New diterpenoid glucosides from Siegesbeckia pubescens

Five new diterpenoids isolated from Siegesbeckia pubescens, pubesides A similar toE, were established as ent-2 alpha ,15,16-trihydroxypimar-8(14)-en-2-O-beta -D-glucopyranoside (1), ent-15,16,19-trihydroxypimar-8(14)-en-19-O-beta -D-glucopyranoside (2), beta -D-glucopyranosyl-ent-15,16-dihydroxypimar-8(14)-en-19-oiclate (3), ent-2-oxo-15,16,19-trihydroxypimar-8(14)-en-19-O-beta -D-gluco-pyranoside (4), ent-2-oxo-15,16,19-trihydroxypimar-8(14)-en-19-O-beta -D-glucopyranoside-15,16-acetonide (5) by 1D and 2D NMR techniques.

Essential Oil from Siegesbeckia pubescens Induces Apoptosis through the Mitochondrial Pathway in Human HepG2 Cells

Siegesbeckia pubescens（SP） has been used as a traditional medicine for the treatment of and inflammatory diseases. However, the activities of SP against hepatocellular carcinoma and the related mechanisms remain unclear. The present study aimed to examine the effects of the essential oil of SP（SPEO） on the proliferation of hepatocellular carcinoma cells and the possible mechanisms. The growth inhibition of Hep G2 cells was analyzed by MTT assay. Hoechst 33258 and fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells. Flow cytometry was used to detect cell apoptosis and cell cycle. The expressions of the target proteins were detected by Western blotting. The results showed that SPEO obviously inhibited the proliferation of Hep G2 cells in a dose-dependent manner. SPEO activated a series of apoptotic proteins in Hep G2 cells, increasing expression levels of Bax, caspase-3 and caspase-9, and decreasing the bcl-2 expression level. SPEO displayed promising anti-hepatocellular carcinoma activities in vitro, partly by inducing apoptosis in Hep G2 cells through activating the mitochondrial pathway.

HPLC-ELSD同时测定腺梗豨莶草中6个二萜成分的含量

为了研究腺梗豨莶草中6个二萜成分(对映海松烯型二萜:奇任醇、Hythiemoside B和豨莶精醇;对映贝壳杉烷型二萜:对映-17,18-二羟基-贝壳杉烷-19-羧酸、对映-16β,17-二羟基-贝壳杉烷-19-羧酸和对映-16α氢-贝壳杉烷-17,19-二羧酸)的含量,本试验建立了高效液相色谱法-蒸发光散射检测器(HPLC-ELSD)同时测定二萜类化合物含量。样品粉碎过筛加甲醇和乙酸乙酯回流提取,蒸发减压除去溶剂,甲醇溶解,0.45μm滤膜滤过,取续滤液进行测定。色谱条件:色谱柱为Waters Symmetry Shield^(TM)RP18柱(250 mm×4.6 mm,5μm),流动相为0.3%甲酸水溶液-乙腈(v/v),梯度洗脱,流速为1.0 mL/min。蒸发光散射检测器漂移管温度为103℃,雾化气流速为3.0 L/min。应用该方法测定腺梗豨莶草样品不同部位中6个二萜类化合物的含量,同时比较叶、枝、茎中对映海松烯型二萜、对映贝壳杉型二萜和总二萜含量的差异。结果显示,6个二萜成分在其线性范围内线性关系良好(r≥0.9992);日内和日间精密度相对标准偏差(RSD)均小于3.5%;回收率介于96.5%~101.5%,RSD均小于2.3%;腺梗豨莶草不同部位(叶、枝、茎)的二萜类化合物含量差异较大。结果表明,本试验所建立的HPLC-ELSD方法简便、准确、重复性好,为腺梗豨莶草药材全面的质量评价和临床应用中最佳药用部位的选择提供了参考。

豨莶草提取物对脑缺血/再灌注大鼠的神经保护作用

目的 研究豨莶草提取物对脑缺血/再灌注大鼠的神经保护作用,并探讨其作用机制。方法 SD大鼠60只,线栓法制备大脑中动脉闭塞(middle cerebral artery occlusion, MCAO)模型,实验分为假手术(Sham)组,模型(MCAO)组,豨莶草低剂量(XXC-L)、中剂量(XXC-M)、高剂量(XXC-H)组,连续给药7天。mNSS评估大鼠神经功能损伤,MRI检测大鼠缺血侧脑梗死体积,Nissl染色检测Nissl阳性细胞数;Western blot检测Bax、Bcl-2、NeuN蛋白表达;免疫荧光染色检测Tunel、NeuN;RT-qPCR检测IL-1β、TNF-α、IL-6 mRNA表达。结果 豨莶草提取物能显著降低MCAO大鼠mNSS评分和缺血侧脑梗死体积(P<0.05,P<0.01),促进缺血侧脑组织Nissl阳性细胞数和NeuN表达、抑制Tunel阳性细胞数(P<0.01);同时,豨莶草提取物能抑制IL-1β、TNF-α、IL-6mRNA及Bax蛋白,促进Bcl-2、NeuN蛋白(P<0.01)。结论 豨莶草提取物对MCAO大鼠具有神经保护作用。

A new ent-pimarane diterpenoid from Siegesbeckia pubescens

One new ent-pimarane diterpenoid ent-16-nor-3-oxo-pimar-8（14）-en-15-al （1） together with four known diterpenoids kirenol （2）, ent-2-oxo-15,16,19-trihydroxypimar-8（14）-ene （3）, darutigenol （4） and darutoside （5） were isolated from the ethanol extract of Siegesbeckia pubescens. The planar structures and relative configurations of these compounds were elucidated by comprehensive spectroscopic analysis.

Efficacy of Xixiancao (Herba Siegesbeckiae Orientalis) on interactions between nuclear factor kappa-B and inflammatory cytokines in inflammatory reactions of rat synovial cells induced by sodium urate

OBJECTIVE:To investigate the interaction between nuclear factor kappa-B(NF-κB)and inflammatory cytokines in synovial cell inflammatory responses induced by sodium urate,and to evaluate the efficacy of Xixiancao(Herba Siegesbeckiae Orientalis)on these interactions.METHODS:The interactions between NF-κB and inflammatory cytokines/mediators in synovial cells in acute gouty arthritis were investigated.We observed the expressions of NF-κB,interleukin(IL)-1β,IL-8,and tumor necrosis factor alpha(TNF-α)in synovial cells at different timepoints in an in vitro model of synovial cell inflammatory responses induced by sodium urate and in an in vivo model of gouty arthritis.Changes in the expressions of NF-κB,IL-1β,IL-8,and TNF-αin synovial cells of all experimental groups were compared and observed after treatment with different doses of Xixiancao(Herba Siegesbeckiae Orientalis)and colchicine.The interactions between NF-κB and IL-1β,IL-8,and TNF-αwere analyzed.Pathological changes in synovial tissues were observed in rats with acute gouty arthritis.RESULTS:Compared with the blank group,the expression levels of NF-κB,IL-1β,IL-8,and TNF-αwere increased significantly at different timepoints in the in vitro model of synovial cell inflammatory responses induced by sodium urate,and in the in vivo model of gouty arthritis.Compared with the model group,the expressions of NF-κB,IL-1β,IL-8,and TNF-αin synovial cells induced by sodium urate were decreased in the different Xixiancao(Herba Siegesbeckiae Orientalis)dose groups and the colchicine group.The effect was more obvious in the high dose Xixiancao(Herba Siegesbeckiae Orientalis)group.The expression of NF-κB in synovial cells was positively correlated with the expressions of IL-1β,IL-8,and TNF-α.Histopathological examination of synovial tissues in the high dose Xixiancao(Herba Siegesbeckiae Orientalis)group and Colchicine group showed that the characteristics of acute gouty arthritis were reduced,and there was a trend towards a positive correlation between NF-κB and inflammatory cytokine expressions.CONCLUSION:The activation of NF-κB is associated with the activation of IL-1β,IL-8,and TNF-αduring the pathogenesis of acute gouty arthritis,leading to the continuation and enhancement of the inflammatory response.Expressions of IL-1β,IL-8,and TNF-αin synoviocytes during acute gouty arthritis effectively inhibit local inflammation.

Chemical Constituents of Siegesbeckia orientalis L.

The aerial parts of Siegesbeckia have been used as the traditional Chinese medicine in the treatment of rheumatic arthritis, hypertension, malaria, neurasthenia, and snakebite. In order to find new and bioactive compounds, the chemical constituents of the aerial parts of S. orientalis L. were investigated and two new compounds, namely β-D-glucopyranosyl-ent-2-oxo-15,16-dihydroxy-pimar-8（14）-en-19-oic-late （compound 1 ） and [1 （10） E,4Z]-8β-angeloyloxy-9α-methoxy-6α, 15-dihydroxy-14-oxogermacra-1 （10）,4,11 （13）-trien-12-oic acid 12,6-1actone （compound 2）, as well as five known ent-pimarane diterpenes （compounds 3-7） were isolated. The structures of the two new compounds were identified by their physicochemical properties and spectral analysis, particularly oneand two-dimensional nuclear magnetic resonance spectral methods.

Study on Therapeutic Mechanism of Anti-Rheumatism Effect of Siegesbeckia Pubescens

Objective:To explore the mechanism of anti-rheumatic effect of the active fraction of Siegesbeckia pubescens(AFSP).Methods:Adjuvant arthritis(AA)model of rat was produced to observe the effect of AFSP on lymphocyte proliferation,interleukin-1 and-2(IL-1,-2)activity,pathologic section of ankle joint,and analgesic effect,in model rat.Results:AFSP could reduce the inflammatory pathologic response of ankle joint.It has good analgesic effect,the analgesic rate being 65%.AFSP could also strengthen T-lymphocyte proliferation,promote IL-2 activity and inhibit IL-1 activity.Compared with the control group,the difference was significant(P<0.01).Conclusion:By means of regulating the immune function of organism,AFSP could improve the local pathologic response to antagonize against rheumatism,therefore,it is an effective anti-rheumatism herbal medicine.

豨莶草调节Nrf2介导的抗氧化途径对去卵巢大鼠骨质疏松症的影响

目的探讨豨莶草对骨质疏松症大鼠抗氧化和保护骨组织的作用机制。方法采用双侧卵巢切除术法制备骨质疏松症大鼠模型,分为模型组(Model)、ML385(Nrf2抑制剂)组、豨莶草组(Hsb)和豨莶草+ML385组(Hsb+ML385),另设置假手术组(Sham),每组12只。给予相应药物干预,连续干预12周。采用Micro CT测定各组大鼠骨密度(Bone mineral density,BMD)及骨小梁微结构参数变化;HE染色观察股骨组织病理学改变;ELISA法检测大鼠血清中抗酒石酸酸性磷酸酶-5b(TRACP-5b)和碱性磷酸酶(Alkaline phosphatase,ALP)水平;化学法检测大鼠血清中活性氧(Reactive oxidative stress,ROS)、超氧化物歧化酶(Superoxide dismutase,SOD)及丙二醛(Malondialdehyde,MDA)水平;采用qRT-PCR和Western bolt分别检测骨组织中Nrf2和HO-1 mRNA和蛋白表达水平。结果与Sham组比较,Model组大鼠骨组织结构破坏明显,BMD、血清ALP水平及SOD活性显著降低(P<0.05),TRACP-5b、MDA及ROS水平显著增加(P<0.05),而骨组织中Nrf2和HO-1 mRNA和蛋白表达水平显著下调(P<0.05)。与Model组比较,Hsb组大鼠骨组织结构损伤明显改善,BMD及血清ALP水平及SOD活性显著升高(P<0.05),TRACP-5b、MDA及ROS水平明显降低(P<0.05),骨组织中Nrf2和HO-1 mRNA和蛋白表达水平显著上调(P<0.05),而ML385组大鼠上述各指标进一步升高/降低。与Hsb组比较,Hsb+ML385组大鼠骨组织结构破坏明显,BMD、血清ALP水平及SOD活性又显著降低(P<0.05),TRACP-5b、MDA及ROS水平又显著增加(P<0.05),而骨组织中Nrf2和HO-1 mRNA和蛋白表达水平又显著下调(P<0.05)。结论豨莶草可以降低氧化应激水平,改善大鼠骨质疏松症,其作用机制可能与激活Nrf2/HO-1信号有关。

豨莶草提取物及其活性成分治疗关节炎的研究进展

豨莶草性寒,味辛、苦,具有祛风湿、利关节的功效,是治疗风湿痹证、骨节疼痛的传统中药之一,也是现代中医临床治疗关节炎的常用中药。近年来的临床研究与基础研究表明,豨莶草提取物及其活性成分主要通过保护软骨细胞、维持破骨细胞与成骨细胞的动态平衡、抑制促炎因子的释放等作用阻止骨性关节炎的发展;豨莶草提取物及其活性成分治疗痛风性关节炎的作用机制主要与减少滑膜炎症与尿酸形成、抗氧化应激反应、调节促炎因子水平等有关。综述豨莶草治疗骨性关节炎和痛风性关节炎两种关节炎的研究进展,可为关节炎发病机制的深入研究和治疗关节炎新药的研发提供参考。

豨莶草对黄嘌呤氧化酶和环氧合酶-2双重抑制作用的虚拟筛选

目的探究豨莶草对黄嘌呤氧化酶(XOD)和环氧合酶-2(COX-2)具有双抑制活性成分及结合机制。方法由TCMSP数据库筛选得出豨莶草所含化合物成分,以黄嘌呤氧化酶、环氧合酶-2为目标,使用Autodock Tools软件对化合物进行虚拟筛选,并使用Pymol软件针对代表性活性成分与黄嘌呤氧化酶、环氧合酶作用模式进行分析。结果豨莶草所含的活性成分与黄嘌呤氧化酶的结合能,在-5.0 kcal·moL^(-1)以下的有5种化合物,与环氧合酶-2的结合能,在-5.0 kcal·moL^(-1)以下的有9种化合物。根据分子间作用力来看,豨莶草中的豆甾醇、豨莶精醇对这两种蛋白的活性位点有氢键相互作用和疏水相互作用。结论豨莶草中的豆甾醇、豨莶精醇成分可能对黄嘌呤氧化酶和环氧合酶-2有双重抑制的效果。

豨莶精醇结构修饰及其抗凝血因子Xa活性研究

豨莶草是我国传统中药,具有广泛的药理作用,比如抗炎镇痛、抗血栓、免疫调节、抗过敏、抗菌、抗疟、抗动脉粥样硬化、抗缺血性脑损伤、抗肿瘤等。化学成分和药理活性研究表明豨莶草中丰富的萜类化合物是豨莶草发挥多种功效的物质基础。豨莶精醇系从豨莶草中分离得到的二萜类化合物,药理作用研究表明豨莶精醇具有良好的抗血小板聚集、抗凝血、改善血液流变学作用。为了发现活性更好、开发价值更高的先导化合物,通过缩酮、氧化、缩合、溴代等系列反应,对豨莶精醇进行了结构修饰研究,得到豨莶精醇衍生物11个(化合物1~11)。根据核磁和质谱数据确定了衍生物的结构,并通过以凝血因子Xa(FXa)为靶标的计算机辅助药物设计结合体外抗FXa试验评价了衍生物的活性,结果显示在豨莶精醇的A环上引入三氟甲基噁唑环(化合物9)或者噻唑环(化合物11)时活性明显提高,IC_(50)值分别为0.71±0.07和0.22±0.03μmol/L,为豨莶精醇结构的进一步优化提供参考,具有一定的研究价值。

豨莶草中奇壬醇的闪式提取工艺研究

优选中药豨莶草中奇壬醇的闪式提取工艺。以奇壬醇的提取率为评价指标,在单因素实验基础上,通过正交实验法优化闪式提取奇壬醇的工艺参数,对比闪式提取、热回流提取、常温浸提三种方法的提取效果。采用HPLC测定奇壬醇含量,流动相为乙腈-0.1%的磷酸水溶液(体积比24∶76),流速1.0 mL/min,检测波长215 nm。最佳闪式提取工艺条件为:乙醇浓度80%、料液比1∶35(g∶mL)、提取时间2.0 min;此时奇壬醇的平均提取率为1.327 mg/g。优选的工艺稳定,提取效率高,能耗少,重复性好的特点,可为豨莶草资源的开发提供重要参考。

基于AKT/FOXO1信号通路探讨豨莶草提取物对冠心病大鼠心肌损伤的保护作用

目的:基于蛋白激酶B(AKT)/叉头框蛋白O1(FOXO1)通路探讨豨莶草提取物对冠心病(CHD)大鼠心肌损伤的影响。方法:从78只大鼠中随机选取66只大鼠复制CHD大鼠模型,其余12只作为对照组。将造模成功的60只大鼠按随机数字表法分为模型组、豨莶草提取物低剂量组、豨莶草提取物高剂量组、阳性药物对照组、豨莶草提取物高剂量+MK2206组,每组12只。各给药组大鼠分别给予相应药物;模型组、对照组大鼠灌胃给予等体积的蒸馏水,同时尾静脉注射100μg/kg生理盐水,1次/d,连续给药4周。观察心脏功能相关指标心率(HR)、平均动脉压(MAP)、左室收缩压(LVSP)的变化;HE染色、Masson染色分别检测心肌组织病理损伤及纤维化程度;ELISA法检测心肌组织中心肌肌钙蛋白Ⅰ(cTnⅠ)、肌红蛋白(Mb)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;TUNEL染色观察心肌细胞凋亡;Western blotting法检测半胱氨酸蛋白酶3(Caspase-3)、Bcl-2相关X蛋白(Bax)、p-AKT、AKT、p-FOXO1、FOXO1蛋白表达情况。结果:与对照组比较,模型组大鼠HR、MAP、LVSP及心肌组织中p-AKT/AKT、p-FOXO1/FOXO1均明显降低(P<0.05),心肌组织病理损伤及纤维化程度严重,cTnI、Mb含量,IL-6、TNF-α水平,心肌细胞凋亡率,以及心肌组织中Caspase-3、Bax蛋白相对表达量均明显升高(P<0.05);与模型组比较,豨莶草提取物低剂量组、豨莶草提取物高剂量组、阳性药物对照组、豨莶草提取物高剂量+MK2206组大鼠上述指标变化均呈相反趋势,且差异有统计学意义(P<0.05);MK2206减弱了高剂量豨莶草提取物对CHD大鼠心肌损伤的保护作用。结论:豨莶草提取物可能通过激活AKT/FOXO1通路对CHD大鼠心肌损伤发挥保护作用。

豨莶草活性部位抗风湿作用机理研究

目的：阐明豨莶草抗风湿作用机理。方法：建立佐剂性关节炎（ａｄｊｕｖａｎｔ ａｒｔｈｒｉｔｉｓ，ＡＡ）大鼠模型。观察豨莶草活性部位对ＡＡ大鼠淋巴细胞增殖反应、白细胞介素一１（ＩＬ－１）、白细胞介素一２（ＩＬ一2）活性的影响，并同时观察ＡＡ大鼠踝关节病理切片，以及豨莶草的镇痛作用。结果：豨莶草可减轻ＡＡ大鼠踝关节炎症等病理反应；有较好的镇痛作用，镇痛率为６５％。豨莶草可增强ＡＡ大鼠Ｔ细胞的增殖功能，促进ＩＬ－２的活性，抑制ＩＬ－１的活性，与对照组比较有显著性差异（Ｐ ＜ ０．０１）。结论：豨荃草可通过调整机体免疫功能，改善局部病理反应而达到抗风湿作用。

毛梗莶化学成分的研究Ⅱ

目的：对毛梗莶（Ｓｉｅｇｅｓｂｅｃｋｉａｇｌａｂｒｅｓｃｅｎｓ）的化学成分进行分离、鉴定研究。方法：采用各种层析色谱技术进行分离。从毛梗莶中分离到了５个化合物，用ＩＲ，ＭＳ，１ＨＮＭＲ，１３ＣＮＭＲ和２ＤＮＭＲ光谱鉴定化合物。结果：经光谱鉴定化合物为Ⅰ对映１６β，１７二羟基贝壳杉１９羧酸（ｅｎｔ１６β，１７ｄｉｈｙｄｒｏｘｙｋａｕｒａｎ１９ｏｉｃａｃｉｄ），化合物Ⅱ为单棕榈酸甘油脂（ｇｌｙｃｅｒａｌｍｏｎｏｐａｌｍｉｔａｔｅ），化合物Ⅲ为对映贝壳杉１６β，１７，１８三醇（ｅｎｔｋａｕｒａｎ１６β，１７，１８ｔｒｉｏｌ），化合物Ⅳ为１８羟基贝壳杉１６烯１９羧酸（１８ｈｙｄｒｏｘｙｈａｕｒａｎ１６ｅｎ１９ｏｉｃａｃｉｄ），化合物Ⅴ为胡萝卜苷（Ｄａｕｃｏｓｔｅｒｏｌ）。结论：化合物Ⅰ、Ⅱ、Ⅲ为首次从该植物中得到。

豨莶草的质量标准研究

目的:探讨豨莶草药材的定性、定量检测方法.方法:采用TLC检定豨莶草中的奇壬醇,HPLC测定豨莶草中奇壬醇的含量.结果:以氯仿-甲醇-甲酸(25:5:1)为薄层展开系统,可以清晰地检测奇壬醇;采用Polaris C18色谱柱,以乙腈-水(25:75)为流动相,可使奇壬醇达到基线分离,豨莶草中奇壬醇平均含量为0.14%,奇壬醇在0.186～14.88 μg呈良好线性关系,平均回收率100.0%,RSD 2.4%.结论:本法可作为豨莶草药材的定性、定量检测方法.

毛梗莶化学成分的研究Ⅰ

目的：对毛梗莶（ＳｉｅｇｅｓｂｅｃｋｉａｇｌａｂｒｅｓｃｅｎｓＭａｋｉｎｏ．）地上部分的化学成分进行分离，并鉴定其化学结构。方法：采用柱层色谱法进行分离，从中分离到６个化合物，用ＩＲ，ＭＳ，１ＨＮＭＲ，１３ＣＮＭＲ和２ＤＮＭＲ等光谱技术鉴定化合物的结构。结果：经光谱鉴定化合物为：化合物Ⅰ大花酸（ｇｒａｎｄｉｆｌｏｒｉｃａｃｉｄ），化合物Ⅱ１６β，１７二羟基贝壳杉烷（１６β，１７ｄｉｈｙｄｒｏｘｙｋａｕｒａｎｅ），化合物Ⅲ阿魏酸（ｆｅｒｕｌｉｃａｃｉｄ），化合物Ⅳ二十七烷醇（ｈｅｐｔａｃｏｓａｎｏｌ），化合物Ⅴβ谷甾醇（βｓｉｔｏｓｔｅｒｏｌ），化合物Ⅵ琥珀酸（ｓｕｃｉｎｉｃａｃｉｄ）。结论：化合物Ⅰ，Ⅱ为ｋａｕｒａｎｅ型二萜类化合物，化合物Ⅲ、Ⅳ、Ⅵ为首次从该植物中分离得到。

豨莶草抗血栓有效组分成分分析

目的:分析豨莶草抗血栓有效组分中化学成分类型及主要成分。方法:采用LC/MS的方法分析有效组分中化学成分类型,采用HPLC法测定有效组分中奇壬醇的含量。结果:有效组分主要化学成分类型为二萜类成分,其中活性成分奇壬醇在组分中的含量大于10%。结论:豨莶草抗血栓有效组分以二萜类成分为主。

常用中药豨莶研究进展

莶中已分离鉴定 42个成分 ,其中 17个是新二萜类化合物。我研究组确定了 17个新成分中的 10个 ,并对