Artemisia annua Chinese name: Qing hoa su English: wormwood, sweet wormwood

1. Artemisia annua Chinese name: Qing hoa su English: wormwood, sweet wormwood

2. Artemisia annua

4. Crystals of artemisinin isolated from A. annua Aug. 19, 2005

5. Artemisinin 94 105.7 172.3

6. Dept-135, and C-13 NMR Spectra of Artemisinin 12a 3 12 10 C12 C3 C12a Artemisinin C10 X

7. 172.6 ppm C-10 105.9 and 79.9 C-3 and C-12a 94.1 (C-12), 50.5 (C-5a) and 45.3 (C-8a) C-3 C-12a C-10 C-12 C-13 NMR spectrum of the crude EtOH ext of A. annua

8. Aug. 19, 2005

9. Aug. 19, 2005 Dihydro-epideoxyareannuin B Confirms presence of Active ingredient in the Tea Artemisinin • EtOH Ext • Artemisinin • Tea (5 g) 1 2 3

10. Dihydro-epideoxyarteannuin B Constituents other than Artemisinin

11. Quantitative Aspects • Artemisinin Levels in A. annua grown in Vietnam • Stages of the growth of the plant • Vegetative growth 5-8 months (from sown seeds) • Mass formation of buds (9 months) • Full blooming (10 months) • Artemisinin highest in 5-months old plant (0.86%) • Content then drops to 0.4% and • Post flowering as low as 0.16% • Essential oil: 1.9% (v/w) just before flowering Source: Planta Medica, 1993 (I.d. 16624)

12. J. Nat. Prod.,68 (1), 133 -149, 2005. Quantitative 1H NMR: Development and Potential of a Method for Natural Products Analysis Guido F. Pauli,*Birgit U. Jaki, andDavid C. Lankin. College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612 Abstract: Based on a brief revision of what constitutes state-of-the-art "quantitative experimental conditions" for 1H quantitative NMR (qHNMR), this comprehensive review contains almost 200 references and covers the literature since 1982 with emphasis on natural products. It provides an overview of the background and applications of qHNMR in natural products research, new methods such as decoupling and hyphenation, and analytical potential and limitations, and compiles information on reference materials used for and studied by qHNMR. The dual status of natural products, being single chemical entities and valuable biologically active agents that need to be purified from complex matrixes, results in an increased analytical demand when testing their deviation from the singleton composition ideal. The outcome and versatility of reported applications lead to the conclusion that qHNMR is currently the principal analytical method to meet this demand. Considering both 1D and 2D 1H NMR experiments, qHNMR has proved to be highly suitable for the simultaneous selective recognition and quantitative determination of metabolites in complex biological matrixes. This is manifested by the prior publication of over 80 reports on applications involving the quantitation of single natural products in plant extracts, dietary materials, and materials representing different metabolic stages of (micro)organisms. In summary, qHNMR has great potential as an analytical tool in both the discovery of new bioactive natural products and the field of metabolome analysis.

14. 1H NMR of Artemisinin

16. Top: 8 month plant extract 77-75I Bottom: Artem. Ref. DHCL

17. Expansion of H-12 region of 77-75I From ratio Artem. mmol = 0.019 Hence Artem. Wt = 5.47 mg Artem % = 0.6% Ref. 2 mg 0.0087 mmol H-12

18. 2 month plant extract 77-76C Ref. 2 mg 0.0087 mmol Integral Ratio 1:1 Calcd Artem. mmol = .009 Hence Artem. Wt = 2.5 mg Artem % = 0.3%

20. Artemisinin 1 mg 282 MW 0.0035 mmol Caffeine 2mg 194 MW 0.0100 mmol DHCL 2mg 230 MW 0.0087 mmol Artemi/Caf. Integral Ratio .037/1 Therefore Artemi = 0.37 x 0.01 = 0.0037 mmol Mass of Artemi. = 0.0037 x 282 = 1.04 mg = 1 mg Artemi/DHCL Integral Ratio 0.37/0.8 = 0.46 Therefore Artemi = 0.46 x 0.0087 = 0.004 mmol Mass of Artemi. = 0.004 x 282 = 1.1 mg = 1 mg Conclusion: Actual Artemi. Added 1 mg Found by QHNMR 1 mg

21. What next?

22. Thank you very much for your attention