雷公藤多糖的提取和含量测定方法研究

目的建立雷公藤中多糖的测定方法,进行不同提取条件时,药物中多糖的含量比较.方法采用苯酚-硫酸比色法,于490nm处测定含量.结果该方法线性关系良好,平均加样回收率为100.5%,RSD＝1.23%.结论本法准确,可作为雷公藤中多糖的质量控制.     

雷公藤多甙抑制小鼠腹腔巨噬细胞产生一氧化氮

雷公藤多甙（ｐｏｌｙｇｌｙｃｏｓｉｄｅｏｆＴｒｉｐｔｅｒｙｇｉｕｍｗｉｌｆｏｒｄｉＨｏｏｋ．ｆ．,ＴＷＰ）临床广泛用于治疗类风湿性关节炎等多种自身免疫性疾病,既可抑制细胞免疫又可抑制体液免疫［１］．但其在免疫抑制过程中对一氧化氮合酶（ＮＯＳ）的影响却...     

Triterpenes from Tripterygium wilfordii Hook

Two new friedelane-type triterpenes, tripterfrielanons A (1) and B (2), along with six known triterpenoids, friedelin (3), canophyllal (4), canophyllalic acid (5), 3-oxo-29-hydroxyfriedelane (6), wilforlide A (7), wilforlide B (8), have been isolated from the EtOH extract of the roots of Tripterygium wilfordii Hook.f. Compounds 4, 5, 6 were isolated for the first time from this plant. The new triterpenes 1 and 2 exhibited mild cytotoxic activity against human Hela cell lines in vitro. The assay showed the IC₅₀ of 1 and 2 were 8.5 and 25 μg/mL, respectively.     

正相HPLC法测定雷公藤药材及其制剂中雷公藤酯甲的含量

目的：建立正相高效液相色谱法对雷公藤药材和雷公藤多苷片中雷公藤酯甲的含量测定方法。方法：采用Alltima Silica色谱柱。流动相：正己烷-异丙醇（95：5）。采用蒸发光散射检测器，漂移管温度：70℃，气流速：1．8L／min。结果：雷公藤酯甲在0．64～3．2μg范围内线性关系良好（r=0．9998），平均回收率为97．98%，RSD=4．31％（n=5）。结论：本方法简便，灵敏，准确，重现性好，可用于雷公藤药材及其制剂的含量测定和质量控制。     

雷公藤多苷对女性生殖内分泌系统的影响

雷公藤多苷(GTW)是一种临床应用广泛的免疫抑制剂,对女性生殖系统功能有明显抑制作用。本文主要从GTW的生殖抑制作用研究概况,及研究中存在的分歧等方面进行综述,以期为GTW的进一步药理研究及临床应用提供参考。     

雷公藤免疫抑制成分蒽醌的研究

目的 :研究雷公藤去皮根正丁醇提取部分中具有免疫抑制作用的化学成分。方法 :雷公藤去皮根水煎液经乙醇沉淀后的正丁醇提取部分 ,经硅胶和SephadexLH -20等柱分离纯化 ,并采用NMR、MS等波谱方法及与标准品对照薄层层析鉴定化学结构 ,免疫抑制活性测定。结果 :分离得到多个化合物 ,其中化合物1为蒽醌类成分大黄素 ,具有较强的免疫抑制作用。结论 :该化合物是首次从雷公藤属植物中分离得到。     

雷公藤在临床治疗类风湿性关节炎中的用药规律探析

目的：利用现代计算机技术与统计学分析相结合的方法探析雷公藤在治疗类风湿性关节炎（rheumatoid arthritis,RA）时的用药规律。方法：全面收集2006年6月至2016年6月国内外期刊公开发表的、含雷公藤且对RA有一定临床疗效的中药处方,建立处方数据库,采用IBM SPSS Modeler 14.1进行频数分析和关联度分析,比较雷公藤在治疗RA时,内服方剂和外用方剂中的配伍规律,以及内服与外用的用药差异。结果：雷公藤治疗RA时,收集的药方共214个,包括外用方55个,内服方159个。口服给药配伍频次较高的是甘草、白芍、青风藤、地黄、鸡血藤、黄芪、当归、独活、薏苡仁、桂枝等,补虚药、祛风湿药、解表药居多;外用方中药物频次较高的包括乳香、没药、川芎、细辛、川乌、白芍、红花、草乌、威灵仙等,祛风湿药和活血化瘀药所占比例较大。经关联度和网络图分析得雷公藤在口服和外用两种给药方式时配伍用药存在一定的差异。结论：文本挖掘能比较客观地总结出雷公藤在治疗RA时的用药规律,为雷公藤的配伍减毒的机制研究和临床应用提供参考。     

In vitro immunosuppressive and cytotoxic activities of Tripterygium wilfordii extract

Tripterygium wilfordii Hook. f. (TW) is a traditional herbal medicine which has been widely used for the treatment of rheumatoid arthritis and other autoimmune diseases. However, adverse reactions of TW such as hepatotoxicity and nephrotoxicity have been frequently reported in clinic. With the aim to evaluate the potency and toxicity of TW, we collected eleven batches of TW from different localities across Chinese mainland, and investigated the inhibition of their methanol extracts on the proliferation of mouse spleen lymphocytes, normal human hepatocyte (L-02) cells and African green monkey kidney (COS-7) cells. TW extracts with three different concentrations were designed as the experimental groups. Our present findings provided consistent evidence that TW had significant concentration-dependent inhibitory action on lymphocytes, L-02 and COS-7 cells. At the concentrations of 0.75 and 1.5?mg/mL, most TW groups showed statistically significant inhibition of lymphocyte proliferation when compared with the control group (p?p?>?0.05). In most test groups, significant toxicities were shown on L-02 cells at 0.6 and 3.0?mg/mL (p?p?相似文献   

基于网络药理学初探雷公藤干预狼疮性肾炎的效-毒作用

目的 运用网络药理学方法探讨雷公藤对狼疮性肾炎(lupus nephritis, LN)的效-毒作用。方法 通过TCMSP平台检索雷公藤活性成分和作用靶点;在CoolGeN、OMIM和Gene Cards数据库中收集LN的疾病基因;借助Cytoscape软件绘制药物-成分-靶点-疾病作用网络,利用STRING数据库进行蛋白相互作用网络分析;运用DAVID数据库对作用靶点进行GO和KEGG富集分析,结合文献探讨预测雷公藤对LN的效-毒作用及其机制。结果 筛选得到雷公藤52个活性成分和与LN相关的38个靶点,大部分成分可通过NOD样受体、p53、TOLL样受体等信号通路调控免疫炎症、细胞增殖和细胞凋亡等过程而对LN起治疗作用,但雷公藤甲素(triptolide)、雷公藤红素(tripterine)、山萘酚(kaempferol)和β-谷甾醇(beta-sitosterol)等可能不利于LN病情好转。结论 雷公藤的大部分成分对LN具有治疗作用,但部分有效成分可能同时存在促进足细胞凋亡、加速肾脏纤维化的副作用。     

雷公藤提取物固体脂质纳米粒水分散体的制备及体外透皮吸收

制备雷公藤乙酸乙酯提取物固体脂质纳米粒水分散体,并初步研究了体外透皮行为.     

HPLC指纹图谱结合主成分分析评价不同产地雷公藤药材质量

目的 建立雷公藤药材的HPLC指纹图谱,并结合主成分分析法(PCA)评价不同产地的雷公藤药材质量。方法 采用HPLC构建雷公藤药材的指纹图谱,并运用PCA法对指纹图谱进行统计分析,以各主要色谱峰的保留时间和峰面积为变量建立得分图和载荷图。结果 标定了雷公藤药材HPLC指纹图谱的18个共有峰,并指认了其中的6个共有峰,分别为雷公藤甲素、雷公藤内酯酮、雷公藤晋碱、雷公藤次碱、雷公藤红素、雷公藤内酯甲,11批雷公藤药材的相似度为0.587~0.913。PCA结果表明,前5个主成分的累积贡献率达到89.603%,选择这5个因子即可对雷公藤药材进行综合评价。根据综合得分可知,来自四川阿坝、四川大巴山、云南西双版纳产地的雷公藤药材质量较好。结论 HPLC指纹图谱结合PCA可以快速、客观地评价不同产地雷公藤药材的质量差异。     

雷公藤根化学成分的分离与鉴定

目的研究雷公藤(Tripterygium wilfordiiHook.f.)根的化学成分。方法采用硅胶柱色谱、Sephadex LH-20、开放ODS柱色谱、HPLC等各种色谱技术对雷公藤根的化学成分进行分离纯化;根据理化常数测定和光谱分析对分离得到的化合物进行结构鉴定。结果从雷公藤根乙醇提取物中分离得到7个化合物,分别鉴定为2α,3α,23-三羟基乌苏烷-12-烯-28-酸(2α,3α,23-trihydroxyurs-12-en-28-oic acid,1)、2α,3α,23-三羟基乌苏烷-12,20(30)-二烯-28-酸(2α,3α,23-trihydroxyurs-12,20(30)-dien-28-oic acid,2)、2α,3α,24-三羟基乌苏烷-12-烯-28-酸(2α,3α,24-trihydroxyurs-12-en-28-oic acid,3)、2α,3α,19α-三羟基乌苏烷-12-烯-28-酸(euscaphic acid,4)、熊果酸(urasolic acid,5)、齐墩果酸(oleanolic acid,6)、2S,2′S-aurantiamide acetate(7)。结论化合物1、2、3、7为首次从雷公藤属植物中分离得到。     

近红外光谱双模型法快速判别雷公藤去皮减毒工艺的水平

目的：利用近红外光谱法和化学计量学分析软件,建立快速判别雷公藤真伪的一致性模型和雷公藤去皮减毒工艺水平的相关系数模型。方法：收集雷公藤切片和打粉样本的近红外漫反射光谱,运用OPUS软件分别建立其一致性模型和相关系数模型,并用非雷公藤样本及去皮减毒不完全样本进行验证。结果：基于近红外光谱法建立了快速判别雷公藤样本的近红外光谱双模型：可以快速、准确地判断被测样本是否为雷公藤;可以判断雷公藤样本的去皮减毒工艺水平。结论：该方法建立的近红外光谱双模型法易于操作、快速有效,可作为判断雷公藤去皮减毒工艺水平的快速筛查方法。     

液相色谱-质谱法测定雷公藤浸膏中4种倍半萜类生物碱含量

目的建立一种灵敏、可靠的雷公藤浸膏中4种倍半萜类雷公藤生物碱含量的高效液相色谱-质谱检测方法。方法雷公藤浸膏样品经氯仿溶解后用稀盐酸（2.0 mol·L~（-1））提取,Waters Oasis.MCX小柱进行净化,以0.05%（V/V）醋酸-醋酸铵溶液（5 mmol·L~（-1））/乙腈（45/55,V/V）为流动相,采用Zorbax SB C_（18）柱（250mm×4.6mm,5μm）分离,应用高效液相色谱/大气压化学电离正离子选择离子监测模式（SIM）测定,雷公藤春碱、雷公藤定碱、雷公藤吉碱和雷公藤次碱的定量离子分别为m/z874、884、858和868。结果 4种雷公藤生物碱的绝对回收率为86.5%～96.0%,质量浓度在1.0～200.0μg·L~（-1）内具有良好线性,批内RSD〈7.4%,批间RSD〈9.3%,定量检出限均为1.0μg·kg~（-1）。结论本方法简便、灵敏、干扰少、特异性好,可用于雷公藤浸膏中4种倍半萜类雷公藤生物碱含量的检测。     

雷公藤皂苷和白介素-10抑制树突状细胞诱导实验性自身免疫性甲状腺炎(英文)

目的:研究雷公藤皂苷和白介素-10(IL-10)在树突状细胞(DC)诱导实验性自身免疫性甲状腺炎(EAT)中的作用.方法:ELISA法检测甲状腺球蛋白抗体水平,TNF_β和NO分别采用生物学方法及Griess法测定,淋巴细胞增殖试验采用MTT掺入法.结果:DC能诱导EAT的发生,表现为甲状腺内出现淋巴细胞浸润并伴有血清中TgAb明显升高;但经雷公藤皂苷和IL-10处理后,DC不能诱导EAT的发生,表现为甲状腺无病理改变,血清中TgAb明显低下.同时,雷公藤皂苷组和IL-10组小鼠TNF_β活性、NO浓度和淋巴细胞增殖能力显著低于树突状细胞组(P<0.05).结论:雷公藤皂苷和IL-10能显著抑制DC诱导EAT的发生.     

LC-MS法同时测定雷公藤浸膏中雷公藤新碱和雷公藤碱戊含量

目的建立一种灵敏、准确的雷公藤浸膏中雷公藤新碱和雷公藤碱戊含量的LC-MS法。方法雷公藤浸膏样品经氯仿溶解后用稀盐酸（2．0mol·L“）溶液提取,上清液移人Oasis。MCX固相萃取小柱进行净化,然后样品在ZorbaxPlusRRHDC18柱（50innl×2．1mm,1．8μm）上,以醋酸铵缓冲液[0．05％醋酸一酸酸铵溶液（5mmol·L-1）]-乙腈（30：70）为流动相进行分离,应用LC—MS法,在大气压化学电离正离子选择离子监测模式（SIM）下测定,雷公藤新碱和雷公藤碱戊的定量离子分别为m／z806和m／z779。结果雷公藤新碱和雷公藤碱戊的绝对回收率为87．0％-97．5％,在含量1．0—200．0μg·kg-1内均具有良好线性,日内RSD均〈9．8％,日间RSD均〈14．7％,定量检出限均为1．0μg·kg-1。结论本方法简便、灵敏、干扰少、特异性好,可用于雷公藤浸膏中雷公藤新碱和雷公藤碱戊含量的监测。     

The Effect of Polyphenols Isolated from Cynanchi wilfordii Radix with Anti-inflammatory,Antioxidant, and Anti-bacterial Activity

Recently, Cynanchi wilfordii Radix has gained wide use in Asian countries as a functional food effective for relieving fatigue, osteoporosis, and constipation, particularly in menopausal disorders. However, its anti-inflammatory and anti-microbial activities have not been explored in detail to date. The anti-inflammatory, antioxidant, and anti-bacterial properties of the Cynanchi wilfordii Radix extracts obtained with water, methanol, ethanol, and acetone were compared. All 4 polyphenol-containing extracts exhibited anti-inflammatory and antioxidant effects. The ethanol extract was found to elicit the most potent reduction of nitric oxide (NO), prostaglandin E₂ (PGE₂), and cytokine (IL-1β, IL-6, IL-10, and TNF-α) levels, as well as inhibit the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a concentration-dependent manner. The evaluation of antioxidant activity also revealed the ethanol extract to have the highest free radical scavenging activity, measured as 85.3±0.4%, which is equivalent to 99.9% of the activity of α -tocopherol. In the assessment of anti-bacterial activity, only ethanol extract was found to inhibit the growth of the Bacillus species Bacillus cereus and Bacillus anthracis. These results show that polyphenols of Cynanchi wilfordii Radix have anti-inflammatory, antioxidant, and anti-bacterial properties that can be exploited and further improved for use as a supplementary functional food, in cosmetics, and for pharmaceutical purposes.     

Norlignans from Pouzolzia zeylanica var. microphylla and their nitric oxide inhibitory activity

Five new compounds, pouzolignan F [4-hydroxy-3-(3,5-dihydroxyphenyl)-2-[bis(4-hydroxy-3-methoxyphenyl)methyl]butyl acetate] (1), pouzolignan G [4-hydroxy-3-(3,5-dihydroxyphenyl)-2-[(4-hydroxy-3,5-dimethoxyphenyl)(4-hydroxy-3-methoxyphenyl)methyl]butyl acetate] (2), pouzolignan H [1,4-dihydroxy-3-(3,5-dihydroxyphenyl)-2-[bis(4-hydroxy-3-methoxyphenyl)methyl]butane] (3), pouzolignan I [1,4-dihydroxy-3-(3,5-dihydroxyphenyl)-2-[(4-hydroxy-3,5-dime thoxyphenyl)-(4-hydroxy-3-methoxyphenyl)methyl]butane] (4), and pouzolignan J [1,4-dihydroxy-3-(3,5-dihydroxyphenyl) -2-[(3,4,5-trimethoxyphenyl)(4-hydroxy-3-methoxyphenyl)methyl]butane] (5), along with two known compounds, indolyl-3-carboxylic acid (6) and uracil (7), were isolated from the aerial parts of Pouzolzia zeylanica (L.) Benn. var. microphylla (Wedd.) W.T.Wang. The structures of these compounds were characterized based on spectroscopic methods, including IR, NMR (¹H–¹H COSY, HSQC, HMBC, and NOESY), and HR-ESI/TOF-MS experiments. All the new norlignans were assayed for inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages.     

Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity.

Acute cyanide toxicity is attributed to inhibition of cytochrome c oxidase (CcOX), the oxygen-reducing component of mitochondrial electron transport; however, the mitochondrial action of cyanide is complex and not completely understood. State-3 oxygen consumption and CcOX activity were studied in rat N27 mesencephalic cells to examine the functional interaction of cyanide and nitric oxide (NO). KCN produced a concentration-dependent inhibition of cellular respiration. Cyanide's median inhibitory concentration (IC50) of oxygen consumption (13.2 +/- 1.8microM) was higher than the CcOX IC50 (7.2 +/- 0.1microM). Based on respiratory threshold analysis, 60% inhibition of CcOX was necessary before oxygen consumption was decreased. Addition of high levels of exogenous NO (100microM S-nitroso-N-acetyl-DL-penicillamine) attenuated cyanide inhibition of both respiration and CcOX. On the other hand, when endogenous NO generation was blocked by an NOS inhibitor (N(omega)-monomethyl-L-arginine ester), the cyanide IC50 for both respiration and CcOX increased to 59.6 +/- 0.9microM and 102 +/- 10microM, respectively, thus showing constitutive, low-level NO production enhanced cyanide inhibition. Laser scanning cytometry showed that cyanide elevated mitochondrial NO, which then was available to interact with CcOX to enhance the inhibition. It is concluded that the rapid, potent action of cyanide is due in part to mitochondrial generation of NO, which enhances inhibition of CcOX. At low mitochondrial oxygen tensions, the cyanide-NO interaction would be increased. Also, the antidotal action of sodium nitrite is partly explained by generation of high mitochondrial levels of NO, which antagonizes the CcOX inhibition.     

The Comet Assay with Multiple Mouse Organs: Comparison of Comet Assay Results and Carcinogenicity with 208 Chemicals Selected from the IARC Monographs and U.S. NTP Carcinogenicity Database

ABSTRACT

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology.

Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organspecific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally.

Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data.

Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.