甘草总黄酮及其成分异甘草素调控M2型巨噬细胞极化的机制研究

考察甘草总黄酮(TFRG)及异甘草素(ISL)对M2型巨噬细胞的调控机制。利用IL-4(60μg·L^-1)诱导鼠源RAW264.7巨噬细胞6 h为M2型巨噬细胞。TFRG及ISL在Trans-well小室迁移实验中减弱因M2型巨噬细胞导致乳腺癌4T1细胞的体外迁移力提高。TFRG和ISL呈剂量依赖性抑制精氨酸酶1(Arg-1)在基因和蛋白水平的表达,并提高血红素加氧酶1(HO-1)的基因表达,同时提高诱导型一氧化氮合酶(iNOS)的蛋白表达,提高microRNA-155及其靶基因之一SHIP1的表达,并降低信号转导及转录活化因子3和6(STAT3/6)蛋白磷酸化水平。TFRG及其成分ISL是甘草抑制巨噬细胞向M2表型极化的活性组分和成分,部分通过调控miR155和STAT信号通路,影响Arg-1等M2型特征物的表达而抑制巨噬细胞M2表型极化后提高的乳腺癌细胞体外迁移力。     

HPLC法测定甘草中甘草素、异甘草素、甘草苷的含量

《中华人民共和国药典》2 0 0 0年版一部收载有甘草 Glycyrrhiza uralensis Fisch.、胀果甘草 G.in-flata Bat.、光果甘草 G. glabra L .3个种。为了保证临床疗效 ,本实验对甘草中甘草素、异甘草素、甘草苷的含量进行了比较。1　仪器与材料Waters高效液相色谱仪 ,Lambdal2紫外可见分光光度计 ( PE) ,Metter AE1 63电子分析天平。乙腈、二水醋酸、甲醇均为分析纯 ,水为重蒸水。甘草购自浙江中医学院实验药厂 ,胀果甘草和光果苷草均购自浙江省金华市医药站药材公司。甘草素、异甘草素、甘草苷 (自制 ,均经光谱学鉴定 ,采用面积归一化法…     

异甘草素的药理作用研究

异甘草素(isoliquiritigenin)为甘草中异黄酮类化合物,近年来其广泛药理活性备受关注.文章对异甘草素国内外药理活性研究文献进行了综述.异甘草素有抗肿瘤、抗氧化、抗炎等作用,并能扩张动脉,对心脏和脑有保护作用.     

酸水解法提取甘草中异甘草素工艺研究

 目的以甘草粉末为原料,研究了酸水解法提取异甘草素的工艺。方法采用正交实验考察盐酸浓度、水解时间、水解温度、料液比(甘草重量:盐酸体积)4个因素对异甘草素提取率的影响,确定酸水解法提取异甘草素的最佳工艺。结果酸水解法提取异甘草素的最佳工艺参数为盐酸浓度1mol·L^-1,水解作用时间2h,水解温度90℃,料液比1∶5。水解后碱中和pH为7,过滤后用体积分数80%乙醇提取滤渣,料液比1∶10,超声20min,提取2次;乙酸乙酯萃取滤液,液液比1∶1,萃取2次。结论酸水解法提取异甘草素的提取率为2.47‰,浸膏中异甘草素含量为3.01%,其提取率和含量分别是索氏提取的8.82和10.03倍,是乙醇超声提取的9.88和9.41倍。     

异甘草素的简便合成研究

目的 研究异甘草素的合成工艺.方法 以间苯二酚和对羟基苯甲醛为原料,通过乙酰化、羟基保护、羟醛缩合及脱保护合成得到了异甘草素,并对关键步骤羟醛缩合反应的条件(反应物的摩尔比、催化剂的种类、反应温度、溶剂和反应时间)进行了优化.结果 以Ba (OH)2为催化剂,乙醇为反应溶剂,对羟基苯甲醛和2,4-二羟基苯乙酮的摩尔比为2.0,温度为78℃的条件下,反应24 h,异甘草素的合成收率最高,达到了86％.结论 异甘草素的合成路线方便,原料简单,产率高.     

HPLC双波长法测定复方甘草合剂中甘草苷、甘草素及异甘草素的含量

目的：研究以HPLC法同时测定甘草合剂中甘草苷，甘草素及异甘草素含量的方法，方法：采用Cromasil C18柱，以甲醇－0．5％冰醋酸为流动相，梯度洗脱，双波长检测，测定波长λ1=276nm,λ2＝372nm，结果：甘草苷在50－1250ng范围内线性关系良好,r=0.9999,甘草素在5．0－125．0ng范围内线性关系良好，r=0.9998，异甘草素在3．0－6．0ng范围内线性关系良好，r=0.999，平均加样回收率，甘草苷为98．30％，RSD为1．01％（n=6)，甘草素为98．01％，RSD为0．68％（n=6)，异甘草素为98．73％，RSD为0．89％（n=6），结论：操作简便，结果可靠，重现性好，可作为该产品质量控制的方法。     

HPLC同时测定甘蒲祛斑凝胶中甘草素、异甘草素、甘草查尔酮A的含量

目的: 建立同时测定甘蒲祛斑凝胶中甘草素、异甘草素、甘草查尔酮A含量的方法。方法: 采用高效液相色谱法,Agilent HC-C₁₈色谱柱 (4.6 mm×250 mm,5 μm),流动相0.05%磷酸水(A)-乙腈(B)梯度洗脱(0~4 min,80%A;4~18 min,80%~65% A;18~22 min,65%~62% A;22~25 min,62% A,25~30 min,62%~55% A;30~35 min,55% A,35～45 min,55%~50% A;45~50 min,50%~55% A;50~60 min,55%~80% A),柱温30 ℃,流速0.8 mL·min^-1,检测波长276,368 nm。结果: 甘草素、异甘草素、甘草查尔酮A的线性范围分别为0.050 4~0.403 2 (r=1),0.039 4~0.315 2 (r=1),1.638~8.192 μg (r=0.999 9);甘草素平均回收率为98.81%,RSD 1.7%;异甘草素平均回收率为99.16%,RSD 1.2%;甘草查尔酮A平均回收率为97.68%,RSD 1.0%。结论: 该方法简便、灵敏度高、专属性强,可用于甘蒲祛斑凝胶中3种指标性成分的含量测定。     

异甘草素对Hela细胞氧化还原状态的影响

目的 探讨异甘草素体外抗氧化作用及其对Hela细胞氧化还原状态的影响,考察异甘草素抑制宫颈癌Hela细胞增殖与活性氧的关系.方法 采用化学方法测定异甘草素对羟自由基,DPPH,超氧阴离子清除率和抑制脂质过氧化的抑制率,考察其抗氧化效果;SRB法测定细胞活力,以细胞内ROS荧光探针DCFH-DA,测定荧光强度以表征细胞内ROS水平,试剂盒测定细胞内SOD和CAT的活力,考察其对细胞内氧化还原状态的影响.结果 异甘草素具有强的体外抗氧化作用,并且呈浓度依赖性地抑制Hela细胞的增殖;随异甘草素浓度变化呈现1～7.5 mg/L抗氧化,高于7.5 mg/L浓度促氧化的变化.结论 异甘草素浓度依赖性地抑制宫颈癌Hela细胞的增殖;异甘草素低浓度时清除细胞内活性氧,高浓度时促进自由基的生成,促进Hela细胞凋亡.     

降香中异甘草素含量的高效液相色谱测定

目的 建立高效液相色谱(HPLC)法测定降香中异甘草素含量的方法.方法 高效液相色谱法,样品用70%的甲醇提取,色谱柱为Waters Surefire C(18)(150 mm×4.6 mm,5μm);流动相为乙腈-1.0%醋酸溶液(32:68),等度洗脱;流速1.0 ml/min;柱温40℃,检测波长350 nm.结果 异甘草素浓度与峰面积呈良好的线性关系(r = 0.999 8),平均回收率98.5%,RSD=2.03%.结论 实验方法 分离度好,快速简捷,重复性好,可以准确地测定降香中异甘草素的含量.     

高效液相色谱法同时测定甘草中甘草酸、甘草苷、异甘草素的含量

目的建立同时测定甘草中甘草酸、甘草苷和异甘草素含量的方法。方法采用高效液相色谱法对甘草粉末的67％甲醇提取物进行分析。用C18反相色谱柱,1％磷酸水-乙腈梯度洗脱,对不同色谱峰分别采用248、276、360、370nm紫外波长检测。结果甘草酸的回归方程为,=1×10^6X＋16220,r^2=1;甘草苷的回归方程为r=2×10^6X＋49444,r^2=0．9995;异甘草素的回归方程为r=1×10^7X＋4．4667,r^2=1。甘草酸、甘草苷和异甘草素的加样回收率分别为98．01％、102．63％、98．18％。结论本方法准确、稳定、可靠,可用于甘草中甘草酸、甘草苷和异甘草素3种成分的同时测定。     

Characterization and immunostimulating effects on murine peritoneal macrophages of oligosaccharide isolated from Panax ginseng C.A. Meyer

Ethnopharmacological relevance

Panax ginseng C.A. Meyer has been the most precious and renowned Chinese herb used in Asian countries for the treatment of various medical disorders.

Aim of the study

The aim of this work was to investigate the activation effect on murine peritoneal macrophages of oligosaccharide from the roots of P. ginseng.

Materials and methods

In this work, the water-extracted oligosaccharide of P. ginseng was (WGOS) isolated and purified from the roots of P. ginseng by hot water extraction, ultrafiltration and gel-permeation chromatography. The monosaccharide composition and degree of polymerization (DP) of WGOS were determined by a combination of acid hydrolysis, high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Phagocytosis of macrophages was measured by uptake of the neutral red by macrophages, nitric oxide (NO) was determined by the Griess method, inducible NO synthase (iNOS) activity was determined by colorimetric method using a reagent kit, and tumor necrosis factor-α (TNF-α) was analyzed by enzyme linked immunosorbent assay (ELISA). The reactive species detection kit was used to measure the reactive oxygen species (ROS) level.

Results

WGOS was composed of glucose and the DP was ranging from 2 to 14. Immunological tests showed that treatment of WGOS significantly increased phagocytosis of macrophages, and promoted NO, TNF-α and ROS production. Furthermore, WGOS dose-dependently stimulated NO formation through the up-regulation of iNOS activity.

Conclusions

Taken together, WGOS possessed high immunopotentiating activity and could be developed as a novel immnunostimulant.     

不同蒸制时间对人参活性成分及其药代动力学行为与抗炎作用的影响研究

选用优质人参分别高压蒸制0,2,4,8 h后制备人参提取物,并利用HPLC研究人参蒸制前后的化学成分发生的变化;采用UFLC-MS/MS多反应监测(MRM) 定量分析,以地高辛为内标物在负离子模式条件下,研究灌服不同蒸制时间的人参提取物的小鼠体内人参皂苷类成分的药代动力学行为差异;以及通过给灌胃人参提取物1周后的小鼠腹腔注射脂多糖(LPS),建立炎症模型,运用酶联免疫法检测炎症模型小鼠血浆中的肿瘤坏死因子α(TNF-α)和白细胞介素-1β (IL-1β)的含量,探讨不同蒸制时间对人参发挥抗炎作用的影响。经测定灌服蒸制8 h的人参提取物可明显降低炎症模型小鼠血浆中的TNF-α和IL-1β含量。研究结果表明人参经蒸制后化学成分发生变化,入血成分相应改变,人参经蒸制8 h后有助于起到抗炎效果,为进一步揭示人参发挥抗炎作用的物质基础和量效关系提供实验依据。     

水杉总黄酮对大鼠血小板聚集、血液流变性的作用及机制探讨

目的 观察水杉总黄酮（FMG）对大鼠血小板聚集活性、血小板5－HT释放反应、血浆NO含量及血液流变性的影响。方法 比浊法测定大鼠血小板聚集活性、荧光光度法测定血小板5－HT释放反应、分光光度法测定血浆NO含量、血液流变全自动分析仪检测血液流变性。结果 FMG抑制大鼠血小板聚集活性，抑制血小板5－HT释放，升高血浆NO含量，降低急性血瘀大鼠的全血比粘度、血浆比粘度、红细胞电泳时间、红细胞压积、红细胞计数、血沉方程K值，改善大鼠的血液流变性。结论 FMG有抗血小板聚集活性改善血液流变性的作用。其抗血小板聚集的作用机制可能与抑制血小板释放反应、增加体内NO合成及Ca^2 拮抗作用有关。     

二黄凝胶贴膏镇痛抗炎作用及机制研究

目的:考察二黄凝胶贴膏的镇痛抗炎作用及其机制,为临床应用提供依据。方法:采用小鼠热板法、小鼠福尔马林致痛法与二甲苯致小鼠耳肿胀法、角叉菜胶致大鼠足肿胀法,贴敷给药7天,分别考察镇痛或抗炎作用,分别测定小鼠或大鼠血清中IL-1β及TNF-α的含量,探索镇痛或抗炎机制。结果:二黄凝胶贴膏4.57g/cm~2能显著减少福尔马林致痛小鼠舔足时间,二黄凝胶贴膏4.15g/cm~2能显著提高热板法小鼠痛阈值、减少福尔马林致痛小鼠舔足时间、降低小鼠血清中TNF-α含量,对IL-1β无影响,二黄凝胶贴膏4.57g/cm~2能显著减小角叉菜胶致大鼠足肿胀度、降低大鼠血清中IL-1β、TNF-α含量。结论:二黄凝胶贴膏具有明显的镇痛、抗炎作用,可能通过影响血清中IL-1β与TNF-α含量来实现。     

Molecular mechanism of anti-inflammatory activity of Pluchea indica leaves in macrophages RAW 264.7 and its action in animal models ofinflammation

Ethnopharmacological relevance: Pluchea indica Less.

(Asteraceae) is a Thai medicinal plant used in traditional medicine for the treatment of hemorrhoids, lumbago, leucorrhoea and inflammation. This study investigated the molecular mechanism of anti-inflammatory activity of Pluchea indica leaf extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and also determined its action in acute inflammation animal models.

Materials and methods

The inhibitory effect of Pluchea indica leaf extract on LPS-induced nitric oxide (NO) production was evaluated by Griess reaction. Protein and mRNA expressions were determined by real time RT-PCR and Western blotting analysis, respectively. Inducible nitric oxide synthase (iNOS) promoter activity was evaluated by iNOS promoter based reporter gene assay. In vivo anti-inflammatory effect was examined in ethylphenylpropiolate (EPP)-induced ear edema and carrageenan-induced paw edema in rat models.

Results

Ethyl acetate fraction of ethanol extract of Pluchea indica leaves (EFPI) exhibited the potent inhibitory effect on NO production in LPS-induced macrophages and also inhibited PGE₂ release. EFPI reduced iNOS mRNA and protein expression through suppressed iNOS promoter activity and nuclear translocation of subunit p65 of nuclear factor-κB, but did not inhibit phosphorylation of the mitogen-activated protein kinases (MAPKs). Moreover, EFPI possessed anti-inflammatory activities on acute phase of inflammation as seen in EPP-induced ear edema and carrageenan-induced paw edema inrats.

Conclusions

These data support the pharmacological basis of Pluchea indica plant as a traditional herbal medicine for treatment of inflammation.     

Antinociceptive and anti-inflammatory activity of carumbelloside-I isolated from Caralluma umbellata

The phytochemical study using Caralluma umbellata (Asclepiadaceae) whole plant allowed the isolation of a novel pregnane glycoside named carumbelloside-I (3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-3beta,14beta -dihydroxypregn-5-en-20-one). Carumbelloside-I was evaluated for both antinociceptive activity and anti-inflammatory activity. The antinociceptive activity was evaluated in mice using the writhing test method, while the anti-inflammatory activity was evaluated in rats using the paw edema test with carrageenin. Carumbelloside-I has significant antinociceptive action. It has no anti-inflammatory activity.     

Topical anti-inflammatory and analgesic activity of kirenol isolated from Siegesbeckia orientalis

Ethnopharmacological relevance

Siegesbeckia orientalis has been traditionally used as a topical anti-inflammatory and analgesic agent.

Aims of the study

Current study was designed to explore the topical anti-inflammatory and analgesic effects of a constituent isolated from Siegesbeckia orientalis (Compositae), in order to validate its folk use.

Materials and methods

Kirenol was isolated from ethanolic extract of Siegesbeckia orientalis. Several topical formulations containing kirenol were investigated for anti-inflammatory and analgesic activities in rat. The effects were studied using carrageenan-induced rat acute inflammation model, complete Freund's adjuvant (CFA)-induced chronic inflammation and formalin test in rats. Piroxicam gel and methyl salicylate ointment were studied as positive control for anti-inflammatory and analgesic activity, respectively.

Results

The anti-inflammatory effect of kirenol 0.4-0.5% (w/w) was similar to the effect of piroxicam gel 4 h after carrageenan injection. The analgesic activity of topical preparation with more than 0.4% (w/w) was observed in the late phase. These effects may be due, at least in part, to the pro-inflammatory cytokine production of IL-1β and TNF-α. The administration of kirenol cream at the dose of 0.3, 0.4 and 0.5% (w/w) significantly inhibited the development of joint swelling induced by CFA, which was auxiliary supported by histopathological studies.

Conclusion

Kirenol has demonstrated its significant potential to be further investigated for its discovery as a new lead compound for management of topical pain and inflammation, although further pharmacological research is necessary to fully understand its mechanism of action. It also supports the potential beneficial effect of topically administered Siegesbeckia orientalis in inflammatory diseases.