UPLC-Q-TOF-MS/MS combined with network pharmacology and molecular docking to explore the pharmacological substances and mechanisms of action of elliptical leaf flower anchor in anti hepatitis
Halenia elliptica D. Don is a plant in the Gentianaceae family, belonging to the Halenia genus. It has a bitter and cold taste and is used as a medicinal herb throughout the plant. It is commonly used in the Tibetan medicine system to treat liver and gallbladder diseases. Clinically used to treat diseases such as hepatitis caused by excessive heat and heat. Although elliptical leaf flower anchor has been used in clinical practice, its effectiveness in treating hepatitis and experimental liver injury has been confirmed. However, there are not many literature reports on its anti hepatitis effect, and its mechanism of action is still unclear. Therefore, further research is needed to clarify its mechanism of action and provide theoretical basis for the development and utilization of Tibetan medicine.

Network pharmacology is an emerging branch of pharmacology that is based on the basic theories of systems biology and network biology. Through the use of relevant databases and algorithms for deep mining, analysis, and summarization, it constructs a network of drug targets and mechanisms that act on diseases. It has the characteristics of integrity and systematicity, and is particularly suitable for the study of drug action mechanisms such as Chinese and Tibetan medicine, which have multiple components, multiple targets, and complex pathways of action. This study intends to use UPLC-Q-TOF-MS/MS technology and network pharmacology research methods to explore the potential mechanism of action of the elliptical leaf flower anchor against hepatitis, and screen its active ingredients and targets, in order to provide new ideas and theoretical basis for further research on the pharmacological mechanism of the elliptical leaf flower anchor against hepatitis.

This study analyzed 40 chemical components of the elliptical leaf flower anchor using UPLC-Q-TOF-MS/MS technology, mainly including flavonoids, terpenes, triterpenes, and alkaloids mainly composed of kaempferol and its glycosides. Among them, J34, J39, J35, J24, J38 and other compounds have relatively high degree values, and four of the top ten compounds in terms of degree values are Koushanketone components, indicating that Koushanketone components may be the main active ingredients of the elliptical leaf flower anchor in anti hepatitis. Zhang et al.’s research also showed that Huagan and its Koushanketone glycosides are the main anti hepatitis components, which promote liver protection by increasing protein synthesis and glycogen and ribonucleic acid synthesis in liver cells. Wang et al. investigated the inhibitory effects and specific mechanisms of two types of ketogenic monomers on the proliferation of HepG2 liver cancer cells, and found that both ketogenic compounds can effectively inhibit the proliferation of HepG2 liver cancer cells, which may be achieved by inducing autophagy. Zhang et al. studied the effect of kaempfer1 on acute liver injury induced by acetaminophen and found that kaempfer1 protects liver cells by inhibiting the JNK pathway to initiate necrosis mode and mitochondrial function. Kaempfer1 can also upregulate GSH in liver cells, thereby achieving hepatoprotective effects. Shakya et al. found that kaempferol reduces the oxidative stress response in mouse liver tissue caused by alcohol and polyunsaturated fatty acids by inhibiting the expression of antioxidant enzymes such as catalase, glutathione peroxidase, and glutathione-S-transferase, thereby providing a certain protective effect against liver damage. There are few literature reports on the other important components with high degree values, and their pharmacological and mechanism of action need further experimental verification.

According to the KEGG pathway enrichment and component target pathway network diagram, it can be seen that PIK3CA, TNF, PIK3R1, MAPK8, LGALS3, MAPK1, MAPK14, STAT3, IL6, PTGS2 and other targets, as well as TNF, Toll like receptor, FoxO, HIF-1, VEGF, FcepsilonRI and other signaling pathways, have higher degree values and more components connected to them, which may be the main potential targets and pathways for the anti hepatitis of the elliptical leaf flower anchor.

PI3K signaling pathway is a key signal transduction pathway that controls cell proliferation, apoptosis and metabolism in cells, and PIK3CA and PIK3R1 have high mutation rates in liver cancer, brain cancer, breast cancer, stomach cancer and colon cancer. Griffith et al. created the tumor genotype spectrum of PIK3CA and PIK3R1 affecting mutation by studying the expression of PIK3CA and PIK3R1 genes in human breast cancer, opening up a new way for gene therapy of cancer. Xiao et al. found that hypothermia pretreatment can inhibit the release and apoptosis of inflammatory cytokines through the PI3K/AKT/FOXO3a pathway, and has a protective effect on ischemia-reperfusion liver injury. Jiang et al. found that epigallocatechin gallate can significantly improve liver injury and pathological morphology in rats with biliary obstruction. Its mechanism of action may be achieved by regulating the PI3K/Akt signaling pathway, promoting the phosphorylation of PI3K and AKT, and inhibiting liver cell apoptosis. Both PIK3CA and PIK3R1 belong to the PI3K signaling pathway. The PI3K/Akt pathway can promote liver fibrosis by activating hepatic stellate cells and fibroblasts. The ketone component in the elliptical leaf flower anchor may mediate the PI3K signaling pathway and inhibit liver cell apoptosis to achieve anti hepatitis effects.

MAPKs are a type of signaling protein that activates and regulates a range of cellular activities. Activated mitogen activated protein kinase (MAPK) participates in the regulation of cell proliferation, differentiation, transformation, and apoptosis by phosphorylating nuclear transcription factors, cytoskeletal proteins, and enzymes, and is closely related to the occurrence of various diseases such as inflammation and tumors. Roman et al. found that in RAW264.7 macrophages, sPGN intensity activates the MAPK1/ERK2 pathway, moderately activates the MAPK8/JNK pathway, and mildly activates the P38 pathway. Among them, the ERK pathway mainly mediates the inflammatory response, and the activation of the P38 pathway under inflammatory conditions has multiple characteristics. MAPK8 gene is highly expressed in tumor tissues of many parts of the human body, such as liver cancer, gastric cancer, and pancreatic cancer. The carcinogenic mechanism is that MAPK8 pathway is first activated by external stimuli and transferred to the nucleus to increase the activity of transcription factors, further promoting the proliferation and differentiation of cancer cells.

TNF, as an important cytokine, can induce various signaling pathways within cells, including proliferation, apoptosis, inflammation, immunity, etc. Wang et al. found that the level of TNF in patients with chronic hepatitis C is significantly higher than that in normal healthy individuals. Tilg et al. found that the levels of serum inflammatory factors TNF – α and IL-6 in patients with chronic hepatitis B can reflect the degree of liver cell damage. As important inflammatory factors, TNF – α and IL-6 play an important role in the occurrence and development of hepatitis B, and are positively related to the severity of the disease. Therefore, current research believes that traditional Chinese medicine can protect the liver by reducing the levels of TNF – α and IL-6 in serum.

In summary, based on the determination and analysis of the chemical components of the elliptical leaf flower anchor, this study used network pharmacology analysis methods to analyze 33 potential anti hepatitis targets of 40 chemical components of the elliptical leaf flower anchor. It was found that the elliptical leaf flower anchor may exert its anti hepatitis effect through biological processes such as leukocyte migration, positive regulation of nitrogen monoxide biosynthesis, platelet activation, positive regulation of extracellular regulatory protease ERK1 and ERK2 cascade, regulation of phosphatidylinositol 3-kinase signal transduction, as well as key signaling pathways such as TNF, PI3K, Toll like receptor, FoxO, HIF-1, VEGF, Fcepslon RI, etc. The molecular docking results confirmed that the predicted main chemical components and key targets have good binding activity, indicating that the prediction in this study is accurate and reliable. However, this study also has certain limitations, such as incomplete databases, so further validation of related target pathways is needed.