱法研究确定了槲皮素和异槲皮素与猕猴桃糖蛋白GP2结合位点更接近色氨酸残基;利用傅里叶红外光谱和紫外光谱研究槲皮素和异槲皮素的加入对糖蛋白GP2构象的变化。