柱前衍生-RP-HPLC法测定青蒿中青蒿素的含量

采用柱前衍生-RP-HPLC法测定10个不同产地的青蒿药材中青蒿素的含量.采用Lichrospher 100 RP-18e(250 mm×4.6 mm,5μm,Merck KgaA,Germany)色谱柱,甲醇-0.01 mol/L醋酸钠-醋酸缓冲液(pH 5.8)(体积比62:38)为流动相;检测波长:260 nm;流速:0.5 mL/min;柱温:25℃.结果表明该法准确重现性好,可以为青蒿质量标准的制订提供科学依据.     

UPLC-UV法测定不同产地青蒿中青蒿素的含量

目的:建立快速简便检测青蒿素的超高效液相-紫外(UPLC-UV)法,并对不同产地青蒿中青蒿素的含量进行检测。方法:色谱柱Agilent Eclipse Plus C18(2.1 mm×50 mm,1.8μm),流动相乙腈/水(45/55),流速1.0mL/min,柱温28℃,波长200 nm。结果:该方法对青蒿素的分离度较好,保留时间缩短为1.5 min。并且,整个分析过程可以在2 min内完成。线性范围0.101 17～10.117μg,进样量与峰面积线性相关,A=109.4C+6.7026,R2=0.9 993(n=9),加样回收率为99.3%(RSD=2.6%,n=6)。结论:UPLC-UV法分析时间短、样品前处理简单、精密度、稳定性、加样回收率等符合分析检测要求,对于青蒿中青蒿素的含量能进行快速准确的分析。     

柱前衍生化HPLC法测定加巴喷丁胶囊中主药的含量

目的:建立柱前衍生化HPLC法测定加巴喷丁胶囊中的主药含量。方法:对加巴喷丁柱前衍生化后进行测定,选择Sinochrom-ODS(250 mm×4.6 mm,5μm)为固定相,0.33 mol.L-1乙酸水溶液-甲醇-乙腈(30∶70∶30)为流动相等度洗脱,流速1.0 mL.min-1,检测波长336 nm。结果:加巴喷丁在1.0～12.0 mg.L-1质量浓度范围内线性关系良好,其平均加样回收率为99.4%(n=9),RSD为0.84%;测得样品中加巴喷丁含量为标示量的99.82%。结论:该法便捷可靠,专属性强,可用于加巴喷丁胶囊中主药的含量测定。     

广西地形对青蒿中青蒿素含量的影响

针对广西青蒿种植中适生地选择的迫切要求,在对广西31个样地155株青蒿中青蒿素含量测定的基础上,分析了青蒿素含量地理变异,不同地形条件下青蒿素含量变异,海拔高度、坡度、坡向等地形因子对青蒿素含量的影响,不同区域内青蒿素含量变异分析,并应用GIS技术开展了基于地形因子的青蒿种植区划分析.得到如下结果和结论:①各样地间的青蒿素含量存在显著性差异(P=0.000<0.05);②海拔高度和坡度是影响青蒿素含量变化的主要地形因子,生长在海拔较高、坡度较大的丘陵、山地区域内的青蒿,青蒿素含量高于生长在海拔较低、坡度较小的平原区域内的(P=0.000<0.05);③桂西南地区内的青蒿素含量最高,桂东南平原区域内的青蒿素含量最低,二者与其他区域的青蒿素含量差异显著(P=0.000<0.05);④青蒿素含量在(0.76%～0.93%)的为最适宜区,主要包括桂东北和西南地区的阳朔、都安、大化、苹果、马山、武鸣、隆安、南宁和崇左等县市.青蒿素含量在(0.61%～0.76%)的为较适宜区域.青蒿素含量在(0.47%～0.61%)的为适宜区.青蒿素含量在(0 32%～0.47%)的为不适宜区,主要包括北海市、钦州市、贵港市、贺州市、梧州市和岑溪市的部分地区.⑤桂北地区地形复杂\"小地形\"、\"小气候\"特征明显,从小尺度范围内研究青蒿的生态适宜性,更能得到较显著的效果.     

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。     

柱前衍生化HPLC法测定黄芪中黄芪甲苷的含量

目的：建立黄芪中黄芪甲苷的柱前衍化高效液相色谱测定法,并用该法对不同产地黄芪中黄芪甲苷的含量进行比较,方法：以吡啶－苯甲酰氯（２．５：１）为衍生化试剂,对黄芪甲苷分子中的羟基进行苯甲酰化,以甲醇－四氢呋喃－水（９０：４：６,０．２％三乙胺）为流动相,ＶＤ３为内标物,在２３０ｎｍ波长处检测。结果黄芪甲苷在０．００４－０．０８０ｍｇ．ｍＬ＾－１范围内呈线性,相关系数为０．９９９９,平均回收率为９４．７     

柱前衍生氨基酸的高效液相色谱分析

综述了目前柱前衍生氨基酸高效液相色谱分析技术的最新进展和现状,分别详细介绍了应用最广、技术最先进的几种柱前衍生氨基酸分析技术和方法。     

柱前衍生HPLC法测定大鼠血浆中的河蚌糖胺聚糖

目的:建立柱前衍生高效液相色谱法(HPLC)用于河蚌糖胺聚糖在大鼠血浆中的含量测定。方法:使用异硫氰酸荧光素(FITC)对河蚌糖胺聚糖进行荧光标记,色谱柱为Shodex SB-804HQ(8.0×300 mm),流动相为流速为0.5 m L·min-1的0.1 mol·L-1的Na H2PO4-Na2HPO4缓冲液(p H 7.8),柱温30℃,激发波长495 nm,发射波长520 nm。结果:FITC对河蚌糖胺聚糖的荧光标记率为70;线性范围为5~120μg·m L-1,批内和批间精密度均小于15.0%,提取回收率为51.5~58.6%,样品在血浆中稳定性良好。结论:血浆中内源性杂质不干扰样品的测定,建立的方法符合生物样本的分析要求。     

2,4—二硝基氟苯柱前衍生法测定氨基酸的改进

本文报道了用２,４－二硝基氟苯柱前衍生反相高效液相色谱法测定氨基酸,１５种常见的氨基酸可得到很好的分离。衍生物很稳定,在室温下保存两个月无明显变化,检测极限可达到５ｐｍｏｌ。本法可应用于食品,药品等样品的氨基酸测定。     

Modulation of artemisinin biosynthesis by elicitors,inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.     

Abscisic acid (ABA) treatment increases artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> by enhancing the expression of genes in artemisinin biosynthetic pathway

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L., is the most effective antimalarial drug. In an effort to increase the artemisinin production, abscisic acid (ABA) with different concentrations (1, 10 and 100 μM) was tested by treating A. annua plants. As a result, the artemisinin content in ABA-treated plants was significantly increased. Especially, artemisinin content in plants treated by 10 μM ABA was 65% higher than that in the control plants, up to an average of 1.84% dry weight. Gene expression analysis showed that in both the ABA-treated plants and cell suspension cultures, HMGR, FPS, CYP71AV1 and CPR, the important genes in the artemisinin biosynthetic pathway, were significantly induced. While only a slight increase of ADS expression was observed in ABA-treated plants, no expression of ADS was detected in cell suspension cultures. This study suggests that there is probably a crosstalk between the ABA signaling pathway and artemisinin biosynthetic pathway and that CYP71AV1, which was induced most significantly, may play a key regulatory role in the artemisinin biosynthetic pathway.     

Enhanced artemisinin accumulation and metabolic profiling of transgenic Artemisia annua L. plants over-expressing by rate-limiting enzymes from isoprenoid pathway

Artemisinin, a natural product isolated from aerial parts of Artemisia annua L. plant, is a potent antimalarial drug against drug-resistant malaria. In recent times, the demand (101–119 MT) for artemisinin is exponentially increasing with the increased incidence of drug-resistant malaria throughout the world, especially African and Asian continents. However, the commercial production of artemisinin-based combination therapies has limitation because of the presence of low concentration of artemisinin in plants. Therefore, transgenic lines of A. annua L. plants over-expressing both HMG-Co A reductase (hmgr) and amorpha-4, 11-diene synthase (ads) genes were developed to enhance the content of artemisinin. The selected transgenic lines (TR4, TR5, and TR7) were found to accumulate higher artemisinin (0.97–1.2%) as compared to the non-transgenic plants (0.63%). The secondary metabolite profiles of these lines were also investigated employing gas chromatography mass spectrometry, which revealed a clear difference in these metabolites in transgenic and non-transgenic lines of A. annua L. at different growth and developmental stages. The major metabolites reported in these lines at pre-flowering stage were related to essential oil and chlorophyll biosynthesis (71.33% in TR5 transgenic lines vs. 61.70% in non-transgenic line). Based on these results, we concluded that over-expression of both hmgr and ads genes in A. annua L. plants results not only increase in artemisinin content, but also enhances synthesis of other isoprenoid including essential oil. It is also evident from this study that the novel artemisinin-rich varieties of A. annua L. could be developed by suppressing essential oil biosynthesis, so that more carbon could preferentially be diverted from mevalonate pathway to artemisinin biosynthesis.     

Optimization of cryopreservation of Artemisia annua L. callus

Cryopreservation of callus tissue of Artimisia annua L. was optimized. Two lines of calli were precultured on MS medium with 5% (v/v) dimethyl sulfoxide, and protected by a cryoprotectant containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, 30% (v/v) glycerol and 13.6% (w/v) sucrose. The highest survival rate of callus A201 reached 87% after it was pretreated at 25°C, cryopreserved by liquid nitrogen, recovered in water bath at 25°C and reloaded at 25°C with 34% (w/v) sucrose solution, and that of callus A202 reached 78% after it was treated as callus A201, except pretreated at 35°C, recovered at 35°C and reloaded with 47.8% (w/v) sucrose solution.     

内生青霉菌对黄花蒿组培苗生长和青蒿素合成的影响

从黄花蒿茎中分离得到了17株内生真菌,其中内生青霉菌(Penicilliumsp.Y2)能有效促进黄花蒿组培苗生长及青蒿素合成。内生青霉菌悬浮培养5d后,分别将培养液与菌丝匀浆后经过高压灭菌处理,或将培养液经过高压灭菌、过滤除菌处理获得3种内生菌诱导子(A、B和C)。结果表明,3种内生菌诱导子对植株生长、抗氧化酶活性及青蒿素合成都有促进作用,诱导子C青蒿素合成诱导效果最好,可促进黄花蒿组培苗的干重增长44.44%、可溶性糖含量提高38.24%,诱导超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性,从而提高青蒿素合成达58.86%,黄花蒿组培苗青蒿素含量达4.701mg.g-1(干重)。     

State of the art of the production of the antimalarial compound artemisinin in plants

For more than three centuries we have relied on the extracts of the bark of Cinchona species to treat malaria. Now, it seems we may be changing to the leaves of a Chinese weed, Artemisia annua, and its active compound artemisinin. Artemisinin-derived drugs have been proved particularly effective treatments for severe malaria, even for multi-drug-resistant malaria. However, this promising antimalarial compound remains expensive and is hardly available on a global scale. Therefore, many research groups have directed their investigations toward the enhancement of artemisinin production in A. annua cell cultures or whole plants in order to overproduce artemisinin or one of its precursors. This article provides a brief review of the state of art of the different aspects in A. annua research.     

Improved growth of Artemisia annua L hairy roots and artemisinin production under red light conditions

Hairy root cultures of Artemisia annua L were cultivated for 30 days under either white, red, blue, yellow or green light. Red light at 660 nm gave the highest biomass of hairy roots (5.73 g dry wt cells l^–1 medium) and artemisinin content (31 mg arteminsinin g^–1 dry cells) which were, respectively, 17% and 67% higher than those obtained under white light.     

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。