Enhancement of the isolation selectivity of isoflavonoid puerarin using oligo-β-cyclodextrin coupled polystyrene-based media

The isolation selectivity of the isoflavonoid puerarin, a well-known traditional Chinese medicine, was studied when native and oligo-β-cyclodextrin (CDP) coupled polystyrene-based macroporous resins were used as adsorbents by static tests. The research results indicated that the CDP coupled resin HPD-100-CDP offered the best adsorption and desorption capacity for puerarin than others and its equilibrium adsorption data at 25 °C fit best to the Freundlich isotherm. The performance of separation of puerarin on HPD-100-CDP column in one step was evaluated. Based on the above experimental data, a novel medium PS-CDP was synthesized and its chromatographic retention behaviors were also explored. ESI-MS/MS, ¹H NMR spectroscopy and UV absorption spectrum were used for the detection and characterization of puerarin in isolated fraction. Under the optimum mobile phase, methanol/acetic acid/water = 5.0/6.6/88.4 (v/v/v), the purity and recovery of puerarin were 95.3% and 86.7%, respectively, by HPLC analysis. In conclusion, the PS-CDP medium can enhance the isolated selectivity of puerarin and it can be applied in preparative scale operations.     

葛根素联合小檗碱干预非酒精性脂肪性肝炎两种动物模型的病理学研究

目的:从病理学的角度探讨葛根素及小檗碱对非酒精性脂肪性肝炎(NASH)的干预作用。方法:制备高脂饮食诱导的SD大鼠NASH模型及蛋氨酸-胆碱缺乏饮食诱导的C57BL/6小鼠NASH模型,各给药组造模并同时灌胃给药,大鼠持续8周后取材,小鼠持续2周后取材。肝组织制成石蜡切片进行HE染色,制成冰冻切片进行油红O染色,经光镜观察摄片后,采用NAFLD活动度积分对各组进行NASH评估。结果:HE及油红O检测提示葛根素联合小檗碱可减轻两种NASH动物模型肝脏的脂肪变性、抑制肝细胞空泡化的发生,减少肝小叶内炎性病灶的数量。给予葛根素联合小檗碱的SD大鼠NAS总分为2.60±0.52(3),给予葛根素联合小檗碱的C57BL/6小鼠NAS总分为2.80±0.79(3),均已排除NASH。结论:葛根素联合小檗碱可显著抑制NASH相关病理变化的发生发展,对NASH具有一定的干预作用。     

葛根素对糖尿病心肌细胞的保护及其机制研究

观察葛根素（Puerarin）对链脲佐菌素（streptozotocin,STZ）诱导的糖尿病大鼠心肌细胞的保护作用,并探讨血小板反应素1（Thrombospondin-1,TSP-1）的表达改变及其作用。雄性SD大鼠45只随机分为三组（n=15）：糖尿病组和葛根素治疗组采用一次腹腔注射链脲佐菌素（STZ）65mg／kg制备糖尿病模型,其中葛根素治疗组于造模后葛根素腹腔注射4周（100mg/kg/day）,正常对照组仅腹腔注射等量生理盐水（6ml／kg）,同样喂养4周。四周后各组大鼠处死。H—E染色及透射电子显微镜观察三组大鼠心肌细胞纤维显微结构和超微结构的病理改变．免疫组化和实时荧光定量PCR法观察大鼠心肌细胞中TSP-1蛋白和mRNA表达的变化．同时利用Langendorff离体心脏灌流法测定各组大鼠心室肌细胞功能。结果发现葛根素治疗组较糖尿病组大鼠的体重增加明显,同时血糖下降,有显著性差异（P〈0．01）。H—E染色显示糖尿病大鼠多处心肌肌丝紊乱伴少量炎症细胞浸润,电镜下发现有线粒体嵴消失溶解,肌丝排列紊乱等病理改变,而葛根素治疗组大鼠偶见上述病理变化。免疫组化显示葛根素治疗组心肌内TSP-1阳性细胞密度小于糖尿病大鼠,TSP-1 mRNA表达也比糖尿病大鼠要低。此外葛根素治疗组大鼠的左室收缩末压（LVSEP）、左心室舒张末期压（LVEDP）等心功能指标均明显低于正常组（P〈0．01）,但较糖尿病组有显著改善（P〈0．01）。上述结果显示葛根素能保护糖尿病大鼠心肌细胞的高糖损伤和维持心室肌细胞的功能,而该机制可能与抑制心肌细胞TSP-1表达的水平有关。     

Protective roles of puerarin and Danshensu on acute ischemic myocardial injury in rats

Ischemic heart diseases have been the leading cause of death in both developed and developing countries over the past decades. The aim of this study was to investigate the cardioprotective effects of the complex preparation (called Shenge), made of puerarin (isolated from Pueraria lobata Ohwi., also called Kudzu) and Danshensu (isolated from the Chinese herb, Salvia miltiorrhiza), on acute ischemic myocardial injury in rats and its underlying mechanisms. The left anterior descending (LAD) coronary artery was occluded to induce myocardial ischemia in the hearts of SD rats. Shenge was injected into the tail vein 15 min after occlusion at doses of 0, 30, 60, or 120 mg/kg body wt. ST elevation was then measured at 60, 120, and 240 min after Shenge administration. The ischemic size, serum levels of creatine kinase isoenzyme-MB (CK-MB), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA), and ST elevation were measured after the rats were sacrificed. Shenge decreased ST elevation induced by acute myocardial ischemia, reduced ischemic size, serum levels of CK-MB, LDH and MDA, and increased serum activity of SOD in a dose-dependent manner. The combined use of puerarin and Danshensu at a ratio of 1:1 showed the most effective activity. In conclusion, Shenge exerts significant cardioprotective effects against acute ischemic myocardial injury in rats, likely through its antioxidant and anti-lipid peroxidation properties, and thus may be an effective and promising medicine for both prophylaxis and treatment of ischemic heart disease.     

一种简便方法从野葛根中提取纯化葛根异黄酮

采用超声法70%乙醇为溶剂从野葛根中提取葛根异黄酮,将其以水为溶剂结合盐析法进行纯化,并且利用分光光度法和HPTLC法分别对超声提取物和纯化的葛根异黄酮产品中总黄酮和葛根素的含量进行了测定.结果表明以70%乙醇为溶剂超声萃取30 min提取葛根异黄酮和纯化葛根异黄酮产品的产率分别为17.8%和4.9%,异黄酮含量分别为42.55%和91.02%,葛根素含量分别为19.54%和67.15%.该方法从野葛根中提取纯化异黄酮具有省时、节约能源、操作方法简便和总异黄酮产品纯度高的优点.     

葛根素对大鼠心室肌细胞动作电位的影响

目的:从电生理角度探讨葛根素抗心律失常的可能机制。方法:采用膜片钳技术记录大鼠心室肌细胞动作电位(AP)、转染的人胚胎肾细胞缓慢延迟整流钾电流(IKs),观察加药前、后葛根素对AP和IKs的影响。结果:0.01、0.1、1 mmol/L葛根素可浓度依赖性地延长动作电位时程,分别使APD50从(71.8±11.8)ms延长至(86.9±10.7)ms、(100.5±14.1)ms和(123.6±25.4)ms;使APD90从(164.6±21.4)ms延长至(188.3±11.5)ms、(221.6±25.7)ms和(278.7±38.2)ms(n=6,均P0.05),而对RMP、APA和APD20无显著影响。此外,0.01、0.1、1 mmol/L葛根素对IKs抑制率分别为(17.8±2.5)%、(40.4±1.9)%和(60.9±3.2)%(n=6,均P0.05)。结论:葛根素可能通过抑制IKs来延长动作电位时程,发挥抗心律失常作用。     

葛根素对U14宫颈癌小鼠血液流变学、脾淋巴细胞增殖及细胞毒性影响

摘要 目的：研究葛根素治疗对U14宫颈癌小鼠血液流变学、脾淋巴细胞增殖活性及对宫颈癌细胞毒性的影响。方法：45只雌性昆明小鼠随机分为对照组、模型组和葛根素组。模型组和葛根素组小鼠通过腋下注射U14小鼠宫颈癌细胞建立U14宫颈癌移植瘤小鼠,并且葛根素小鼠通过葛根素灌胃进行治疗,对照组和模型组小鼠给予等量生理盐水。比较各组小鼠血流变学、脾淋巴细胞增殖活性及对宫颈癌细胞毒性。结果：经葛根素治疗的葛根素组宫颈癌小鼠肿瘤重量显著低于模型组小鼠（P<0.05）,葛根素治疗宫颈癌小鼠的抑瘤率是（42.91±12.91）%。宫颈癌小鼠低切/高切全血粘度、血浆粘度值以及血细胞比容均显著升高（P<0.05）,而葛根素治疗可显著降低宫颈癌小鼠低切/高切全血粘度、血浆粘度值以及血细胞比容（P<0.05）。宫颈癌小鼠脾脏重量、脾脏指数和脾淋巴细胞体外增殖能力均显著下降（P<0.05）,而葛根素治疗可显著提高宫颈癌小鼠脾脏重量、脾脏指数和脾淋巴细胞体外增殖能力（P<0.05）。此外,经葛根素治疗的宫颈癌小鼠脾淋巴细胞对U14宫颈癌细胞细胞毒性显著高于模型组宫颈癌小鼠（P<0.05）。结论：葛根素治疗可降低U14宫颈癌小鼠血液粘度、改善血流变性质,并且可以提高脾淋巴细胞的增殖活性和对宫颈癌细胞的杀伤力。     

葛根素对帕金森病细胞模型的保护作用研究

目的:研究葛根素对帕金森病细胞模型的保护作用及其具体的作用机理.方法:用0.16mM的MPP+处理PC12细胞48h建立帕金森病细胞模型.实验分为对照组、损伤组和保护组,损伤组用MPP+(0.16mM)处理PC12细胞;保护组用葛根素提前预处理PC12细胞1h,后加MPP+.检测PC12细胞存活率、Caspase-3活性及ERβ的转录活性.结果:葛根素能够抑制caspase-3的激活,且其依赖于ERβ的表达,雌激素受体拮抗剂ICI182,780可阻断上述效应;其次葛根素可提高ERβ的转录活性.结论:葛根素对MPP+诱导损伤的PC12细胞具有抗细胞凋亡的保护作用,且具体的作用机理可能依赖于ERβ介导的经典的基因组作用模式.     

葛根素对肥胖型高血压大鼠血压和血管功能影响的实验研究

目的:本研究旨在观察饮食中添加葛根素对肥胖型高血压大鼠的心血管代谢指标的影响,尤其关注其对于血压和血管功能的效应。方法:①自发性高血压大鼠24只,分正常饮食对照组(8只)、高脂饮食组(8只)、高脂饮食+葛根素组(8只),大鼠先进行1周的适应性喂养,1周后进行干预,干预时间为14周;②每周测1次体重、鼠尾血压;③实验结束时空腹取血浆测血脂、血糖值,取胸主动脉观察主动脉的内皮依赖性及非内皮依赖性舒张功能。结果:①葛根素可防止高脂饮食导致的自发性高血压大鼠体重的增加及血压、血糖的升高,与高脂饮食组比较,P<0.05或P<0.01。②长期葛根素喂养可有效防止高脂饮食导致的高血压大鼠的血脂水平升高;③长期的葛根素喂养可显著改善肥胖型高血压大鼠的血管舒张功能及降低血压。结论:葛根素可有效改善肥胖型高血压大鼠的相关代谢指标,并可明显降低血压及改善血管功能,提示葛根素对肥胖型高血压有较好的防治作用,值得进一步深入研究。     

葛根素预防雌激素缺乏性骨质疏松的机制探讨

目的观察葛根素对去卵巢大鼠股骨骨密度、颌骨骨密度和血清中肿瘤坏死因子（TNF-α）、C反应蛋白（CRP）及雌二醇（E2）水平的影响。方法 3月龄雌性SD大鼠随机分为假手术组（sham group）、单纯去势组（OVX group）、去势＋雌二醇治疗组（OVX-estrogen group）和去势＋葛根素治疗组（OVX-puerarin group）。给药3个月后测量各组大鼠股骨和颌骨骨密度,血清中TNF-α和CRP及雌二醇（E2）水平。结果 OVX-puerarin组较sham组体重明显增加（P〈0.01）;较OVX-estrogen组的TNF-α（P〈0.01）、CRP（P〈0.05）及E2（P〈0.01）水平显著下降;其下颌骨和股骨远端骨密度与OVX组、sham组之间无显著性差异,但其股骨近端骨密度较OVX-estrogen组明显增加（P〈0.05）。结论葛根素作为抗炎因子,能够显著降低TNF-α和CRP的水平,但并不会提高雌激素的水平;葛根素对卵巢切除所致的骨质疏松症有一定的治疗作用,但会明显提高机体体重。