基于HPLC指纹图谱、多指标成分含量测定结合化学计量学的怀牛膝质量评价＊

目的 本实验采用HPLC方法建立来自3个产地15批怀牛膝药材的指纹图谱，并对β-蜕皮甾酮，25R-牛膝甾酮，25S-牛膝甾酮进行含量测定，同时通过化学计量学的方法对15批怀牛膝进行质量评价。方法 选用Waters SunFire C₁₈（150 mm×4.6 mm，5 μm）色谱柱，检测波长280 nm，以乙腈-0.1%甲酸水梯度洗脱，流速为1.0 mL·min^-1，柱温25℃，进样量10 μL。以β-蜕皮甾酮峰为参照峰，运用软件《中药色谱指纹图谱相似度评价系统（2012版）》对15批怀牛膝药材进行相似度分析，使用Metabo Analyst 5.0网站进行聚类热图分析，使用SIMCA 14.1软件进行PCA和PLS-DA分析。结果 标定了31个共有指纹峰，指认了3个已知成分，并对其进行含量测定，化学计量学分析结果将15批怀牛膝药材样品分为3类，并且筛选了不同产地间潜在的差异性成分。结论 该方法具有良好的精密度、重复性以及稳定性，可为怀牛膝的质量评价与控制提供科学依据及参考。     

不同规格怀牛膝不同极性部位HPLC指纹图谱

目的研究并比较不同规格怀牛膝不同极性部位的HPLC指纹图谱,探索其内部质量差异,为该药材的规格标准完善及临床用药提供参考。方法将怀牛膝用体积分数75%乙醇水浴回流提取,得体积分数75%乙醇回流提取物,用40 mL水溶解后,依次用石油醚、三氯甲烷、乙酸乙酯、正丁醇进行萃取,得到各萃取相及萃取后的水相,浓缩至浸膏,采集各部位HPLC指纹图谱;运用相似度、综合聚类法进行数据分析,同时对其不同部位的指纹图谱进行比对分析。结果石油醚、氯仿部位均标定了11个共有峰,乙酸乙酯部位标定了10个共有峰,正丁醇部位标定了19个共有峰,水部位标定8个共有峰;氯仿部位指纹图谱相似度差异较大,其他部位指纹图谱相似度差异较小,均在0.9以上;石油醚部位指纹图谱差异主要体现在峰高,乙酸乙酯部位、氯仿部位、水部位的化学成分种类及峰高均存在差异;综合聚类分析能将不同规格怀牛膝区分开。结论不同规格怀牛膝内部质量存在差异;实验中所建立的HPLC指纹图谱可以全面反映不同规格怀牛膝的化学成分分布,为不同规格怀牛膝的整体质量评价提供参考。     

不同基原土牛膝的性状显微比较研究

目的 建立3种不同基原的土牛膝的性状及显微鉴别方法。方法 在植物分类学鉴定基础上，对不同基原的土牛膝进行鉴定，并使用性状和显微研究方法，对采集的土牛膝进行比较分析。结果 通过研究发现，3种土牛膝可以通过横切面显微特征区分，粉末显微特征区别不明显。结论 土牛膝不同基原可以通过性状、显微进行鉴别,通过对二者性状、显微的细致研究，为相关的标准修订与提高提供依据。     

Achyranthes bidentata polysaccharide enhances growth performance and health status in weaned piglets

This study is conducted to investigate the effects of Achyranthes bidentata polysaccharide (ABP) as a dietary additive on growth performance, plasma parametre profile and the mRNA abundances of IGF-1 and IL-1β in liver, jejunal mucosa and mesentery lymph node. A total of 200 three-hybreded (Landrace×Yorkshire×Duroc) piglets weaned at 28 days of age were allocated into five dietary treatment groups on the basis of body weight and litter of origin in a complete randomised design. Five diets were tested for 35 days, including the basal control diet, the antibiotic treatment diet (basal control diet+500 mg/kg flavomycin), and three ABP treatment diets, in which ABP were added to basal control diet with 500, 1000 and 1500 mg/kg, respectively. There was higher average daily feed intake (ADFI) in animals fed with 500 mg/kg ABP when compared with animals in other groups (P<0.05). However, there was no significant deference in ADFI among animals in control group, antibiotics group (500 mg/kg flavomycin), high dose ABP supplementation group (1000 and 1500 mg/kg ABP) (P>0.05). Flavomycin (500 mg/kg) and different dose of ABP supplementation significantly increased average daily gain (P<0.05). Compared with animals in control group, there was low FiG in animals fed with 500 mg/kg flavomycin, 500, 1000 and 1500 mg/kg ABP, respectively (P<0.05). Flavomycin (500 mg/kg) and different dose of ABP supplementation significantly decreased the diarrhoea frequency of weaned piglets (P<0.05). Moreover, there was lower diarrhoea frequency in animals fed with different dose of ABP compared with animals fed 500 mg/kg flavomycin (P<0.05). Supplementation of ABP increased plasma concentrations of hormones, antibodies, and alkaline phosphatase (P<0.05) and IL-1β mRNA abundance in liver, jejunal mucosa and lymph nodes. These findings indicate that ABP is effective in improving growth performance and defending capacity, which suggests that ABP can be used as a diet additive for weanling piglets.     

牛膝活性提取物对大鼠海马神经元生长的促进作用

目的:研究牛膝活性提取物(ABPPk)对体外培养的大鼠海马神经元生长的促进作用.方法:以体外原代培养的胎鼠海马神经元为模型,通过微管蛋白(β-tubulinⅢ)免疫荧光染色,采用Leica Qwin软件测量不同浓度ABPPk处理24 h对海马神经元突起延伸和突起分支的影响;通过突触结合蛋白(SYN)和突触后致密蛋白(PSD95)双标免疫荧光染色,采用LeicaQwin软件测量ABPPk(250 ng/ml)处理7d对海马神经元突触形成的影响;通过免疫印迹定量分析不同浓度ABPPk作用24 h对海马神经元生长相关蛋白(GAP-43)表达水平的影响,作用7d对PSD-95表达水平的影响,以及ABPPk(250 ng/ml)作用不同时间对磷酸化胞外信号调节激酶(ERK1/2)水平的调节.分析ABPPk对海马神经元的作用与ERK通路的关系.结果:ABPPk可有效促进海马神经元的突起延伸和突触形成,免疫印迹结果显示,ABPPk能显著增加海马神经元GAP-43和PSD-95蛋白表达,作用5 min即能显著上调ERK磷酸化水平,作用15 min ERK磷酸化水平达到峰值,PD98059可抑制该过程.结论:ABPPk能促进体外培养的海马神经元突起生长和突触形成,上调GAP-43和PSD-95蛋白水平,其作用可能与ERK通路的激活有关.     

Antiobesity,hypolipidemic, antioxidant and hepatoprotective effects of Achyranthes aspera seed saponins in high cholesterol fed albino rats

Introduction

Numerous herbal medicines have been recommended for the treatment of different diseases. Achyranthes aspera, Linn. (Family: Amaranthaceae), popularly known as Charchitta or Pitpapra, is commonly used by traditional healers for the treatment of fever, malaria, dysentery, asthma, arterial hypertension, pneumonia, and diabetes. The root extract is well reputed for its insect molting hormonal activity. This investigation was conducted to evaluate the effects of saponins from Achyranthes aspera seeds on the serum lipid profile of albino rats fed a high cholesterol diet.

Material and methods

Hypolipidemic, antioxidant and hepatoprotective activities of these saponins were tested as described previously. To determine the mechanism underlying the observed effects, serum antioxidant status was assessed according to ABTS (2,2’-azino-bis-3-ethylbenzo-thiazoline-6-sulfonic acid), superoxide dismutase and ferric ion reducing antioxidant power (FRAP) assays in saponin-treated hyperlipidemic animals. Liver enzyme levels were determined to reveal any possible hepatotoxicity.

Results

Four-week oral administration of A. aspera seed saponins produced a significant (p < 0.05) decrease of total cholesterol, total triglycerides and LDL-C and a significant increase of HDL-C level in hyperlipidemic rats. Treatment with A. aspera seed saponins also showed a significant (p < 0.01) improvement of serum antioxidant status in tested animals. No significant hepatotoxicity was produced by such treatment as the serum liver enzyme activity remained unaltered.

Conclusions

Saponins from A. aspera seeds possess antihyperlipidemic and antioxidant properties which might lead to improvement of serum lipid profile and blood antioxidant status. Our findings support the folkloric use of this indigenous plant in the treatment of hyperlipidemia. However, its exact mechanism of action remains to be elucidated.     

基于网络药理学探究络石藤-牛膝药治疗关节炎的作用机制

目的 基于网络药理学分析“络石藤-牛膝”药对治疗关节炎（Arthritis）的潜在作用机制。 方法 通过检索中草药系统药理学数据库平台（TCMSP）进行药对“络石藤-牛膝”有效成分及靶基因筛选;以“Arthritis”为关键词,在GeneCards数据库、NCBI基因数据库以及OMIM数据库进行人类基因检索并获得关节炎相关基因;将筛选出的药对靶点与疾病靶点输入韦恩图制作软件Venny2.1,获得共有靶点;运用STRING数据库进行PPI网络构建,Cystoscape构建“药物-成分-靶点-疾病”网络;运用R语言软件对关键靶基因进行基因本体论（GO）功能富集分析及与基因组百科全书（KEGG）通路的集分析。 结果 从TCMSP中筛选获得络石藤9种化合物成分,94个靶点;牛膝获得21种化合物成分,205个靶点。Venny映射得到128个共有靶点,STRING数据库筛选出5个基因簇和4个核心基因构建PPI网络。GO功能富集分析显示,“络石藤-牛膝”药对涉及2329条生物过程,150项分子功能相关,60项细胞组成相关。通过KEGG通路富集分析,“络石藤-牛膝”药对治疗关节炎富集到154条信号通路。 结论 基于网络药理学,从有效成分、基因靶点、信号通路等方面初步验证了“络石藤-牛膝”药对治疗关节炎的作用机制。     

土牛膝治疗急性咽喉炎的实验研究

目的观察土牛膝提取物对大白兔急性咽喉炎的治疗作用。方法将18只大耳白黑眼兔(WHBE)随机分为3组,分别给予相当于临床用药量的10倍、20倍的土牛膝提取液及等体积蒸馏水,连续灌胃7 d,然后建立家兔声带炎症模型,观察、测试各组家兔炎症发生情况。结果各土牛膝剂量组急性咽喉炎好转度好于模型组,且呈明显量效关系。结论土牛膝提取物可以显著抑制家兔急性咽喉炎的症状。     

中药怀牛膝的液相色谱指纹图谱研究

目的：建立怀牛膝药材液相色谱指纹图谱检测方法,为其质量控制提供方法。方法：以蜕皮甾酮为参照物,采用RP-HPLC法,色谱柱为Kromasil-C18（250mm×4.6mm,5μm）,流动相为乙腈-0.4%磷酸水溶液梯度洗脱,检测波长为254nm。结果：该方法可使怀牛膝药材中各成分较好分离,并根据检测结果确定了14个共有指纹峰,建立了怀牛膝HPLC指纹图谱共有模式。结论：建立的指纹图谱方法简便可靠,可以作为怀牛膝药材质控的重要方法。     

人IL-6 mRNA的实时定量检测法

目的：建立TaqMan-MGB技术实时定量检测人IL-6 mRNA的方法，并用于检测人外周血单个核细胞在牛膝多糖诱导下IL-6 mRNA的表达。方法：（1）抽取人外周静脉血，EDTA抗凝，用淋巴细胞分离液分离外周血单个核细胞，用Trizol裂解液提取总RNA，将总RNA逆转录为cDNA，PCR扩增IL-6后纯化PCR产物，与pMD 18-T载体进行连接，转化宿主菌DH-5α。提取重组质粒DNA，作为实时荧光定量检测的标准品。建立TaqMan-MGB技术实时定量检测IL-6 mRNA的方法。（2）终浓度分别为0、100、200、400、800和1 600 mg/L的牛膝多糖作用于人外周血单个核细胞，定量检测IL-6 mRNA的表达。结果：（1）酶切分析、PCR扩增以及测序结果均证实IL-6 cDNA成功重组到pMD 18-T载体上。（2）TaqMan-MGB技术检测IL-6 mRNA线性范围广；灵敏度高；特异性好；重复性好。（3）牛膝多糖作用于人外周血单个核细胞后IL-6 mRNA表达增高。结论：TaqMan-MGB技术能较为精确地定量IL-6 mRNA。