Discovery of the key active compounds in Citri Reticulatae Pericarpium (Citrus reticulata "Chachi") and their therapeutic potential for the treatment of COVID-19 based on comparative metabolomics and network pharmacology

Abstract

Edible herbal medicines contain macro- and micronutrients and active metabolites that can take part in biochemical processes to help achieve or maintain a state of well-being. Citri Reticulatae Pericarpium (CRP) is an edible and medicinal herb used as a component of the traditional Chinese medicine (TCM) approach to treating COVID-19 in China. However, the material basis and related mechanistic research regarding this herb for the treatment of COVID-19 are still unclear. First, a wide-targeted UPLC-ESI-MS/MS-based comparative metabolomics analysis was conducted to screen for the active metabolites of CRP. Second, network pharmacology was used to uncover the initial linkages among these metabolites, their possible targets, and COVID-19. Each metabolite was then further studied via molecular docking with the identified potential SARS-CoV-2 targets 3CL hydrolase, host cell target angiotensin-converting enzyme II, spike protein, and RNA-dependent RNA polymerase. Finally, the most potential small molecule compound was verified by in vitro and in vivo experiments, and the mechanism of its treatment of COVID-19 was further explored. In total, 399 metabolites were identified and nine upregulated differential metabolites were screened out as potential key active metabolites, among which isorhamnetin have anti-inflammatory activity in vitro validation assays. In addition, the molecular docking results also showed that isorhamnetin had a good binding ability with the key targets of COVID-19. Furthermore, in vivo results showed that isorhamnetin could significantly reduced the lung pathological injury and inflammatory injury by regulating ATK1, EGFR, MAPK8, and MAPK14 to involve in TNF signaling pathway, PI3K-Akt signalling pathway, and T cell receptor signaling pathway. Our results indicated that isorhamnetin, as screened from CRP, may have great potential for use in the treatment of patients with COVID-19. This study has also demonstrated that comparative metabolomics combined with network pharmacology strategy could be used as an effective approach for discovering potential compounds in herbal medicines that are effective against COVID-19.

Keywords: COVID-19; Citri Reticulatae Pericarpium; Network Pharmacology; comparative metabolomics; isorhamnetin.

FIGURE 1

The strategy to discover potential compounds against COVID-19 based on comparative metabolomics and network pharmacology.

FIGURE 2

Clustering heatmap of metabolites (A–C) and volcano plots for the comparison group GCP1 versus GCP3 (D), GCP1 versus GCP5 (E), GCP1 versus GCP10 (F).

FIGURE 3

Differential metabolite analysis based on orthogonal signal correction and partial least squares-discriminant analysis (OPLS-DA) and principal component analysis for each comparison group. (A–C) OPLS-DA model plots for the comparison group GCP1 versus GCP3, GCP1 versus GCP5, GCP1 versus GCP10. 3D PCA plots for the comparison group GCP1 versus GCP3, GCP1 versus GCP5, GCP1 versus GCP10 (D–F).

FIGURE 4

Venn diagram showing the overlapping and unique differential metabolites between the comparison groups.

FIGURE 5

The structure of the effective ingredients in Citri Reticulatae Pericarpium.

FIGURE 6

GO enrichment analysis of the intersection of Citri Reticulatae Pericarpium. (A) Biological process. (B) Cellular component. (C) Molecular function.

FIGURE 7

Construction of the target network of Citri Reticulatae Pericarpium. (A) Component-target network. (B) Venn diagram between constituents-related targets and COVID-19-related targets. (C) Network of nine constituents and overlapped COVID-19-related targets. (D) The PPI network. (E) KEGG analysis of Citri Reticulatae Pericarpium. The sizes and colors are correlated to the degrees of targets in the network: larger sizes and deeper purple colors mean a higher degree of target correlation.

FIGURE 8

Molecular docking of the potential active Citri Reticulatae Pericarpium compound isorhamnetin with SARS-CoV-2 protein targets (A) 3CL, (B) ACE2, (C) S1, and (D) RdRp and the positive control ebselen with SARS-CoV-2 protein targets (E) 3CL, (F) RdRp, and (G) S1.

FIGURE 9

In vitro validation of isorhamnetin. (A) Effects of isorhamnetin on the viability of A549 cells. Effect of isorhamnetin on the production of IL-6 (B), IL-1β (C), TNF-ɑ (D) in LPS-induced A549 cells. The results represent the mean ± SEM (n = 3), # p < 0.05, ## p < 0.01 versus Control; *p < 0.05, **p < 0.01 cersus LPS.

FIGURE 10

Effects of isorhamnetin on histopathological changes in lung tissues of LPS-induced lung-inflammatory mice. (A) HE staining of lung tissues (n = 6). The images were taken with eyepiece at 20× and 40×. (B) W/D ratio of lung tissues (n = 6). (C) Lung injury score (n = 6). ^# p < 0.05, ^## p < 0.01 versus Control; * p < 0.05, ** p < 0.01 versus LPS.

FIGURE 11

Isorhamnetin suppresses lung inflammation-related targets induced by LPS. Core target-associated markers examined by western blot (n = 3). # p < 0.05, ## p < 0.01 versus Control; * p < 0.05, ** p < 0.01 versus LPS.

FIGURE 12

Isorhamnetin suppresses lung inflammation-related targets induced by LPS. (A) Core targets-associated markers examined by immunofluorescence (n = 3). Effect of isorhamnetin on the production of IL-6 (B), IL-1β (C), TNF-ɑ (D) in LPS-induced mice (n = 6). ^# p < 0.05, ^## p < 0.01 versus Control; * p < 0.05, ** p < 0.01 versus LPS.