Predictive Analysis of Anti hepatitis B Virus Quality Markers of Artemisia annua Based on Network Pharmacology and Fingerprint
Artemisia rupestris L., which belongs to the genus Artemisia of Compositae, is a commonly used folk medicine of Xinjiang Uygur and Kazak, and is mainly distributed in the grasslands, hillsides or forests of Tianshan Mountains, Altay Mountains and other places in Xinjiang. It is now included in the Uyghur Medicine Volume of the “Drug Standards” of the Ministry of Health, the “Uyghur Pharmacopoeia” (Volume One), and the “Chinese Materia Medica” (Uyghur Medicine Volume). Yizhihao has the functions of clearing heat and detoxifying, dispelling wind and cooling blood, anti HBV, antiviral, liver protection and enzyme reduction, regulating immunity, and promoting digestion. It is mainly used clinically for viral colds and hepatitis, and has significant therapeutic effects. It is widely used in folk medicine, especially in Uyghur hospitals. According to the prescription survey of Xinjiang Uyghur Hospital, the dosage of Yizhihao can be regarded as the largest in the formulation of Uyghur medicine, and the variety of drug preparations is diverse. During the COVID-19, the Xinjiang Health Commission organized experts to formulate and print the “Xinjiang version” of the Chinese medicine prevention and treatment plan in combination with the regional characteristics of Xinjiang. Compound Yizhihao Granules was selected as the first choice medicine to inhibit respiratory inflammation and fight viruses.

This article establishes the fingerprint spectrum of Artemisia scoparia and identifies the chromatographic peaks as reference standards. At the same time, network pharmacology is used to study the interaction between various components in Artemisia scoparia and hepatitis B virus (HBV), aiming to provide a basis for comprehensive quality control of Artemisia scoparia in Xinjiang, and to explore the possible pharmacological substance basis and mechanism of action of Artemisia scoparia in treating HBV from a holistic perspective. This lays the foundation for the study of quality markers of Artemisia scoparia in Xinjiang, and provides theoretical basis for further cell biology experiments and animal model experiments in the later stage.

This study is based on the quality marker (QMarker) proposed by Academician Liu Changxiao, and predicts and analyzes the quality markers of Artemisia annua through network pharmacology and fingerprint analysis. The combination of network pharmacology and fingerprint technology is used to evaluate the quality of traditional Chinese medicine, effectively avoiding the drawbacks of evaluating the quality of traditional Chinese medicine based on the content of a single component. In the study, fingerprint spectra were constructed for 18 batches of Artemisia annua samples from different origins and batches. Similarity evaluation, HCA, and OPLS-DA analysis were used to comprehensively evaluate them. The scores showed that there were differences in the comprehensive quality of Artemisia annua medicinal materials from different origins, with obvious regional differentiation. This further demonstrates that this method can be easily, quickly, and reliably applied to the quality control of Artemisia annua.
Through the analysis of the “active component target” and “component target pathway” network diagram constructed by network pharmacology, it can be concluded that Artemisia argyi can regulate the key targets in PPI network, such as AKT1, EGFR, SRC and ESR1, and act on key signal pathways, such as PI3K-AKT signal pathway, Rap1 signal pathway and Ras signal pathway, to play a pharmacodynamic role in the treatment of hepatitis B virus. EGFR is a receptor tyrosine kinase that triggers signaling events crucial for cellular processes, including the Ras mitogen activated protein kinase (MAPK), PI3K Akt, and JAK-STAT pathways. After stimulation with its ligand epidermal growth factor (EGF), EGFR automatically phosphorylates to activate these signaling pathways. So far, it has been reported that EGFR plays a key role in the entry mechanisms of different types of viruses, such as human cytomegalovirus, hepatitis C virus (HCV), and respiratory syncytial virus. Chen et al.’s research suggests that IFN – α may inhibit the phosphorylation levels of mTOR and p70S6K by regulating the PI3K/AKT pathway, thereby alleviating fibrosis and liver function damage caused by HBV. Liu et al reported that the X protein binding protein of hepatitis B virus is highly expressed in hepatoma cells, which may promote the proliferation and migration of hepatoma cells by activating PI3K/Akt signaling pathway.
In the process of screening active ingredients, the highest degree values are quercetin (degree=187), β – sitosterol (degree=109), luteolin (degree=100), stigmasterol (degree=86), upland cotton glycoside (degree=53), artemisic acid (degree=51), quercetin (degree=49), acacetin (degree=37), kaempferol (degree=28), and quercetin (degree=20), all of which have a degree greater than 10 times the median. Due to the widespread presence of quercetin, β – sitosterol, carotenoids, and stigmasterol in various traditional Chinese medicinal materials, these components are widely present. It has multiple biological activities, so it cannot be used as a quality indicator component to distinguish the quality of medicinal materials.
This article selects biomarkers for the safety and effectiveness of traditional Chinese medicine based on the five principles of quality indicators proposed by Academician Liu Changxiao: measurability, uniqueness, effectiveness, correlation with traditional Chinese medicine theory, and traceability. The chemical composition measurability is one of the key factors for determining the Q-Marker of traditional Chinese medicine. The content of flavonoids and sesquiterpenes in Artemisia scoparia in Xinjiang is relatively rich. Our research group has conducted extensive studies on the determination of Artemisia scoparia components in the early stage, including quantitative detection of common components in Artemisia scoparia medicinal materials and their compound Artemisia scoparia capsules and Artemisia scoparia nasal drops from different origins and harvest periods. The detectable components include Artemisia scoparia ketone acid, chlorogenic acid, luteolin, vitexin, 6-demethoxy4 ‘- O-methyl artemisin ketone, and artemisin. Based on this, the flavonoids and sesquiterpenes in Artemisia scoparia meet the connotation of “measurability” in Q-Marker and can be used as candidate Q-Marker. However, the network pharmacology screening showed that the content of artemisia, paeoniflorin, hesperidin, and acacetin in Artemisia scoparia was relatively low. From the perspective of component measurability, they are not suitable as landmark components for evaluating the quality of Artemisia scoparia.
To further clarify the quality markers of Artemisia scoparia, HPLC was used to study 18 batches of Artemisia scoparia from different origins and batches, identifying 18 common peaks. Through comparison with reference standards, 8 common peaks were identified, namely chlorogenic acid, rutin, aucubin, luteolin, artemisic acid, apigenin, quercetin, and artemisin; The similarity ranges from 0.868 to 0.984, indicating that the chemical composition similarity of Artemisia scoparia from different origins and batches is high, but there are certain differences; Through OPLS-DA analysis, it was found that the common peaks rutin, quercetin, artemisia ketone acid, artemisia argyi su, and apigenin are important differential components of different batches of artemisia medicinal materials.
In summary, according to the results of network pharmacology, the acidity values of luteolin and artemether rank high, and these two components can be used as Q-markers for artemether; According to the analysis of the common peaks and content results in the fingerprint spectrum, chlorogenic acid, artemisic acid, apigenin, paeoniflorin, and rutin have high contents. However, chlorogenic acid and rutin are widely present in most Chinese medicinal materials and cannot be used as quality indicator components. The content of artemisinin acid in artemisia is high, and it has been reported that artemisinin acid has liver protection, anti-virus, anti hepatitis B virus and other effects, which can be used as the Q-Marker of artemisia. Chiang et al.’s study showed that apigenin has good antiviral activity against herpes simplex virus, adenovirus, hepatitis B virus, etc., and can be considered as a potential Q-marker for Artemisia annua. Li et al. found that luteolin has inhibitory effects on Enterococcus faecalis, and it can interfere with virus replication and inhibit virus induced cell apoptosis, making it a potential Q-marker for Artemisia annua. Based on the analysis of the effectiveness of chemical components, fingerprint spectra, and chemical stoichiometry results, it is preliminarily predicted that one branch artemisia ketone acid, apigenin, luteolin, and paeoniflorin are potential Q-markers in one branch artemisia. The determination of their quality markers provides a scientific basis for the comprehensive control and evaluation of the quality of one branch artemisia in Xinjiang, and provides theoretical reference for the further development and application of one branch artemisia medicinal materials in Xinjiang. However, considering the differences in the update status of the five databases used in this study and the mechanisms for predicting targets, there are still certain limitations, and further validation of the pathways and targets through pharmacological experiments is needed to further determine the accuracy of the prediction results in this study.