四种苦豆子生物碱对巨噬细胞产生肿瘤坏死因子α的影响

目的：研究苦参碱（matrine MT)、氧化苦参碱（oxymatrine OMT)、槐果碱（sophocarpine SC)、槐定碱（sophoridine SRI)四种苦豆子生物碱对小鼠腹腔巨噬细胞产生肿瘤坏死因子的影响,来探讨它们的抗炎、调节免疫作用机理。方法：用LPS刺激体外培养的小鼠腹腔巨噬细胞,使之剂量依赖性地产生肿瘤坏死因子,观察四种苦豆子生物碱对巨噬细胞产生肿瘤坏死因子影响。结果：在一定剂量范围内,四种苦豆子生物碱均能剂量依赖性地抑制巨噬细胞经LPS刺激产生TNFα。结论：四种苦豆子生物碱均抑制小鼠腹腔巨噬细胞生成TNFα,这可能是它们抗炎、免疫调节作用的机制之一。     

六种苦豆子生物碱对炎症介质白三烯的影响

目的：研究苦参碱(matrine MT)、氧化苦参碱(oxymatrine OMT)、槐果碱(sophocarpine SC)、氧化槐果碱(oxysophocarpine OSC)、槐定碱(sophoridine SRI)、氧化槐定碱(oxysophoridine OSRI)6种苦豆子类生物碱对炎症介质白三烯巴和白三烯134的影响，来研究其抗炎作用机制。方法：以大鼠腹腔白细胞为材料，采用高效液相色谱法(HPLC)测定白细胞内白三烯C4和白三烯B4水平。结果：SRI对LTC4的生物合成影响很小，但对LTB4的生物合成有明显的抑制作用。其余5种生物碱对LTC4和LTB4的生物合成均有明显的抑制作用，且有良好的剂量-效应关系。结论：6种苦豆子生物碱对白三烯(LTC4和LTB4)的生物合成均具有不同程度的抑制作用，这可能是它们抗炎作用的机制之一。     

苦豆子总碱及4种单体生物碱对幽门螺杆菌的体外抑菌作用研究

目的 探讨苦豆子总碱、苦参碱、苦参素、槐定碱、槐果碱体外抑制幽门螺杆菌(Helicobacter pylori,Hp)的作用.方法 选用5种苦豆子生物碱,纸片扩散法粗测抑菌浓度范围;依据粗测范围,液体倍比稀释法测定5种生物碱的最小抑菌浓度(MIC).结果 苦豆子总碱的MIC为32 mg/ml,在体外对Hp有一定的抑制作用.苦参碱、苦参素的MIC为64 mg/ml,槐定碱的MIC为128 mg/ml,体外抑菌活性微弱.槐果碱在体外对Hp无明显抑制作用.结论苦豆子总碱有可能成为替代抗生素的抗Hp候选药物.     

苦豆子总碱及四种生物单碱体外触杀阴道毛滴虫的实验研究

目的 :探索苦豆子总碱、苦参碱、氧化苦参碱、槐果碱、槐定碱体外触杀阴道毛滴虫的作用 ,为治疗滴虫性阴道炎寻找更为有效的中草药。方法 :将苦豆子总碱及其四种生物单碱稀释为 4 0 0、 2 0 0、 10 0、 5 0 μg/ml的不同浓度对体外培养的阴道毛滴虫进行杀虫实验 ,并与对照药(甲硝唑 )比较 ,确定疗效。结果 :苦豆子总碱、氧化苦参碱在高浓度 (4 0 0 μg/ml)时对阴道毛滴虫的生长、繁殖有一定的抑制作用 ;槐果碱、槐定碱无抑制作用 ;苦参碱 4 0 0、 2 0 0、 10 0 μg/ml对阴道毛滴虫具有明显的触杀作用 ,与甲硝唑比较两者差异无显著性 (P >0 0 5 ) ;与其他四种生物碱各个浓度比较均有显著性差异 (P <0 0 5 )。结论 :苦参碱有明显的触杀阴道毛滴虫的作用 ,是一种高效具有开发价值的生物碱。     

RP-HPLC法测定苦豆子总碱中苦参碱与氧化苦参碱、槐果碱与氧化槐果碱含量

目的:建立测定苦豆子总碱中氧化型生物碱与还原型生物碱含量的反相高效液相色谱法。方法:采用C18键合硅胶反相柱,以乙腈-0.05 mol/L磷酸二氢钾水溶液(三乙胺2.0 m L/L)为流动相,梯度洗脱,检测波长205 nm,柱温30℃,流速1.0 m L/min。结果:氧化苦参碱、氧化槐果碱、苦参碱、槐果碱分离良好,氧化苦参碱平均回收率为99.10%,RSD为1.15%;苦参碱平均回收率为98.76%,RSD为1.64%;氧化槐果碱平均回收率为100.12%,RSD为1.86%;槐果碱平均回收率为98.35%,RSD为2.01%。结论:所建立的测定苦豆子总碱中氧化型与还原型生物碱含量方法专属性强,灵敏度高,能有效控制苦豆子总碱质量。     

苦豆子种子中生物碱的分离及lehmannine的结构确定

目的：研究苦豆子种子中生物碱的分离及槐果碱转移氢化制备苦参碱的研究。方法：利用离子交换,萃取和柱层,并结合转移氢化增大不同生物碱理化性质差异进行分离。结果：从苦豆子种子中分得5种生物碱,其结构经元素分析,IR,MS,1HNMR和13CNMR光谱鉴定为氧化苦参碱,氧化槐定碱,苦参碱,槐定碱和lehannine,结论：首次从苦豆子种子中分和lehmannine。     

HPLC同时测定苦豆子中槐定碱、氧化槐果碱和氧化苦参碱的含量

目的：建立同时测定苦豆子中槐定碱、氧化槐果碱及氧化苦参碱含量的HPLC测定法。方法：色谱柱为Phenomenex Gemini C₁₈(46 mm×250 mm,5 μm),以甲醇(A)-02%磷酸水(B)为流动相,检测波长205 nm,流速1 mL·min^-1,柱温30 ℃。结果：在线性范围内3个对照品的标准曲线呈良好的线性关系(r≥0999 7)。平均回收率分别为1000%,970%,970%。结论：可通过HPLC同时测定苦豆子中槐定碱、氧化槐果碱和氧化苦参碱的含量,并可用该方法进行苦豆子药材的质量控制。     

苦参碱、氧化苦参碱、槐定碱对巨噬细胞活性及分泌肿瘤坏死因子-α的影响

目的观察苦参碱、氧化苦参碱和槐定碱对巨噬细胞RAW264.7活性及分泌肿瘤坏死因子-α(TNF-α)的影响。方法6孔培养板培养巨噬细胞RAW264.7,不同浓度的苦参碱、氧化苦参碱和槐定碱与巨噬细胞RAW264.7共培养1d,采用MTT法检测不同浓度的苦参碱、氧化苦参碱和槐定碱对巨噬细胞RAW264.7活性的影响;收集培养液,采用ELISA方法检测每份培养液中TNF-α的含量。结果不同浓度的苦参碱、氧化苦参碱和槐定碱对巨噬细胞RAW264.7活性有不同程度的抑制作用,不同浓度的苦参碱、氧化苦参碱和槐定碱对巨噬细胞RAW264.7分泌TNF-α有抑制作用,浓度越大,抑制作用越强,苦参碱对巨噬细胞RAW264.7分泌TNF-α的抑制作用更明显。结论苦参碱、氧化苦参碱和槐定碱能抑制巨噬细胞RAW264.7的活性及TNF-α的分泌,苦参碱的抑制作用更明显。     

苦豆子抗内毒素效应的实验研究

目的:研究苦豆子中提取的苦豆子总碱及氧化苦参碱、槐定碱、槐果碱对内毒素肺损伤小鼠模型的干预作用及其作用机制。方法:以清开灵注射液作为本研究的阳性对照药物。分别连续3天腹腔注射给予ICR小鼠各个生物碱及清开灵,均末次给药后1 h腹腔注射LPS(E.coil,055∶B5)9 mg/kg造成小鼠内毒素性肺损伤,观察小鼠一般状况、白细胞数量、肺W/D比值、肺组织病理形态学改变,以及免疫组织化学法检测肺组织CD14和清道夫受体(SR-A)的表达,放免法检测血清肿瘤坏死因子(TNF-α)和白细胞介素-6(IL-6)含量。结果:预防性给予内毒素肺损伤小鼠苦豆子总碱和三种苦豆子单体碱,均不同程度改善了模型鼠的一般状况,并可不同程度地升高外周血白细胞,降低肺脏W/D比值,减轻肺组织病理改变,降低模型鼠肺组织CD14表达,增加SR-A表达,降低血清中TNF-α和IL-6的水平。结论:苦豆子总碱和三种苦豆子单体碱可不同程度地减轻内毒素肺损伤小鼠的病理损害,它们的抗内毒素机制可能与调节LPS的识别受体,并进而影响下游炎症因子表达有关。     

苦豆子不同提取物对小鼠急性毒性的“谱-毒”关系研究

目的建立苦豆子提取物HPLC化学指纹图谱,测定苦豆子在不同提取物对小鼠的半数致死量(LD_(50)),分析其"谱-毒"关系。方法采用75%乙醇回流法(ER)、水煎煮法(WD)、75%乙醇超声法(EU)和水超声法(WU)分别制备苦豆子提取物,并建立其HPLC指纹图谱,测定不同提取物的LD_(50);运用指纹图谱相似度评价系统软件分析不同提取方法下苦豆子提取物化学成分与LD_(50)之间的关系。结果 ER、WD、EU、WU 4种提取方法制备的苦豆子提取物LD_(50)分别为38.397、24.994、18.536、19.957g/kg;苦豆子提取物对小鼠脏器的眼观病变主要表现在肝、肾,其中以ER提取物毒性最大。苦豆子提取物HPLC 10个共有峰可分为2类,第4、10号峰及氧化苦参碱、氧化槐果碱与LD_(50)呈显著负相关。结论构建了苦豆子"谱-毒"关系分析方法,其未鉴定出的第4、10号峰及氧化苦参碱和氧化槐果碱为毒性反应的主要化学成分。     

电针对关节炎大鼠脊髓背角环氧合酶-2表达的影响

目的 本工作试图通过观察慢性炎症痛大鼠脊髓背角环氧合酶（COX）-2蛋白表达的变化，探讨脊髓背角COX-2是否与电针镇痛的作用机制有关。方法 在佐剂性关节炎大鼠的模型上，采用行为学和免疫组化技术两种方法，观察电针治疗后不同时相点关节炎大鼠的痛敏评分及脊髓背角COX-2蛋白的表达，并与正常组、模型组和药物组加以对照。结果 电针能明显减小关节炎大鼠痛敏评分的绝对值，多次电针具有显著的累积效应；并且随着电针治疗次数的增加，大鼠脊髓背角COX-2免疫阳性细胞数显著下降。结论 电针阳陵泉和昆仑两穴对佐剂性关节炎大鼠有明显的镇痛作用，并同时抑制其脊髓背角COX-2蛋白的表达。     

Anti-inflammatory compounds of “Qin-Jiao”, the roots of Gentiana dahurica (Gentianaceae)

Ethnopharmacological relevance

“Qin-Jiao” is a well-known traditional Chinese medicinal (TCM) herb having been used generally for fighting rheumatoid arthritis (RA) since ancient times. The root of Gentiana dahurica Fisch (Gentianaceae) is one of the four officially validated “Qin-Jiao” as listed in the Chinese Pharmacopoeia. In addition, it is a common Tibetan medicinal herb used for the treatment of tonsillitis, urticaria, and RA, while the flowers have been used as a Mongolian herb for curing cough sore throat and eliminating the phlegm due to its anti-inflammatory effect.

Aim of the study

The aim of the study was to characterize the anti-inflammatory compounds in “Qin-Jiao”, on the basis of detailed investigation on not only the phytochemical study of Gentiana dahurica, but also the bioactive evaluation on compounds obtained presently and previously from different “Qin-Jiao” origins and Gentiana species.

Materials and methods

The ethanol extract of air-dried roots of Gentiana dahurica was suspended into H₂O and extracted with EtOAc and n-BuOH, successively. Repeated column chromatography (CC) and semi-preparative HPLC were carried out on each of the fractions. The isolated compounds were determined by detailed spectroscopic analysis and acidic hydrolysis. Anti-inflammatory activities of 18 isolates, together with 12 typical compounds obtained previously by our group from the other “Qin-Jiao” origins (Gentiana crassicaulis, Gentiana straminea) and Gentiana rigescens, were tested by inhibitory effects on LPS-induced NO production in macrophage RAW264.7 cells and TPA-induced cyclooxygenases-2 and -1 (COXs-2/1) production on zebrafish model.

Results

A new lignan glycoside (1) was identified, together with 20 known compounds, including 10 iridoid glycosides (2–11), three steroids (12–14), four lignans (15–18), one phenylpropanoid (19) and two triterpenes (20–21). Anti-inflammatory bioassay showed that only compound 21 displayed potential inhibitory effect on NO production (IC₅₀=16.85 μM), while 20 tested compounds had inhibitory activities on COXs-2/1. Among them, the triterpenoid 21 was the most active compound with an inhibitory value of 78% at a concentration of 30 μM. All the tested compounds showed no cytotoxicity on five human cancer cell lines (40 μM) and zebrafish (30 μM), except for 21 displaying weak cytotoxicity on human myeloid leukemia HL-60 (IC₅₀=16.43 μM).

Conclusion

Most of compounds particularly iridoid glycosides from “Qin-Jiao” display potential inhibitory effect on COXs-2/1. The results support the historical importance of the well-known TCM herb, “Qin-Jiao”, having been commonly used for fighting RA. As major components, the bioactive iridoid glycosides should play important role in the anti-inflammatory effect of “Qin-Jiao”. Although further research will be required to evaluate the selective activities of the COXs-2/1 inhibitors, this work validates the medicinal use of “Qin-Jiao” and provides information for different “Qin-Jiao” origins having different treating effects on RA.     

Anti-inflammatory effect of Ajuga bracteosa Wall Ex Benth. mediated through cyclooxygenase (COX) inhibition

Ethnopharmacological relevance

Ajuga bracteosa Wall Ex Benth. (Labiateae) is described in Ayurveda for the treatment of rheumatism, gout, palsy and amenorrhea.

Aim of the study

The aim of present investigation is to study anti-inflammatory activity of Ajuga bracteosa, to understand possible mechanism of action and to identify the constituents responsible for its activity.

Materials and methods

The anti-inflammatory activity of 70% ethanolic extract was evaluated in TPA-induced mouse ear edema assay and in vitro cyclooxygenase (COX)-1 and COX-2 inhibitory activity was determined using EIA kits employing appropriate reference standards. Aajugarin I, lupulin A, withaferin A, reptoside and 6-deoxyharpagide were isolated from the 70% ethanolic extract by silica gel column chromatography.

Results

The 70% ethanol extract of whole plants of Ajuga bracteosa showed a significant (p < 0.05) and dose-dependent anti-inflammatory activity in an acute inflammation model at the dose of 0.5 and 1.0 mg/ear. The extract also exhibited a strong in vitro COX-1 and COX-2 inhibitory activity at 25 and 50 μg/mL concentration. Among the isolated compounds 6-deoxyharpagide exhibited highest COX-2 inhibition while rest of the compounds exhibited weak to moderate COX-1 and COX-2 inhibition at 30 μM concentration.

Conclusions

The results suggest that the 70% ethanol extract of Ajuga bracteosa possesses promising anti-inflammatory activity, which is possibly mediated through inhibition of COX-1 and COX-2 enzymes. The isolated constituents could be responsible in part for its anti-inflammatory and COX inhibitory activity. The study supports traditional use of Ajuga bracteosa for inflammatory diseases.     

Effect of isolated fractions of Harpagophytum procumbens D.C. (devil's claw) on COX‐1, COX‐2 activity and nitric oxide production on whole‐blood assay

The present study evaluates the effect of isolated fractions of Harpagophytum procumbens (devil's claw) on cyclooxygenase (COX‐1 and COX‐2) activities and NO production using a whole blood assay. The activity of COX‐1 was quantified as platelet thromboxane B₂ production in blood clotting and COX‐2 as prostaglandin E₂ production in LPS‐stimulated whole blood. Total NO₂^?/NO₃^? concentration was determined by Griess reaction in LPS stimulated blood. Assays were performed by incubation of isolated fractions obtained by flash chromatography monitored with HPLC, TLC and identified by ¹HNMR, containing different amounts of harpagoside with blood from healthy donors. Indomethacin and etoricoxib were the positive controls of COX‐1 and COX‐2 Inhibition. Data shows that fraction containing the highest concentration of harpagoside inhibited indistinctively COX‐1 and COX‐2 (37.2 and 29.5% respectively) activity and greatly inhibited NO production (66%). In contrast the fraction including iridoid pool increased COX‐2 and did not alter NO and COX‐1 activities. The fraction containing cinnamic acid was able to reduce only NO production (67%). Our results demonstrated that the harpagoside fraction is the main responsible for the effect of devils claw on these enzyme activities. However, other components from devil's claw crude extract could antagonize or increase the synthesis of inflammatory mediators. Copyright © 2010 John Wiley & Sons, Ltd.     

Cyclooxygenase inhibitory flavonoids from the stem bark of Semecarpus anacardium Linn

The ethyl acetate extract of stem bark of Semecarpus anacardium showing in vivo anti-in fl ammatory activity in carrageenan induced rat paw edema assay was investigated in order to identify its active compounds. Chemical investigation of the ethyl acetate extract of S.anacardium afforded 3,4,2',4'-tetrahydroxychalcone (butein) and 7,3',4'-trihydroxy fl avone. Evaluation of COX-1 inhibitory activity of 3,4,2',4'-tetrahydroxychalcone and 7,3',4'-trihydroxy fl avone provided the IC(50) values of 28.4 and 36.7 micro M respectively. Further investigation of these compounds for COX-2 inhibitory activity revealed moderate potency towards this enzyme.     

环氧合酶在脑胶质瘤中的表达及意义

目的探讨环氧合酶(COX-2)在脑胶质瘤中的表达及意义。方法采用免疫组化的方法检测56例脑胶质瘤中COX-2蛋白表达,分析COX-2表达水平与胶质瘤恶性程度的相关性。结果①对照组与胶质瘤组COX-2阳性表达率分别为9%和68%;②低恶性与高恶性度肿瘤COX-2表达不同;③胶质瘤恶性程度与COX-2染色深浅及阳性细胞数百分比之间呈正相关。结论COX-2可能参与了胶质瘤从低恶性度向高恶性转化的发展过程,对实体脑肿瘤的生长及侵袭有重要作用,是高级别肿瘤区别于低级别肿瘤的一个重要病理学特点。     

Oligomerized grape seed proanthocyanidins ameliorates isoproterenol-induced cardiac remodeling in rats: role of oxidative stress

The effects of oligomerized grape seed proanthocyanidins (GSP) on haemodynamics, cardiac hypertrophy and fibrosis as well as apoptosis signal-regulating kinase 1 (ASK1) and nuclear factor-κB (NF-κB) cascades in isoproterenol (Iso)-induced cardiac remodelling (CR) rat model were investigated, in addition, the serum SOD activities and MDA content were assayed. Rats were treated with Iso to induce CR and were given distilled water or GSP for 1 week. Control rats received vehicle instead of Iso. Administration of GSP markedly alleviated the elevation of the left ventricle weight (LVW)/body weight (BW), heart weight (HW)/body weight (BW) ratio and cross-sectional area of cardiomyocytes, decreased collagen deposition in the heart, and improved the haemodynamic index. Meanwhile, treatment with GSP significantly ameliorated oxidative stress by improving SOD activities and decreasing MDA formation. Moreover, GSP apparently inhibited the expression ASK1, NF-κB and its targeted gene - COX-2. These findings suggest that administration of GSP has the potential to attenuate Iso-induced CR by repressing oxidative stress and inhibiting the activation of the cellular signaling cascades involving the ASK1 and NF-κB pathways, at least in part, providing a molecular mechanism for the cardioprotective effect of GSP.     

女贞子对正常大鼠肝脏COX活性的影响及其DNA甲基化调节机制的初步研究

探讨女贞子对大鼠肝脏细胞色素c氧化酶(cytochrome c oxidase,COX)活性的影响及可能的DNA甲基化机制。实验采用女贞子的水提物按2.0 g·kg~(-1)和6.0 g·kg~(-1)灌胃大鼠30 d,处死大鼠后,取肝脏测定COX活性、COX的亚基之一COX4I1蛋白浓度、测定Cox4i1,Dnmt3a,Tet1基因mRNA表达水平、TET1和DNMT3A的蛋白水平以及检测Cox4i1DNA甲基化频率。结果显示女贞子低剂量和高剂量能显著性升高COX的活性和COX4I1的浓度(P0.05);有降低TET1蛋白和DNMT3A的蛋白表达量的趋势;但未显著改变Cox4i1,Dnmt3a,Tet1基因mRNA表达。与空白对照组相比,女贞子高剂量组甲基化频率有降低的趋势但未达到显著水平。该研究表明:女贞子对大鼠肝脏的COX酶活性有促进作用,其可能通过促进COX4I1的蛋白量增加实现;女贞子对DNMT3A的蛋白表达有降低趋势,其可能是Cox4i1DNA甲基化频率有降低趋势的原因。女贞子对于DNMT3A和TET1的蛋白表达可能有同向调节作用,具体机制仍不明确。     

Anti‐inflammatory Activities of Eleven Centaurea Species Occurring in the Carpathian Basin

Our study aimed at the identification of anti‐inflammatory activities of different fractions of C. sadleriana extract after per os administration in rats, the identification of the active compounds of the plant and the investigation of the in vitro anti‐inflammatory activities of Centaurea species native to or cultivated in the Carpathian Basin. The aerial parts of Centaurea sadleriana Janka have been used in Hungarian folk medicine to treat the wounds of sheep. Methanol extract of C. sadleriana was fractioned by solvent‐solvent partitioning. The n‐hexane fraction was further fractionated and the anti‐inflammatory activities of certain subfractions were confirmed in vivo in rats. The n‐hexane and chloroform fraction of the methanol extract of C. sadleriana exhibited remarkable COX‐1 and COX‐2 inhibiting effects in vitro. Chromatographic separation of the fractions led to the identification of the active subfractions and 11 compounds (α‐linolenic acid, γ‐linolenic acid, stigmasterol, β‐sitosterol, campesterol, vanillin, pectolinarigenin, salvigenin, hispidulin, chrysoeriol and apigenin). The in vitro screening for anti‐inflammatory activities of further Centaurea species occurring in the Carpathian Basin (C. adjarica, C. bracteata, C. cataonica, C. cynaroides, C. dealbata, C. indurata, C. macrocephala, C. melitensis, C. nigrescens, C. ruthenica) revealed considerable COX‐1 and COX‐2 inhibitory activities. Because C. sadleriana is an endangered species native only to the Carpathian Basin, the investigation of more prevalent species is reasonable. Copyright © 2012 John Wiley & Sons, Ltd.