LC-MS/MS法测定大鼠口服人参皂苷Rg1,Re,Rb1和Rd的药动学

 目的建立检测大鼠血浆中人参皂苷Rg₁,Re,Rb₁和Rd的分析方法,并研究这4种成分在大鼠体内药动学特征。方法采用LC-MS/MS测定4种人参皂苷的血药浓度,用DAS2.0软件对结果进行统计分析。结果质谱采用MRM方式进行检测,人参皂苷Rg1和Rb1的线性范围为2～1000μg·L^-1,相关系数为0.997和0.998,人参皂苷Re的线性范围为1～1000μg·L_-1,相关系数为0.998,人参皂苷Rd的线性范围为1.6～1000μg·L^-1,相关系数为0.9940。这4种成分的最低检测限分别为2,1,2和1.6μg·L^-1,提取回收率为75.69%到98.79%,方法的精密度、准确度和稳定性均符合要求。结论本方法操作简单、专属性强、灵敏度高、准确性好,可用于人参皂苷类成分的体内测定。     

UPLC测定参麦注射液中人参皂苷Rg1、人参皂苷Re、人参皂苷Rb1的含量

目的：采用超高效液相色谱法分离测定参麦注射液中人参皂苷Rg₁、人参皂苷Re、人参皂苷Rb₁的含量。方法：采用ACQUITY UPLC HSS T3 （2.1 mm×50 mm,1.8 μm）色谱柱,流动相乙腈（A）-0.05%磷酸（B）,梯度洗脱（0~5 min,19%A;5~12 min,19%~28%A;12~18 min,28%~40%A）,流速0.3 mL·min^-1,检测波长203 nm,柱温25 ℃。结果：人参皂苷Rg₁、人参皂苷Re和人参皂苷Rb₁分别在0.020 96~0.209 6,0.017 64~0.176 4,0.023 88~0.238 8 g·L^-1与相应峰面积呈良好线性关系,平均加样回收率（n=6）分别为100.12%,100.08%,99.51%,RSD分别为1.60%,2.03%,1.15%。结论：与HPLC相比,UPLC在不影响分离效果的情况下可显著提高参麦注射液中人参皂苷Rg₁、人参皂苷Re、人参皂苷Rb₁的分析速度,改善分析效果,同时可减少溶剂的消耗。该方法简便、快捷、准确、重复性好,可代替HPLC法用于参麦注射液的多指标质量控制。     

10批市售人参药材的质量分析

目的:比较10批市售人参药材中人参皂苷Rg1、Re和Rb1的含量测定,分析评价其质量状况.方法:采用HPLC法测定10批市售人参药材中人参皂苷Rg1、Re和Rb1的含量测定,色谱柱为Agilent-ZORBAX Eclispe XDB-C18色谱柱,以乙腈-水为流动相进行梯度洗脱,柱温30℃,体积流量1.0 mL/min,检测波长203 nm.结果:人参中人参皂苷Rg1、Re和Rb1的平均含量依次是0.23％.0.15％,0.18％,均低于《中国药典》标准规定的含量,合格率为60％.结论:市售人参质量差异较大,应加强对中药质量的监督管理,以进一步提高中药材质量.     

微米红参的制备及含量测定

目的:优化微米红参的制备工艺,测定其主要人参皂苷含量。方法:采用单因素法对影响红参微米化因素进行筛选,以平均粒径和粒度分布为考察指标,优化微米红参化生产的最佳工艺;采用高效液相色谱法测定人参皂苷Rg1,Re,Rb1的含量。结果:最佳工艺为风袋长度20 cm,粉碎时间为40 min,单次进料量为500 g;测得样品中人参皂苷Rg1,Re,Rb1的含量分别2.06,1.77,2.41 mg.g-1,微米红参人参皂苷含量测定显著高于红参普通粉与切片。结论:微米红参制备工艺合理、稳定;建立的含量测定方法适用于微米红参;微米红参利于人参皂苷成分溶出。     

HPLC测定温阳振衰颗粒中人参皂苷Rg_1、人参皂苷Re、人参皂苷Rb_1含量

目的采用反相高效液相色谱法同时测定温阳振衰颗粒中人参皂Rg_1、人参皂苷Re、人参皂苷Rb_1的含量。方法采用Agilent TC-C18色谱柱(4.6 mm×250 mm,5μm),流动相为乙腈-0.05%磷酸水,梯度洗脱(0~35 min,19%乙腈;35~58 min,19%~29%乙腈;58~70 min,28%乙腈;70~100 min,29%~40%乙腈),流速1.0 m L/min,检测波长203 nm,柱温35℃。结果人参皂苷Rg_1、人参皂苷Re、人参皂苷Rb_1的进样量分别在0.22~2.2μg、0.22~2.2μg、0.26~2.6μg范围内与峰面积呈良好的线性关系,相关系数分别为0.999 8、0.999 8、0.999 1;人参皂苷Rg_1、人参皂苷Re、人参皂苷Rb_1的平均回收率分别为98.04%、96.58%、96.75%。结论本方法准确、灵敏度高、重复性好,可作为温阳振衰颗粒的质量控制方法。     

超声辅助酶解人参总皂苷制备人参稀有皂苷Compound K的研究

该实验采用超声辅助酶解人参总皂苷以制备人参稀有皂苷Compound K。以转化率为指标,通过单因素考察超声功率、超声时间等超声预处理因素和pH、温度、底物浓度、酶用量、反应时间等酶解因素对转化率的影响,并应用响应面法试验优化制备工艺;采用MS,¹H,¹³C-NMR鉴定酶解产物。结果表明,超声辅助酶解反应的最适条件为超声功率250 W,超声时间15 min,酶解pH 5.5,酶解温度50℃,酶解时间36 h,酶用量-底物4：5,底物质量浓度1.0 g·L^-1;反应产物相对分子质量622.4,核磁图谱证实产物为人参稀有皂苷Compound K。在此条件下,以人参总皂苷计,人参稀有皂苷Compound K转化率为6.91%。该工艺反应条件温和,转化率较高,工艺简单可靠,适合工业化生产。     

制备工艺对参麦注射液质量的影响

目的:考察不同制备工艺对参麦注射液质量的影响。方法:采用3种生产工艺制备参麦注射液,用HPLC测定红参药材以及不同工艺制备的红参中间体和参麦提取液的指纹图谱,并测定人参皂苷Rg₁,Re总量和Rb1含量以及参麦提取液中总皂苷含量。结果:3个不同工艺的红参中间体和参麦提取液在指纹图谱和人参皂苷含量上都有显著的差异。结论:不同的提取工艺对参麦提取液质量有很大的影响,用HPLC指纹图谱法可以快速准确检测参麦提取液的质量差异。     

石柱参与林下山参及传统园参成分差异的比较研究

目的:比较石柱参与林下山参及传统园参成分差异。方法:采用高效液相色谱法测定人参皂苷Re、Rg1、Rb1;采用可见紫外分光光度法测定人参总皂苷的含量。结果:石柱参与林下山参人参总皂苷的含量相当,但高于抚松普通参和集安边条参的含量;石柱参中人参皂苷Re、Rg1、Rb1的含量均高于林下山参和抚松普通参和集安边条参。结论:石柱参人参皂苷的含量较高,可为其内在质量评价提供理论依据。     

人参皂苷类成分对戊巴比妥钠损伤心肌细胞ATP酶及相关离子的影响

目的:探讨人参皂苷类成分对戊巴比妥钠损伤心肌细胞的影响。方法:将培养至第5日的乳鼠大鼠心肌细胞暴露于0.8%戊巴妥钠5min制备大鼠乳鼠心肌细胞损伤模型;选用人参皂苷Rg1为代表,MTT法确定作用浓度;然后选取最佳浓度作为人参皂苷Rg1、Rb1、Re及阳性药物西地兰的作用浓度,作用时间为24h,试剂盒测定心肌细胞内Na+、K+、Ca2+、Mg2+浓度和细胞膜上Na+-K+-ATP酶、Ca2+-ATP酶、Ca2+-Mg2+-ATP酶活性。结果:在1×10-8M剂量下,人参皂苷Rg1、Rb1、Re均能不同程度升高模型细胞Ca2+-ATP酶、Ca2+-Mg2+-ATP酶的活性,降低Na+-K+-ATP酶的活性,同时纠正因造模引起的细胞内Na+、K+、Mg2+、Ca2+等离子浓度的异常。结论:人参皂苷Rg1、Rb1、Re对心衰心肌细胞具有一定治疗作用。     

RP-HPLC测定西洋生晒参和西洋红参中人参皂苷含量

 目的RP-HPLC测定西洋生晒参和西洋红参中人参皂苷含量。方法利用Agilent 1100 Series高效液相色谱仪进行测定。色谱柱为德国Nudeosil-C₁₈(4·6mm×150mm,5μm);流动相为乙腈-水梯度洗脱,流速为1.5mL·min^-1;检测波长203nm。结果西洋生晒参和西洋红参均含有较高的人参皂苷Rb1,Re,与西洋生晒参比较,西洋红参在保留时间27～28min处的Re,51min的Rb1及64min处的一未知化合物的含量均有明显变化,此点在以往的西洋红参研究中鲜见报道。结论本方法简便可靠、快速、重现性好,适用于西洋生晒参和西洋红参中人参皂苷含量测定,为进一步研究西洋红参的成分及其药理作用提供了参考数据。     

Investigation of plant extracts in traditional medicine of the Brazilian Cerrado against protozoans and yeasts

Aim of the study

To investigate the activities of the 217 plant extracts in traditional medicine of the Brazilian Cerrado against protozoans and yeasts.

Materials and methods

Plant extracts were prepared by the method of maceration using solvents of different polarities. The growth inhibition of chloroquine-resistant Plasmodium falciparum strain (FcB1) was determined by measuring the radioactivity of the tritiated hypoxanthine incorporated. Activity against Leishmania (Leishmania) chagasi and Trypanosoma cruzi was measured by the MTT colorimetric assay. The antifungal tests were carried out by using the CLSI method. The active extracts were tested also by cytotoxicity assay using NIH-3T3 cells of mammalian fibroblasts.

Results

Two hundred and seventeen extracts of plants were tested against Plasmodium falciparum. The eleven active extracts, belonging to eight plant species were evaluated against L. (L.) chagasi, Trypanosoma cruzi, yeasts and in NIH-3T3 cells. The results found in these biological models are consistent with the ethnopharmacological data of these plants. The ethyl acetate extract of Diospyros hispida root showed IC₅₀ values of 1 μg/mL against Plasmodium falciparum. This extract demonstrated no toxicity against mammalian cells, resulting in a significant selectivity index (SI) of 435.8. The dichloromethane extract of Calophyllum brasiliense root wood was active against Cryptococcus gattii LMGO 01 with MIC of 1.95 μg/mL; and Candida albicans ATCC 10231 and Candida krusei LMGO 174, both with MIC of 7.81 μg/mL. The same extract was also active against Plasmodium falciparum and L. (L.) chagasi with IC₅₀ of 6.7 and 27.6 μg/mL respectively. The ethyl acetate extract of Spiranthera odoratissima leaves was active against Cryptococcus gattii LMGO 01 with MIC of 31.25 μg/mL, and against Plasmodium falciparum with IC₅₀ of 9.2 μg/mL and Trypanosoma cruzi with IC₅₀ of 56.3 μg/mL.

Conclusion

The active extracts for protozoans and human pathogenic yeasts are considered promising to continue the search for the identification and development of leading compounds.     

Effect of feeding Mucuna pruriens on helminth parasite infestation in lambs

Ethnopharmacological relevance

Mucuna pruriens is a tropical legume anecdotally reputed to have anthelmintic properties. This study was conducted to examine the validity of such claims.

Aim of the study

The aim of this study was to determine if ingestion of Mucuna seeds reduces helminth parasite infestation in lambs.

Materials and methods

Thirty-six Dorper × Katahdin ram lambs were assigned to three treatments, a cottonseed meal based control diet, a diet in which Mucuna replaced cottonseed meal and the control diet with levamisole (7.5 mg/kg body weight) administration. All diets were isonitrogenous and isocaloric. The 12 lambs in each treatment were assigned randomly to 4 pens, each containing 3 lambs. Lambs were trickle infected three times per week by gavage with infectious Haemonchus contortus larvae (2000 larvae/lamb) for 3 weeks.

Results

Levamisole treatment decreased fecal egg counts by 87% and abomasal worm counts by 83%. Mucuna intake did not statistically affect fecal egg counts or abomasal worm counts, though numerical (P > 0.10) reductions of 7.4% and 18.1%, respectively were evident. Anemia indicators, feed intake, and lamb growth were unaffected by treatment.

Conclusions

Levamisole reduced the Haemonchus parasite burden in lambs significantly but feeding Mucuna reduced the burden by levels unlikely to eliminate the clinical effects of parasitism.     

Screening of the topical anti-inflammatory activity of the bark of Acacia cornigera Willdenow, Byrsonima crassifolia Kunth, Sweetia panamensis Yakovlev and the leaves of Sphagneticola trilobata Hitchcock

Ethnopharmacological relevance

An investigation of topical anti-inflammatory activity was undertaken on plants used in Central America traditional medicine.

Aim of study

Four herbal drugs used in the folk medicine of Central America to treat inflammatory skin affections (Acacia cornigera bark, Byrsonima crassifolia bark, Sphagneticola trilobata leaves and Sweetia panamensis bark) were evaluated for their topical anti-inflammatory activity.

Materials and methods

Petroleum ether, chloroform and methanol extracts were obtained for herbal medicines and then extracts were tested on Croton oil-induced ear dermatitis model in mice.

Results

Almost all the extracts reduced the Croton oil-induced ear dermatitis in mice and the chloroform ones showed the highest activity, with ID₅₀ (dose giving 50% oedema inhibition) values ranging from 112 μg/cm² (Byrsonima crassifolia) to 183 μg/cm² (Sphagneticola trilobata). As reference, ID₅₀ of the non-steroidal anti-inflammatory drug indomethacin was 93 μg/cm².

Conclusions

Lipophilic extracts from these species can be regarded as potential sources of anti-inflammatory principles.     

中国石斛属植物文献计量研究

石斛是珍稀濒危中药材,目前正处于快速发展阶段。为全面了解我国石斛属植物研究的历史和发展现状,作者以1954～2010年"中国知网中国学术期刊网络出版总库"收录的石斛研究文献为依据,采用文献计量学的原理和方法,对我国石斛属植物研究文献从文献年代分布、期刊分布与被引频率、主题分布、研究对象分布、作者与研究机构分布等方面进行了统计与分析。结果表明,我国石斛研究明显分为起步(2个)、停滞、平稳发展、快速上升5个阶段;期刊分布存在离散性与集中性并存的现象,已形成核心期刊研究群,并以《中国中药杂志》、《中草药》和《陕西中医》为代表;研究主题广泛涉及临床与药理、组织培养与种苗繁育、成分分析等多个领域,已经形成比较稳定的研究机构和团队,但研究对象差异显著,以铁皮石斛、金钗石斛和霍山石斛最为集中。我国石斛属植物的研究已取得显著成果,但种植产业发展缓慢,供需矛盾突出,预计种苗繁育与人工种植、产品开发、化学与药理等方面是未来的研究热点,其文献报道仍将进一步上升。     

乌拉尔甘草HMGR基因cDNA的克隆与序列分析

目的:对乌拉尔甘草3.羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3methylglutary CoA reductase,HMGR)的cDNA克隆并进行序列分析.方法:根据NCBI数据库中的豆科其他物种HMGR的cDNA保守区设计引物,利用同源扩增和cDNA末端快速扩增技术从甘草根中获得目的基因;利用BLAST进行序列比对,ORF Finder寻找开发阅读框,Prosite分析蛋白质的基本结构域,Clustal x比对已有HMGR的氨基酸序列,并构建进化树.结果:得到1个全长为1 842 bp的HMGR的cDNA序列,含有1 722 bp的开放阅读框(open reading frame,ORF),编码573个氨基酸,具有HMGR家族的特异序列,推测的氨基酸序列与豌豆、蒺藜苜蓿的氨基酸序列一致性分别为84%,76%.结论:对甘草HMGR基因的cDNA进行了克隆,为进一步研究3-羟基-3-甲基戊二酰辅酶A在甘草酸生物合成途径中的作用提供了理论依据.     

Experimental study on anti-inflammatory activity of a TCM recipe consisting of the supercritical fluid CO2 extract of Chrysanthemum indicum, Patchouli Oil and Zedoary Turmeric Oil in vivo

Ethnopharmacological relevance

Chrysanthemum indicum (Compositae) Linné, Pogostemon cablin (Blanco) Benth and Curcuma wenyujin (Zingiberaceae) Y. H. Chen et C. Ling are three of the extensively used herbal remedies among traditional Chinese medicines for the purpose of anti-inflammation. A traditional Chinese medicine (TCM) recipe named CPZ consisting extracts of the above three herbs, has shown noteworthy anti-influenza activity, which is closely related to its anti-inflammatory feature.

Aim of this study

To investigated the anti-inflammtory activity of CPZ in vivo for a further exploration of the recipe's anti-inflammatory properties.

Materials and methods

The anti-inflammatory property of CPZ on acute inflammation was evaluated by inflammatory models of dimethylbenzene (DMB)-induced ear vasodilatation and acetic acid-induced capillary permeability enhancement in mice, as well as the carrageenan-induced paw edema rat model, in which inflammation-related cytokine including prostaglandin E₂ (PGE₂), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) in the edematous paw tissue were determined by enzyme linked immunosorbent assay (ELISA). Moreover, effect of CPZ on chronic inflammation was observed through granuloma formation in rats subjected to cotton pellet implantation.

Results

CPZ (340, 170, and 85 mg/kg for mice, p.o.) not only decreased the DMB-induced ear vasodilatation but also attenuated capillary permeability under acetic acid challenge in mice. And the significant inhibition on carrageenan-induced paw edema was observed. Further more, the ELISA results showed that CPZ (170, 85, and 42.5 mg/kg for rats, p.o.) could up-regulate the level of IL-1β in the edema paw tissue of rats significantly while down-regulate that of PGE₂, but no apparent effect on TNF-α or NO was observed in the test. Besides, CPZ had a certain degree of restraining effect on the cotton pellet-induced granuloma formation in rats and the highest dose of 170 mg/kg even showed a significant suppression on it.

Conclusion

The above results indicated that CPZ possessed a potent anti-inflammatory activity, which is indicated to be closely associated with its regulation on IL-1β and PGE₂ thereby mediating the inflammatory response acting at an appropriate level.     

Regulation effects on abnormal glucose and lipid metabolism of TZQ-F,a new kind of Traditional Chinese Medicine

Aim of the study

Based on the recipe of the traditional anti-diabetic formula TZQ, we developed TZQ-F, a new formula including 8 fractions isolated from Red Paeony root, Mulberry leaf, Lotus leaf, Danshen root and Hawthorn leaf with a good quality assurance. The study was aimed at fraction preparation and effects of the fractions on abnormal glucose and lipid metabolism.

Materials and methods

The active fractions were obtained by macroporous resin, ion-exchange resin and polyamide resin column chromatographies. HPLC analyses were used for quality control. In vitro mechanism study included DPPH radical scavenging, AGEs formation inhibition, α-glucosidase inhibition and lipase inhibition, and rats on high-fat diet were used for in vivo study.

Results

In vitro mechanism study showed that among the 8 fractions, three of them had inhibition effects on intestinal disaccharase, three with inhibition effects on lipase, and five with effects of free radical scavenging. In vivo study showed that after 4 weeks of treatment, TZQ-F significantly decreased the levels of serum total cholesterol, TG, glucose, LDL-C and HDL-C in rats on high-fat diet. Consistent with the in vitro and in vivo results, histology study demonstrated that TZQ-F alleviated hepatic steatosis induced by high-fat diet.

Conclusions

TZQ-F possesses the potential regulation effects on abnormal glucose and lipid metabolism.     

常用藏药“蒂达”(藏茵陈)基原物种药用合理性及资源利用价值评价

目的:对“蒂达”各品种不同基原植物药用合理性及其资源利用价值作出评价.方法:采用HPLC指纹图谱共有特征峰、结合SPSS聚类分析方法,评价“蒂达”不同品种基原植物的成分组成相似性.结果:“蒂达”不同品种涉及的来源有龙胆科獐牙菜属、花锚属、扁蕾属及肋柱花属共15种1变种33个样品,指纹图谱全谱相似度较差;以10个共有峰成分的相对面积聚类,其基原植物可分别聚为4个组;上述4个属的属间成分组成差异较大;其中獐牙菜属不同种间成分组成变化较大,而其他各属种间成分组成相似度较高.结论:上述龙胆科4属植物均含有具有保肝利胆生物活性的环烯醚萜类、(山)酮类及三萜类成分,藏医学将其归为“蒂达”一类药材使用具有一定的物质基础;鉴于“蒂达”类药材不同基原各品种间在成分组成上差异较大,分别制定各品种的HPLC指纹图谱,是鉴别和控制各品种质量的有效手段,可有效控制各基原品种质量.     

厚朴与凹叶厚朴群体遗传学研究

目的:对厚朴与凹叶厚朴的群体遗传学进行研究,为中药厚朴的质量控制提供分子生药学方面的依据。方法:对厚朴与凹叶厚朴15个居群应用2个叶绿体基因间序列psbA-trnH和trnL-trnF进行PCR扩增并测序,计算厚朴与凹叶厚朴单倍型频率,用程序HaploNst分析遗传多样性和遗传结构,应用TCS version 1.13软件构建单倍型网状进化树。结果:厚朴与凹叶厚朴均无特有单倍型存在,但单倍型频率存在显著差异,已开始出现遗传分化的趋势,NST略大于GST。结论:厚朴与凹叶厚朴在遗传上已出现遗传分化的趋势,但尚未完全分化成彼此独立的单系。     

Antifungal activity of Brazilian medicinal plants involved in popular treatment of mycoses

A survey of medicinal plants used to treat common mycoses was done in the Curituba district, Sergipe State, Brazil. One hundred inhabitants were interviewed by health agents and traditional healers. Four different plants were the most cited (more than 50% of the citations): Ziziphus joazeiro, Caesalpinia pyramidalis, Bumelia sartorum and Hymenea courbaril. The aqueous extracts obtained following traditional methods and using different parts of these plants, were submitted to drop agar diffusion tests for primary antimicrobial screening. Only the water infusion extract of Ziziphus joazeiro and Caesalpinea pyramidalis presented a significant antifungal activity against Trichophyton rubrum, Candida guilliermondii, Candida albicans, Cryptococcus neoformans and Fonsecaea pedrosoi, when compared to the antifungal agent amphotericin B. The minimal inhibitory concentration (MIC) of the bioactive extracts was evaluated by the microdilution method. Best activity with a MIC of 6.5 microg/ml for both extracts was observed against Trichophyton rubrum and Candida guilliermondii. Ziziphus joazeiro and Caesalpinea pyramidalis extracts presented also low acute toxicity in murine models. The present study validates the folk use of these plant extracts and indicates that they can be effective potential candidates for the development of new strategies to treat fungal infections.