Project number: PZW-AAF4 Post-harvest Storage Stability of Artemisinin and Flavonoids in Artemisia annua A Major Qualifying Project Report: Submitted to the Faculty Of the WORCESTER POLYTECHNIC INSTITUTE In partial fulfillment of the requirements for the Degree of Bachelor of Science by _________________________ _________________________ Meredith Ghilardi Jason Purnell Date: Approved: ________________________ Prof. Pamela J. Weathers, Major Advisor Acknowledgements We would like to thank Dr. Pamela Weathers for support and guidance for this project. We would also like to thank Dr. Melissa Towler for all of her help and guidance in the lab. We would like to thank the graduate students Sibo Wang and Liwen Fei for their help in the lab. 1 Contents Acknowledgements ..................................................................................................................... 1 Abstract ....................................................................................................................................... 3 A. The Problem and Its Significance. ....................................................................................... 4 A.1.Artemisinin Biosynthesis. ................................................................................................. 9 A.2.Microbial Synthesis of Artemisinin. ............................................................................... 12 A.3. Peroxidases and Relationship to Artemisinin. ............................................................... 12 A.4. Catalase and its Relation to Artemisinin. ...................................................................... 14 A.5. Flavonoid Biosynthesis and Presence in A. annua. ....................................................... 15 A.6. Flavonoids and Malaria. ................................................................................................ 18 A.7. Other Flavonoid Activities. ........................................................................................... 19 B. Hypothesis: ........................................................................................................................ 21 C. Objectives: ......................................................................................................................... 21 D. Methods: ............................................................................................................................... 22 D.1.Plant material. ................................................................................................................. 22 D.2. Drying methods. ............................................................................................................ 23 D.3. Peroxidase Assay. .......................................................................................................... 24 D.4. Total Flavonoids Assay. ................................................................................................ 26 D.5. Artemisinin Assay. ........................................................................................................ 26 D.6. Statistical Analysis......................................................................................................... 27 E. Results: ............................................................................................................................... 28 E.1: Effect of Storage on Flavonoid Content of Dried Leaves. ............................................. 28 E.2. Stability of AN and Flavonoids After Rehydration. ...................................................... 30 F.Discussion and Conclusions. ................................................................................................. 32 G.Recommendations for Future Work. ..................................................................................... 34 Literature Cited ......................................................................................................................... 35 Appendix A: .............................................................................................................................. 41 2 Abstract Artemisinin in combination with another drug is the best current antimalarial therapy. Dried leaves of Artemisia annua from which artemisinin is extracted and purified may offer a better treatment. We used GC-MS and the AlCl3 assay to measure the stability of artemisinin and flavonoids in dried leaves after wetting and with long term dry storage. Neither 24 hour water immersion nor long term dry storage affected artemisinin stability. Short-term drying showed a drop in flavonoid stability initially and then a gradual increase in total flavonoid levels after sixteen weeks of storage. 3 A. The Problem and Its Significance. In 2012, about 3.4 billion people, nearly half of the estimated global population, lived at risk of malaria. Currently, 104 countries and territories consider malaria to be an endemic (WHO, 2013). About 1.2 billion of this estimated population was considered to be at high risk, with their regions reporting more than one case in every 1,000 people. Most lived in either South-East Asia or sub-Saharan Africa. The World Health Organization (WHO) estimated that there were about 207 million cases of malaria worldwide, with 80% of reported cases occurring in sub-Saharan Africa (WHO, 2013). The WHO also estimated that there were 627,000 lethal cases in 2012, 90% of which occurred in sub-Saharan Africa. The vast majority of lethal cases, as high as 77%, were in children under five years of age. Children under five faced more risk than adults due to having a developing immune system that leaves them more susceptible to infection. Many also do not have access to proper nutrition, especially if they live in the poorer parts of the region. Effective treatment for children has also brought new issues to light. Many are not old enough to swallow pills and non-encapsulated medications are difficult to deliver properly as young children do not like to eat anything with a bitter taste, which is a common property of prospective therapies formed around edible Artemisia annua. Malaria is caused by a genus of parasites called Plasmodium, of which five species are known; P. vivax, P. ovale, P. malaria, P. knowlesi, and the most virulent, P. falciparum (CDC, 2013). This parasite relies on two hosts; the primary host is the female Anopheles mosquito that carries sporozoites and inoculates the secondary human host during feeding. The Anopheles mosquito is part of what places equatorial regions at a higher risk due to its reproductive patterns, which require the non-moving bodies of water and warm, humid climate that are found at latitudes near the equator throughout the year. Most sporozoites follow an asexual reproduction cycle with two stages, exo-erythrocytic and the erythrocytic (CDC, 2013). Once introduced the sporozoites are transported through the blood stream and mature in the liver into schizonts, which reproduce until they become large enough to rupture the host cell. Abbreviations AN, artemisinin; POD, peroxidase; APX, ascorbate peroxidase; GPX, Guaiacol Peroxidase; WHO, World Health Organization; ADS, Amorpha-4,11-Diene Synthase; FPP, Farnesyl Pyrophosphate; FPPS, Farnesyl Diphosphate Synthase; SERCA, Sarco/Endoplasmic Reticulum; DHAA, Dihydroartemisinic Acid; ACT, Artemisinin-Based Combination Therapies; PACT, plant-based ACT 4 This causes a release of merozoites that in turn move into the red blood cells and develop into ring-stage trophozoites. Once mature, the trophozoites either form schizonts to continue the erythrocytic cycle or form gametocytes that progress into a sporogenic cycle, which occurs after being transferred to a new mosquito host. It is the stages of the cycle that are present in the blood stream that can cause fever and other flu-like symptoms as well as hemolytic anemia (CDC, 2013). The P. falciparum species is the most likely species to progress past these common symptoms to the severe and potentially fatal form of the disease called cerebral malaria that affects the central nervous system. The drugs of choice for treating malaria have commonly been chloroquine, mefloquine, or combinations of quinine with tetracycline, however most parasite strains have become resistant to many of these treatments (Phyo et al., 2012). This resistance spurred an increased interest in the plant Artemisia annua L., the source of a traditional Chinese medicine called ‘Qing Hao’ that has been used for centuries to treat fevers and other malaria symptoms. In the 1970s it was found that the active drug responsible for the antimalarial properties was artemisinin (AN) (Willcox et al., 2004), a sesquiterpene lactone, which contains an endoperoxide bridge that is essential to the efficacy of artemisinin against Plasmodium species (Figure 1). Further research has also shown that AN can be used in combination with other drugs to treat some cancers as well as leishmaniasis (Sen et al., 2007). AN is currently the most effective treatment for malaria in the form of ACTs, or Artemisinin-based Combination Therapies. While these therapies lack the drug resistance of older methods, they tend to be too expensive for those in the areas of highest risk of infection like sub-Saharan Africa. Due to the low solubility of AN many of these treatments use semi-synthetic derivatives of AN such as artemether, arteether, or artesunate (Bégué et al., 2005). 5 Figure 1: Chemical Structure of Artemisinin In a 2013 report on malaria, the WHO reported that resistance