LC-MS/MS metabolomics profiling of Glechoma hederacea L. methanolic extract; in vitro antimicrobial and in vivo with in silico wound healing studies on Staphylococcus aureus infected rat skin wound

Abstract LC-MS/MS analysis of Glechoma hederacea L. methanolic extract (GHME), revealed the identification of 25 metabolites. Ursolic acid (1), 2α-hydroxyursolic acid or corosolic acid (2), 2β-hydroxyursolic acid or epi-corosolic (3), luteolin 7-O-β-D-glucopyranoside (4) and rosmarinic acid (5) were isolated and identified using spectroscopy. Antibacterial activity of GHME against multi drug resistance Staphylococcus aureus clinical isolates was measured. Minimum inhibitory concentrations (MICs) were ranged from 62.5 to 500 µg/ml. In vivo wound healing potential of 2%, and 5% GHME prepared hydrogels were criticized on Staphylococcus aureus infected wound rat model. 5% GHME prepared hydrogel treated group showed significant (p < 0.05) shrinkage of their colony forming unit/ml (CFU/ml) values in comparison with standard Fucidin. Meanwhile, wound closure associated with full re-epithelization and hair follicles proliferation was noticed after ten days of treatment. Finally, among the GHME isolated compounds, luteolin 7-O-β-D-glucopyranoside (4) exhibited the highest molecular docking score (–9.6 kcal/mol) against matrix metalloproteinase-8 target (MMP-8). Graphical Abstract


Introduction
The medicinal plant Glechoma hederacea L. (kakidooshi in Japanese), frequently named ground ivy is a perennial creeping herb (Lamiaceae family) with expanded Asian, European, and American distribution. Traditionally, it has been used to cure inflammation, gastrointestinal and renal defects (Kumarasamy et al. 2003).
Staphylococcus aureus is a common clinical infectant of wounds. It acquires resistance to multiple antimicrobials, complicating the healing prosses (Attallah et al. 2021). Ghosh and Gaba (2013) reviewed the contributions of safe and less resistible folk medicinal plants in wound healing through their phytochemicals humidifying, antioxidant, antimicrobial, anti-inflammatory, and healing bio-phases stimulation properties.
Our study is established for further LC-MS/MS phytochemical estimation of Glechoma hederacea L. methanolic extract (GHME). Separation of active ingredients from the targeted plant was achieved. Unexplored in vitro antimicrobial activity of GHME against multidrug resistant S. aureus clinical isolates and in vivo wound healing activity of its formulated hydrogels were examined on wounded rat model infected by this bacterium. Decisively, wound healing molecular docking of its isolated metabolites against matrix metalloproteinase-8 target was done.

Result and discussion
2.1. Phytochemical investigation 2.1.1. LC-MS/MS categorization of GHME (supplementary material) Negative and positive mode total ion chromatogram mass analysis of GHME defined twenty-five known secondary metabolites in comparison to previous data (Supplementary material Figure S1(A,B); Table S1).

Isolation of metabolites from GHME (supplementary material)
Isolated compounds are illustrated in Figure 1.

In vivo wound healing (supplementary material)
We investigated the antimicrobial susceptibility of S. aureus clinical isolates (n ¼ 35) using Kirby-Bauer method and their antimicrobial resistance profile (Supplementary  material Table S2). In vitro antibacterial activity of GHME against S. aureus clinical isolates by disc agar diffusion method was carried out (Attallah et al. 2021). The resultant MIC values were ranged from 62.5 to 500 mg/ml via broth microdilution method. The in vivo wound healing potentiation of 2%, and 5% GHME prepared hydrogels were evaluated on five S. aureus infected skin wound of rat groups (Supplementary material Figure S2 (A,B) via pre, and post treatment's (CFU/ml) counting. After ten days of treatment, significant (p < 0.05) deflated CFU/ml values was observed for both concentrations of GHME hydrogel (Supplementary material Figure S3). Histopathological examinations were preformed (Supplementary material Figure S4 (A-E)). Full epithelized and closure of S. aureus infected rats skin wound was noticed through histopathological evaluation of 5% GHME hydrogel treated group (Supplementary material Figure S4D), nearby to that of Fucidin (Supplementary material Figure S4E).

Molecular docking study (supplementary material)
The role and importance of the isolated compounds (1-5) in wound healing process were specified. The interactions between each component and the target of interest MMP-8 (PDB ID code 5H8X) (Tauro et al. 2016) were performed by molecular docking calculations on the active site of MMP-8 as summarized in (Supplementary material  Table S3 and Figures S5(A-F)). The docking protocol's reliability (Xiao et al. 2018) was tested by comparing the crystallized inhibitor's best docking pose with its bound conformation at (Supplementary material Figure S5A (Left)), the root mean square deviation (RMSD) was determined to be 0.798. The best three components alignment with co-crystalized BF471 (catechol inhibitor) was shown in (Supplementary material Figure S5A (Right)).luteolin 7-O-b-D-glucopyranoside (4) conceded high wound healing molecular docking score (-9.6 kcal/mol) to MMP-8 enzyme (Supplementary material Figure S5B), among other GHME isolates (1)-(3) & (5). It forms nine H-bonds, through phenolic and carbonyl groups of flavone part and hydroxyl groups of sugar part with the GLY79, LEU81, HIS83, ALA84, LEU114, TYR137, ASN139 and ARG143. In addition, there are two electrostatic interactions between flavone ring and GLU119. Furthermore, a face-to-face aromatic p-p-stacking of flavone and phenyl ring with the imidazole and benzene rings of HIS118 and TYR140 respectively is confirmed. Besides, few hydrophobic interactions between aromatic rings with various hydrophobic amino acid residues have been confirmed.
Contributions of flavonoids and phenolic acids in field of wound healing were inspired by their anti-inflammatory, antimicrobial, and antioxidant activities (Tsala et al. 2013). In view of this study, GHME is a healer candidate of S. aureus infected rats skin wound. Our findings are in coherence with those reports, which exposed to the in vivo wound healing effects of either rosmarnic, ursolic acids or luteolin (Naika et al. 2016;K€ uba et al. 2021). Moreover, our wound healing in silico study of GHME isolates against MMP-8 is complementary to thoses studies of rosmarinic acid's antimicrobial, ursolic acid's wound enhancing and corosolic acid's anti-inflammatory effects (Abd El Wahed et al. 2015;Li et al. 2020;Chen et al. 2021). Yet, further research is required to conclude GHME anti-S.aureus mechanism of action.

Conclusion
LC-MS/MS analysis of GHME, revealed identification of 25 metabolites. Ursolic acid (1), 2a-hydroxyursolic acid or corosolic acid (2), 2b-hydroxyursolic acid epi-corosolic (3), luteolin 7-O-b-D-glucopyranoside (4) and rosmarinic acid (5) were isolated and identified by spectroscopy. In vivo wound healing potential of the prepared 2% and 5% of GHME hydrogel concentrations on Staphylococcus aureus infected wound rat model was highlighted, which adopted a significant (P < 0.05) shrinkage of their CFU/ml values. 5% G. hederacea L. hydrogel concentration showed remarkable wound closure associated with full re-epithelization and hair follicles proliferation. Among the isolated compounds, luteolin 7-O-b-D-glucopyranoside had the highest molecular docking score (-9.6 kcal/mol) against the matrix metalloproteinase-8 target, which helps to resolve inflammation and allows for the development of new strategy for addressing wound healing abnormalities.