Exploring the Anti H1N1 Active Components and Mechanisms of Naked Purple Beads Based on UPLC-Q-Exactive-Orbitrap-MS, Network Pharmacology, and Experimental Validation
The traditional Chinese medicine, Callicarpa nudiflora Hook.&Arn., is a dried leaf of the plant Callicarpa nudiflora Hook.&Arn. in the family Verbenaceae. Naked Purple Beads are mainly distributed in Hainan, Guangxi, Guangdong and other regions, with the authentic production area being Wuzhishan District in Hainan Province. Naked Flower Purple Pearl is the only newly added traditional Chinese medicine in the 2020 edition of the Pharmacopoeia of the People’s Republic of China. Its unilateral preparations, Naked Flower Purple Pearl Tablets and Naked Flower Purple Pearl Capsules, are widely used to treat respiratory and digestive bleeding and bacterial infectious inflammation caused by excessive blood heat and toxicity. Phytochemists have identified over 300 compounds from naked purple pearls, including phenylpropanoids, flavonoids, triterpenoids, diterpenes, iridoids, volatile oils, and more. Modern pharmacological research has confirmed that naked flower purple pearl has anti-inflammatory, antithrombotic, anti-tumor, antibacterial, and hepatoprotective activities. In the process of screening traditional Chinese medicine with antiviral activity, it was found that the extract of naked flower purple pearl has good antiviral activity (such as H1N1 influenza virus, enterovirus 71, etc.). Influenza A virus infection is a common cause of pneumonia related deaths. Severe infection with influenza A virus can cause bilateral lung infiltration and hypoxemia, known as acute respiratory distress syndrome, which is one of the main causes of death caused by influenza A virus. This experiment focuses on the anti H1N1 activity of naked flower purple bead. The chemical composition of the effective parts of naked flower purple bead was identified using ultra-high performance liquid chromatography quadrupole electrostatic field orbitrap MS, and the anti H1N1 mechanism of naked flower purple bead was predicted and experimentally verified using network pharmacology. The mechanism of action of naked flower purple bead against H1N1 was preliminarily explored.

callicarpa nudiflora
The influenza A virus has developed varying degrees of resistance to commonly used M2 channel inhibitors and NA inhibitors. Therefore, finding new drugs or treatment methods for anti influenza A has become an urgent problem to be solved. Traditional Chinese medicine has played an important role in the clinical treatment of influenza A virus. The holistic concept in traditional Chinese medicine theory systematically regulates patients as an organic whole, while network pharmacology explores the interaction between the biological functions of each node in the network and diseases by constructing a drug component target pathway disease network, which is consistent with the holistic concept in traditional Chinese medicine. This experiment first screened the main anti H1N1 active sites of the traditional Chinese medicine Naked Flower Purple Pearl, and analyzed the sites to identify 34 chemical components. Combining literature and databases, 67 overlapping targets of drugs and diseases were identified. Through enrichment analysis of cross targets, we speculate that the important biological process of EACN against H1N1 may be the positive regulation of protein phosphorylation and response to oxidative stress. The KEGG enrichment analysis results showed that 19 compounds in EACN act on the influenza A virus pathway through 11 targets. The drug affects the M2 channel of H1N1 virus by acting on PLG and PRSS1, and can also affect the virus release process by acting on the neuraminidase (NA) pathway through PLG. PLG and PRSS1 may be key targets for EACN to directly act on H1N1. EACN can inhibit the proliferation of H1N1 by affecting its shedding and release processes. In the drug ingredient target pathway disease network, RELA and NF – κ B1 are the two targets with the highest degree values. The NF – κ B signaling pathway in which they are located can promote the expression of factors such as IL1 β, TNF – α, IL-6, and promote inflammatory response, suggesting that EACN may act on key targets such as RELA and NF – κ B1 to regulate the NF – κ B signaling pathway and reduce the damage of inflammation to the body. According to the results predicted by network pharmacology, EACN can regulate T cell signaling pathways, affect Th1 and Th2 differentiation, and exert immune regulatory effects. Therefore, this study conducted animal experiments to verify the inhibitory effect, anti-inflammatory effect, and immune regulatory effect of EACN on H1N1 proliferation. The experimental results showed that EACN can improve the weight loss and lung index increase caused by H1N1 infection, reduce the viral load in mouse lung tissue, alleviate pathological damage in mouse lung tissue, lower the levels of TNF – α, IFN – γ, IL-1 β in peripheral blood serum, and increase the level of serum antibody IgG. This study first used in vitro experiments to screen the active extraction sites of Naked Purple Pearl against H1N1, and predicted the active ingredients of EACN against H1N1 using UPLC-Q-Hexactive-Orbitrap-MS and network pharmacology. The predicted results were verified by animal experiments, and potential active ingredients of EACN against H1N1 were screened, including quercetin, myricetin, luteolin, kaempferol, quercetin, apigenin, and acacetin. It was preliminarily demonstrated that Naked Purple Pearl can exert anti H1N1 effects through multiple pathways such as direct virus inhibition, anti-inflammatory effects, and immune regulation. In subsequent experiments, it is necessary to conduct in vitro and in vivo experiments to validate the selected monomeric compounds and further investigate the mechanism of EACN’s anti H1N1 effect at the mRNA and protein levels.