反相高效液相色谱法测定青蒿中青蒿酸的含量

本文建立了反相高效液相色谱快速测定青蒿中青蒿酸含量的方法。色谱条件为:Diamonsil C18色谱柱(250 mm×4.6 mm,5μm),柱温为(30±1)℃,流动相采用乙腈与0.2%磷酸水溶液混合液(体积比65:35),流速1 mL/min,检测波长220 nm。标准曲线回归方程:Y=8.784573×10-8X-6.443559×10-5,r=0.9997,青蒿酸回收率为102.4%。实验证明该方法稳定可靠、精密度高、重现性好、简单可行,适用于青蒿酸的分析检测。     

蒙药材刺柏叶中槲皮苷的含量测定

本实验建立了蒙药材刺柏叶中槲皮苷的HPLC含量测定方法.色谱条件为:Diamonsil C18(4.6 mm ×250 mm,5μm)和Shim-Pack C18(4.6 mm× 250 mm,5μm),流动相:甲醇-0.01 mol/L磷酸二氢钾溶液-冰醋酸(40∶ 60∶ 1.5),检测波长:254 nm,流速:1.0 mL/min,柱温:30℃.槲皮苷进样量在40.2 ng ～603.0 ng范围内线性关系良好(r =0.9998),其平均加样回收率为100.45％,RSD为0.69％(n=6).本方法简便易行,结果准确,重复性好,可用于刺柏叶中槲皮苷的含量测定.     

HPLC法测定金菊五花茶颗粒中绿原酸的含量

目的:建立HPLC法测定金菊五花茶颗粒中绿原酸的含量。方法:色谱柱:Eclipse XDB-C18(150mm*4.6mm,5μm)流动相:甲醇-0.15%磷酸溶液(20:80)流速:1.0ml/min,检测波长:327nm。结果:绿原酸线性范围:0.026~0.260μg(r=0.9997),加样回收率为:99.20%,RSD=1.25%(n=6)。结论:本法操作简便,准确,可用于测定金菊五花茶冲剂中绿原酸的含量。     

高效液相色谱法测定重组人干扰素α-2b注射液中乙二胺四乙酸二钠含量

目的:利用高效液相色谱(HPLC)法测定重组人干扰素α-2b注射液中EDTA二钠(乙二胺四乙酸二钠)的含量。方法:将EDTA二钠与氯化铁溶液于70℃水浴中反应20 min左右;色谱柱为SunFire C18(250 mm×4.6 mm,5μm,Waters),流动相为5%甲醇+95%0.64 g/L四丁基溴化铵和4.1 g/L三水合乙酸钠混合液,用冰乙酸调pH值至4.0,流速1 mL/min,检测波长254 nm。结果:该测定方法线性范围为0.025~0.5 g/L,线性关系良好(r=0.9997),加样回收率为98.27%(n=9,RSD=2.55%)。结论:本方法准确、快速、可靠,可用于重组人干扰素α-2b注射液中EDTA二钠含量的测定。     

高效液相色谱法直接测定当归中阿魏酸的含量

建立了一种高效液相色谱法直接测定当归中阿魏酸的含量。色谱柱为Shim-pack CLC-ODS(6.0 mm i.d×150 mm,5μm),流动相为甲醇—1.0%冰醋酸水溶液(体积比为25∶75),流速为1.0 mL/min,检测波长为313 nm。研究结果表明:阿魏酸的线性范围为1.0~10.0 mg/L,回归方程为A=13.352 8C 0.013 6(r=0.998 5),最小检测限为10 ng,加样平均回收率为94.67%,相对标准偏差(RSD)为1.57%。方法操作简便、快速、灵敏、准确。     

HPLC测定7种龙胆科植物花中龙胆苦苷与獐牙菜苦苷的含量

目的:建立7种龙胆花中龙胆苦苷和獐牙菜苦苷含量测定的HPLC方法。方法:采用微波辅助动态回流法进行提取,色谱条件:Fusion-RP 80 A C18柱(150 mm×4.6 mm,5μm);流动相:甲醇-0.2%磷酸溶液梯度洗脱(0~25 min:15%~30%);流速:1 mL/min;柱温:30℃;检测波长240 nm。结果:7种龙胆花中獐牙菜苦苷和龙胆苦苷的色谱峰与共存组分完全达到基线分离,线性范围分别为0.105~0.945μg(r=0.999 9),0.3~0.7μg(r=0.999 9),平均加样回收率分别为97.8%(RSD=1.02%),98.9%(RSD=1.51%)。结论:所建立的方法测定快速,结果准确可靠。     

蜜楝花及果实中吴茱萸碱、吴茱萸次碱及内酯含量积累变化研究

分析测定了不同生长时期蜜楝花及果实中吴茱萸生物碱及吴茱萸内酯含量及其积累变化。色谱条件为:色谱柱为Inertsil ODS-3C_(18)柱(4.6×150 mm,5μm),以乙腈-四氢呋喃-水(41∶1∶58)为流动相,流速1.0 mL/min,检测波长225 nm,柱温30℃。蜜楝中吴茱萸内酯在49.65~248.25μg/mL范围内呈良好的线性关系,Y=1955.4X-5577.3(R~2=0.9996),平均加样回收率102.70%,RSD为3.00%(n=6);吴茱萸碱在8.88~88.80μg/mL范围内呈良好的线性关系,Y=89913X+73191(R~2=0.9990),平均加样回收率103.99%,RSD为2.18%(n=6),吴茱萸次碱在8.25~49.49μg/mL范围内呈良好的线性关系,Y=56127X-51856(R~2=0.9990),平均加样回收率103.50%,RSD为1.48%(n=6)。随着发育阶段的推移,蜜楝花、果实中吴茱萸碱、吴茱萸次碱及吴茱萸内酯的含量逐渐增加,在近成熟的果实中三种成分含量最高。研究结果表明,蜜楝果实中生物碱和吴茱萸内酯的含量丰富,可作为提取制备吴茱萸生物碱及吴茱萸内酯成分的一种植物资源,具有很好的开发利用价值。     

HPLC测定双金花茶中免疫活性分子山奈酚的含量

目的建立HPLC测定双金花茶中免疫活性分子山奈酚含量的方法。方法采用Kromasil C18(4.6 mm×200 mm,5μm)色谱柱;流动相:甲醇∶0.1%醋酸水溶液(70∶30);流速:1.0 ml/min;检测波长:370 nm;柱温:25℃。结果山奈酚在进样量0.04～0.40μg(r=0.9999)范围内呈良好线性关系,平均加样回收率为100.00%,RSD为0.07%(n=6)。结论 HPLC法简便易行,准确可靠,可用于双金花茶中免疫活性分子山奈酚含量测定。     

RP-HPLC法测定葫芦巴中葫芦巴皂苷B的含量

本文采用Kromasil C18(250×4.6 mm,5μm),乙腈-0.2%磷酸溶液(83:17)为流动相,流速1.0 mL/min,DAD检测器,检测波长203 nm,对葫芦巴中葫芦巴皂苷B含量进行测定,能将葫芦巴皂苷B与杂质分开,葫芦巴皂苷B在1.60~11.20μg范围内峰面积与其浓度线性关系良好(R=0.99996),样品的平均加样回收率为99.83%,RSD为1.47%(n=6)。该法测定样品快速准确,重现性好。     

高效液相色谱法测定甜菊糖苷中甜菊醇的含量

建立高效液相色谱法测定甜菊糖苷中甜菊醇的含量。色谱条件:色谱柱为Phenomenex C18(4.6 mm×250 mm,5μm);流动相为乙腈-水(50∶50);流速1.0 mL/min;检测波长213 nm;柱温30℃;进样量10μL。线性范围为1.046μg/mL~52.3μg/mL(r=0.9997),加标平均回收率为96.60%,RSD为0.51%(n=6)。本方法准确度高、精密度高、重复性好、简捷易操作,可以作为甜菊糖苷中甜菊醇含量的测定方法。     

TLC-densitometric analysis of artemisinin for the rapid screening of high-producing plantlets of Artemisia annua L

A simple TLC-densitometric technique has been developed for the rapid and accurate analysis of artemisinin in a large number of Artemisia annua plantlets cultured in vitro. This new analytical method is based on the structural conversion of artemisinin on a silica gel layer by ammonia vapour to form 10-azadesoxyartemisinin, a chromophore-containing compound (lambdamax 320 nm) that can be detected by UV-based TLC densitometry. The TLC system was evaluated quantitatively in terms of product stability, precision, accuracy and calibration. Good linearity was obtained in the range of 0.01-0.12 microg artemisinin. The technique appeared to be accurate and sensitive as compared with the complicated pre-column reaction-HPLC technique. Among 90 samples of A. annua plantlets, the artemisinin content in the leaves appeared to be highly variable, ranging from 0.02 to 0.67% w/w dry weight. These results demonstrate that densitometric TLC can be a cheap and simple technique for the accurate screening of high-artemisinin-producing plants.     

Simple and rapid physico-chemical methods to examine action of antimalarial drugs with hemin: its application to Artemisia annua constituents

Malaria is a major health problem in many countries and according to an estimate of the WHO, more than 500 million infections occur per year. Artemisinin, a sesquiterpene from Artemisia annua L., has received considerable attention as a promising and potent antimalarial drug for its stage speciticity, its rather low toxicity, effectiveness against drug-resistant Plasmodium species and activity against cerebral malaria. From recent studies it seems that hemin is primarily involved in the antimalarial activity of the constituents of Artemisia annua L. Thus, the interaction of a compound with hemin may represent a crucial screening test to define its efficacy. In this study the interaction between artemisinin and hemin was investigated by UltraViolet/Visible (UV/Vis) spectrophotometry and High Performance Liquid Chromatography/Diode Array Detector/Mass Spectrometry (HPLC/DAD/MS). In addition, some flavonols isolated from Artemisia annua L. were also tested to investigate their possible role in the interaction between artemisinin and hemin. These two simple physico-chemical methods can be useful as rapid and widespread screening methods for the search of other alkylating antimalarial constituents from natural sources or for the evaluation of the activity of semisynthetic analogues of artemisinin.     

Nitric oxide potentiates oligosaccharide-induced artemisinin production in Artemisia annua hairy roots

The purpose of the present study was to characterize the generation of nitric oxide （NO） in Artemisia annua roots induced by an oligosaccharide elicitor （OE） from Fusarium oxysporum mycelium and the potentiation role of NO in the elicitation of artemisinin accumulation. The OE （0.3 mg total sugar/mL） induced a rapid production of NO in cultures, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 8 h of OE treatment. Artemisinin content in 20-day-old hairy roots was increased from 0.7mg/g dry wt to 1.3 mg/g dry wt by using the OE treatment for 4d. In the absence of OE, the NO donor sodium nitroprusside （SNP） at 10, 50 ~1 and 100 ~1 enhanced the growth of hairy roots, but had no effect on artemisinin synthesis, The combination of SNP with OE increased artemisinin content from 1.2 mg/g drywt to 2.2 mg/g dry wt, whereas the maximum production of artemisinin in cultures was 28.5 mg/L, a twofold increase over the OE treatment alone. The effects of SNP on the OE-induced artemisinin were suppressed strongly by the NO scavenger 2-（4- carboxyphenyl）-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide （cPTIO）. The results suggest that NO can strongly potentiate elicitor-induced artemisinin synthesis in A. annua hairy roots.     

Production of artemisinin by hairy rot cultures of Artemisia annua L in bioreactor

Hairy root cultures of Artemisia annua L were cultivated in four different culture systems: a flask, a bubble column, a modified bubble column and a modified inner-loop airlift bioreactor. The artemisinin contents of hairy root cultures in the bubble column and the modified inner-loop airlift bioreactor were higher than that in the modified bubble column. The growth rate and hairy root distribution in the modified inner-loop airlift bioreactor were better than those in other bioreactors, and dry weight and artemisinin production reached to 26.8 g/L and 536 mg/L after 20 days.     

青蒿过氧化物酶的克隆及其在体外对青蒿素生物合成的影响

用RACE方法从青蒿(Artemisia annua L.)高产株系001中克隆了一个过氧化物酶.将此基因在大肠杆菌BL21(DE3)pLysS细胞中进行原核表达得到重组蛋白(APOD1),表达的蛋白分别以抗坏血酸、愈创木酚为底物进行过氧化反应,结果显示,APOD1催化愈创木酚的活力是抗坏血酸的1.8倍左右,由此表明,克隆的APOD1类属于植物经典过氧化物酶(第三大类过氧化物酶).经与其他植物过氧化物酶同源性比较分析,推测APOD1的氨基酸序列与白羽扇豆(Lupinus albus)、辣根菜(Armoracia rusticana)、小麦(Triticum aestivum)、烟草(Nicotiana tabacum)和蕃茄(Lycopersicon esculentum)的一致性分别为42.0%、36.2%、38.9%、33.6%和32.8%.Northern杂交分析表明,此基因在青蒿的根、茎和叶中均有表达.加入APOD1至青蒿细胞提取液有利于青蒿酸向青蒿素的生物转化,但APOD1并不能直接以青蒿酸作为氧化底物.     

促进黄花蒿发根青蒿素合成的内生真菌诱导子的制备

应用酸解法对黄花蒿(ArtemisiaannuaL.)内生胶孢炭疽菌(Colletotrichumgloeosporioides)菌丝体进行提取,在黄花蒿发根培养系统中比较了各制备提取物的青蒿素诱导活性。活性提取物经过SephadexG25层析后,部分纯化的内生菌寡糖提取物(MW<2500)可显著促进发根青蒿素的合成,培养23d的发根经诱导子(0.4mg/mL)处理4d后,青蒿素产量可达13.51mg/L,比同期对照产量提高51.63%,诱导作用与诱导子浓度、作用时间相关。内生菌寡糖诱导子的制备和使用,在青蒿素生物技术生产研究中为首次应用。     

青蒿素对蔬菜种子发芽和幼苗生长的化感效应

试验以菜豆、豇豆、大白菜和小白菜为对象,用不同浓度的青蒿素浸种,研究了黄花蒿产生的化感物质??青蒿素对蔬菜种子发芽及幼苗生长的影响。结果表明,青蒿素对蔬菜种子发芽和幼苗生长的化感作用表现出浓度效应和品种差异,即浓度越高,抑制作用愈强,尤以豇豆种子发芽率和小白菜生长的表现最为明显,前者的发芽率可降低75.00%,后者的苗高降幅高达88.37%,且胚根停止生长。青蒿素抑制同季和后季作物的种子发芽和幼苗生长,有利于扩大黄花蒿的生存空间,增强生存竞争优势。在黄花蒿?蔬菜种植体系中,选择抗化感作用较强的大白菜和菜豆可提高土地利用率和整体生产水平。用青蒿素浸种后,蔬菜幼苗的根系活力降低,菜豆和豇豆叶绿素含量提高,而大、小白菜降低,均可视为妨碍生长的生理原因。此外,青蒿素浸种还提高蔬菜种子可溶性糖和游离氨基酸含量,推测青蒿素对种子水解酶活性的影响较小,但抑制合成酶催化的生化反应,导致代谢紊乱,抑制幼苗生长。     

Quantification of artemisinin in Artemisia annua extracts by 1H-NMR

Artemisinin is a polycyclic sesquiterpene lactone that is highly effective against multidrug-resistant strains of Plasmodium falciparum, the etiological agent of the most severe form of malaria. Determination of artemisinin in the source plant, Artemisia annua, is a challenging problem since the compound is present in very low concentrations, is thermolabile and unstable, and lacks chromophoric or fluorophoric groups. The ain of this study was to develop a simple protocol for the quantification of artemisinin in a plant extract using an (1)H-NMR method. Samples were prepared by extraction of leaf material with acetone, treatment with activated charcoal to remove chlorophylls and removal of solvent. (1)H-NMR spectra were measured on samples dissolved in deuterochloroform with tert-butanol as internal standard. Quantification was carried out using the using the delta 5.864 signal of artemisinin and the delta 1.276 signal of tert-butanol. The method was optimised and fully validated against a reference standard of artemisinin. The results were compared with those obtained from the same samples quantified using an HPLC-refractive index (RI) method. The (1)H-NMR method gave a linear response for artemisinin within the range 9.85-97.99 mm (r(2) = 0.9968). Using the described method, yields of artemisinin in the range 0.77-1.06% were obtained from leaves of the A. annua hybrid CPQBA x POP, and these values were in agreement with those obtained using an HPLC-RI.     

An ultra performance liquid chromatographic method for determining phytosterol uptake by Caco-2 cells

A simple method for the determination of cellular uptake of phytosterols by Caco-2 cells has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC-UV). UPLC-UV was established using an ODS column, acetonitrile/H(2)O (9:1, v/v) as a mobile phase, and a detection wavelength at 210 nm. As analytes, β-sitosterol, campesterol, stigmasterol, and brassicasterol were selected based on the abundance in foods and the similarity of their structures. A linear relation was observed between the peak area and the amount of sterol injected from 50 to 2000 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.5% (n=6). This method was applied to the determination of cellular uptake of phytosterols by Caco-2 cells. Recovery tests showed that phytosterols were extracted from the cell lysates by chloroform and determined by UPLC-UV with a recovery rate of more than 80.2% and an RSD of less than 11.3% (n=3). When Caco-2 cells were incubated with phytosterols at 37°C, their uptake was increased with time in a concentration-dependent manner. This method will be useful for the simultaneous determination of cellular phytosterols in an in vitro intestine model.     

高效液相色谱法检测发酵液中木糖和木糖醇

建立高效液相色谱检测发酵液中木糖和木糖醇含量的分析方法。色谱柱为HypersilNH2 柱 (4 .6mmi.d.× 2 5 0mm ,5 μm) ,柱温 3 5℃ ,流动相为乙腈—水 (80∶2 0 ) ,流速 1 .0mL .min 1,示差折光检测器检测。木糖和木糖醇在 3 .0～ 60mg.mL 1范围内 ,峰面积与其浓度线性关系良好 (г=0 .9995 ) ;平均回收率分别为 96.0 7% (n =5 ,RSD =0 .5 1 % )和 97.47% (n =5 ,RSD =1 .1 3 % )。方法简便、快速、准确。