来自植物青蒿（Artemisia annua L）的青蒿素，作为青蒿素联合疗法（ACT），是目前治疗疟疾的最佳疗法，这种疾病尤其在发展中国家影响儿童和成人。传统上，中国人将青蒿作为茶用于治疗“发热”。近年来的研究表明，青蒿茶浸提物和口服该植物的干叶具有预防和治疗效果。在青蒿叶中存在的复杂化学物质基质似乎增强了青蒿素的生物利用度和功效。尽管这些植物化学物质在抗疟疾活性方面约比青蒿素弱1000倍，但主要是包括其他青蒿素化合物、萜类化合物（主要是单萜和倍半萜）、类黄酮类和多酚酸的小分子。此外，青蒿叶的多糖成分可能增强了青蒿素的生物利用度。啮齿动物的药代动力学研究显示，在接受青蒿叶治疗的Plasmodium chabaudi感染小鼠中，T1/2和Tmax更长，Cmax和AUC更大，而在健康小鼠中则相对较小。在健康小鼠中，与纯青蒿素喂养的小鼠相比，接受干叶青蒿素的小鼠血清中青蒿素水平要高出40倍以上。人体试验数据显示，当以干叶形式给予时，只需要40倍于纯青蒿素的剂量即可获得治疗效果。尽管青蒿素联合疗法对许多疟疾患者仍然负担不起，但考虑到A. annua干叶片生产的成本估计比青蒿素联合疗法低几个数量级，尽管生产能力有所提高。考虑到2000多年来，这种植物一直被用于传统中医治疗发热，并且没有青蒿素耐药性的明显出现，证据表明应将经济实惠的A. annua干叶片纳入用于抗击疟疾和其他青蒿素敏感疾病的药物库。

简介
疟疾每年造成近一百万人死亡，尤其在非洲和儿童中尤为突出，近三十亿人受到其影响。目前，从青蒿（Artemisia annua L.）中提取的青蒿素（见图1）与另一种抗疟疾药物一同使用（即青蒿素联合疗法，ACT）作为首选治疗方法，以减缓药物耐药性的出现。尽管有这些努力，但青蒿素耐药性正在出现，而且持续性和/或无症状的疟疾也可能在疾病传播中起到作用。此外，对于发展中国家来说，ACT的成本高昂，供应不足。

Artemisia annua（青蒿植物的单一克隆，约在花蕾形成时高约2米），青蒿素和基于植物的青蒿素联合疗法片剂。

青蒿素是一种萜类内酯，主要产生和储存于青蒿（Artemisia annua）植物的具有腺毛的叶片和花蕾中，这是一种被认可为安全的药用草本植物。该植物还产生了40多种类黄酮化合物、许多多酚类化合物以及各种其他萜类化合物，包括单萜、倍半萜、二萜和三萜。正如后文所讨论的，这些化合物中许多具有微弱的抗疟疾活性，并且根据转录组分析，许多似乎也是在腺毛中产生和/或储存的，这些腺毛也包含青蒿素。

我们和其他人提出了直接食用青蒿的建议，可以是作为茶浸提物，也可以是通过口服叶片。与口服纯青蒿素相比，我们发现植物材料的存在显著增强了健康和感染Plasmodium chabaudi的小鼠血清中青蒿素的出现。由于干叶中天然存在大量温和的抗疟疾化合物，我们将这种口服干叶治疗称为基于植物的青蒿素联合疗法（pACT）。这些整株植物的方法类似于中国人使用该植物进行的2000多年的传统用法。

要使用类似草药这样的复杂材料生产出具有治疗效果的药物，需要满足一些关键因素：草药产品必须具有治疗效果；草药中关键化学成分的含量必须是可验证的一致的；生产成本也必须是经济有效的。在这里，我们总结并更新了我们最近的一篇评论，该评论涉及青蒿对疟疾的影响，并进一步讨论了pACT的生物利用度和治疗效果，以及如何以一致的剂量廉价地生产这样的草药制剂。

青蒿（Artemisia annua）的预防性使用
茶浸提物、其化学成分和体外研究
直到最近，据我们所知，很少有进行良好控制的研究来检验青蒿茶浸提物中青蒿素和其他化合物的提取、回收和稳定性。van der Kooy等人进行了一项系统研究，对青蒿治疗性茶浸提物的制备进行了研究，并显示了将干燥的青蒿叶浸提的近93%的青蒿素提取出来的情况，但仅在特定条件下。最佳制备方法是：每升9克干重叶片，100°C浸泡5分钟。随后，在室温下储存茶浸提物显示，青蒿素浓度在24小时以上的时间内是稳定的，这对于疟疾流行地区而言是重要的，因为那里没有制冷设备。青蒿素的水溶解度约为50毫克/升，因此从热水茶浸提物中提取的青蒿素量是合理的。其他使用相同提取方案的研究也测量了茶中青蒿素和一些关键类黄酮的提取和稳定性。发现青蒿素在室温下稳定性长达48小时；然而，一些类黄酮的提取效果较差，并且在室温下不稳定。

Carbonara等人检测到了青蒿茶浸提物中的一系列酚类化合物，包括每克干重约0.06毫克的环胞烯醇，浸提条件是约为van der Kooy等人提出的最佳条件（每升9克干重叶片）的4-10倍（约每升38克干重）。茶中大多数测量到的酚类化合物在室温下在浸提后的48小时内保持不变。更近期，Suberu等人在一升青蒿茶中鉴定了毫克级别的酚酸、类黄酮和倍半萜，所有这些化合物在微摩尔或更低的浓度下表现出IC50值（表1）。实际上，茶浸提物本身的IC50值分别为7.6和2.9纳摩尔/升，对氯喹（CQ）敏感的HB3和CQ不敏感的Dd2株的疟原虫来说，比单纯的青蒿素更好，表明茶中成分之间存在协同作用。显然，如果茶浸提物要成为一种治疗选择，那么它必须是经过一致可靠的制备和摄入。正如van der Kooy等人建议的那样，理想情况下，每天应制备一升茶浸提物，并在24小时内均匀地饮用约250毫升，持续几天。

茶浸提物临床试验
Ogwang等人在乌干达进行了一项随机临床试验，测试了青蒿茶作为预防疟疾的方法，共有132名年龄在18至60岁之间的成年农场工人参与了为期12个月的试验。茶浸提物每周消耗一次，每次成人用量为2.5克干叶，茶中含有55-100毫克青蒿素/升。在9个月的时间内追踪疟疾情况，同时在12个月的时间内追踪不良临床效果。在使用青蒿茶的人群中，发生与发热相关的就医事件减少了80%。事实上，一些患者报告称使用青蒿茶超过7年，没有发生疟疾或严重不良事件。尽管这项研究表明每周一次的青蒿茶浸提物消费可能提供预防性保护，但该研究中没有包括儿童或老年人，因此需要进行更多的临床试验，涉及不同人群和年龄组。作者认为，由于单周剂量有效，所以除青蒿素外的其他化合物可能发挥了预防作用，因为青蒿素本身的血浆半衰期较短。

青蒿茶的治疗应用
茶浸提物
Mueller等人和Blanke等人的研究报告了青蒿（cv. Artemis）茶对人类疟疾患者的疗效，但结果有时存在冲突。他们的茶浸提物中含有47-94毫克青蒿素/升，但奎宁治疗的对照组中再发病率要低得多，因此，茶治疗患者中寄生虫再现被归因于再发病而不是再感染。在Blanke等人的试验中，包括了一个安慰剂茶组，在接受茶治疗的患者中，再发病率始终低于那些接受500毫克纯青蒿素治疗的患者。然而，De Donno等人最近表明，在1升青蒿茶浸提物中加入5克干叶对氯喹耐药（W2）和氯喹敏感（D10）株的疟原虫均有效，IC50值分别为5.60纳摩尔/升和7.08纳摩尔/升，这与Suberu等人的研究结果一致。这些后来的体外研究表明，茶应该是有效的，那么为什么与早期的人体试验存在差异呢？制备方法对尽可能保持植物的生化完整性至关重要。最近的体外研究可能使用了更一致地制备的茶浸提物，而不是早期的人体试验，因此浸提物的化学成分变化和植物原料的变化可能解释了不同的反应。

认为茶是单一疗法的论点缺乏实证，考虑到现在已经确认的青蒿茶及其成分的化学复杂性和相关的抗疟疾活性。尽管动物和人体的治疗性茶试验数据相关性较好，但遗憾的是，它们并不支持使用青蒿茶治疗疟疾，因为动物和人体数据都是负面的，青蒿素剂量不易控制，而茶中其他潜在的协同成分也不易控制或提取。尽管如此，茶的使用可能在疟疾预防中起到一定作用，以减少不同社区中疟疾的发生率，或在暂时缓解疟疾方面起到作用，主要是为了预防昏迷或“争取时间”，让来自农村地区的感染者前往存有青蒿素联合疗法的医院或诊所。

Dried leaf A. annua - pACT
最近，Elfawal等人测量了被感染寨卡疟原虫（P. chabaudi）的小鼠的寄生虫血症，这些小鼠分别以两种不同剂量（0.6或3.0毫克青蒿素；24和120毫克/千克）纯青蒿素作为鼠饲料或作为pACT饲料。通过pACT投递的青蒿素至少比纯青蒿素更有效，并且反应持续时间更长，能够降低寄生虫血症。除青蒿素外，在青蒿（Artemisia annua）中已经鉴定出600多种植物化学物质，但目前对这些化学物质的化学性质、制备方法（采收、干燥、储存等）以及整体生物利用度缺乏信息。

在科学文献中，使用干叶青蒿进行的临床试验很少，除了由Mueller等人在刚果民主共和国进行的试验外，几乎没有其他的发表。尽管世界卫生组织不鼓励进行整株植物或茶浸提物的临床试验，一些非洲大学一直在进行他们自己的试验，其中许多试验没有发表，也没有通过聚合酶链反应（PCR）进行结果评估，正如后来的ACT临床试验所做的那样（C. Kasongo与P. Lutgen的个人通讯）。这些试验中的许多使用了青蒿浸提物，与对照组或其他抗疟疾药物进行了比较，如青蒿酸阿莫地喹，显示出浸提物的敏感性更高，而且治疗失败较少。例如，在刚果民主共和国，有54名患疟疾的志愿者接受了为期10天的治疗，用含有青蒿干叶粉末的胶囊进行治疗。每位患者服用15克含有15毫克青蒿素的干叶（叶中青蒿素含量为0.1%）。

在中非的Bangui进行了一项旨在预防术后严重疟疾的研究，将青蒿（Artemisia annua）干叶粉末装入胶囊中，共有25名患者参与，其中22名为1-16岁的儿童。治疗持续时间为3-4天，每天服用0.4-0.5克的青蒿。

迄今为止最具临床定义意义的pACT疗效研究是在肯尼亚西部的Suba地区的国际昆虫生理学和生态学中心（ICIPE）Mbita Field校区进行的。这是ICIPE与肯尼亚医学研究所之间的合作项目，并且是一项开放标签的、非随机的临床试验，主要旨在评估逐渐增加剂量的pACT片剂的疗效、安全性和耐受性。这些片剂是由坦桑尼亚的一个名为Natural Uwemba System for Health的非政府组织制造的，使用了在坦桑尼亚高地（海拔2000-2200米）种植的青蒿的混合物。叶子在开花前刚好被收割，然后在阴凉处干燥约3周，然后压碎、粉碎、均质化，并在常温下压制成500毫克的片剂。片剂强度良好，不需要任何赋形剂。通过带有二极管阵列检测器的高效液相色谱法（HPLC），对随机选取的100片片剂批次的己烷提取物进行分析，结果显示片剂中青蒿素的含量一致，为0.74% ± 0.06%（即每片约含有3.7毫克）。

试验的四个队伍中，每队有12名年龄在15至56岁之间（平均23.42岁）的同意接受的疟疾患者。根据Giemsa染色的血液涂片，寄生虫血症为0.02%-4%，血红蛋白水平>8 mg/dL。每个队伍接受了四种递增剂量的青蒿片剂，分别为第一天两次2-5片，接下来的5天每天两次1-4片（见表2）。治疗一周后，不同队伍的三名患者的血涂片中出现了寄生虫的再现；然而，所有剂量在临床和寄生虫学上对疟疾的消退都是有效的，第28天复发率为9%-20%，且没有可测量的毒性。

与通常的纯青蒿素大剂量治疗相比，这种治疗通常在第1天给予1000毫克，接下来的第2-7天每天给予500毫克，该剂量给了227名疟疾患者[39]。测得的pACT治愈率也与或高于其他使用纯青蒿素[40,41]（如青蒿酸、青蒿醚等[42]）的结果相当。此外，使用pACT的积极治疗反应似乎在第二个测试剂量水平之后与剂量无关（见表2[20]）。尽管ICIPE[20]试验中使用的口服剂量远低于任何茶叶研究，但再发率远低于茶叶研究，通常优于使用纯青蒿素的研究[39]（见表2）。事实上，通过pACT提供的总青蒿素约100毫克用于全疟疾治疗的效果，比Giao等人使用的4000毫克纯青蒿素效果更好[39]（见表2）。这种40倍的差异与Weathers等人的早期药代动力学研究结果相吻合，该研究表明将药物作为pACT给予时，其生物利用度提高了45倍。

这些结果表明，pACT中天然植物化学物质的混合物在口服片剂中尤为重要。结果也与中国关于感染P. berghei的小鼠的研究一致，该研究比较了纯青蒿素与粗制的青蒿提取物的效果[43]，以及Elfawal等人和Weathers等人的研究[23][22]。在所有三项研究中，所施用的产品青蒿素水平相当，但粗制制剂和pACT在减少寄生虫血症方面至少比纯青蒿素高出3.5倍，这表明在提取物和口服干叶中的非青蒿素成分可能具有协同作用。

口服青蒿素
赵等人早期的药代动力学研究表明，口服或直肠给药的双氢青蒿素在人体内的生物利用度比青蒿素更高。口服给药的双氢青蒿素的Cmax、Tmax和T1/2分别为0.13-0.71 mg/L、1.33 h和约1.6 h；而纯青蒿素的这些值分别为0.09 mg/L、1.5 h和2.27 h。Alin等人比较了口服青蒿素和青蒿素-氯喹联合疗法用于治疗恶性疟疾的效果。感染和未感染的患者的药代动力学参数相似。单剂量后，青蒿素的生物利用度未发生变化。有趣的是，当比较治疗失败与成功时，药代动力学表现相似，表明仅测量青蒿素药代动力学的研究不足以预测治疗成功。Ilet等人还回顾了恶性疟疾患者中青蒿素的药代动力学，报告了9.1 mg/kg的剂量，与Alin等人的剂量相当。Cmax和Tmax值与Alin等人的报道并没有太大差异。

注：Cmax（最大血浆浓度）是指在药物给药后血浆中药物浓度达到的最高水平，通常表示为浓度单位（如毫克/升）。

        Tmax（达到最大血浆浓度的时间）是指在药物给药后血浆中药物浓度达到最大值所需的时间。通常表示为时间单位（如小时）。

在Ilet等人对青蒿素及其衍生物的药代动力学参数的回顾中，口服纯青蒿素剂量在健康受试者中约为6-11 mg/kg，Cmax为0.15-0.39 mg/L。剂量似乎没有太大影响。Ashton等人的早期研究比较了每人250、500和1000 mg的青蒿素剂量，Cmax和T1/2均表现出剂量依赖性增加，分别为0.21、0.45和0.79 mg/L和1.38、2.0和2.8 h，但Tmax保持相对稳定在2.3-2.8 h。

饮食是口服药物的重要考虑因素，Dien等人比较了青蒿素口服剂量在空腹和饭后的情况，空腹和非空腹受试者的Cmax值相似。青蒿素与食物一起摄入似乎并不影响青蒿素的吸收。相比之下，Weathers等人后来进行的一项啮齿动物研究观察到，当青蒿素作为复杂的植物材料pACT的一部分摄入时，小鼠的血清中的药物量约增加了45倍，而口服纯药物后60分钟内在血清中检测不到青蒿素。然而，当青蒿素与小鼠饲料一起摄入时，即使在60分钟后，也可以在血清中检测到青蒿素。在Ashton等人的研究中，青蒿素以9.1 mg/kg的剂量每天连续给药7天，分别在第1、4、7和21天进行测量。在第1天，血浆中的Cmax和T1/2与使用类似剂量的其他研究中的数据相似。然而，在第4天和第7天，Cmax减小，而T1/2增加，表明尽管青蒿素每天给药7天，但在第一剂后可能难以被吸收或在第一剂后降解。在第三次给药后，Cmax从0.31降至0.11 mg/L，而T1/2从3.0增至4.8 h。这些结果表明，青蒿素可能被代谢或在体内其他部位积聚。

在肝脏中，细胞色素P450（CYP450）酶将青蒿素代谢为去氧青蒿素、去氧双氢青蒿素、9,10-二氢去氧青蒿素以及一种名为“结晶7”的代谢物。Svensson等人利用人类肝微粒体显示，CYP450的活性，尤其是CYP2B6的活性，与青蒿素血清水平的降低呈负相关。在Ashton等人研究的间歇给药中，CYP450的水平在给药14天后被允许下降，然后再给药，此时的Cmax从0.11增至0.20 mg/L，而T1/2从4.8减至2.7 h。通常情况下，青蒿素在体内的最大浓度随着剂量增加而增加，T1/2在口服纯青蒿素的报道试验中大约为1.4-4.8小时，所以增加和延长青蒿素的治疗可能会减少再发率。

茶叶提取物中的青蒿素
除了Räth等人[16]外，关于人体内口服茶叶提取物中青蒿素的药代动力学的报道很少。在Räth等人的研究中，青蒿素的Cmax在摄入后0.6小时为0.24 mg/L。含有94.5 mg青蒿素的茶叶提取物的Cmax相当于250 mg纯青蒿素的剂量，但Tmax明显较短，为0.6小时，而纯青蒿素的Tmax为2.8小时。与纯青蒿素相比，茶叶提取物中青蒿素的较短半衰期可能解释了观察到的更高再发率。尽管茶叶提供的青蒿素似乎更易于被吸收，但其较短的T1/2（约为0.9小时）与纯青蒿素的T1/2（约为2小时）相比，表明每天给药两次以上可能更有益；实际上，建议每天给药四次。

临床茶叶提取物试验中不可接受的高再发率归因于低血浆浓度，几乎比传统剂量（60公斤人体重500毫克或8.3毫克青蒿素/公斤）的纯青蒿素低40%。尽管未指明，但据估计，茶试验剂量约为1.5 mg/kg，接近赵等人使用的1.1 mg/kg纯青蒿素剂量，远低于传统上认为的8.3 mg/kg的药理有效剂量。尽管如此，茶叶剂量的青蒿素Cmax为0.24 mg/L，几乎是纯青蒿素（Cmax=0.13 mg/L）的两倍，如赵等人所测量。A. annua茶叶对在塞内加尔皮金收集的40个恶性疟疾病原体的抗疟活性也很强（平均IC50为0.095 µg/mL）。

干叶（pACT）给药的青蒿素
到目前为止，尚无人体pACT的药代动力学研究。在一项对喂食青蒿素的健康小鼠进行的小型 PK 研究中，通过 pACT 递送的青蒿素比作为纯药物递送的青蒿素多约 45 倍 [21]。最近，在健康小鼠和受 P. chabaudi 感染的小鼠中，以 100 mg/kg 体重剂量的青蒿素治疗健康小鼠和 P. chabaudi 感染小鼠，对青蒿素及其肝脏代谢物之一脱氧青蒿素的药代动力学进行了 120 分钟的比较[22] 。在pACT治疗的健康小鼠中，青蒿素的一阶消除速率常数估计为0.80/h，相应的T1/2为51.6分钟。Cmax和Tmax分别为4.33 mg/L和60分钟。AUC为299.5 µg·min/mL。青蒿素的一阶吸收速率常数估计为1.39/h。相比之下，在感染小鼠中，pACT处理后的AUC更大，为435.6 µg·min/mL。感染小鼠的血清中的青蒿素水平在研究期内持续上升。因此，消除半衰期T1/2不能确定，因此Cmax和Tmax只能估计为≥6.64 mg/L和≥120分钟。然而，感染小鼠中的青蒿素和去氧青蒿素水平相对于健康小鼠有所增加。

注 ：T1/2通常代表药物的半衰期，即药物在体内消失一半所需的时间。半衰期是描述药物消除速度的重要参数，它可以帮助确定药物的给药方案和剂量。半衰期越长，药物在体内停留的时间越长，通常需要更少的剂量来维持治疗效果。反之，半衰期较短的药物可能需要更频繁的剂量或更高的剂量才能保持疗效。

     AUC代表"曲线下面积"（Area Under the Curve），在药物学中，它是药物浓度随时间变化的曲线下方的面积。AUC可以提供关于药物在体内曝露的总量的信息，通常用于评估药物的生物利用度（bioavailability）和药代动力学（pharmacokinetics）。对于给定的剂量和给药途径，AUC值越高，通常表示药物在体内的吸收和分布越好。

青蒿素的一阶吸收速率常数（k）是描述药物从给药部位（例如口服）到达血液循环的速率的参数。具体来说，这个值表示每小时有多少药物被吸收到血液中。在这种情况下，1.39/h 表示在每小时内大约有1.39倍的青蒿素从给药部位吸收到血液中。

总的来说，在健康受试者中，青蒿素浓度下降，去氧青蒿素水平升高，而在感染小鼠中，青蒿素水平在研究期内持续上升，而去氧青蒿素水平先下降，然后保持稳定，因此感染似乎减缓了小鼠将青蒿素代谢为去氧青蒿素的能力。A. annua中的许多化合物抑制P. falciparum和CYP34A。在研究中使用的高剂量（100 mg/kg）中，血清中的青蒿素和去氧青蒿素的测量几乎相等，表明使用了过量的青蒿素。

植物材料的存在影响了青蒿素的药代动力学。当青蒿素剂量为100 mg/kg体重时，60分钟内未检测到小鼠血清中的青蒿素。然而，当存在植物材料时，如小鼠饲料或A. annua pACT，青蒿素在血清中的水平升高至2.44和4.32 µg/mL，分别表明即使是小鼠饲料，植物材料的存在也对青蒿素在血液中的出现产生了重大积极影响。据我们所知，这是关于动物或人类口服A. annua的唯一药代动力学数据。

青蒿素以外的治疗化合物在青蒿中的作用

类黄酮
青蒿富含精油、香豆素、多酚、多糖、皂苷、萜类化合物和类黄酮。青蒿中的类黄酮和其他化合物的含量随着发育阶段的不同而变化，有些在全盛期时含量最高[57]。青蒿中有40多种类黄酮[13]，至少有11种，包括青蒿素、卡斯提辛、菊黄素、菊黄醇-D、酢苣菊素、乌檀酮、山奈酚、芦丁、杨梅素、槲皮素和芸香苷，据报道对疟原虫疟疾具有微弱的治疗效果(Table 1[52–54,58])。这些类黄酮中的一些被证明能够使青蒿素对体外疟原虫的IC50提高多达50%，表明它们具有协同作用(Table 1[52])。Elford等人[53]还表明，虽然卡斯提辛（5-羟基-2-（3-羟基-4-甲氧基苯基）-3,6,7-三甲氧基色素-4-酮）与青蒿素呈现协同作用，但与氯喹没有协同作用，这表明了不同的相互作用机制。将卡斯提辛与青蒿素结合使用抑制了寄生虫介导的运输系统，该系统控制了疟疾感染红细胞中肌醇和L-谷氨酰胺的内流。这些类黄酮与青蒿素之间明显的协同作用表明，类黄酮对于青蒿（无论是整片干叶还是茶叶）的有效使用可能是重要的。

许多类黄酮具有抗疟原虫作用，在体外抑制P. falciparum在肝细胞中的生长，正如报道的膳食类黄酮[54]。据我们所知，尚无关于青蒿输送类黄酮的药代动力学的报告。一些类黄酮据报道具有较长的血浆半衰期；例如，在青蒿和大多数水果中发现的槲皮素，其血浆半衰期为27小时[59]。槲皮素 [2-(3,4-二羟基苯基)-3,5,7-三羟基-4H-香豆素]，也在大蒜中发现，抑制寄生虫的生长，并且对不同品系的疟原虫表现出差异性活性(Table 1[54,58])。芸香苷，是槲皮素的鲁丁糖苷 [α-L-鼠李糖苷-(1→6)-β-D-葡萄糖苷]，显示出类似的结果，表明糖基成分对抗疟疾活性影响不大(Table 1[58])。已知类黄酮在体内存在时间超过5天；这可能解释了奥格旺等人[30,31]报道的青蒿茶浸出物每周一次剂量引起预防性作用。许多膳食类黄酮在体外抑制疟原虫的生长，但是据报道，其在饮食中的含量不足以提供对疟疾的保护[54]。然而，像青蒿这样富含类黄酮（例如，高达0.6%）的植物可能会与青蒿素协同作用，以防止经常食用时的疟疾。

黄酮槲皮素 [2-(3,4-二羟基苯基)-5,7-二羟基-4-香豆素] 在艾迪拜阿中的含量达到0.0023%干重，并且已用于治疗多种疾病，包括咳嗽、腹泻、痢疾、糖尿病、癌症和疟疾。尽管槲皮素的IC50值约为11 µmol/L[54]，并且是青蒿中发现的活性最强的抗疟疾黄酮之一，但无法将其在研究之间进行比较，如Ganesh等人所指出的[58]（见表1）。不同黄色素对抗疟疾的反应似乎受到被测试的疟原虫品系的影响。槲皮素还通过抑制寄生虫的脂肪酸合成，阻止寄生虫生长超过幼年滋养体期阶段。这种抗疟疾活性的机制似乎与抑制寄生虫脂肪酸生物合成有关。这些脂质是寄生虫用来将血红素解毒为血红素的必需物质[60]。独立于人体宿主，顶复门虫寄生虫使用脂肪酸生物合成途径。途径中的酶，如NADPH依赖性β-酮酰-ACP还原酶（FabG），是潜在的抗疟靶点。在30种研究的黄酮中，槲皮素和槲皮素对这些酶的抑制作用具有最低的IC50值，并且还对多种品系的P. falciparum表现出体外活性[60]。

异生维特辛（5,7-二羟基-2-(4-羟基苯基)-6-[(2S,3R,4R,5S,6R)-3,4,5-三羟基-6-(羟甲基)氧杂环-2-基]色素-4-酮）是另一种类黄酮，是芸香素的6-C-葡萄糖苷，在青蒿茶浸出液中的浓度高达100毫克/升，并且具有微量的抗疟疾活性(Table 1[19,28])。异生维特辛抑制了脂质过氧化和黄嘌呤氧化酶活性，并保护细胞免受ROS损伤，其总LD50 > 400 µmol/L[61]。

萜类化合物
柠檬烯（1-甲基-4-(1-甲基乙烯基)-环己烯）是“桉叶素”组成部分之一，包括1,8-桉叶素（尤加利醇）、柠檬烯、肉豆蔻烯、α-蒎烯、β-蒎烯、香叶烯和α-桉叶醇[62]；其中许多影响特定阶段的疟原虫物种。例如，柠檬烯通常在青蒿中以每千克7毫克的含量存在[14]，并抑制疟原虫的异戊二烯生物合成[63]，以及在环和滋养体阶段的发育[64]。尤加利醇影响滋养体阶段[65]。柠檬烯还抑制了P. falciparum的蛋白异戊二烯化，停止了治疗48小时内的寄生虫发育[64]。在这些试验中，对体外疟原虫的IC50为2.27 mmol/L，比van Zyl等人测得的533 µmol/L的IC50高出一倍多[55]。柠檬烯及其代谢物在血浆中至少保留48小时[66]，因此药代动力学是有利的，这对于消除配子体和疟疾传播是重要的。

挥发性单萜烯α-蒎烯（4,6,6-三甲基双环[3.1.1]庚-3-烯）在植物中的含量可达到干重的0.05%，其IC50为1.2 µmol/L，与奎宁的0.29 µmol/L相当[55]。尤加利醇（1,8-桉叶醇）在青蒿的挥发油中可能占到30% [0.24%-0.42% (V/DW)]，并且强烈抑制前炎症细胞因子肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6和IL-8[68]。对早期滋养体阶段的耐氯喹和敏感氯喹疟原虫品系都有影响[65]。

尤加利醇（1,3,3-三甲基-2-氧杂双环[2,2,2]辛烷）也是挥发性的，在吸入或口服后迅速进入血液[69,70]。在IC50为0.02 mg/mL且毒性低（约25 mg/mL的LD50）的情况下，口服或吸入都是合理的[65,71]。事实上，尤加利醇的浓度可以在60分钟内达到15 µg/mL[69]，表明其可能用作抗疟疾吸入剂。

青蒿酮（3,3,6-三甲基-1,5-庚二烯-4-酮）是某些品种的青蒿的主要成分，但几乎没有研究。像姜黄素[72]这样的其他酮类化合物已被认为是β-血红素合成的抑制剂，因此青蒿酮可能起着类似的作用，并影响血红素晶体的形成。尽管血红蛋白对红血球内的裂殖子中寄生虫的存活和增殖至关重要，但它会留下像血红素这样的有毒碎片。寄生虫随后将血红素中的Fe2+氧化为Fe3+形成血红素，这是一种无毒的不溶性聚合物晶体，称为β-血红素（也称为赫莫因），它还抑制了细胞介导的对寄生虫的免疫。青蒿叶水提取物抑制了β-血红素的合成[73]。

挥发油通常含有大量的单萜烯，这些单萜烯可能增强青蒿素的抗疟疾效果，甚至逆转了对青蒿素的P. berghei的观察到的耐药性[74]。单萜烯倾向于在青蒿的盛花期更高[75]，但在高温或阳光下干燥或将干燥的叶子压缩成片剂时会急剧减少[13,76]，特别是在干燥叶片中。虽然单萜烯具有一定的抗疟疾潜力，但大多数单萜烯相当挥发，因此它们在治疗中可能不如非挥发性黄酮类、酚酸和较高分子量的倍半萜重要。

与α-蒎烯和尤加利醇不同，樟脑（1,7,7-三甲基双环[2.2.1]庚烷-2-酮）没有报道抗疟活性，但可能占青蒿挥发油的43.5%。考虑到樟脑比尤加利醇或α-蒎烯的挥发性要低（熔点分别为204°C、176°C和155°C，闪点分别为54°C、49°C和33°C），它可能在增强从胃肠道到血液的疏水分子（如青蒿素）的运输中发挥作用[21,22]。樟脑也可能影响胸腺细胞的存活率，并通过产生T细胞来帮助发展疟疾免疫力[79]。在50 µg/mL的浓度下，樟脑增加了培养胸腺细胞的存活率[80]。

倍半萜烯香叶醇（3,7,11-三甲基-1,6,10-十二烯-3-醇）的IC50为0.99 µmol/L，阻断了寄生虫的红细胞内期间发育阶段（Table 1[55]）。巴西亚马逊盆地的印第安人用三色堇叶蒸气治疗疟疾；香叶醇被确定为导致裂殖子期100%生长抑制的活性成分[81]。香叶醇水平随测试的培养品种而异，埃塞俄比亚植物中发现了最高含量之一[82]。青蒿素类化合物的其他倍半萜烯最近才被显示出在微摩尔/L水平上具有抗疟活性，与植物中发现的其他化合物类似(Table 1[19])。这些青蒿素类化合物被提取到青蒿茶中，根据它们的相对浓度和目标寄生虫品系的不同，与青蒿素显示出不同的相互作用。例如，对于CQ敏感的疟原虫HB3品系，arteaninn B与青蒿素显示出加成相互作用，而对于CQ不敏感的Dd2品系，相互作用则是协同的。

酚酸
迷迭香酸（（2“R”）-2-[[(2“E”)-3-(3,4-二羟基苯基)-1-氧代-2-丙烯基]]氧基]-3-(3,4-二羟基苯基)丙酸）和绿原酸（（1S,3R,4R,5R）-3-{[(2Z)-3-(3,4-二羟基苯基)丙-2-烯酰基]氧基}-1,4,5-三羟基环己烷羧酸）是在各种青蒿品种中发现的强抗氧化剂[56]。在Caco-2研究中，这些酸显著抑制了CYP3A4的活性，这是一种负责将青蒿素代谢为脱氧青蒿素的肝P450之一，这是药物的非活性形式[50]。这些和其他酚酸存在于青蒿茶浸出液中[19]。两种酚酸的IC50约为65 µmol/L（Table 1），并且还显著减少了白细胞介素IL-6和IL-8的分泌，因此增强了抗疟活性并减少了炎症[56]。

青蒿中常见的其他化合物可能影响pACT的有效性
尽管其他药用植物种类的药代动力学更好地研究，但是青蒿的药代动力学在最近的一些研究中获得了一定程度的了解。然而，大多数药代动力学的数据来自单独的类黄酮或挥发性单萜烯或酚酸。截至目前，青蒿在体内的药代动力学尚未研究清楚。

在许多研究中，虽然已经确定了活性成分，但它们在青蒿的浓度范围非常广泛，因此无法对它们的活性浓度-效应关系进行比较。例如，有报道称，尤加利醇的IC50为1-2 mmol/L，樟脑为2-3 mmol/L，α-蒎烯为5-20 mmol/L，青蒿素为0.15-2.5 mmol/L，而槲皮素为11 mmol/L。最后，尽管青蒿提取物具有较低的IC50，但某些化合物与药物在人体内的浓度之间可能存在几个数量级的差异，这表明其在体内的活性可能无法解释。

因此，我们不能依靠青蒿的单一化合物提供理论上的最低活性剂量，而且通常不能保证在治疗中达到足够的治疗浓度。此外，许多活性成分的疗效被寄生虫的抗药性所减弱。虽然许多化合物都对CQ和青蒿素耐药的品系有不同程度的抑制作用，但它们在高浓度下也会受到影响[82]。

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生产考虑

与传统提取方法相比，对于植物全叶制剂（pACT）生产和提取青蒿素的生产成本，由于生产成本通常是严格保密的秘密，公开可比较的成本估算很少。然而，可以从de Vries等人的研究中估算成本，他们报告从含有0.6%青蒿素的青蒿植物中回收1公斤青蒿。随着单元操作（unit ops）数量的增加，下游加工成本和产品损失也会增加，这一事实通常没有得到广泛认识。事实上，对于生物技术过程，回收率可以在9%至51%之间。举例来说，如果一个4步过程的每一步都是95%的高效率，那么整个过程的最终效率大约是81%，而一个步骤的95%效率的单步过程则具有95%的整体回收率。提取青蒿素（eAN）与pACT-AN的描述过程步骤如图2所示。从收获的干叶到准备包装或转化为交付药物（如蒿甲醚或青霉素）的材料，pACT有一个单元操作，而eAN有八个。提取溶剂和其他化学品显然不再是成本的一部分。因为eAN有8个单元操作而pACT只有一个，而且eAN的至少有两个单元操作涉及大量的热量，所以pACT的能源成本至少减少了90%。劳动力、利息、折旧和维护成本也受到单元操作数量的影响，因此我们估计，减少了七个单元操作步骤后，这些成本将减少约88%。虽然可能存在更好的提取过程，但根据de Vries等人的分析，我们对于pACT生产成本的估计约比提取青蒿素的成本少了30%。de Vries等人提供的数据是基于0.6%的青蒿素含量，因此如果收获更高产量的培育品种，成本将按比例下降。此外，成本再次下降，因为pACT无需将青蒿素转化为蒿甲醚或青霉素；这些转化是必要的，因为它们比纯青蒿素具有更高的生物利用度，而这对于pACT并不是一个问题。

植物基青蒿素联合疗法生产与从干燥收获的叶子中提取的青蒿素到可用于包装（植物基青蒿素联合疗法）或转化为蒿甲醚或青蒿酯（提取青蒿素）的产品之间的比较。 AN：青蒿素； eAN：提取青蒿素； pACT：基于植物的青蒿素联合疗法。

表3
基于植物源青蒿素联合疗法治疗成年患者的预估数量

对于含有A. annua培育品，根据不同的干叶吨位治疗的患者数量

注：
1. 假设：每位成年患者需要100毫克青蒿素（AN）连续6天进行治愈；在0.7%和1.4%的AN情况下，单个成年人总疟疾治疗所需的干叶分别约为15克和73克；
2. 低于东非所有地区报告的平均2.5 T/ha；
3. 等于在美国马萨诸塞州Stow进行的A. annua SAM（圣麻田间试验）的最大获得量。A. annua：青蒿。

A. annua的干叶产量在全球各地存在差异。“在东非，平均产量为2.5吨/公顷（范围为0.75-4.2）…”[112]。根据我们的田间试验[113]、报道的东非A. annua叶片平均产量[112]以及肯尼亚人体试验中使用的剂量[20]，可以估算出干叶产量，并根据生物质中青蒿素的含量，估算出pACT能够治疗的成年患者数量（见表3）。

当前的ACT药物与pACT
根据肯尼亚人体疟疾试验[20]中获得的用药信息，每位成年患者在6天内总共需要约100毫克青蒿素进行疟疾治疗，因此对于含有0.7%青蒿素的A. annua叶片，需要15克干叶进行6天的治疗过程。在每公顷收获2吨干叶的情况下，可以为127,260名成年患者提供疟疾治疗（见表3）。对于含有1.4%青蒿素的叶片，只需要7.5克干叶，因此从每公顷产出2吨叶片的土地上，可以为两倍数量的患者提供治疗（见表3）。显然，选择含有更高青蒿素水平的品种将大大增加每公顷可治疗的患者数量。

根据《消除疟疾倡议》，从一吨纯化青蒿素可以提供176万名成年患者使用青蒿甲醚/氯喹进行治疗，以及250万名成年患者使用富马酸阿替米司汀/阿莫地喹进行治疗[114]（见表4）。使用同样一吨青蒿素等效物，但通过含有0.7%青蒿素含量的pACT给药，将收获约142.8吨干A. annua叶。根据肯尼亚人体疟疾试验中的用药数据（见表2[20]），每位患者需要15克干叶，因此可以治疗8.64百万名成年患者，约为当前任何一种ACT药物的四倍。因此，pACT的实际成本主要取决于干叶的成本及其青蒿素含量。

估计目前的青蒿素联合治疗与基于植物的青蒿素联合治疗可治疗的患者数量

注：假设pACT进行6天的治疗，每位患者接受15克干叶进行完整的疟疾治疗，对于含有0.7%青蒿素的叶片。要获得与提取药物1吨等量的青蒿素，需要收获142.8吨含有0.7%青蒿素的干A. annua叶。 AL：青蒿甲醚/氯喹；AS/AQ：富马酸阿替米司汀/阿莫地喹；pACT：基于植物的青蒿素联合治疗。

尚未发表的Rich和Weathers实验室的数据表明，pACT可以防止青蒿素耐药性的出现；植物本身似乎就起到了自己的联合疗法（pACT）的作用。这将消除当前使用的联合疗法中包含辅助药物的必要性。辅助药物的成本至少与青蒿素部分的药物相当[6]。因此，消除额外的辅助药物可能会导致至少额外50%的成本降低，因此最终的pACT成本降低保守估计远低于当前的ACT疗程成本。

考虑到A. annua是无毒的，安全的口服，剂量可能不需要针对儿童进行调整。另一方面，青蒿素叶味苦，因此掩盖味道，也许是用糖，应该有助于儿科治疗。我们最近的模拟消化研究显示，向pACT中添加蔗糖（蔗糖）并不会显著改变消化后释放的青蒿素量，并且还可以使释放的黄酮类化合物的量增加一倍[115]。

与新兴的青蒿素来源或其他新型抗疟疾药物的比较
至少有三种其他新兴的抗疟疾治疗技术：合成青蒿素[116]，通过基因工程微生物生产的半合成青蒿素（SSA）[117]，以及一种单剂量药物OZ439[118]。2013年初，赛诺菲/PATH药物开发计划宣布他们将能够在2014年生产多达60吨的SSA，价格约为每千克400美元，取决于数量；赛诺菲现在拥有SSA的世界卫生组织预认证[119]。尽管价格与目前约每千克550美元的价格相比并没有便宜多少，但供应将更或多或少是无限的。尽管大量SSA的生产可能看似有利，但也存在一些严重的劣势，每种新合成抗疟疾药物和pACT的一些优势和劣势比较见表5。

表5
新兴抗疟疾治疗技术与植物基青蒿素联合疗法的比较

 青蒿素：Artemisinin

质量保证考虑

农业质量
种植青蒿的传统和成本最低的方法是使用种子，在发展中国家，农民更喜欢将一季的种子保存到下一季。然而，即使是优质的种子也会导致A. annua植物在代代之间差异很大（请参阅Ferreira等人的综述）。青蒿的茎插芽在约两周内很容易生根，因此建议通过根插芽的无性繁殖来消除这种变异性。虽然这种繁殖方法对于大型种植园来说不划算，但对于几公顷或受控环境农业来说，它是有效的。考虑到仅种植几公顷青蒿就能治疗大量患者（见表3），建议通过根插芽的无性繁殖。由于pACT疗法涉及直接消费植物的干叶，所以收获的叶材必须保持清洁，这在受控环境农业和遵循良好农业规范的情况下是最容易做到的，尤其是应用于新鲜农产品。然而，受控农业可能会导致农业就业机会的减少，这是一个需要在当地评估的问题。或者，必须在田间收获和收获后储存过程中非常小心，以免影响产品的质量。世界卫生组织已经为了提取青蒿素而专门制定了良好的农业实践标准，用于一般的药用植物，以及用于减少草药药品污染。

化学一致性和定量化
为了向患者提供可靠的治疗剂量，收获的青蒿干叶必须具有可靠和一致的成分。无性繁殖提供了所需的一致性。最近，我们展示了在实验室、田间和家庭花园中在三年时间内种植的植物的青蒿素含量的单个克隆（SAM）为1.38% ± 0.26%（w/w）。因此，尽管培养和环境条件有所不同，但可以实现主要治疗成分的一致水平。此外，收获的叶材的含量当然不是成品的保证，例如，压缩的叶片片剂。Weathers等人的分析表明，尽管在片剂压缩后青蒿素含量非常稳定，但其他成分却变化很大。例如，虽然黄酮类化合物在片剂压缩后增加了，但更易挥发的单萜烯类化合物却显著减少。因此，监测入库的收获材料的组成和最终产品的组成配置是至关重要的。

已经使用复杂且昂贵的分析方法来分析青蒿中的许多成分，但这些方法并不是测量和保证产品质量的必要条件。青蒿素很容易提取，然后可以使用各种薄层色谱法进行定量，并使用对甲醛茴香醛染料进行可视化。其他关键成分如黄酮类化合物也可以使用薄层色谱法进行分离，并使用紫外光下加氯化铝试剂进行可视化。也可以使用廉价的可见光谱法通过加氯化铝法来定量总黄酮类化合物，使用槲皮素作为廉价的标准物质。据我们所知，目前没有廉价而可靠的分光光度法可用于测量复杂植物提取物中的青蒿素。

社会经济利益

其他疾病
青蒿素及其衍生物也对许多病毒[129]、各种人类癌细胞系[130–133]以及一些被忽视的热带疾病，包括血吸虫病[134]、利什曼病[135,136]、锥虫病[137]和一些牲畜疾病[133,138]具有有效性。

尽管在公共卫生重要性方面排在疟疾之下，但血吸虫病、利什曼病和锥虫病每年的感染估计分别约为2400万、130万（其中0.3万是内脏型，1万是皮肤型）和30000[139]。这些疾病以及许多其他疾病对青蒿素类药物治疗有响应。尽管IC50约比疟原虫高出约1000倍，但通过口服pACT[20–22]，青蒿素的明显更高的生物利用度可能会减少治疗所需的药物量。目前，pACT尚未在体内针对除疟疾以外的其他疾病进行过测试。

对于艾滋病病毒/获得性免疫缺陷综合症（HIV/AIDS）患者，疟疾治疗更加复杂。疟疾和HIV共感染在非洲造成了主要的健康负担，主要是因为“现在已经明确，HIV感染会导致更高的疟疾发病率和更严重的疟疾表现”。免疫系统受损，艾滋病患者更容易感染疟疾，而且对疟疾治疗的反应也较慢。此外，在Tusting等人的荟萃分析中，社会经济发展与更好的疟疾治疗结果强相关。最近，青蒿素已经展示出抗HIV活性，因此口服该草药的干叶不仅可以治疗疟疾，还可以提高HIV/AIDS患者的福祉。

农业、就业和自主权
青蒿在75多个国家种植。2011年，约有163吨青蒿素从主要位于中国、越南和东非（包括马达加斯加）的种植园和小规模持有者农场提取出来；价值约为每公斤550美元。随着Sanofi开始生产半合成青蒿素，预计2014年将生产60吨，预计价格约为每公斤400美元。随着这种新型青蒿素的生产，荷兰皇家热带研究所预测，天然青蒿素的市场将大幅动荡，从而破坏了农民的安全。热带研究所进一步担心，“制药公司将积累对生产过程的控制和权力；青蒿生产者将失去一种收入来源；在疟疾流行的地区进行的本地生产、提取和（可能）制造ACT的工作将转移到西方制药公司的主要生产地点”，破坏了这些已经贫困的国家脆弱的经济体系。中国和非洲的小规模农户的平均种植面积约为0.2公顷，因此，尽管实施pACT可能不需要像提取青蒿素那样多的农业土地，但它仍然可以帮助小规模持有者获得一种收入来源。我们估计，可以建造成本小于50000美元的本地微型制造厂，并生产质量控制的pACT片剂，其成分可轻松验证。我们的整体方法，如图3所示，可以实现对疟疾和可能对其他青蒿素敏感性疾病的地方控制，同时提高人口的社会经济地位。

植物性青蒿素联合疗法生产总体方案。 pACT：基于植物的青蒿素联合疗法； TLC：薄层色谱法。

结论

越来越多的证据表明使用青蒿叶片治疗疟疾以及可能的其他疾病的治疗效果。植物中复杂的抗寄生虫化合物混合物似乎解释了其治疗活性，动物和人类试验支持了这一说法。同时，显而易见的是，使用青蒿素联合疗法的成本仅为当前或新兴抗疟疾疗法的一小部分。同样，最近的证据表明，持续性和/或无症状疟疾的存在可能需要更具预防性的方法来使用青蒿素联合疗法，甚至是青蒿茶。考虑到在2000多年的历史中，这种植物被用于中医传统治疗发热，而青蒿素抗药性并没有明显出现，综合考虑所有累积证据，主张将青蒿素联合疗法纳入抗击疟疾的药物库中，极有可能也包括其他疾病的治疗。

核心提示
从植物青蒿（Artemisia annua L.）中提取的青蒿素及其衍生物是目前最佳的抗疟疾治疗药物，以青蒿素联合疗法（ACT）的形式使用。然而，在疟疾流行的发展中国家，青蒿素的可获得性和成本是一个问题。口服青蒿叶干更有效地治疗疟疾，而叶子的茶浸液具有预防作用。生产和交付茶和青蒿叶干片的成本远低于ACT。

致谢
本研究得到了部分来自伍斯特理工学院和马萨诸塞大学临床与转化科学中心的支持；部分支持来自国家中医药中心的奖励号码NIH-R15AT008277-01。

作者贡献：Weathers PJ、Towler M、Hassanali A、Lutgen P和Engeu PO共同参与了文章的撰写；Hassanali A、Lutgen P和Engeu PO提供了临床数据；Weathers PJ和Towler M进行了实验室和野外样本的分析。

贡献者信息：
Pamela J Weathers，生物与生物技术系，伍斯特理工学院，01609，美国伍斯特。
Melissa Towler，生物与生物技术系，伍斯特理工学院，01609，美国伍斯特。
Ahmed Hassanali，肯尼亚肯亚塔大学纯与应用科学学院，20100，内罗毕，肯尼亚。
Pierre Lutgen，IFBV-BELHERB，邮政信箱98，L-6908尼德兰文，卢森堡。
Patrick Ogwang Engeu，自然化疗研究所，卫生部，邮政信箱4864坎帕拉，乌干达。

Artemisinin from the plant Artemisia annua (A. annua) L, and used as artemisinin combination therapy (ACT), is the current best therapeutic for treating malaria, a disease that hits children and adults especially in developing countries. Traditionally, A. annua was used by the Chinese as a tea to treat “fever”. More recently, investigators have shown that tea infusions and oral consumption of the dried leaves of the plant have prophylactic and therapeutic efficacy. The presence of a complex matrix of chemicals within the leaves seems to enhance both the bioavailability and efficacy of artemisinin. Although about 1000-fold less potent than artemisinin in their antiplasmodial activity, these plant chemicals are mainly small molecules that include other artemisinic compounds, terpenes (mainly mono and sesqui), flavonoids, and polyphenolic acids. In addition, polysaccharide constituents of A. annua may enhance bioavailability of artemisinin. Rodent pharmacokinetics showed longer T1/2 and Tmax and greater Cmax and AUC in Plasmodium chabaudi-infected mice treated with A. annua dried leaves than in healthy mice. Pharmacokinetics of deoxyartemisinin, a liver metabolite of artemisinin, was more inhibited in infected than in healthy mice. In healthy mice, artemisinin serum levels were > 40-fold greater in dried leaf fed mice than those fed with pure artemisinin. Human trial data showed that when delivered as dried leaves, 40-fold less artemisinin was required to obtain a therapeutic response compared to pure artemisinin. ACTs are still unaffordable for many malaria patients, and cost estimates for A. annua dried leaf tablet production are orders of magnitude less than for ACT, despite improvements in the production capacity. Considering that for > 2000 years this plant was used in traditional Chinese medicine for treatment of fever with no apparent appearance of artemisinin drug resistance, the evidence argues for inclusion of affordable A. annua dried leaf tablets into the arsenal of drugs to combat malaria and other artemisinin-susceptible diseases.

Keywords: Malaria, Infectious disease, Artemisia annua, Artemisinin, Combination therapy, Artemisinin combination therapy

INTRODUCTION

Nearly three billion people are affected by malaria with almost a million deaths annually, especially in Africa and amongst children[1]. Currently extracted from Artemisia annua (A. annua) L., artemisinin (Figure 1) is delivered in concert with another antimalarial drug [artemisinin combination therapy (ACT)] as the preferred treatment to slow emergence of drug resistance. Despite these efforts, artemisinin resistance is appearing[2] and persistent and/or asymptomatic malaria may also be playing a role in disease transmission[3–5]. Moreover, for developing countries ACT is costly and the supply is inadequate[6–9].

Artemisia annua (single clone of Artemisia annua cultivar at approximately 2 m height at floral bud formation), artemisinin and plant-based artemisinin combination therapy tablets.

Artemisinin is a sesquiterpene lactone that is produced and stored in the glandular trichomes that are mainly on the leaves and floral buds of A. annua, a GRAS medicinal herb[10–12]. The plant also produces > 40 flavonoids[13], many polyphenols, and a variety of other terpenes including mono-, sesqui-, di-, and triterpenes[14]. As discussed later, many of these have weak antimalarial activity, and, based on transcriptome analyses, many also seem to be produced and/or stored in the glandular trichomes that also contain artemisinin[15].

We and others proposed direct consumption of A. annua either as a tea infusion[16–19] or by oral consumption of the leaves[20–24]. In contrast to the oral consumption of pure artemisinin, we showed that the presence of plant material significantly enhanced appearance of artemisinin in the serum of healthy and Plasmodium chabaudi-infected mice[22]. Because of the plethora of mild antimalarial compounds naturally present in the dried leaves of the plant, we have termed this orally consumed dried leaf therapeutic plant-based artemisinin combination therapy, or pACT. These whole plant approaches are similar to the more than 2000 year traditional use of the plant by the Chinese[25].

To produce a therapeutically effective drug using a complex material like a medicinal plant requires that a number of key factors be met: the medicinal herbal product must be therapeutically effective; levels of key chemical components in the herb must be verifiably consistent; production must also be cost effective. Here we summarize and update our recent review[26] on the effects of A. annua on malaria and further discuss the bioavailability and therapeutic efficacy of pACT and how such an herbal drug could inexpensively be produced with a consistent dose.

PROPHYLACTIC USE OF A. ANNUA

Tea infusion, its chemistry, and in vitro studies

Until recently, there have been, to our knowledge, few well-controlled studies examining extraction, recovery, and stability of artemisinin and other compounds in A. annua tea infusion. A systematic study of preparations of A. annua therapeutic tea infusion was performed by van der Kooy et al[27] and showed that nearly 93% of available artemisinin was extracted from dried A. annua leaves, but only under certain conditions. Best preparation method was: 9 g DW leaves/L, for 5 min at 100 °C. Subsequent storage of the tea infusion at room temperature showed that artemisinin concentration was stable for > 24 h, important for malaria-endemic locations where there is no refrigeration. Artemisinin water solubility is approximately 50 mg/L[27], so the amount of artemisinin recovered from hot water tea infusions is reasonable. Other studies using the same extraction protocol also measured extraction and stability of artemisinin and some key flavonoids in the tea. Artemisinin was found to be stable at room temperature for up to 48 h[28] ; however, some flavonoids were poorly extracted and not stable at room temperature[29].

Carbonara et al[28] detected an assortment of phenolics, including 0.06 mg/g DW cirsilineol, in an A. annua tea infusion prepared at about a 4–10 fold higher proportion (approximately 38 g DW/L) than that proposed as optimal (9 g DW/L) by van der Kooy et al[27]. Most of the measured phenolics in the tea remained constant at room temperature for 48 h post-infusion. More recently, Suberu et al[19] identified milligram amounts of phenolic acids, flavonoids, and sesquiterpenes in a liter of A. annua tea, all of which demonstrated IC50 values in the micromolar or less range (Table 1). Indeed, the IC50 of the tea infusion itself was 7.6 and 2.9 nmol/L for the chloroquine (CQ)-sensitive HB3 and CQ-insensitive Dd2 strains of P. falciparum, respectively, and better than artemisinin alone suggesting synergism of constituents in the tea mixture. Clearly if a tea infusion is to be a therapeutic option, it must be consistently and reliably prepared and ingested. As suggested by van der Kooy et al[27], ideally a liter of tea infusion would be prepared daily and consumed in equal aliquots of about 250 mL over 24 h for several days.

 

Tea infusion clinical trials

Ogwang et al[30,31] tested Artemisia tea as a prophylaxis against malaria in 132 adult farm workers, aged 18–60 years, for 12 mo in a randomized clinical trial in Uganda. Tea infusion was consumed once a week at 2.5 g dried leaves per adult infusion dose with 55–100 mg artemisinin/L. Malaria was tracked for 9 mo while adverse clinical effects were tracked for 12 mo. Among those who used Artemisia tea there were 80% fewer fever-related hospital visits. Indeed, some patients reported using A. annua tea for > 7 years with no incidence of malaria and no serious adverse events. Although this study suggested that once weekly consumption of A. annua tea infusion may offer prophylactic protection, there were no children or elderly in the study, so additional clinical trials need to be conducted with different populations and age groups. Authors argued that since a single weekly dose was effective, compounds other than artemisinin may have played the prophylactic role since artemisinin itself has short plasma half-life.

 

THERAPEUTIC USE OF A. ANNUA

Tea infusion

Reports on the efficacy of A. annua (cv. Artemis) tea on human malaria patients by Mueller et al[17,32] and Blanke et al[33] yielded at times conflicting results. Their tea infusions contained 47–94 mg artemisinin/L, but recrudescence was much lower in the quinine-treated control group, so parasite reappearance in the tea-treated patients was ascribed to recrudescence and not re-infection[17]. In the Blanke et al[33] trial that included a placebo tea, recrudescence was consistently lower in the tea patients than in those treated with 500 mg pure artemisinin. More recently, however, De Donno et al[34] showed that 5 g dried leaves in one liter of A. annua tea infusion was effective against both CQ-resistant (W2) and CQ-sensitive (D10) strains of P. falciparum with IC50 values of 5.60 nmol/L and 7.08 nmol/L, respectively, results also consistent with those of Suberu et al[19] as already highlighted. These latter in vitro studies suggested that tea should be efficacious, so why the discrepancy with the earlier human trials? Preparation methodology is crucial for preserving as much biochemical integrity of the plant as possible[27]. The more recent in vitro studies likely used more consistently prepared tea infusions than the earlier human trials, so variations in chemical composition of the infusions and in the plant source material could explain the different responses.

The argument that tea is a monotherapy is unsubstantiated considering the now well-established chemical complexity and related antiplasmodial activity of tea infusions of A. annua and its components. Although data from therapeutic tea trials in animals and in humans correlate well, unfortunately, they do not support use of A. annua tea for treating malaria because animal and human data are comparably negative, the artemisinin dose is not easily controlled, and other potentially synergistic components in the tea are not readily controlled or extracted. Nevertheless, use of the tea could play a role in malaria prophylaxis to reduce incidence of malaria in different communities, or in temporary relief from malaria, mainly in prevention of coma or “to buy time” to enable an infected person from a rural area to travel to a hospital or clinic stocked with ACT.

 

Dried leaf A. annua - pACT

Recently, Elfawal et al[23] measured parasitemia in mice infected with P. chabaudi that were fed two different doses (0.6 or 3.0 mg artemisinin; 24 and 120 mg/kg) of either pure artemisinin in mouse chow or as pACT. Artemisinin delivered via pACT was at least five times more effective, and with a longer lasting response, than pure artemisinin in reducing parasitemia. Excluding artemisinin there are > 600 phytochemicals that have been identified in Artemisia annua[35], but there is currently a lack of information on the chemistry, effect of the preparation method (harvesting, drying, storage, etc.), and overall bioavailability of these chemicals[36]

Clinical trials using dried leaf A. annua are scarce in the scientific literature and few, other than those in Democratic Republic of Congo by Mueller et al[17,32], are published. Despite the fact that WHO does not encourage either whole plant or tea infusion clinical trials[37], some African universities have been conducting their own trials, many of which have not been published nor results assessed by polymerase chain reaction (PCR) as later done for clinical trials with ACTs (personal comm from C. Kasongo to P. Lutgen). Many of these trials used A. annua infusions, and compared to controls or even other antimalarial drugs, e.g., artesunate-amodiaquine, showed significantly greater sensitivity of the infusion with fewer late therapeutic failures. For example, in Democratic Republic of Congo, 54 malaria-infected volunteers were treated for 10 d with capsules containing powdered leaves of A. annua. Each patient was given 15 g dried leaves containing 15 mg of artemisinin (artemisinin content in leaves = 0.1%[38]

In a study aimed at preventing severe post-operative malaria at Bangui, Central Africa, powdered leaves of A. annua were administered in capsules to 25 patients, 22 of them children aged 1–16 years[24]. Treatment duration ranged from 3–4 d with a dose of 0.4–0.5 g/d of A. annua

The most clinically definitive study to date of pACT efficacy was conducted at the International Centre of Insect Physiology and Ecology (ICIPE) Mbita Field campus, Suba District, in Western Kenya. This was a collaborative project between ICIPE and Kenya Medical Research Institute[20]Table 2[39]) and was an open-label, non-randomized clinical trial mainly targeted to assess efficacy, safety, and tolerance of increasing doses of pACT delivered as tablets. The tablets were made by a Tanzania-based NGO, Natural Uwemba System for Health, from a hybrid of A. annua grown in the Tanzania highlands (2000–2200 m altitude). Leaves were harvested just before flowering, dried for approximately 3 wk under shade, then crushed, powdered, homogenized, and pressed into 500 mg tablets under ambient temperature. Tablets were robust with no excipient required. Using HPLC with diode array detector, analysis of hexane extracts of randomly selected batches of 100 tablets showed artemisinin content of the tablets was consistent at 0.74% ± 0.06% (i.e., approximately 3.7 mg per tablet).

The four cohorts of the trial each had 12 consenting patients aged 15–56 years (average 23.42) with P. falciparum malaria. Based on Giemsa-stained blood smears counted against 200 wbc, parasitemia was 0.02%-4% and hemoglobin levels > 8 mg/dL. Each cohort received one of four increasing numbers of A. annua tablets, ranging from 2–5 tablets twice on day 1, followed by 1–4 tablets twice daily for the next 5 d (Table 2). A week following the treatments, three patients scattered throughout different cohorts showed re-appearance of parasites in blood smears; however, all doses were effective in clinical and parasitological regression of malaria, with 9%-20% recrudescence at day 28 and no measurable toxicity.

Compared to the usual large pure artemisinin doses of 1000 mg on day 1 followed by 500 mg on each of days 2–7 that were administered to 227 malaria patients[39]Table 2). The measured pACT cure rate also was comparable to or exceeded other results using pure artemisinin[40,41], and similar levels of artemisinin (artesunate, artemether, etc.[42]). Furthermore, the positive therapeutic response using pACT appeared somewhat independent of dose beyond the second level of dose tested (Table 2[20]). Although oral doses used in the ICIPE[20] trials were far less than any tea studies, levels of recrudescence were much lower than tea and often better than in studies using pure artemisinin[39] (Table 2). Indeed, about 100 total mg of total artemisinin delivered via pACT for a full malaria treatment yielded a better recrudescence rate than the 4000 mg of pure artemisinin used by Giao et al[39] (Table 2). This 40-fold difference correlates well with the early pharmacokinetic studies by Weathers et al[21] that showed 45-fold enhanced bioavailability of the drug when delivered as pACT.

These results suggest that the natural phytochemical blend in pACT is important especially when orally administered as tablets. The results are also consistent with a study in China on mice infected with P. berghei, which compared the effects of pure artemisinin with crude A. annua extracts[43], and the studies by Elfawal et al[23] and Weathers et al[22]. In all three studies the administered products had comparable levels of artemisinin, but crude preparations and pACT were at least 3.5 times more effective in reducing parasitemia than pure artemisinin, suggesting a synergistic role for non-artemisinin constituents in the extracts and orally consumed dried leaves.

COMPARATIVE PHARMACOKINETICS AND BIOAVAILABILITY

Orally delivered artemisinin

When given orally or rectally, dihydroartemisinin showed higher bioavailability in humans than artemisinin in an early pharmacokinetic study by Zhao et al[44]. The Cmax, Tmax, and T1/2 for orally delivered dihydroartemisinin were 0.13–0.71 mg/L, 1.33 h, approximately 1.6 h, respectively; for pure artemisinin they were 0.09 mg/L, 1.5 h, and 2.27 h, respectively. Alin et al[45] compared orally delivered artemisinin and artemisinin-mefloquine combination therapy for treatment of P. falciparum malaria. Infected and uninfected patients had similar pharmacokinetic parameters. After a single dose, bioavailability of artemisinin was not altered. Interestingly, pharmacokinetics were similar when comparing treatment failures with successes, suggesting that studies that only measure artemisinin pharmacokinetics were inadequate for predicting therapeutic success[45]. Ilet et al[46] also reviewed artemisinin pharmacokinetics in patients with falciparum malaria and reported a dose of 9.1 mg/kg, which was comparable to that of Alin et al[45]. Cmax and Tmax values did not differ much from those reported by Alin et al[45].

In the Ilet et al[46] review of pharmacokinetic parameters of artemisinin and its derivatives, oral pure artemisinin doses ranged from about 6–11 mg kg/L in healthy subjects and Cmax was 0.15–0.39 mg/L. Dose seemed to have no major effect. An earlier study by Ashton et al[47] compared increasing artemisinin doses of 250, 500, and 1000 mg per person and both Cmax and T1/2 showed dose-dependent increases of 0.21, 0.45, and 0.79 mg/L, and 1.38, 2.0, and 2.8 h, respectively, but Tmax remained relatively constant at 2.3–2.8 h.

Diet is an important consideration for any orally delivered drug, and when Dien et al[48] compared artemisinin oral doses given with and without food, Cmax values were similar between subjects who fasted and those who did not. Food consumption along with artemisinin did not seem to affect artemisinin absorption. In contrast, a later rodent study by Weathers et al[21] observed that when artemisinin was consumed as part of a complex plant material, pACT, approximately 45-fold more drug entered the serum of mice than orally administered pure drug. Similarly, when pure artemisinin was fed to mice, it was not detectable in the serum after 60 min. However, artemisinin was detected in the serum when consumed in conjunction with mouse chow, which consists of a variety of plant materials including soy, oats, wheat, alfalfa, beet pulp, corn, etc[22].

In a study by Ashton et al[49], artemisinin at 9.1 mg/kg was given daily for 7 d, and measurements taken on days 1, 4, 7, and 21. On day 1 plasma Cmax and T1/2 were similar and comparable to data from other studies using a similar dose. On day 4 and 7, however, Cmax decreased, while T1/2 increased, indicating that although artemisinin was delivered daily for 7 d, it was either not readily absorbed or it degraded after the first dose. After the third dose, Cmax fell from 0.31 to 0.11 mg/L, and T1/2 increased from 3.0 to 4.8 h. These results suggested that either artemisinin was metabolized or accumulated elsewhere in the body.

In the liver, cytochrome P450 (CYP450) enzymes metabolize artemisinin to deoxyartemisinin, deoxydihydroartemisinin, 9,10-dihydrodeoxyartemisinin, and a metabolite named “crystal 7”[50]. Extended artemisinin dosing may not be beneficial as shown by Svensson et al[50] using human liver microsomes where activity of CYP450s, CYP2B6 in particular, correlated with decreasing artemisinin serum levels. In intermittent dosing studied by Ashton et al[49], the P450 levels were allowed to decline for 14 d before delivery of another dose, and Cmax rose from 0.11 to 0.20 mg/L, and T1/2 decreased from 4.8 to 2.7 h. Generally, maximum concentration of artemisinin in the body increased with increasing doses with T1/2 ranging from about 1.4–4.8 h for reported trials using oral pure artemisinin. Thus, increased and extended artemisinin treatment may reduce recrudescence.

Tea infusion delivered artemisinin

Other than Räth et al[16], there are few reports on the pharmacokinetics of tea infusion artemisinin delivered in humans. In the Räth et al[16] study, artemisinin Cmax was 0.24 mg/L at 0.6 h post consumption. Tea infusion containing 94.5 mg artemisinin had a Cmax equivalent to a dose of 250 mg pure artemisinin, but at a significantly shorter Tmax, 0.6 h vs 2.8 h[47]. Compared to pure artemisinin, the shorter half-life of artemisinin in the tea infusion may account for the observed higher recrudescence. Although tea-delivered artemisinin seemed more bioavailable, its shorter T1/2 of 0.9 h compared with about 2 h for pure artemisinin, suggested that more than two doses per day may be more beneficial; indeed, four doses a day were recommended.

The unacceptably high recrudescence rates in clinical tea infusion trials were attributed to low plasma concentrations, almost 40% lower than that for traditional doses (500 mg per person of 60 kg or 8.3 mg artemisinin/kg) of pure artemisinin. Although not specified, tea trial doses have been estimated at about 1.5 mg/kg, close to the 1.1 mg/kg dose of pure artemisinin used by Zhao et al[44], which is far below the 8.3 mg/kg that is traditionally accepted as pharmacologically effective. Nevertheless, the Cmax of 0.24 mg/L artemisinin for the tea dose is nearly twice that for pure artemisinin (Cmax = 0.13 mg/L) as measured by Zhao et al[44]. A. annua tea also showed potent antiplasmodial activity against 40 field isolates of P. falciparum collected in Pikine, Senegal (mean IC50 0.095 µg/mL[51]).

Dried leaf (pACT) delivered artemisinin

There are as yet no pharmacokinetic studies of pACT in humans. In a small PK study of healthy mice fed artemisinin there was about 45-fold more artemisinin delivered via pACT than when delivered as the pure drug[21]. More recently, pharmacokinetics of artemisinin and one of its liver metabolites, deoxyartemisinin, were compared over 120 min in healthy and P. chabaudi-infected mice treated with dried A. annua leaves at a 100 mg/kg body weight dose of artemisinin[22]. In pACT-treated healthy mice, the first order elimination rate constant for artemisinin was estimated to be 0.80/h, corresponding to a T1/2 of 51.6 min. Cmax and Tmax were 4.33 mg/L and 60 min, respectively. The AUC was 299.5 µg min/mL. The first order absorption rate constant was estimated at 1.39/h. In contrast, the AUC for pACT-treated infected mice was greater at 435.6 µg·min/mL. Serum levels of artemisinin in the infected mice continued to increase over the 120 min of the study period. As a result, the elimination half-life, T1/2 could not be determined, so Cmax and Tmax could only be estimated at ≥ 6.64 mg/L and ≥ 120 min, respectively. Nevertheless, both Cmax and Tmax of artemisinin were greater in infected than in healthy mice.

Generally, artemisinin concentrations decreased with a concomitant rise in deoxyartemisinin levels only in healthy subjects[22]. In contrast, artemisinin levels in infected mice continued to rise over the study period whilst deoxyartemisinin levels fell and then leveled, so infection seemed to retard the capacity of the mice to process artemisinin into deoxyartemisinin over the two-hour period. Many compounds in A. annua inhibit P. falciparum[52–55] and CYP34A[56]. At the high (100 mg/kg) dose used in the study, nearly equal amounts of artemisinin and deoxyartemisinin were measured in the serum, indicating that an excessive dose of artemisinin was used.

The presence of plant material affected artemisinin pharmacokinetics. At 60 min no artemisinin was detected in serum of mice fed pure artemisinin at 100 mg/kg body weight. When plant material was present, however, as mouse chow or A. annua pACT, artemisinin level in the serum rose to 2.44 and 4.32 µg/mL, respectively, demonstrating that the presence of plant material, even mouse chow, had a major positive impact on the appearance of artemisinin in the blood[22]. To our knowledge, these are the only data available on pharmacokinetics for orally delivered A. annua in animals or humans.

 

NON-ARTEMISININ THERAPEUTIC COMPOUNDS IN A. ANNUA

Flavonoids

A. annua is rich in essential oils, coumarins, polyphenols, polysaccharides, saponins, terpenes, and flavonoids. The levels of flavonoids and other compounds in A. annua change with developmental growth stage, with some being highest during full bloom[57]. There are > 40 flavonoids[13], and at least 11, including artemetin, casticin, chrysoplenetin, chrysoplenol-D, cirsilineol, eupatorin, kaempferol, luteolin, myricetin, quercetin, and rutin, are reported to have weak therapeutic efficacy against falciparum malaria (Table 1[52–54,58]). Some of these flavonoids were shown to improve the IC50 of artemisinin against P. falciparum in vitro by as much as 50%, suggesting synergy (Table 1[52]). Elford et al[53] also showed that while casticin (5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-3,6,7-trimethoxychromen-4-one) showed synergism with artemisinin, it did not synergize with chloroquine, suggesting a different interactive mechanism. Combining casticin with artemisinin inhibited parasite-mediated transport systems that control influx of myoinositol and L-glutamine in malaria-infected erythrocytes. These apparent synergistic actions between flavonoids and artemisinin suggest that flavonoids are likely to be important for efficacious use of A. annua consumed either as whole dried leaves or as tea.

Many flavonoids have antiplasmodial effects and inhibit P. falciparum growth in liver cells in vitro as reported for dietary flavonoids[54]. To our knowledge, there are no reports on pharmacokinetics of A. annua delivered flavonoids. Some flavonoids are reported to have long plasma half-lives; e.g., quercetin, found in A. annua and most fruits, has a plasma half-life of 27 h[59]. Quercetin [2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H–chromen-4-one], also found in garlic, inhibits parasite growth with differential activity against different strains of Plasmodium (Table 1[54,58]). Rutin, which is a rutinose [α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranose] glycoside of quercetin, showed similar results, suggesting that the sugar moiety did not significantly affect antimalarial activity (Table 1[58]). Flavonoids are known to persist in the body for > 5 d; this may explain the once a week dose inducing a prophylactic effect from A. annua tea infusion that was reported by Ogwang et al[30,31]. Many dietary flavonoids inhibit Plasmodium growth in vitro, but amounts in the diets are reportedly insufficient to offer protection against malaria[54]. Plants such as A. annua with high concentrations of flavonoids (e.g., up to 0.6%) may, however, work in concert with artemisinin to prevent malaria when consumed regularly.

The flavone luteolin [2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-chromenone] comprises up to 0.0023% DW in Artemisia[14] and has been used for a variety of ailments including cough, diarrhea, dysentery, diabetes, cancer, and malaria. Although luteolin has an IC50 value around 11 µmol/L[54] and is one of the more active antiplasmodial flavonoids found in A. annua, one cannot compare its role between studies as indicated by Ganesh et al[58] (see Table 1). The antimalarial response of different flavonoids seems to be affected by the strain of Plasmodium being tested. Luteolin also prevents completion of a full intra-erythrocytic cycle by inhibiting progression of parasite growth beyond the young trophozoite stage. The mechanism of this antiplasmodial activity seems to be related to the inhibition of parasite fatty acid biosynthesis. These lipids are required by the parasite to detoxify heme into hemozoin[60]. Independent of the human host, apicomplexan parasites use a fatty acid biosynthetic pathway. Enzymes in the pathway, like the NADPH-dependent b-ketoacyl-ACP reductase (FabG), are potential antimalarial targets. Among 30 flavonoids studied, luteolin and quercetin had the lowest IC50 values for the inhibition of these enzymes and also showed in vitro activity in the sub-micromolar range against multiple strains of P. falciparum[60].

Isovitexin {5,7-dihydroxy-2-(4-hydroxyphenyl)-6-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) oxan-2-yl]chromen-4-one} is another flavone, the 6-C-glucoside of apigenin, that was found in A. annua tea infusion at > 100 mg/L with micromolar antiplasmodial activity (Table 1[19,28]). Isovitexin inhibits lipid peroxidation and xanthine oxidase activity and protects cells from ROS damage with an overall LD50 > 400 µmol/L[61].

Terpenes

Limonene (1-Methyl-4-(1-methylethenyl)-cyclohexene) is part of the “cineole cassette” that includes 1,8-cineole (eucalyptol), limonene, myrcene, α-pinene, β-pinene, sabinene, and α-terpineol[62]; many of these affect particular stages of Plasmodium species. For example, limonene is often present at 7 mg/kg in A. annua[14] and inhibits isoprenoid biosynthesis in Plasmodium[63] and development at the ring and trophozoite stages[64]. Eucalyptol affects the trophozoite stage[65]. Limonene also arrests protein isoprenylation in P. falciparum, halting parasite development within 48 h of treatment[64]. The IC50 against in vitro Plasmodium in these trials was 2.27 mmol/L, more than twice the IC50 of 533 µmol/L measured by van Zyl et al[55]. Limonene and its metabolites remain in the plasma for at least 48 h[66], so the pharmacokinetics is favorable, which is important for elimination of gametocytes and malaria transmission.

The volatile monoterpene α-pinene (4,6,6-trimethylb-ficyclo[3.1.1]hept-3-ene) is present in the plant at levels up to 0.05% of dry weight[14]; it has an IC50 of 1.2 µmol/L, in the range of quinine at 0.29 µmol/L[55]. Eucalyptol (1,8-cineole) may comprise up to 30% [0.24%-0.42% (V/DW)] of the essential oil in A. annua[67] and is a strong inhibitor of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8[68]. Both chloroquine-resistant and chloroquine-sensitive Plasmodium strains are affected at the early trophozoite stage[65].

Eucalyptol (1,3,3-Trimethyl-2-oxabicyclo[2,2,2]octane) is also volatile and rapidly enters the blood when delivered either as an inhalant or orally [69,70]. At an IC50 of 0.02 mg/mL and low toxicity (LD50 of approximately 25 mg/mL), either oral or inhalation delivery is reasonable[65,71]. Indeed eucalyptol concentrations can reach 15 µg/mL in 60 min[69] suggesting its possible use as an antimalarial inhalant.

Artemisia ketone (3,3,6- trimethyl-1,5-heptadien-4-one), a major constituent of some cultivars of A. annua, has barely been studied. Other ketones like curcumin[72] have been implicated as inhibitors of β-hematin synthesis, so artemisia ketone may play a similar role and affect hemozoin formation. Although hemoglobin is required for Plasmodium survival and multiplication in merozoites inside the red blood cell, it leaves toxic debris like heme. The parasite subsequently oxidizes Fe2+ in heme to Fe3+ forming hematin, a nontoxic insoluble polymeric crystal called β-hematin (also known as hemozoin), which also inhibits cell-mediated immunity against the parasite. Water extracts of A. annua inhibit hemozoin synthesis[73].

Essential oils often contain a large amount of monoterpenes that may enhance the antimalarial effect of artesunate and even reverse the observed resistance of P. berghei against artesunate[74]. Monoterpenes tend to be higher in the pre-flowering phase of A. annua[75], but are drastically reduced by high drying temperatures or drying in the sun[13,76] and, of particular concern, during compression of dried leaves into tablets[77]. Although monoterpenes have some antimalarial potential, most are rather volatile and thus they may be therapeutically less important than the nonvolatile flavonoids, phenolic acids, and higher molecular weight sesquiterpenes.

Unlike α-pinene and eucalyptol, camphor (1,7,7-Trim ethylbicyclo[2.2.1]heptan-2-one) has no reported antimalarial activity, but it may comprise as much as 43.5% of the essential oil of A. annua[78]. Considering camphor is less volatile than either eucalyptol or α-pinene (melting points of 204 °C, 176 °C, and 155 °C, and flash points of 54 °C, 49 °C, and 33 °C, respectively), it may instead play a role in enhanced transport of hydrophobic molecules like artemisinin from pACT across the intestinal wall into the bloodstream[21,22]. Camphor may also affect thymocyte viability and aid in developing malaria immunity through production of T-cells[79]. At 50 µg/mL, camphor increased viability of cultured thymocytes[80].

The sesquiterpene nerolidol (3,7,11-Trimethyl-1,6,10-dodecatrien-3-ol) has an IC50 of 0.99 µmol/L and arrests development of the intraerythrocytic stages of the parasite (Table 1[55] ). Indians of the Amazon basin in Brazil treated malaria using the vapors of the leaves of Viola surinamensis; nerolidol was identified as the active constituent leading to 100% growth inhibition at the schizont stage[81]. Nerolidol levels vary with the cultivar tested, with one of the highest values found in plants from Ethiopia[82]. There is a greater concentration of this sesquiterpene in stems than leaves of A. annua[83].

Other sesquiterpenes found in the artemisinin biosynthetic pathway were only recently shown to have antiplasmodial activity at µmol/L levels, similar to that of other compounds found in the plant (Table 1[19]). These artemisinic compounds were extracted into A. annua tea infusions and showed varying interactions with artemisinin depending on their relative concentrations and the target parasite strain. For example, arteannuin B showed an additive interaction with artemisinin against the CQ-sensitive Plasmodium HB3 strain, while against the CQ-insensitive Dd2 strain the interaction was synergistic.

Phenolic acids

Rosmarinic ((2”R”)-2-[[(2”E”)-3-(3,4-Dihydroxyphenyl)-1-oxo-2-propenyl]]oxy]-3-(3,4-dihydroxyphenyl) propanoic acid) and chlorogenic ((1S,3R,4R,5R)-3-{[(2Z)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihydroxycyclo-hexanecarboxylic acid) acids are strong antioxidants found in a wide variety of A. annua cultivars[56]. In Caco-2 studies, these acids significantly inhibited activity of CYP3A4, one of the hepatic P450s responsible for metabolism of artemisinin to deoxyartemisinin, an inactive form of the drug[50]. These and other phenolic acids are present in A. annua tea infusion[19]. Both phenolic acids have an IC50 of about 65 µmol/L (Table 1) and also significantly reduced secretion of cytokines IL-6 and IL-8, and thus enhanced antimalarial activity while reducing inflammation[56].

Other compounds often found in A. annua and that may affect pACT efficacy

Although polysaccharides in other medicinal plants have been more extensively studied, they seem to have been rather overlooked in A. annua, probably because most Artemisia extracts are obtained using organic solvents and polysaccharides are only soluble in water. Polysaccharides extracted from Artemisia iwayomogi showed hydroxyl radical scavenging activity three times stronger than glutathione or caffeic acid, and ROS inhibition was twice as strong as ascorbic acid[84]. In A. iwayomogi, more polysaccharides were found in stems than in leaves and their solubility was also higher from stem than from leaf tissue[84].

The combination of polysaccharides with lipophilic molecules like artemisinin may lead to a higher bioavailability of the antimalarial constituents when delivered via A. annua, which may explain the lower effective therapeutic dose against malaria observed for pACT than for pure artemisinin[20,23,26]. Indeed, Han[85] showed that ginseng polysaccharides had preventive and curative antimalarial activities and synergized with artesunate in malaria-infected mice. Sulfated polysaccharides inhibited the in vitro invasion of merozoites into erythrocytes and interfered with merozoite surface protein[86–88]. Heparin and other sulfated polysaccharides have been shown to inhibit blood-stage growth of plasmodium[89,90]. Some sulfated polysaccharides inhibited the formation of rosettes between infected red blood cells (iRBC) and uninfected RBCs, as well as adhesion of iRBCs to placental chondroitin sulfate A, which is linked to severe disease outcome in pregnancy-associated malaria[91].

Saponins, common in many plants, have an important role in human and animal nutrition and are reportedly present in A. annua, but only as measured in alcoholic extracts using the nonquantitative foaming test[92,93] (Weathers, unpublished). These soap-like amphiphilic (lypo- and hydrophilic) bioactive compounds are mainly produced by plants. Recently, there has been interest in the clinical use of saponins as chemotherapeutic agents[94], and as adjuvants for vaccines[95]. At very low doses saponins are efficient, have hemolytic properties, produce 40–50 Å pores in erythrocyte membranes, and modulate the sodium pump and ATPase[96]. Saponins also have a hypoglycemic effect mainly by inhibiting intestinal permeability and absorption of glucose and may therefore inhibit the growth of P. falciparum, which needs glucose to grow[97]. Better identification, quantification, and investigation into the role of saponins in pACT efficacy are warranted.

The coumarin, scopoletin (7-hydroxy-6-methoxychromen-2-one), also known for its antinociceptive properties[98,99], is commonly found in most Artemisia species at, for example, about 0.2% (w/w) in a Luxembourg cultivar. Known for its anti-oxidant, hepatoprotective, and anti-inflammatory activities, scopoletin scavenging capacity for hydroxyl radical, DPPH, superoxide anion, hydrogen peroxide, and Fe2+ chelating activity is almost at the level of α-tocopherol (Vitamin E)[100].

Although not antiplasmodial, scopoletin inhibits TNF-α, IL-6, and IL-8 at millimolar concentrations, and is thus likely one of the major anti-inflammatory and antipyretic constituents of A. annua[101]. Coumarins can activate lymphocytes, thereby stimulating immunological functions[102]. Indeed, scopoletin induced cell proliferation in normal lymphocytes with an immunomodulatory effect[101]. In uninfected erythrocytes internal Na concentration is much lower than external concentration, but the K concentration is higher; in infected blood cells this situation is drastically reversed[103]. Scopoletin significantly stimulated erythrocyte membrane ATPases at 0.1 µmol/L, in particular Na-K-ATPase vs Ca-ATPase or Mg-ATPase[104], so scopoletin may affect malaria infection. A significant hormetic effect was also noticed; stimulation was higher at scopoletin concentrations of 10 µg/mL than at 1 or at 100 µg/mL. In addition scopoletin also inhibited ADP-platelet aggregation at a range of 0.1 to 5 µmol/L and improved blood rheology[105].

Scopoletin may also affect the interaction between malaria and uric acid. Cyclical fevers and high levels of inflammation characterize malaria and this likely aids parasite clearance. Excessive and persistent inflammation, on the other hand, can lead to severe malaria[106]. In the cytoplasm of their parasitophorous vacuole, Plasmodium-infected erythrocytes contain uric acid precipitates that are released upon erythrocyte rupture. Uric acid precipitates are mediators for inflammatory cytokines IL-6, IL-8, and are considered a danger signal for innate immunity. Uric acid is also the causative agent in gout. These precipitates could offer a novel molecular target for anti-inflammatory therapies in malaria. Scopoletin inhibits the activity of xanthine oxidase in hyperuricemic mice after peritoneal administration, and this hypouremic effect is fast and dose-dependent[107].

Toxicology

Although many of the compounds in A. annua have not been tested for their toxicity in, a survey of available MSDS data showed that the LD50 levels for orally administered compounds in rodents ranged from about 160 mg/kg for quercetin to > 8000 mg/kg for nerolidol. The artemisinin LD50 measured via oral dose in a mouse was 4228 mg/kg. Therefore, at the estimated amounts of dried leaves of pACT that may be orally consumed by a malaria patient, most of the compounds reported thus far in A. annua are at concentrations that are orders of magnitude below their LD50 toxicity values.

Toxicology of the dried leaf tablets used in the Kenyan human trial measured the following components: serum levels of urea, serum proteins, creatinine, γ-glutaryl transferase, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, or alkaline phosphatase levels, hemoglobin, and pre- and post-electrocardiograms[20]. Compared to levels prior to treatment with pACT, there was no significant change post-treatment.

 

PRODUCTION CONSIDERATIONS

Production comparisons with traditional extraction

Because production costs are usually closely held secrets, there are few cost estimates that are publicly available to compare pACT production with extracted artemisinin. However, costs can be estimated from a study by de Vries et al[108] where they reported a 1 kg recovery of artemisinin from A. annua containing 0.6% artemisinin. Downstream processing costs and product losses increase with increasing number of unit operations (unit ops), a fact often not generally appreciated[109]. Indeed for biotechnology processes, recovery can be anywhere from 9%-51%[110]. As an example, if each step of a 4 step process is 95% efficient, then the overall process has a final efficiency of about 81%, while a single step process at 95% efficiency has a 95% overall recovery. The described process steps for extracted artemisinin (eAN) vs pACT-AN are shown in Figure 2. From the point of harvested dried leaves to material ready for packaging or conversion to the delivered drug (e.g., artesunate or artemether), pACT has one unit op and eAN has eight[108]. Extraction solvents and other chemicals are clearly no longer part of the cost. Because there is one vs eight unit ops for eAN and at least two of the eAN unit ops involve significant amounts of heat, pACT energy cost is significantly reduced by at least 90%. Costs for labor, interest, depreciation, and maintenance are all also affected by the number of unit ops[109], so we estimated that with seven fewer unit op steps those costs would reduce by approximately 88%. Although better extraction processes may be in play[111], using the de Vries et al[108] analysis our estimate of cost reduction for producing pACT is about 30% less than the cost of producing eAN. Data provided by de Vries et al[108] was based on 0.6% artemisinin content, so if a higher producing cultivar was harvested, costs would drop proportionately. Moreover, cost drops again because with pACT there is no need to convert artemisinin to artesunate or artemether; those conversions were necessary because they have higher bioavailability than pure artemisinin, which is not an issue with pACT[21,22].

Comparison between plant-based artemisinin combination therapy production and extracted artemisinin from dry harvested leaves to product ready either for packaging (plant-based artemisinin combination therapy) or conversion to artemether or artesunate (extracted artemisinin). AN: Artemisinin; eAN: Extracted artemisinin; pACT: Plant-based artemisinin combination therapy.

The de Vries et al[108] process cost estimation focuses on a production yield of 1 kg of artemisinin from 500 kg dried leaves, so per Giao et al[39] that amount of pure artemisinin would treat only 250 patients. Based on the data shown in Table 3 from Kenyan or WPI A. annua at 0.7 and 1.4% artemisinin, 15 and 7.5 g DW leaves, respectively, are required for a total adult pACT treatment; so from 500 kg leaves, 33300 and 66600 patients could be treated, respectively. This represents more than a 130-fold increase in patients treated compared to pure artemisinin with proportionate reduction in price.

Table 3

Estimated numbers of adult patients treatable from plant-based artemisinin combination therapy1

For A. annua cultivar containing	Number of patients treated at various dry leaf tonnage
	2 T/ha2	3 T/ha	4 T/ha3	5 T/ha
0.7% artemisinin/g DW (Kenyan cultivar)	127260	190890	254520	318150
1.4% artemisinin/g DW (WPI cultivar)	254520	381780	509040	636300

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1Assumptions: each adult needs 100 mg artemisinin (AN) over 6 d for a cure; at 0.7% and 1.4% AN that is approximately 15 and 73 g DW leaves, respectively, for a single adult total malaria treatment;

2Below the average of 2.5 T/ha reported for all of East Africa;

3Equal to the maximum obtained growing A. annua SAM in the Stow, MA, United States field trials. A. annua: Artemisia annua.

A. annua dry leaf production varies around the globe. “In East Africa yields average 2.5 T/ha (range = 0.75–4.2)…”[112]. Based on our field trials[113], the reported average A. annua leaf production in E. Africa[112], and the doses used in the Kenyan human trial[20], one can estimate the amount of dry leaf production, and depending on the amount of artemisinin in the biomass, estimate possible number of adult patients that could be treated with pACT (Table 3).

Current ACT drugs vs pACT

Using the dosing information obtained from the Kenyan human malaria trial[20], each adult needs about 100 mg artemisinin total over 6 d for a malaria treatment, so for A. annua leaves with 0.7% artemisinin, 15 g of dried leaves would be needed for a 6 d treatment course. At 2 ton of dried leaves harvested per hectare, 127260 adult patients could be treated for malaria (Table 3). For leaves containing 1.4% artemisinin, only 7.5 g of dried leaves are required, so from a hectare of land producing 2 tons of leaves twice as many patients could be treated (Table 3). Clearly choosing cultivars that have higher levels of artemisinin in their leafy biomass will dramatically increase the number of patients that can be treated from 1 ha.

According to Roll Back Malaria, from one ton of purified artemisinin current ACT therapy can provide 1.76 million adult malaria treatments using artemether/lumefantrine, and 2.5 million adult treatments using artesunate/amodiaquine[114] (Table 4). Using the same one ton artemisinin equivalent, but delivering the drug via pACT with 0.7% artemisinin content, one would have harvested about 142.8 tons of dried A. annua leaves. Assuming 15 g dried leaves per patient from the dosing data in the Kenyan human malaria trial (Table 2[20]), 8.64 million adult patients could be treated, about a four-fold increase over either of the current ACT drugs. The actual cost of pACT, therefore, mainly depends on the cost of the dried leaves and their artemisinin content.

Table 4

Estimated number of patient treatments by current artemisinin combination therapy vs plant-based artemisinin combination therapy

Combination therapy drug	Adult treatments per ton of artemisinin
AL1	1.76 million
AS/AQ1	2.5 million
pACT leaves with 0.7% artemisinin	8.6 million

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1http://www.rollbackmalaria.org/partnership/wg/wgprocurementsupply/docs/psmwg_ppACT-API.pdf p.2 [cited May 27, 2014];

2Assumes a 6 day treatment with pACT, with each patient receiving 15 g dried leaves per full malaria treatment for leaves with 0.7% artemisinin. To obtain an amount of artemisinin equal to 1 T of the extracted drug, one would have to harvest 142.8 tons of dried A. annua leaves containing 0.7% artemisinin AL: Artemether/lumefantrine; AS/AQ: Artesunate/amodiaquine; pACT: Plant-based artemisinin combination therapy.

As yet unpublished data from the Rich and Weathers labs demonstrated that pACT prevents emergence of artemisinin drug resistance; the plant itself seems to function as its own ACT (pACT). This would obviate the need for inclusion of a co-drug as used in currently administered ACTs. The co-drug costs at least as much as the artemisinic portion of the drug[6]. Consequently, elimination of the added co-drug could result in at least an additional 50% reduction in cost, so that the final pACT cost reduction is conservatively estimated to be far below that of a current course of ACT therapy.

Considering that A. annua is nontoxic and safe to consume orally, dose may not have to be adjusted for children. On the other hand, the leaves taste bitter, so masking the taste, perhaps with sugar, should help with pediatric treatment. Our recent simulated digestion study showed that adding table sugar (sucrose) to pACT did not significantly alter the amount of artemisinin released after digestion, with the added benefit of doubling the amount of flavonoids released[115].

Comparison with emerging artemisinin sources or other newer antimalarial drugs

There are at least three other emerging antimalarial therapeutic technologies: synthetic artemisinin[116], semi-synthetic artemisinin (SSA) production from genetically engineered microbes[117], and a single dose drug, OZ439[118]. In early 2013, Sanofi/PATH Drug Development Programme, announced they would have the capacity to produce up to 60 MT of SSA in 2014 at about $400/kg, depending on quantity; Sanofi now has WHO prequalification for its SSA[119]. Although not much cheaper than the current price of about $550/kg[120], supply would be more or less unlimited. Despite what might seem as an advantage to large amounts of SSA production, there are also some serious disadvantages, and comparison of some advantages and disadvantages for each of these new synthetic antimalarial drugs and pACT is noted in Table 5.

Table 5

Comparison of emerging antimalarial therapeutic technologies with plant-based artemisinin combination therapy

Technology	Advantages	Disadvantages
Synthetic AN[116]	Fully synthetic method giving AN = compound Lowers AN cost compared to extraction	Requires co-drug to obviate emergence of AN drug resistance Not yet in production Needs sophisticated process Likely all under Western control Challenging patient compliance due to multiday dosing
Semi-synthetic AN[117]	Semi-synthetic method giving authentic AN Lowers AN cost compared to extraction	Requires co-drug to obviate emergence of AN drug resistance Production began via Sanofi Needs sophisticated process Likely all under Western control Challenging patient compliance due to multiday dosing
OZ439[118]	Single dose cure insures patient compliance In successful Phase 2 trials Mechanism of action not the same as AN Probably low cost due to full synthesis	Requires co-drug to obviate emergence of AN drug resistance Not yet in production Needs sophisticated process Likely all under Western control
pACT[20–24]	Has its own in planta co-drug to obviate emergence of AN drug resistance Very low cost Very consistent product Can be used to treat other diseases Can be locally owned, produced, managed, and distributed	Not yet in production Likely to meet push back from pharmaceutical industry Challenging patient compliance due to multiday dosing

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AN: Artemisinin.

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QUALITY ASSURANCE CONSIDERATIONS

Agricultural quality

The traditional and least costly method for cultivating A. annua uses seeds and in developing countries farmers prefer to save seeds from one growing season to the next. However, seed generated plants of A. annua will vary widely from generation to generation even with high quality starting stock (see review by Ferreira et al[10]). Stem cuttings of A. annua readily root in about two weeks, so clonal propagation via rooted cutting is recommended to eliminate this variability. Although this method of propagation is not cost effective for large plantations, it would work for a few hectares or for controlled environment agriculture. Given the large numbers of patients that could be treated from growing just a few hectares of A. annua (Table 3), clonal propagation by rooted stem cuttings is recommended. Since pACT therapy involves the direct consumption of the dried leaves of the plant, harvested leaf material must be kept clean, which is easiest to do in controlled environment agriculture and following Good Agricultural Procedures[121], particularly as applied to fresh produce[122]. However, controlled agriculture would probably result in loss of agricultural jobs, a concern to be assessed locally. Alternatively, great care must be taken during field harvest and post-harvest storage, so as not to affect the quality of the product. WHO has established good agricultural practices specifically for A. annua for purposes of artemisinin extraction[123], for general medicinal plants[124], and to minimize contamination of herbal medicines[125].

Chemical consistency and quantification

To deliver a reliable dose of therapeutics to a patient, the dried leaves of harvested A. annua must have a reliable and consistent composition. Clonal propagation provides the required consistency. Recently we showed that of 10 crops harvested from vegetative and early flowering plants grown over three years under diverse conditions in the lab, field, and home garden, the artemisinin content of a single clone of A. annua (SAM) was 1.38% ± 0.26% (w/w)[77]. Thus, despite variations in culture and environmental conditions, a consistent level of the main therapeutic constituent can be achieved. Moreover, the content of harvested leaves is certainly not a guarantee of finished product, e.g., compressed leaf tablets. Analyses by Weathers et al[77] showed that although artemisinin content was very stable after tablet compression, other constituents vaied significantly. For example, although flavonoids increased with tablet compression, the more volatile monoterpenes decreased substantially. Thus, it is critical to monitor the composition profile of both incoming harvested material as well as the final product.

Complex and expensive analytical procedures have been used to analyze the many products found in A. annua, but they are not necessary to measure and assure product quality. Artemisinin is easily extracted and then can be quantified using a variety of thin layer chromatography (TLC) methods and visualized with p-anisaldehyde stain[126,127]. Other key constituents like the flavonoids are also readily separated using TLC and visualized under either UV ± AlCl3 reagent[128]. Total flavonoids also can be quantified using inexpensive visible spectroscopy via the AlCl3 method with quercetin used as an inexpensive standard. To our knowledge no inexpensive, reliable spectrophotometric assay is available to measure artemisinin in complex plant extracts.

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SOCIOECONOMIC BENEFITS

Other diseases

Artemisinin and its derivatives are also effective against a number of viruses[129], a variety of human cancer cell lines[130–133], and several neglected tropical diseases including schistosomiasis[134], leishmaniasis[135,136], trypanosomiasis[137], and some livestock diseases[133,138].

Although they rank below malaria in terms of public health importance, schistosomiasis, leishmania, and trypanosomiasis result in estimated annual infections of about 240 million, 1.3 million (0.3 visceral and 1.0 cutaneous), and 30000, respectively[139]. These diseases along with many others respond to treatment with artemisinins. Although the IC50 is about 1000-fold greater than for Plasmodium sp., the greater apparent bioavailability of artemisinin via oral pACT[20–22] would likely reduce the amount of drug required for treatment. At present, pACT has not been tested in vivo for diseases other than malaria.

Malaria treatment is further complicated for Human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients. Malaria and HIV co-infection represents a major health burden in Africa mainly because it is now “well established that HIV infection results in a higher incidence and more severe manifestations of malaria”[140]. With a weakened immune system, AIDS patients are more susceptible to malaria and also respond slower to malaria therapy[140–142]. Furthermore, in a meta-analysis by Tusting et al[143], socioeconomic development strongly correlated with better malaria therapeutic outcomes. Recently, A. annua has demonstrated anti HIV activity[126,144] and thus oral consumption of the dried leaves of this herb will not only treat malaria, but should also enhance the well-being of HIV/AIDS patients.

Agriculture, jobs and self-determination

A. annua is grown in more than 75 countries[145]. In 2011 about 163 MT of artemisinin were extracted from plantations and small stakeholder farms mainly located in China, Vietnam, and Eastern Africa including Madagascar; value was about $550/kg[120]. With the advent of the production of semi synthetic artemisinin by Sanofi, 60 MT were projected for 2014 with an anticipated price of about $400/kg[119]. As this new source of artemisinin becomes available, the Netherlands Royal Tropical Institute projected that the market for natural Artemisia will significantly destabilize, undermining the security of farmers. The Tropical Institute was further concerned that “pharmaceutical companies will accumulate control and power over the production process; Artemisia producers will lose a source of income; and local production, extraction and (possibly) manufacturing of ACT in regions where malaria is prevalent will shift to the main production sites of Western pharmaceutical companies”, disrupting the fragile economics of these already impoverished countries[120]. The average small stakeholder crop area is about 0.2 ha in China and Africa[120], so while implementation of pACT may not require as much agricultural land as for extracted artemisinin, it could still help provide small stakeholders with a source of income. We have estimated that localized micro manufacturing plants could be constructed for < $50000 USD, and produce quality-controlled pACT tablets with readily verifiable contents. Our overall approach, schematically illustrated in Figure 3, leads to local control of malaria and possibly other artemisinin susceptible diseases while also improving the socioeconomic status of the populations.

 

Figure 3

Overall scheme for plant-based artemlslnln combination therapy production. pACT: Plant-based artemisinin combination therapy; TLC: Thin layer chromatography.

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CONCLUSION

Evidence is mounting for the therapeutic efficacy of the use of dried leaves of A. annua, pACT, to treat malaria and possibly other diseases. The complex mixture of antiparasitic compounds in the plant seems to account for its therapeutic activity with animal and human trials supporting this claim. It is also clear that the cost of using pACT is a fraction of that for any other current or emerging antimalarial therapeutic. Likewise, the recent evidence of persistent and/or asymptomatic malaria suggests that a more prophylactic approach to malaria using pACT or even A. annua tea may be warranted. Considering that for > 2000 years this plant was used in traditional Chinese medicine for treatment of fever with no apparent appearance of artemisinin drug resistance, taken together the cumulative evidence argues for inclusion of pACT into the arsenal of drugs to combat malaria, and very likely, other diseases.

Core tip

Artemisinin, extracted from the plant Artemisia annua (A. annua) L., and artemisinin derivatives are the current best antimalarial therapeutics and are delivered as artemisinin combination therapy (ACT). Availability and cost are problematic for the developing world where malaria is endemic. Oral consumption of A. annua dried leaves is more effective than the pure drug. A tea infusion of the leaves has prophylactic effects. Cost of producing and delivering the tea and A. annua dried leaf tablets is much more affordable than ACT.

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Acknowledgments

Supported by Worcester Polytechnic Institute and University of Massachusetts Center for Clinical and Translational Science partially; partially by Award Number NIH-R15AT008277-01 from the National Center for Complementary and Alternative Medicine

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Footnotes

Author contributions: Weathers PJ, Towler M, Hassanali A, Lutgen P and Engeu PO all participated in writing the article; Hassanali A, Lutgen P and Engeu PO provided clinical data; Weathers PJ and Towler M conducted analyses of lab and field samples.

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Contributor Information

Pamela J Weathers, Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609, United States.

Melissa Towler, Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609, United States.

Ahmed Hassanali, School of Pure and Applied Sciences, Kenyatta University, Nairobi 20100, Kenya.

Pierre Lutgen, IFBV-BELHERB, PO Box 98, L-6908 Niederanven, Luxembourg.

Patrick Ogwang Engeu, Natural Chemotherapeutics Research Institute, Ministry of Health, PO Box 4864 Kampala, Uganda.

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