Total flavonoids in Artemisia absinthium L. and evaluation of its anticancer activity
Authors: He Meizhu , Yasin Kamarya , Ma Zihang , Li Jinyao , Xia Lijie 
Published: June 2023

Abstract
Aim of the study:
To optimize the extracting parameters of total flavonoids in Artemisia absinthium L. by ultrasound-assist combined with enzymatic hydrolysis and to combine molecular docking and network pharmacology to predict its core constituent targets and thus evaluate its antitumor activity.
Methods
Reaction surface methodology was used to investigate the univariate effects of enzyme ratio, enzyme amount, enzyme hydrolysis temperature, enzyme hydrolysis time, pH, solid-liquid ratio, ethanol concentration, and sonication temperature on total flavonoid yield in A. absinthium L.. On this basis, a three-factor, three-level experimental approach was adopted with solid-liquid ratio, enzymatic hydrolysis temperature, and ethanol concentration as independent variables and total flavonoids as response values, and then combined with Box-Behnken design (BBD) to optimize the extraction conditions. The quantitative and qualitative analysis of the main components was performed by UHPLC-MS. The inhibitory effect of flavonoids and their active components on the proliferation of cervical cancer HeLa cells was detected by MTT. Molecular docking and network pharmacology were used to predict the molecular mechanism of the main active components (Cynaroside and Astragalin) and to decipher the overall mechanism of total flavonoids against cervical cancer.. pharmMapper and SwissTargetPrediction databases were used to sort out the potential targets of the main chemical components. Targets related to cervical cancer were collected from OMIM and DrugBank.
Results
The content of total flavonoids in A. absinthium L. reached 3.80 ± 0.13%, and the main components included Astragalin, Cynaroside, Ononin, Rutin, Kaempferol-3-O-rutinoside, Diosmetin, Isorhamnetin, and Luteolin. Cynaroside and Astragalin exert their cervical cancer inhibitory functions by regulating several signaling proteins (e.g. EGFR, STAT3, CCND1, IGFIR, ESR1). GO and KEGG analyses showed that the anti-cancer of both compounds was associated with ErbB signaling pathway and FoxO signaling pathway. MTT results showed that total flavonoids of A. absinthium L. and its active components (Cynaroside and Astragalin) significantly inhibited the growth of HeLa cells in a concentration-dependent manner with IC50 of 396.0 ± 54.2 µg/mL and 449.0 ± 54.8 µg/mL, respectively.
Conclusion
The optimal process conditions for obtaining total flavonoids by ultrasound-assisted enzymatic digestion were: enzyme ratio 3:2, enzyme dosage 2%, enzymatic hydrolysis temperature 45℃, enzymatic hydrolysis time 105 min, pH 3.5, solid-liquid ratio 1:15, ethanol concentration 85%, sonication temperature 30℃. Results from network pharmacology and molecular docking indicate that EGFR and SRC are the key targets of the two core components of total flavonoids against cervical cancer, the optimal procedure for extracting total flavonoids from A. absinthium L. was fixed and the total flavonoids showed good anti-cervical cancer activity. Furthermore, its active components can mediate apoptosis by inducing the accumulation of ROS.