甘草酸与三价铬的配位反应研究

中药配合物新药是中药新药研制的新思路。本文通过正交实验和单因素实验研究了溶剂、温度、pH值对甘草酸与铬(Ⅲ)离子配位的影响,并利用红外光谱研究反应物的比例对配位反应的影响。结果表明,最佳的配合反应条件是pH 5.0、乙醇浓度10%(v/v)和温度30℃;反应物比例不同,所得配合物的红外光谱基本一致。     

芹菜素-钐配合物的合成及其抗小鼠高尿酸血症研究北大核心CSCD

秉承中药配位化学理论,以具有一定抗炎、抗痛风活性的芹菜素(AP)为配体,以稀土金属钐(Ⅲ)离子为配位中心,设计合成芹菜素-钐配合物(AP-Sm),以期提高抗高尿酸血症活性。采用紫外(UV)、红外(IR)、氢核磁共振(1H NMR)、电导法、差热-热重分析(TG-DTA)等技术对配合物的化学结构进行表征。考察配合物对酵母浸粉联合氧嗪酸钾诱导的高尿酸血症小鼠模型中尿酸、黄嘌呤氧化酶及超氧阴离子水平的影响。结果表明,芹菜素与钐(Ⅲ)离子配位生成了配合物,配合物组成式为:Sm(C_(15)H_9O_5)_3. 2H_2O。芹菜素A环的5-OH和C环的4位C=O与钐(Ⅲ)离子形成了配合物,且芹菜素与钐(Ⅲ)离子的配位比为3。抗高尿酸血症活性研究发现,芹菜素-钐配合物对高尿酸血症小鼠黄嘌呤氧化酶的抑制作用、清除超氧阴离子能力、降低血清尿酸水平及促进尿酸排泄能力均优于芹菜素。综上说明芹菜素与钐(Ⅲ)离子配位后,所得配合物抗高尿酸血症活性增强。     

芹菜素-钐配合物的合成及其抗小鼠高尿酸血症研究

秉承中药配位化学理论,以具有一定抗炎、抗痛风活性的芹菜素(AP)为配体,以稀土金属钐(Ⅲ)离子为配位中心,设计合成芹菜素-钐配合物(AP-Sm),以期提高抗高尿酸血症活性。采用紫外(UV)、红外(IR)、氢核磁共振(1H NMR)、电导法、差热-热重分析(TG-DTA)等技术对配合物的化学结构进行表征。考察配合物对酵母浸粉联合氧嗪酸钾诱导的高尿酸血症小鼠模型中尿酸、黄嘌呤氧化酶及超氧阴离子水平的影响。结果表明,芹菜素与钐(Ⅲ)离子配位生成了配合物,配合物组成式为:Sm(C_(15)H_9O_5)_3. 2H_2O。芹菜素A环的5-OH和C环的4位C=O与钐(Ⅲ)离子形成了配合物,且芹菜素与钐(Ⅲ)离子的配位比为3。抗高尿酸血症活性研究发现,芹菜素-钐配合物对高尿酸血症小鼠黄嘌呤氧化酶的抑制作用、清除超氧阴离子能力、降低血清尿酸水平及促进尿酸排泄能力均优于芹菜素。综上说明芹菜素与钐(Ⅲ)离子配位后,所得配合物抗高尿酸血症活性增强。     

CO_2超临界萃取葛渣中葛根素的研究

采用正交试验法优化CO2超临界提取葛渣中葛根素的工艺研究。在单因素试验的基础上,选择萃取投料量、萃取温度、萃取压力、萃取时间四个因素,进行L9(34)正交试验设计,评估了这些因素对葛根素提取率的影响。实验结果表明最佳工艺参数为投料量50 g,萃取温度55℃,萃取压力16 MPa,萃取时间90 min。在此条件下,葛渣中葛根素的萃取率为82.5%。     

浙江产蝮蛇Agkistrodon halys(Pallas)蛇毒磷酸二酯酶的酶学性质及其对大分子核酸的作用

1.以双对硝基苯磷酸钠为底物,测得蝮蛇毒磷酸二酯酶的最适pH在10左右,最适温度在60℃左右;酶在37℃和60℃的Km分别为5.65×10~(-4)M和7.97×10~(-4)M。 2.酶在pH5—10.5范围和50℃以下稳定。 3.钙离子和镁离子对酶活力有激活作用,但钡离子、镁离子、锌离子、二价铅离子、钴离子、二价和三价铁离子、锰离子、镍离子等都有不同程度地抑制作用。 4.除乙酸根离子外,碳酸根,硫酸根,乙二胺四乙酸根、磷酸根,钼酸根,酒石酸根,柠檬酸根,亚硝酸根,巴比妥酸根和硫代硫酸根等阴离子都有不同程度地抑制作用。 5.蝮蛇毒磷酸二酯酶能充分水解RNA和DNA但后者的水解速度比前者快许多。     

高聚物型色谱柱在高效液相色谱法中一步分析制备葛根素及大豆苷元的应用

建立了使用以聚苯乙烯-二乙烯基苯(PS-DVB)为基质的高聚物型色谱柱一步分离制备葛根提取液中的葛根素和大豆苷元的方法。采用MKF-RP-HH色谱柱(300 mm×7.8 mm,8μm),葛根提取液经70%乙醇溶解稀释,以水(A)和甲醇(B)为流动相进行梯度洗脱,流速1.0 m L/min,检测波长250 nm,柱温30℃。结果表明:葛根提取液中的2种有效成分葛根素和大豆苷元均与杂质达到了较好的分离效果,且柱效大于2 100 N/m,优于常规的C-18硅胶色谱柱(柱效:1 000 N/m)。该高聚物型色谱柱不仅可用于上述分析,还可用作半制备柱制备得葛根素和大豆苷元纯品,葛根素在2~50μg/m L范围内线性关系良好,回归方程为y葛根=1.342 2x-1.018 4,线性相关系数为0.999;大豆苷元在2~20μg/m L范围内线性关系良好,回归方程为y大豆=2.275x+0.869 5,线性相关系数为0.999;葛根素和大豆苷元的纯化得率分别可达95.9%和78.5%。     

葛根素对小鼠心肌细胞CVB3诱导心率失常的影响及其作用机制

本研究旨在确定葛根素对CVB3诱导心肌炎的治疗作用,并探讨其分子机制。通过测量生理指标和超声心电图检查,探讨葛根素对VMC小鼠心脏功能的影响;借助ELISA检测血清中H2S、TNF-α、hs-CRP和cTnI水平,并结合Western blotting分析葛根素对L-10、STAT3、NF-κβp65和NF-κβp50表达水平的影响。ELISA检测结果显示,葛根素处理可降低血清TNF-α、hs-CRP和cTnl水平,上调IL-10和STAT3的表达,并升高血清H2S含量;Western blotting分析结果表明,葛根素处理下调了NF-κβp65和NF-κβp50的表达水平,并降低P13K和AKT在心脏组织中的磷酸化水平。此外,葛根素处理还增加了心脏组织中Iκβα的表达水平。综上,葛根素通过激活CSE/H2S途径和IL-10/STAT3途径,并抑制NF-κβ信号传导途径来抑制VMC的发展。     

葛根素对冷激诱导的高血压小鼠的影响

目的：检测葛根素灌胃对冷激诱导的高血压小鼠的血压血脂及肾脏结构的影响。方法：小鼠分为正常对照组、冷激对照组、冷激葛根素饲喂组（2,5,10ms／kg bw3组）（n=12）。以寒冷刺激（4±2℃）建立小鼠高血压模型,每天定时灌胃葛根素治疗,对照组给予聚乙烯吡咯烷酮溶液。连续饲喂18d．检测各组小鼠血压、血脂含量,石蜡切片观察肾脏结构。结果：冷激对照组小鼠血压显著高于正常对照组（P〈0．01）,葛根素饲喂组血压显著低于冷激对照组（P〈0．05）。冷激对照组TG含量显著高于正常对照组（P〈0．01）,而冷激饲喂组TG含量明显降低（P〈0．05）;冷激饲喂组TC含量与冷激对照组相比有所降低但无统计学意义;2mg／kg BW葛根素组LDL-C显著低于冷激对照组（P〈0．01）,各组间HDL-C含量比较无统计学意义,但葛根素饲喂组HDL-C／LDL-C值显著高于冷激对照组。冷激对照组肾小管上皮细胞水肿,内腔极度缩小,肾小球明显胀大,肾小囊腔显著变窄,葛根素饲喂组肾小管水肿基本消失,内腔明显增大,肾小球、肾小囊结构趋于正常。结论：葛根素灌服具有降血压、降血脂、改善病变肾脏结构的作用。     

不依赖连接反应的高通量克隆方法

以脱脂菜籽粕酶解的复合氨基酸为主要原料,螯合率为考察指标,采用L16(4 5)正交试验设计,考察了pH值、温度、时间和配位比对螯合率的影响.结果显示,影响因素的高低顺序为:配位比＞pH值＞时间＞温度.结果表明,复合氨基酸与铜螯合的主要影响因素为pH值和配位比,且配位比的影响达到极显著水平.最佳工艺条件为:时间50 min,温度50℃,配位比2∶1,pH为9,此条件下的复合氨基酸螯合铜的螯合率为94.59％,氨基酸含量为30.2％.     

酸水解法从葛根中提取分离葛根素和大豆苷元

本文报道了葛根黄酮的提取精制方法,以６０％乙醇为溶剂,６０℃温浸６ｈ的优化工艺条件下,采用逆流萃取法从葛根中提取葛根黄酮,其收得率为１９．２８％,含量５１．０５％。葛根黄酮提取物采用５％盐酸水解４ｈ,酸水解液用乙酸乙酯萃取放置析出葛根素,乙酸乙酯相经水洗、浓缩和重结晶可得大豆苷元。采用酸水解法可以将葛根黄酮中的葛根素及大豆苷元衍生物水解成葛根素和大豆苷元,有利于葛根素、大豆苷元的分离及产率的提高;该方法从葛根中提取分离葛根素和大豆苷元具有操作简便、产品纯度和产率高、成本低的特点。葛根素和大豆苷元产率分别为１．３６％和０．４５％,纯度为９８．３２％和９１．２５％。     

In vitro monitoring of nanogram levels of puerarin in human urine using flow injection chemiluminescence

A rapid and sensitive chemiluminescence (CL) determination of puerarin with the flow injection technique was presented. It was found that puerarin could enhance the CL generated from luminol-KIO(4) system significantly. The increment of CL intensity was linear over the concentration of puerarin ranging from 0.3 to 100.0 ng mL(-1) (r(2)=0.9963), offering a detection limit as low as 0.1 ng mL(-1) (3 sigma). At a flow rate of 2.0 mL min(-1), one analysis cycle, including sampling and washing, could be accomplished in 20 s with a relative standard deviation of less than 5.0%. The experimental results demonstrated that the excretive amounts of puerarin reached its maximum in 3 h after taking 250 mL green drink containing 275 mg puerarin, and the puerarin excretive ratio during 24 h was 9.70% in the body of volunteers.     

葛根素对正常产后小鼠泌乳作用的影响

摘要 目的：探究葛根素对产后正常小鼠泌乳作用的影响及其机制,并初步探究葛根素对产后正常小鼠的安全性。方法：将雌、雄KM小鼠以3:1比例合笼配种,得到孕鼠饲养至分娩。分娩后的小鼠随机分为正常对照组、葛根素低剂量（18 mg?kg^-1）、高剂量组（72 mg?kg^-1）,每组8只。从产后第3 d起,每天灌胃一次,共10 d。观察小鼠每日泌乳量变化,ELISA法检测血清中催乳素（PRL）、孕酮（P4）、雌二醇（E₂）含量,HE染色观察乳腺、肝、肾、子宫、卵巢组织病理学形态,Western Blot法检测乳腺组织中催乳素受体（PRLR）、酪氨酸激酶 2（JAK2）和信号传导与激活因子5a（STAT5a）的表达。结果：与正常对照组相比,从给药的第6天起,葛根素低剂量组的泌乳量显著升高（P＜0.05）;葛根素低、高剂量组均可见乳腺小叶内腺泡明显变大,分泌物明显增多,且低剂量组更为明显;葛根素低、高剂量组血清PRL水平明显升高（P＜0.01或P＜0.05）;葛根素低剂量组PRLR的蛋白表达明显增加（P＜0.01）,而葛根素高剂量组PRLR、JAK2的蛋白表达明显降低（P＜0.01）。葛根素低剂量组PRLR、JAK2、STAT5a的蛋白表达明显高于葛根素高剂量组（P＜0.01或P＜0.05）。结论：葛根素低剂量对产后正常小鼠有一定促进泌乳作用,高剂量时对泌乳作用不明显。葛根素低、高剂量均未对产后正常小鼠的肝、肾、卵巢和子宫产生明显的病理学改变。     

Anti–coronavirus disease 2019 (COVID-19) targets and mechanisms of puerarin

The present study aimed to uncover the pharmacological function and underlying mechanism of puerarin as a potential treatment for COVID-19, using an in silico methodology, including network pharmacology and molecular docking. The pivotal targets of puerarin to treat COVID-19 were identified and included the epidermal growth factor receptor (EGFR), tumour necrosis factor (TNF), tumour protein p53 (TP53), caspase 3 (CASP3), RELA proto-oncogene (RELA), Fos proto-oncogene (FOS), caspase 8 (CASP8), prostaglandin-endoperoxide synthase 2 (PTGS2), interleukin 2 (IL2), protein kinase CB (PRKCB), B cell lymphoma/leukaemia gene-2 (BCL2), protein kinase CA (PRKCA), nitric oxide synthase 3 (NOS3) and peroxisome proliferator–activated receptor gamma (PPARG). Functionally, the anti–COVID-19 action of puerarin was associated with the suppression of oxidative stress and inflammatory cascades, and cell apoptosis. The signalling pathways of puerarin to treat COVID-19 included modulation of the pathways of apoptosis, IL-17 signalling, mitogen-activated protein kinase (MAPK) signalling and TNF signalling. Molecular docking data illustrated the binding capacity of puerarin with COVID-19 and the effective anti–COVID-19 activity of puerarin. Taken together, our current network pharmacology–based findings revealed the pharmacological role of puerarin in the treatment of COVID-19. Furthermore, the bioinformatic findings elucidated that some of these pivotal targets might serve as potential molecular markers for detecting COVID-19.     

Determination of puerarin in biological samples and its application to a pharmacokinetic study by flow‐injection chemiluminescence

A simple and sensitive flow injection–chemiluminescence (FI–CL) method has been developed for the determination of puerarin, based on the fact that puerarin can greatly inhibit CL of the luminol–H₂O₂–haemoglobin system. The inhibition of CL intensity was linear to the logarithm of the concentration of puerarin in the range 0.08–10.0 μg/mL (r² = 0.9912). The limit of detection was 0.05 μg/mL (3σ) and the relative standard deviation (RSD) for 1.0 μg/mL (n = 11) of puerarin solution was 1.4%. Coupled with solid‐phase extraction (SPE) as the sample pretreatment, the determination of puerarin in biological samples and a preliminary pharmocokinetic study of puerarin in rats were performed. The recoveries for plasma and urine at three different concentrations were 89.2–110.0% and 91.4–104.8%, respectively. The pharmacokinetics of puerarin in plasma of rat coincides with the two‐compartment open model. The T_1/2α, T_1/2β, CL/F, V_Z/F, AUC_{(0 – t)}, MRT_{(0 – ∞)}, T_max and C_max were 0.77 ± 0.21 h, 7.55 ± 2.64 h, 2.43 ± 1.02 L/kg/h, 11.40 ± 3.45 L/kg, 56.67 ± 10.65 mg/h/L, 5.04 ± 2.78 h, 1.00 ± 0.35 h and 19.70 ± 4.67 μg/mL, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

The protective effects of puerarin in cardiomyocytes from anoxia/reoxygenation injury are mediated by PKCε

Puerarin is an isoflavone isolated from traditional Chinese medicine Ge‐gen (Radix Puerariae). Clinical studies have confirmed the cardioprotective effects of puerarin; however, the mechanisms underlying these effects are still unclear. On the basis of previous findings, we hypothesized that puerarin protects cardiomyocytes from ischemia–reperfusion injury via the protein kinase C epsilon (PKCε) (a critical cardioprotective protein) signalling pathway. Neonatal rat primary cardiomyocytes were preconditioned with puerarin or puerarin plus εV1‐2, a selective PKCε inhibitor, prior to anoxia/reoxygenation (A/R) treatment. Western blot analysis showed that expression and activity of PKCε protein in puerarin preconditioned group were both increased compared with the control or A/R group. Subsequent assays showed that preconditioning with puerarin could increase the viability of neonatal rat primary cardiomyocytes treated with A/R, decreased the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, cell necrosis and apoptosis induced by A/R injury. However, the protective effects of puerarin completely disappeared in the group pretreated with puerarin plus εV1‐2. Thus, for the first time, we revealed the protective effects of puerarin in cardiomocytes from anoxia/reoxygenation injury are mediated by PKCε. Copyright © 2014 John Wiley & Sons, Ltd.     

葛根素注射液中葛根素含量的HPLC测定

建立了一种简便、准确、可靠的葛根素测定方法。采用SymmetryC18(3.9mm×150mm,5μm)柱,流动相采用甲醇-水(体积比为3525),流速为1.00ml·min-1,检测波长为250nm。进样量在0.0368μg～0.589μg范围内线性关系良好,(r=0.9994)。该方法简便快速,准确可靠,可用于葛根素注射液的含量测定。同时,测定结果表明两种产品在含量上有差别,因此,保证和进一步提高葛根素注射液质量,并且规范质量标准势在必行。     

葛根素减轻部分由过氧亚硝基阴离子导致的糖尿病大鼠晶状体上皮细胞凋亡

本研究观察葛根素是否减轻部分由过氧亚硝基阴离子（peroxynitrite,ONOO^-）导致的糖尿病大鼠晶状体上皮细胞（lens epithelium cell,LEC）凋亡。采用大鼠腹腔注射链脲佐菌素（streptozotocin,STZ）的方法建立糖尿病动物模型。36只大鼠作为对照组,腹腔注射生理盐水;其他72只大鼠腹腔注射STZ（45mg/kg）后分为STZ组和STZ＋葛根素组,每组36只。STZ注射3d后,STZ＋葛根素组大鼠每天腹腔注射葛根素（140mg／kg）。于实验开始后第20、40和60天用裂隙灯检查晶状体的形态学变化后处死动物。用流式细胞仪检测LEC凋亡,用免疫组化方法检测晶状体中ONOO^-的标志物——硝基酪氨酸（nitrotyrosine,NT）的表达,用基因芯片分析技术检测LEC凋亡相关基因iNOS等的表达。结果发现,对照组大鼠晶状体均透明,各项指标基本正常;STZ组大鼠第20天时即出现晶状体混浊,40-60d期间混浊不断加重;STZ＋葛根素组大鼠20-40d时晶状体混浊呈加重趋势,但40-60d以后明显减轻。对照组LEC轻度凋亡,而STZ组凋亡细胞呈持续性增长,STZ＋葛根素组大鼠20-40d时细胞凋亡呈增长趋势,但40-60d以后明显下降。对照组大鼠晶状体NT未见明显表达;STZ组大鼠NT表达明显加强;STZ＋葛根素组大鼠20-40d时NT表达呈增长趋势,但40-60d以后明显下降。对照组凋亡相关基因未见明显变化,STZ组凋亡相关基因iNOS表达明显上调。其他凋亡相关基因如BCL-2、SOD表达明显下调,但NF-κB和TNFR1-FADD-caspase信号转导途径明显上调;STZ＋葛根素组凋亡相关基因表达则呈相反改变。上述结果表明,在糖尿病大鼠晶状体中有ONOO^-的标志物NT表达,证明糖尿病大鼠LEC凋亡部分由ONOO^-诱导,这可能是氧化损伤导致白内障形成的新途径。葛根素能够部分逆转ONOO^-对LEC的致凋亡作用,提示葛根素可能是治疗糖尿病性白内障的有效药物,其治疗机制可能与葛根素直接抑制凋亡和对抗ONOO^-对糖尿病大鼠LEC的损伤有关。     

氯通道在葛根素注射液致溶血反应中的作用

目的：观察阻断和激活氯通道对葛根素注射液致家兔溶血反应的影响,探讨氯通道在葛根素注射液溶血反应中的作用。方法：将不同浓度的葛根素注射液（0.75、1.5、3、6、12 mg/ml）与家兔红细胞悬液共孵育6 h,使用细胞图像记录系统观测葛根素注射液是否诱导红细胞溶血,酶标仪、流式细胞仪检测红细胞溶血率,并观测激活和阻断氯通道对葛根素注射液溶血作用的影响。结果：葛根素注射液可引起家兔红细胞体外溶血,在所观察的1.5 mg/ml~12 mg/ml范围内,溶血效应呈浓度依赖性增强（n=3,P<0.01）。氯通道阻断剂Tamoxifen （20 μmol/L）、ATP （10 mmol/L）可有效抑制葛根素注射液的溶血作用（n=3~5,P<0.01）;而使用低浓度ATP （50 μmol/L）激活氯通道,则显著增强葛根素注射液的溶血作用（n=4,P<0.01）。结论：葛根素注射液的体外溶血效应呈浓度依赖性,氯通道激活与葛根素注射液诱导的溶血反应密切相关。     

In vitro enzymatic modification of puerarin to puerarin glycosides by maltogenic amylase

Puerarin (daidzein 8-C-glucoside), the most abundant isoflavone in Puerariae radix, is prescribed to treat coronary heart disease, cardiac infarction, problems in ocular blood flow, sudden deafness, and alcoholism. However, puerarin cannot be given by injection due to its low solubility in water. To increase its solubility, puerarin was transglycosylated using various enzymes. Bacillus stearothermophilus maltogenic amylase (BSMA) was the most effective transferase used compared with Thermotoga maritima maltosyl transferase (TMMT), Thermus scotoductus 4-alpha-glucanotransferase (TS4alphaGTase), and Bacillus sp. I-5 cyclodextrin glucanotransferase (BSCGTase). TMMT and TS4alphaGTase lacked acceptor specificity for puerarin, which lacks an O-glucoside linkage between D-glucose and 7-OH-daidzein. The yield exceeded 70% when reacting 1% puerarin (acceptor), 3.0% soluble starch (donor), and 5U/100 microL BSMA at 55 degrees C for 45 min. The two major transfer products of the BSMA reaction were purified using C(18) and GPC chromatography. Their structures were identified as alpha-d-glucosyl-(1-->6)-puerarin and alpha-D-maltosyl-(1-->6)-puerarin using ESI+ TOF MS-MS and 13C NMR spectroscopy. The solubility of the transfer products was 14 and 168 times higher than that of puerarin, respectively.     

Puerarin inhibits the migration of osteoclast precursors and osteoclastogenesis by inhibiting MCP-1 production

ABSTRACT

Puerarin inhibits osteoclastogenesis and cells migration. This study aims to explore whether puerarin prevents osteoclastogenesis by inhibiting osteoclast precursors (OCPs) migration. The results showed that puerarin reduced MCP-1 production in OCPs, while inhibiting OCPs migration based on MCP-1. Puerarin reversed MCP-1-promoted osteoclastogenesis. CCR2 overexpression didn’t increase osteoclastogenesis with puerarin. Therefore, puerarin prevents OCPs migration by reducing MCP-1, whereby inhibiting osteoclastogenesis.