Ultraperformance convergence chromatography is an environmentally friendly analytical technique for dramatically reducing the use of organic solvents compared to conventional chromatographic methods. In this study, a rapid and sensitive ultraperformance convergence chromatography method was firstly established for quantification of thymol and carvacrol, two positional isomers of a major bioactive in the volatile oil of Thymi herba, the dried leaves and flowers of Thymus mongolicus or Thymus przewalskii, known in China as “Dijiao.” Using a Trefoil^TM CEL1 column, thymol and carvacrol were separated in less than 2.5 min and resolution was enhanced. The method was validated with respect to precision, accuracy, and linearity according to the National Medical Products Administration guidelines. The optimized method exhibited good linear correlation (r = 0.9998−0.9999), excellent precision (relative standard deviations (RSDs) < 1.50%), and acceptable recoveries (87.29–102.89%). The limits of detection for thymol and carvacrol were 1.31 and 1.57 ng/L, respectively, while their corresponding limits of quantification were 2.63 and 3.14 ng/L. Finally, the quantities of the two compounds present in 16 T. mongolicus and four T. przewalskii samples were successfully evaluated by employing the developed method. It is hoped that the results of this study will serve as a guideline for the quality control of Thymi herba.

Artemisinin (ART) is a highly effective antimalarial agent isolated from the traditional Chinese herb Qinghao. Metabolism of ART and its derivatives in the body is one of the most pressing issues for pharmaceutical scientists. Herein, an efficient in vitro microorganism model for simulation of metabolism of ART in vivo was developed employing Cunninghamella elegans. Metabolites in the microbial transformation system and plasma of mice pre-administrated ART orally were analyzed by ultra-performance liquid chromatography (UPLC)-electrospray ionization (ESI)-quadrupole time-of-flight (Q-TOF)-mass spectrometry (MS^E) combined with UNIFI software. Thirty-two metabolites were identified in vitro and 23 were identified in vivo. After comparison, 16 products were found to be common to both models including monohydroxylated ART, dihydroxylated ART, deoxyartemisinin, hydroxylated deoxyartemisinin, hydroxylated dihydroartemisinin (DHA), and hydroxylated deoxy-DHA. These results revealed that C. elegans CICC 40250 functioned as an appropriate model to mimic ART metabolism in vivo. Moreover, an overall description of metabolites of ART from C. elegans CICC 40250 has been provided. Notably, DHA was detected and identified as a metabolite of ART in mouse plasma for the first time.

Malaria chemotherapy is greatly threatened by the recent emergence and spread of resistance in the Plasmodium falciparum parasite against artemisinins and their partner drugs. Therefore, it is an urgent priority to develop new antimalarials. Plasmepsin V (PMV) is regarded as a superior drug target for its essential role in protein export. In this study, we performed virtual screening based on homology modeling of PMV structure, molecular docking and pharmacophore model analysis against a library with 1,535,478 compounds, which yielded 233 hits. Their antimalarial activities were assessed amongst four non-peptidomimetic compounds that demonstrated the promising inhibition of parasite growth, with mean IC₅₀ values of 6.67 μM, 5.10 μM, 12.55 μM and 8.31 μM. No significant affection to the viability of L929 cells was detected in these candidates. These four compounds displayed strong binding activities with the PfPMV model through H-bond, hydrophobic, halogen bond or π-π interactions in molecular docking, with binding scores under −9.0 kcal/mol. The experimental validation of molecule-protein interaction identified the binding of four compounds with multiple plasmepsins; however, only compound 47 showed interaction with plasmepsin V, which exhibited the potential to be developed as an active PfPMV inhibitor.

Dihydroartemisinin (DHA) and its’ derivatives have been employed as the most powerful first-line drugs for malarial treatment for several decades. The metabolism of DHA has not been studied clearly. Previous reports were focused on the pharmacokinetics procedure of DHA in healthy rats. The metabolites of DHA in red blood cells (RBC), especially in the RBC from Plasmodium-infected models, have rarely been studied. The Plasmodium species parasitize inside RBC, and these cells should be the final place where DHA performs its activity. In this study, the profile of DHA metabolites in biosample (blood, plasma, and RBC) of the infected and healthy mice was investigated with UPLC-Q-TOF-MS and UNIFI platform to gain insight into DHA metabolism. Results show that a total of 25 metabolites were successfully identified in infected (30 in healthy) blood, 27 in infected (27 in healthy) plasma, and 15 in infected (22 in healthy) RBC. Results show that hydroxylation, OH-dehydration, and glucuronidation reactions were important in the metabolic pathway in vivo. Significantly, DHA metabolites inside RBC were identified for the first time. 8-Hydroxy (8-OH) DHA, 4α-OH deoxy ART, and 6β-OH deoxy ART were identified in vivo for the first time.

10-deoxoartemisinin is a semisynthetic derivative of artemisinin that lacks a lactone carbonyl group at the 10-position, and has stronger antimalarial properties than artemisinin. However, 10-deoxoartemisinin has limited utility as a therapeutic agent because of its low solubility and bioavailability. Hydroxylated 10-deoxoartemisinins are a series of properties-improved derivatives. Via microbial transformation, which can hydroxylate 10-deoxoartemisinin at multiple sites, the biotransformation products of 10-deoxoartemisinin have been investigated in this paper. Using ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS^E) combined with UNIFI software, products of microbial transformation of 10-deoxoartemisinin were rapidly and directly analyzed. The hydroxylation abilities of nine microorganisms were compared using this method. All of the microorganisms evaluated were able to hydroxylate 10-deoxoartemisinin, and a total of 35 hydroxylated products were identified. These can be grouped into dihydroxylated 10-deoxoartemisinins, monohydroxylated 10-deoxoartemisinins, hydroxylated dehydrogenated 10-deoxoartemisinins, and hydroxylated hydrogenated 10-deoxoartemisinins. Cunninghamella echinulata and Cunninghamella blakesleeana are able to hydroxylate 10-deoxoartemisinin, and their biotransformation products are investigated here for the first time. Cunninghamella elegans CICC 40250 was shown to most efficiently hydroxylate 10-deoxoartemisinin, and could serve as a model organism for microbial transformation. This method could be used to generate additional hydroxylated 10-deoxoartemisinins for further research.

Dihydroartemisinin (DHA) is a sesquiterpene endoperoxide with prominent antimalarial efficacy, which was discovered by Professor Youyou Tu through the reduction of artemisinin in the 1970s. It is always a challenging work for scientists to investigate the metabolites of DHA in the red blood cells due to the complicated matrix background. As a bottleneck, the investigation of metabolites, especially exploring the pharmacodynamic material in the red blood cell, is necessary and significant for metabolism research of antimalarial agent. Recently, microbial transformation provides a green and economical means for mimicking mammal metabolism and synthesis active metabolites, based on which is one efficient route for drug discovery. In this study, a strain from Cunninghamella was employed as an efficient tool to explore active metabolites of DHA in erythrocyte. Microbial transformation products of DHA by Cunninghamella elegans CICC 40250 were detected and analyzed by ultra-performance liquid chromatography (UPLC)-electrospray ionization (ESI)-quadrupole time-of-flight (Q-TOF)-mass spectrometry (MS^E), and the main products were isolated and identified. The antimalarial activity of the isolated products was also screened in vitro. Totally, nine products were discovered through UPLC-ESI-QTOF-MS^E, and three main products with novel chemical structures were isolated for the first time, which were also detected in red blood cells as the metabolites of DHA. After evaluation, 7β-hydroxydihydroartemisinin (M1) exhibited a good antimalarial activity with an IC₅₀ value of 133 nM against Plasmodium falciparum (Pf.) 3D7. The structure and stereo-configuration of novel compound M1 were validated via X-ray single crystal diffraction. Microbial transformation was firstly employed as the appropriate model for metabolic simulation in erythrocyte of DHA. Three novel metabolites in erythrocyte were obtained for the first time through our microbial model, and one of which was found to show moderate antimalarial activity. This work provided a new research foundation for antimalarial drug discovery.