Genome-Scale Metabolic Reconstruction, Non-Targeted LC-QTOF-MS Based Metabolomics Data, and Evaluation of Anticancer Activity of Cannabis sativa Leaf Extracts

Abstract

Over the past decades, Colombia has suffered complex social problems related to illicit crops, including forced displacement, violence, and environmental damage, among other consequences for vulnerable populations. Considerable effort has been made in the regulation of illicit crops, predominantly Cannabis sativa, leading to advances such as the legalization of medical cannabis and its derivatives, the improvement of crops, and leaving an open window to the development of scientific knowledge to explore alternative uses. It is estimated that C. sativa can produce approximately 750 specialized secondary metabolites. Some of the most relevant due to their anticancer properties, besides cannabinoids, are monoterpenes, sesquiterpenoids, triterpenoids, essential oils, flavonoids, and phenolic compounds. However, despite the increase in scientific research on the subject, it is necessary to study the primary and secondary metabolism of the plant and to identify key pathways that explore its great metabolic potential. For this purpose, a genome-scale metabolic reconstruction of C. sativa is described and contextualized using LC-QTOF-MS metabolic data obtained from the leaf extract from plants grown in the region of Pesca-Boyaca, Colombia under greenhouse conditions at the Clever Leaves facility. A compartmentalized model with 2101 reactions and 1314 metabolites highlights pathways associated with fatty acid biosynthesis, steroids, and amino acids, along with the metabolism of purine, pyrimidine, glucose, starch, and sucrose. Key metabolites were identified through metabolomic data, such as neurine, cannabisativine, cannflavin A, palmitoleic acid, cannabinoids, geranylhydroquinone, and steroids. They were analyzed and integrated into the reconstruction, and their potential applications are discussed. Cytotoxicity assays revealed high anticancer activity against gastric adenocarcinoma (AGS), melanoma cells (A375), and lung carcinoma cells (A549), combined with negligible impact against healthy human skin cells.

Keywords: Cannabis sativa; anticancer activity; metabolomic validation; plant genome-scale metabolic reconstruction; secondary metabolism.

Figure 1

Iterative workflow from the bottom-up process in C. sativa reconstruction.

Figure 2

Stoichiometric matrices of the implemented model. The y axis represents the number of metabolites, and the x axis represents the chemical reactions.

Figure 3

Reactions and compounds of the C. sativa metabolic reconstruction grouped by pathways according to PlantSEED.

Figure 4

Molecular functions stored in the KO (KEGG Orthology) database containing orthologs of experimentally characterized genes/proteins in the reference genome of Cannabis sativa.

Figure 5

Pathway of secondary metabolism in the biosynthesis of cannabinoids and terpenes in C. sativa [63]. Enzymes related to functional annotation are illustrated in green.

Figure 6

Secondary metabolism of C. sativa, classified by five major metabolite types: terpenes (green), fatty acids (pink), phenolic compounds (red), N-compounds (blue), and cannabinoids (yellow).

Figure 7

Bar diagram with the number of compounds identified in C. sativa leaf extract obtained from legal cultivation from Clever Leaves, via LC–QTOF MS/MS. In blue: electrospray negative ionization mode; in orange: electrospray switching polarity mode; in gray, electrospray positive ionization mode; in yellow, electrospray switching polarity mode.

Figure 8

Cytotoxicity analysis of the C. sativa leaf extract on different carcinoma and healthy cell lines. Results for carcinoma cell lines (A549, AGS and A375) after 24 (A) and 72 h of exposure (B). Results for healthy cell lines (Vero and HFF) after 24 (C) and 72 h of exposure (D).