In 1972, Nobel laureate Youyou Tu’s research team conducted clinical trials on the dried material of Artemisia annua L. from Beijing extracted by ether and then treated with alkali (called “ether neutral dry”), which showed that artemisinin was not the only antimalarial component contained. The biosynthesis of sesquiterpenoids in A. annua has increased exponentially after unremitting cultivation efforts, and the plant resources are now quite different from those in the 1970s. In consideration of emerging artemisinin resistance, it is of great theoretical and practical value to further study the antimalarial activity of A. annua and explore its causes. The purpose of this study is to clarify scientific questions, such as “What ingredients are synergistic with artemisinin in A. annua?”, and “Are there non-artemisinin antimalarial ingredients in A. annua?”. In this paper, Beijing wild A. annua was used as a control and two representative cultivation species of A. annua were selected to evaluate the antimalarial activity of the herbal medicine. The antimalarial activity of different extracts on mice was studied using the Peters’ four-day suppressive test. UPLC-Q-TOF-MS was used to obtain mass spectrum data for all samples, and a UNIFI platform was used for identification. A multivariate statistical method was used to screen the different compounds with positive correlations. The antimalarial activity of different components from the ether extract and alkali treatments was determined and antimalarial components other than artemisinin were obtained. A total of 24 flavonoids, 68 sesquiterpenoids and 21 other compounds were identified. Compounds associated with differential antimalarial activity were identified. The material basis for the antimalarial activity of A. annua was clarified. The antimalarial components of A. annua include two categories: first, artemisinin and non-artemisinin antimalarial active components, of which the non-artemisinin antimalarial active components may include 5α-hydroperoxy-eudesma-4(15),11-diene; second, several antimalarial synergistic ingredients in A. annua, including arteanniun B, arteanniun B analogues and polymethoxy flavonoids.

Objective: To develop an accurate and rapid ultra-performance liquid chromatography (UPLC) coupled with a photodiode array (PDA) method for the simultaneous determination of artemisinin (Art), arteannuin B (Art B), arteannuin C (Art C), dihydroartemisinic acid (DHAA) and artemisinic acid (AA) in Artemisia annua L. Methodology: Chromatography separation was performed on an ACQUITY UPLC BEH C18 Column with isocratic elution; the mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B) (A:B = 40:60, v/v). Data were recorded at an ultraviolet (UV) wavelength of 191 nm for Art, Art C, DHAA and AA, and 206 nm for Art B. Results: The calibration curves of the five sesquiterpene components were all linear with correlation coefficients more than 0.9990. The linear ranges were 31.44–1572 μg/mL, 25.48–1274 μg/mL, 40.56–2028 μg/mL, 31.44–1572 μg/mL and 26.88–1396 μg/mL for Art, Art B, Art C, DHAA and AA, respectively. The precision ranged from 0.08% to 2.88%, the stability was from 0.96% to 1.66%, and the repeatability was all within 2.42% and had a mean extraction recovery of 96.5% to 100.6%. Conclusion: The established UPLC–PDA method would be valuable for improving the quantitative analysis of sesquiterpene components in Artemisia annua L.