NMR and GC-MS based metabolic profiling, total phenolic content, antibacterial, antioxidant, anticancer and In-silico antiviral activity of Origanum ramonense plant

Abstract

Origanum ramonense is a rare and underexplored aromatic (Lamiaceae family) native to the Mediterranean region, but despite its prospects as a medicinal plant, there is a shortage of spectrometry based metabolic profiling of this plant. Thus, the objective of this study was to carry out a detailed investigation of the metabolic profile of Origanum ramonense extracts using multiple solvents (methanol, methanol/water, ethyl acetate, dichloromethane/methanol, and hot water) of varying polarity, and further assess its bioactive potential. Using analytical tools such as NMR and GC/MS, we identified functional groups of plant metabolites and further employed multiple assays such as DPPH (antioxidant activity) and the Folin-Ciocalteu method to understand the plant's bioactivity. The extracts were found to be rich with polyphenolic compounds (17.8-107.2 mg Gallic acid per gram extract) and had strong antioxidant activity (IC50 5.8-128.5). The promising bioactivity was validated not only by results for in-vitro anticancer and (MCF-7 and HeLa cells) antibacterial tests (S. aureus and S. pneumoniae) but also in-silco molecular docking further showed the potential of antiviral activity of the extracted metabolites against SARS-CoV. These findings highlight O. ramonense as a valuable source of natural antioxidants and bioactive compounds, underscoring the need for further research into its medicinal properties.

Keywords: Origanum ramonense danin; Bioactivity; GC-MS; Metabolic profiling; Molecular docking; NMR.

Fig. 1

(a) Comparison of the Number of Publications on the Genus Origanum vs. Origanum ramonense. The Data and graph obtained from Web of science. (b) Original picture of Origanum ramonense plant grown in Palestine.

Fig. 2

A stack plot of the ¹D ¹H NMR spectra of Origanum ramonense plant extracts using five different solvents (i.e., (a) Ethyl acetate; (b) Methanol/water; (c) Dichloromethane /methanol; (d) Methanol; (e) Hot water).The inserted image is the magnified NMR Spectrum of the downfield Region (δ 6.0 to 10.0 ppm). All measures captured using NMR at 800 MHz resolution.

Fig. 3

(a) Functional group ratio comparison of Origanum ramonense obtained using multiple solvents of varying polarity. (b) Line graph comparison to demonstrate the highest percentage of each functional group obtained from Origanum ramonense using different solvents.

Fig. 4

(a) The ¹³C 1D spectra of Origanum ramonense obtained using 600 MHz solid-state NMR. (b) Functional group comparison of the Origanum ramonense plant leaves obtained from ¹³C solid-state NMR spectra.

Fig. 5

(a) Total ion chromatogram of the metabolites obtained from GC-MS analysis of the O. ramonense plant using different solvents; (b) The PCA score plot of the O. ramonense plant extracted in hot water, methanol, and methanol/water, ethyl acetate, dichloromethane/methanol ; (c) Heat map of the O. ramonense plant depicting the different and similar metabolites obtained from the five different solvents; (d) VIP (Variable Importance in Projection) score chart of the most significant metabolites obtained from the five different solvents; (e) Enrichment analysis pie chart illustrating the proportion of each chemical group relative to the total number of metabolites identified by GC-MS analysis. The colors in the pie chart represent different chemical groups, highlighting the distribution within the total of metabolites.

Fig. 5

(a) Total ion chromatogram of the metabolites obtained from GC-MS analysis of the O. ramonense plant using different solvents; (b) The PCA score plot of the O. ramonense plant extracted in hot water, methanol, and methanol/water, ethyl acetate, dichloromethane/methanol ; (c) Heat map of the O. ramonense plant depicting the different and similar metabolites obtained from the five different solvents; (d) VIP (Variable Importance in Projection) score chart of the most significant metabolites obtained from the five different solvents; (e) Enrichment analysis pie chart illustrating the proportion of each chemical group relative to the total number of metabolites identified by GC-MS analysis. The colors in the pie chart represent different chemical groups, highlighting the distribution within the total of metabolites.

Fig. 5

(a) Total ion chromatogram of the metabolites obtained from GC-MS analysis of the O. ramonense plant using different solvents; (b) The PCA score plot of the O. ramonense plant extracted in hot water, methanol, and methanol/water, ethyl acetate, dichloromethane/methanol ; (c) Heat map of the O. ramonense plant depicting the different and similar metabolites obtained from the five different solvents; (d) VIP (Variable Importance in Projection) score chart of the most significant metabolites obtained from the five different solvents; (e) Enrichment analysis pie chart illustrating the proportion of each chemical group relative to the total number of metabolites identified by GC-MS analysis. The colors in the pie chart represent different chemical groups, highlighting the distribution within the total of metabolites.

Fig. 6

(a) Dose-dependent DPPH (%) radical scavenging activity of O. ramonense plant extracts obtained using different solvents (hot water, methanol/water, methanol); (b) Dose-dependent DPPH (%) radical scavenging activity of O. ramonense plant extracts obtained using different solvents (ethyl acetate and dichloromethane/methanol) ; (c) Dose-dependent standard gallic acid reference curve used to compute the quantity of phenolic content in the plant extracts (mg of gallic acid equivalent (GAE)/gram of dry weight plant). The Pearson correlation coefficients (r) obtained from the graphs are greater than 0.9 for all solvents. The samples were analyzed in triplicates.

Fig. 7

Bar diagram summarizing and comparing the TPC and IC₅₀ values of plant extracts from multiple solvents.

Fig. 8

(a) Antimicrobial activity of the different extracts according to the diameters (mm) of the zone of inhibition; (b) Agar disc showing the antimicrobial activities of metabolites from methanol/water and ethyl acetate extracts by inhibition zones (mm) on Staphylococcus aureus. (c) Agar disc showing antimicrobial activity of metabolites from methanol/water and ethyl acetate extracts by inhibition zones (mm) on Streptococcus pneumoniae.

Fig. 9

Structural representation of SARS-CoV active sites and its interaction with the metabolites.

Fig. 10

Diagram summarizing the bioactivity of various metabolites categorized into three main groups: anticancer, antimicrobial, and antioxidant activities.