Effect of Artemisia annua L. as Substitute to Sulfonamides (Sodium Sulfadimerzine) on Coccidiosis and Growth Performance in Rabbits

Coccidiosis is a disease caused by protozoa of the genus Eimeria which seriously affects young rabbits. Treatment based on the use of anticoccidial drugs is increasingly ineffective due to the rapid emergence of resistant strains of coccidia and the high cost of drugs. Consumer demand for rabbit products without chemical residues led to a growing interest in the use of medicinal plants as an alternative treatment for coccidiosis. The present study was carried out during the period of August to December 2020 to assess the anticoccidial effect of hydro-ethanolic extract of leaves of Artemisia annua L., in young rabbits. The antiparasitic efficacy of Artemisia extract was tested on 15 young rabbits (whose age varied between 7 and 9 weeks) divided into 5 lots of 3 animals. The average weight of these animals was 790 g. The results of this study show that the feces samples and the weight of young rabbits before administration of the treatment and the coprological examination (every 7 days for 4 weeks) show a fecal excretion reduction rate (FECRT) of 55.13% in the lot treated by sulfonamide. On the other hand, in animals received treatments extract of the leaves of Artemisia annua L., the average FECRT is evaluated at 69.64%, 79.22%, and 96.36% for respective doses of 400, 800 and 1200 mg/kg bodyweight and proves their anticoccidial effect. Furthermore, the variation in mean Eggs Per Gram (EPG) of coccidia and the average weekly weight gain (AWWG) of each lot were significant in the lots treated with hydro-ethanolic extract (P 0.05). The greatest reductions in oocystal excretion and weight gain obtained were those of lot 5, treated at 1200 mg/kg of hydro-ethanolic leaves extract of Artemisia annua L.

黄花蒿(Artemisia annua L.)提取物对两种病原真菌的生物活性

以石油醚Ⅰ(30℃～60℃)、石油醚Ⅱ(60℃～90℃)、乙醇和丙酮等4种溶剂对黄花蒿的根、茎、叶进行初步提取,采用生长速率法测定不同提取物对玉米小斑病菌、棉花枯萎病菌的抑菌活性。结果表明,黄花蒿提取物对玉米小斑病菌的生物活性好于棉花枯萎病菌;黄花蒿叶的提取物抑菌效果最好,根的抑菌效果最差;叶的石油醚(60℃～90℃)提取物对玉米小斑病菌EC50为156.32mg/L;而叶的丙酮提取物对玉米小斑病菌的EC50为82.37mg/L。

HPLC法同时测定黄花蒿水提浸膏中青蒿素、青蒿乙素、猫眼草黄素和猫眼草酚D的含量

目的建立高效液相色谱(HPLC)法同时测定黄花蒿水提浸膏中青蒿素、青蒿乙素、猫眼草黄素、猫眼草酚D的含量。方法采用Agilent ZORBAX SB-C18(250 mm×4.6 mm,5μm)色谱柱;流动相:乙腈(A)-纯水(B),梯度洗脱;体积流量:0.8 mL·min^(-1);柱温:30℃;进样量:10μL;检测波长:210 nm。结果青蒿素、青蒿乙素、猫眼草黄素和猫眼草酚D进样量分别在1.6088~16.088μg(r=0.9999)、0.0141~0.1414μg(r=1)、0.1851~1.8509μg(r=0.9999)、0.1441~1.4414μg(r=0.9999)范围内与峰面积呈良好的线性关系,平均加样回收率(n=6)分别为102.44%、97.82%、95.07%、95.55%,RSD分别为1.12%、1.44%、1.29%、1.53%。结论该方法简便准确、可靠稳定、重复性好,可用于同时测定黄花蒿水提浸膏中青蒿素、青蒿乙素、猫眼草酚D、猫眼草黄素的含量。

The AaBBX21–AaHY5 module mediates light-regulated artemisinin biosynthesis in Artemisia annua L.

The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood(Artemisia annua L.).Light was previously shown to promote artemisinin production,but the underlying regulatory mechanism remains elusive.In this study,we demonstrate that ELONGATED HYPOCOTYL5(HY5),a central transcription factor in the light signaling pathway,cannot promote artemisinin biosynthesis on its own,as the binding of AaHY5 to the promoters of artemisinin biosynthetic genes failed to activate their transcription.Transcriptome analysis and yeast two-hybrid screening revealed the B-box transcription factor AaBBX21 as a potential interactor with AaHY5.AaBBX21 showed a trichome-specific expression pattern.Additionally,the AaBBX21–AaHY5 complex cooperatively activated transcription from the promoters of the downstream genes AaGSW1,AaMYB108,and AaORA,encoding positive regulators of artemisinin biosynthesis.Moreover,AaHY5 and AaBBX21 physically interacted with the A.annua E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1(COP1).In the dark,AaCOP1 decreased the accumulation of AaHY5 and AaBBX21 and repressed the activation of genes downstream of the AaHY5–AaBBX21 complex,explaining the enhanced production of artemisinin upon light exposure.Our study provides insights into the central regulatory mechanism by which light governs terpenoid biosynthesis in the plant kingdom.

Transcriptional regulation of artemisinin biosynthesis in Artemisia annua L.

Artemisinin, also known as qinghaosu, a sesquiterpene endoperoxide lactone isolated from the Chinese medicinal plant Artemisia annua L., is the most effective antimalarial drug which has saved millions of lives.Due to its great antimalarial activity and low content in wild A. annua plants, researches focused on enhancing the artemisin yield in plants became a hotspot. Several families of transcription factors have been reported to participate in regulating the biosynthesis and accumulation of artemisinin.In this review, we summarize recent investigations in these fields, with emphasis on newly identified transcription factors and their functions in artemisinin biosynthesis regulation, and provide new insight for further research.

Artemisia annua glandular secretory trichomes: the biofactory of antimalarial agent artemisinin

Artemisinin, the key ingredient of first-line antimalarial drugs, has large demand every year. The native plant, which produces small quantities of artemisinin, remains as its main source and thus results in a short supply of artemisinin. Intensified efforts have been carried out to elevate artemisinin production. However, the routine metabolic engineering strategy, via overexpressing or down-regulating genes in artemisinin biosynthesis branch pathways, was not very effective as desired. Glandular secretory trichomes, sites of artemisinin biosynthesis on the surface of Artemisia annua L.(A. annua), are the new target for increasing artemisinin yield. In general, the population and morphology of glandular secretory trichomes in A. annua(Aa GSTs) are often positively correlated with artemisinin content. Improved understanding of Aa GSTs will shed light on the opportunities for increasing plant-derived artemisinin. This review article will refresh classification of trichomes in A. annua and provide an overview of the recent achievements regarding Aa GSTs and artemisinin. To have a full understanding of Aa GSTs,factors that are associated with trichome morphology and density will have to be further investigated, such as genes,micro RNAs and phytohormones. The purpose of thisreview was to(1) update the knowledge of the relation between Aa GSTs and artemisinin, and(2) propose new avenues to increase artemisinin yield by harnessing the potential biofactories, Aa GSTs.

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase （AalCS1） gene from Artemisia annua

Isochorismate synthase(ICS) is a crucial enzyme in the salicylic acid(SA) synthesis pathway. The full-length complementary DNA(cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site(MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction(qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography(HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.

Rol genes enhance content of artemisinin and other secondary metabolites in Shennong hybrid of Artemisia annua

Objective: Artemisia annua is the chief source of artemisinin, a potent antimalarial agent, in which other bioactive phytochemicals are also present. Due to low levels of bioactive compounds including artemisinin and flavonoids, it is necessary to increase the level of the secondary metabolites by regulating the expression of rol genes in the plant.Methods: A hybrid variety of A. annua(Hyb1209 r, Shennong) developed by the Centre for Novel Agricultural Products, University of York, UK, was selected to produce transgenics of rolB and rolC genes. Genetic transformation was carried out via Agrobacterium tumefaciens GV3101 harboring rolB and rolC genes of Agrobacterium rhizogenes cloned separately. HPLC was used for the qualitative and quantitative analysis of flavonoids and artemisinin. Furthermore, thin layer chromatography(TLC) was also used to analyze artemisinin content.Results: Comparative analysis via HPLC revealed considerable enhancement in the phytochemical content of transgenic A. annua plants as compared to the wild type plant. Transgenics of rolB gene showed an average increase of 321% in rutin, 97.2% in caffeic acid, and 218.4% in myricetin, respectively. In the case of rolC gene transgenics, an average increase of 197.5% in rutin, 76.3% in caffeic acid, and 209.3%in myricetin was observed. Transgenics of rolB and rolC genes showed a 14.3%–28.6% and 2.8%–12.7% increase in artemisinin content respectively by HPLC analysis. TLC analysis showed that an average 142.2%and 110.2% enhancement in artemisinin for rolB and rolC transgenics respectively, compared with the wild type. An enhanced production of total flavonoids(average 30.2% and 25.5% increase in rolB and rolC transgenics, respectively) and total phenolics(average 34.3% and 25.8% increase in rolB and rolC transgenics, respectively) was observed as a result of transformation. Transformed A. annua plants showed improved free radical scavenging activity(average 46.5% and 29.1% increase in rolB and rolC transgenics,respectively) and total reducing power(average 32.7% and 26.4% increase in rolB and rolC transgenics,respectively) compared with untransformed plant.Conclusion: rolB and rolC genes were effective for developing A. annua plants with an enhanced level of phytochemicals.

黄花蒿中青蒿素含量的RP-HPLC法测定

建立黄花蒿中青蒿素的高效液相色谱测定方法,采用柱前衍生RP-HPLC法测定黄花蒿中青蒿素的含量,采用ZORBAXXDB-C18(4.6mm×150mm,5μm)色谱柱,甲醇-0.01mol/L醋酸钠-醋酸缓冲液(pH5.8,体积比62∶38)为流动相;检测波长:260nm;流速:0.8mL/min;柱温:30℃。对广西、沈阳、北京、郑州、苏州和杭州等不同产地的野生黄花蒿样品、以及同一产地不同采集时间黄花蒿样品进行检测,结果表明不同地区青蒿素含量差异很大,同一地区不同采集时间黄花蒿样品的青蒿素含量也有差异。该法准确可靠、重现性好,能准确地反映青蒿素含量的检测。

RP-HPLC法测定青蒿中青蒿素的含量

目的建立青蒿(黄花蒿)干叶中青蒿素的含量测定方法,为甲醇提取青蒿素的工业化生产工艺提供依据。方法采用正交试验设计,优选测定条件,采用Shimadzu Shim-pack VP-ODS C18柱(250 mm×4.6 mm,5μm),流动相为乙腈-水(55∶45),流速为1.0 mL.min-1,检测波长为210 nm。结果最佳的含量测定条件为加40倍量甲醇,超声提取3次,每次50 min。青蒿素在1～40μg范围内与峰面积呈良好的线性关系(r=0.99998),平均加样回收率为99.8%,RSD为1.0%(n=6)。结论该方法简便、准确,适用于黄花蒿中青蒿素的含量测定。

黄花蒿中青蒿素的柱前衍生HPLC-UV分析

研究衍生化条件对HPLC-UV法测定黄花蒿青蒿素含量的影响。建立了黄花蒿青蒿素的柱前衍生HPLC-UV测定方法:即青蒿素标准品溶液(或黄花蒿提取浸膏)以体积分数为95%乙醇补充(溶解)至15.0mL,再加入质量分数为0.2%的NaOH8.0mL,45℃水浴30min后冷却至室温,加入0.08mol/L的HAc10mL摇匀,以95%乙醇定容至50mL容量瓶中;HPLC分离,260nm紫外检测。所得方法检测线性范围为0～200mg/L,相对标准偏差(RSD)值为1.40%,平均回收率(n=5)为100.1%。用该方法测定了黑龙江引种的黄花蒿提取物样品,稳定性RSD值为4.35%,重现性RSD值为4.96%。证明该方法简单、稳定、重现性好。

衍生化条件的优化及不同产地青蒿中青蒿素含量的柱前衍生化-HPLC法测定

Derivation conditions of pre-column derivation-HPLC method used for determinating artemisinin content were compared and selected,and artemisinin content in above-ground part of Artemisia annua L.from seventeen locations was compared by optimal pre-column derivation-HPLC method.The optimal derivation condition is selected via comparing of 0.2% NaOH solution addition(3,4,5,6 and 7 mL),derivation temperature(30 ℃,35 ℃,40 ℃,45 ℃ and 50 ℃) and derivation time(0,2,5,10,20,40 and 60 min) during derivation process.The optimal derivation condition is adding 5 mL 0.2% NaOH solution,then insulating in 40 ℃ water bath for 10 min.Differences of artemisinin content in A.annua from seventeen locations are obvious,the general trend is that artemisinin content decreases gradually with location from south to north,in which artemisinin content in A.annua from Youyang of Chongqing is the highest with a value of 7.08 mg·g-1.The method is simple and accurate with good reproducibility,and can be used to determinate artemisinin content in medicinal material of A.annua.

Studies on the biosynthesis of arteannuin——Ⅳ.The biosynthesis of arteanniuin and arteannuin B by the leaf homogenate of Artemisia annua L.

Arteannuic acid and(-)11R,13-d(?)ydroarteannuic acid are the key intermediates in the biosynthesis of arteannuin by the leaf homogenate of Artemisia annua L.,and arteannuic acid and epoxyarteannuic acid are the intermediates of arteannuin B.and epoxyarteannuic acid can not be transformed into arteannuin by the homogenate.

黄花蒿提取物生产工艺条件及其对乳中共轭亚油酸(CLA)生成的调控作用

黄花蒿乙醇提取物含有多种营养活性物质,主要包括萜类、倍半萜类、芳香族类、脂肪酸类、甾体类、黄酮类、生物碱类等化合物。反刍动物日粮中添加黄花蒿乙醇提取物能够增加其乳中共轭亚油酸(CLA)含量。综述了黄花蒿乙醇提取物的主要营养活性物质、提取工艺条件、营养活性物质的检测方法及黄花蒿提取物在动物日粮中的适宜添加量,旨在为生产CLA乳提供参考。

UPLC-MS/MS法检测青蒿中青蒿素的含量

采用UPLC-MS/MS法检测青蒿中青蒿素的含量,色谱柱Waters ACQUITY BEH C18(50 mm×2.5mm,1.9μm),以甲醇和水梯度洗脱,甲醇-水体积比为40%(0 min)-70%(4.5 min)-40%(5.0 min)-40%(5.5 min),流速0.30 mL/min,柱温30℃,进样量3.0μL,离子模式(ESI+)分析,青蒿素母离子为m/z=283.3。子离子m/z=151.2,m/z=209.4。结果表明,在上述条件下,青蒿素与杂质分离完全,无明显干扰,检出限达到0.174 ng,平均回收率为99.38%,精密度良好,青蒿中青蒿素含量为(1 045.19±27.55)mg/kg,该法适用于青蒿素分析。

黄花蒿bHLH转录因子基因家族鉴定及光调控分析

目的:基于黄花蒿全基因组及转录组数据,进行AabHLH基因家族生物信息学分析及在不同光处理下的基因表达分析。方法:基于基因隐马科夫模型(HMM)对AabHLH基因进行鉴定,利用Pfam、GSDS、MEME等在线分析软件对基因结构、分子进化及保守结构域等进行分析。结果:全基因组水平共鉴定出99条黄花蒿AabHLH基因,包含49对复制基因对;基于系统发育树将其分为13个亚族,所有亚族AabHLH蛋白亚细胞定位于细胞核且均为亲水性蛋白,同一亚族基因具有相似的外显子-内含子结构及保守基序。基因表达模式分析发现,AabHLH基因在不同水平上响应蓝光、红光、远红光、白光调控,推测其中6个亚族基因为光照条件下,7个亚族基因为非光照条件下青蒿素合成途径调控基因。结论:在全基因组水平鉴定了AabHLH基因,为深度解析AabHLH基因功能及其对光调控下青蒿素生物合成调控机制提供参考。

青蒿UPLC指纹图谱的建立与多种成分含量测定

目的:建立30批青蒿样品的UPLC指纹图谱及有机酸类、黄酮类、香豆素类等成分的含量测定方法。方法:采用Agilent Eclipise Plus C18(50 mm×2.1 mm,1.8μm)色谱柱;以0.1%磷酸溶液-甲醇为流动相,梯度洗脱;建立指纹图谱;并对青蒿中有机酸类、黄酮类、香豆素类等9个成分进行含量测定。结果:建立了青蒿UPLC指纹图谱及同时测定多成分含量的方法。9种成分在测定范围内线性关系良好,不同产地的青蒿药材质量存在一定差异。江苏盱眙的青蒿综合评价质量最优。结论:本实验建立的方法准确可靠,可同时测定青蒿中东莨菪内酯、滨蒿内酯、异绿原酸A、异绿原酸B、异绿原酸C、木犀草素、芹菜素、猫眼草黄素、艾黄素的含量。

The transcription factors TLR1 and TLR2 negatively regulate trichome density and artemisinin levels in Artemisia annua

The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density;however,the factors affecting trichome development are largely unknown.Here,we demonstrate that the A.annua R2R3 MYB transcription factor TrichomeLess Regulator 1(TLR1)negatively regulates trichome development.In A.annua,TLR1 overexpression lines had 44.7%–64.0%lower trichome density and 11.5%–49.4%lower artemisinin contents and TLR1-RNAi lines had 33%–93.3%higher trichome density and 32.2%–84.0%higher artemisinin contents compared with non-transgenic controls.TLR1 also negatively regulates the expression of anthocyanin biosynthetic pathway genes in A.annua.When heterologously expressed in Arabidopsis thaliana,TLR1 interacts with GLABROUS3a,positive regulator of trichome development,and represses trichome development.Yeast two-hybrid and pull-down assays indicated that TLR1 interacts with the WUSCHEL homeobox(WOX)protein AaWOX1,which interacts with the LEAFY-like transcription factor TLR2.TLR2 overexpression in Arabidopsis and A.annua showed that TLR2 reduces trichome development by reducing gibberellin levels.Furthermore,artemisinin contents were 19%–43%lower in TLR2-overexpressing A.annua plants compared to controls.These data indicate that TLR1 and TLR2 negatively regulate trichome density by lowering gibberellin levels and may enable approaches to enhance artemisinin yields.

HPLC-ELSD法测定黄花蒿提取物中青蒿素

目的 建立HPLC-ELSD法对黄花蒿提取物中青蒿素的测定方法。方法 采用HPLC-ELSD法。色谱柱：Inertsil ODS-3（150mm×4．6mm，5μm）；流动相为乙腈-水（69：31，用三氟醋酸调pH至3）；体积流量：1．0mL／min；检测器漂移管温度：45C；载气体积流量：1．0L／min；分流：开；柱温：25℃。结果 青蒿素在0．5893～5．8935mg／mL时面积的常用对数值与其质量浓度的常用对数值线性关系良好（r=0．989）；样品平均回收率为98．71％（RSD为0．42％，n=6）。结论 该方法灵敏、准确可靠，可作为黄花蒿提取物中青蒿素的定量方法。

基于UHPLC-MS/MS的药用植物内源性激素含量检测方法及黄花蒿不同器官激素的测定

目的激素是调控植物活性次生代谢产物合成途径的核心调控因子,内源激素结构多样、含量极低,检测难度高,缺乏高效、准确的检测方法。本研究利用超高效液相色谱串联质谱(UHPLC-MS/MS)技术构建了对黄花蒿中茉莉酸、细胞分裂素、赤霉素、生长素、脱落酸等多类植物内源性激素高效定量检测方法。方法使用Agilent Poroshell 120 EC-C_(18)(2.7μm,3.0×150 mm)色谱柱,流动相为0.05%甲酸水-乙腈溶液,梯度洗脱,采用多反应监测模式验证方法可行性,并对黄花蒿不同器官中植物激素进行定量分析。结果本方法中检测的16种植物激素均呈良好线性,加样回收率、精密度和稳定性均符合植物样品的测定要求。利用此方法测定青蒿不同器官的激素含量,结果表明脱落酸、茉莉酸、茉莉酸甲酯均在叶中含量较高,赤霉素和生长素在根部含量高,细胞分裂素在叶和茎中含量高。结论青蒿不同器官的激素测定结果可进一步印证脱落酸、茉莉酸、茉莉酸甲酯在调控青蒿素合成中起着重要作用的相关报道,同时表明该方法检测性良好。本文构建的植物内源性激素的检测方法为进一步探究植物生长发育、抵御外界胁迫的相关机制及次生代谢产物的合成等方面提供了手段。