Over the past decades, Colombia has suffered from complex social problems related to illicit crops, including forced displacement, violence, and environmental damage, among other consequences for vulnerable populations [1]. Considerable effort has been made in Colombia to address this issue by creating a regulatory framework for import, export, cultivation, extraction, and research activities, especially of Cannabis sativa [2,3]. When the contingency caused by the coronavirus began, the former Minister of Health authorized Resolution 315 of 2020, which updates the lists of precursor drugs subject to state control and gives free access to the sale of master formulations (preparations made for medical indications) in order to eliminate some access barriers for research, medical, and scientific use [4]. In addition, two years later, Resolution 227 of 2022 was approved, regulating the use of medicinal C. sativa (non-psychoactive components) in food, beverages, and dietary supplements. Furthermore, since the beginning of this year, the national government, through Resolution 2808 of 2022, decided to include magistral preparations of C. sativa medicines within the health benefits plan for patients with pathologies such as refractory epilepsy, fibromyalgia, sleep and appetite disorder, cachexia due to cancer, insomnia, chronic pain, neuropathic pain, and pain associated with cancer, in order to address those public health concerns [5]. These laws laid the groundwork for the cultivation of C. sativa plants, the emergence of the medical cannabis industry, and safe access to medical and scientific use, among other developments. Hence, the current regulatory framework promotes scientific knowledge of C. sativa and allows for the exploration of potential markets for its alternative uses [6].

The field of research related to C. sativa has been expanding at an accelerated rate [7] thanks to the biotechnological capacity hidden in the plant. It is estimated that C. sativa can produce approximately 750 specialized secondary metabolites [8,9,10]. Some of the most relevant are monoterpenes, sesquiterpenoids, triterpenoids, essential oils, flavonoids, phenolic compounds (known as polyphenols [7]), lignans, stilbenoid derivatives, alkaloids, amino acids, spiro-indans, steroids, and glycoproteins, mainly due to their anticancer properties [8,11,12,13,14]. Previous studies have shown a synergy among the metabolic compounds of the plant that, as a whole, show different behavior compared to the individual performance of each metabolite due to the “entourage effect” [15,16]. It is established that C. sativa chemotypes’ rich cannabinoid and terpenoid content offer better pharmacological activities that are able to broaden clinical applications and improve therapeutic issues [17,18,19]. In the same way, remarkable anticancerogenic activity has been demonstrated for C. sativa extracts against different carcinoma cell lines such as melanoma [20], ovarian [21], prostate [22], breast [23], and pancreatic cancer [16]. These studies have revealed a reduction in tumor growth and promotion of apoptosis and autophagy in carcinoma cells [15,23,24,25]. At the taxonomic level, chemotypes are grouped in terms of the relative amounts of their main compounds, the cannabinoids. Drug-type plants (chemotype I) contain high concentrations of the most prevalent cannabinoid known for its psychotropic capacity, (-)-trans-∆9-tetrahydrocannabinol, or D9-THC. When the cannabinoid content corresponds mostly to the second most abundant cannabinoid in the C. sativa plant, cannabidiol, CBD, it corresponds to chemotype III [26]. Finally, chemotype II, which is very scarce, is defined as a balanced content of the two main cannabinoids [27].

For all these reasons, it is critical to understand plant metabolism on a system-wide level to identify metabolic pathways involved in the production of key metabolites, characterize specific phenotypes influenced by environmental factors, and explore alternative uses of the leaf, such as nutraceuticals.

In the last two decades, Genome-Scale Metabolic (GEM) reconstructions have become a fundamental tool taking advantage of the development of high throughput data of omics technologies to study and understand the complex interactions of organisms [28]. Regarding the development of omics technologies in C. sativa, the first sequenced and assembled genome was produced in 2011 by Grassa et al. [29] and since then, publications based on whole-genome sequencing and population studies [30,31,32], transcriptomics [33], proteomics [34], and metabolomics have resolved compelling questions about the chemotype of the plant and its relationship with geography or characteristic markers [9]. Additionally, studies of C. sativa on the metabolic response of the plant under different degrees of stress [35], its potential uses in different industries [14,16], and particularly nutraceuticals [36,37], such as evaluation of anti-malarial activity [38], observation of in vivo antioxidant effects [39], and pathogen resistance [30], among others, stand out.

Meanwhile, in plant systems biology, genome-scale modeling has advanced considerably thanks to the reconstruction of Arabidopsis thaliana, Zea mays, Oryza sativa, and Saccharum officinarum, among others [40], which have proven accurate predictions focused on specific aspects of central carbon metabolism. For Arabidopsis thaliana, GEM modeling has evolved from the production of biomass components observed in experimental data to the inclusion of compartments (cytosol, plastid, mitochondrion, peroxisome, and vacuole), calculation of cell maintenance energy costs, description of photosynthetic processes, integration of secondary metabolism pathways, gene expression, proteomic data, and multi-tissue models [41]; as an example, Scheunemann et al. used the Plant SEED scheme to obtain reconstructions and subsequently integrate transcriptomics data extracted from different plant tissues [42].

Here we present a Genome-Scale Metabolic (GEM) reconstruction of C. sativa with an analysis of non-targeted LC-QTOF-MS (Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry)-based metabolomics data and evaluation of cytotoxicity and anticancer activity of leaf extracts, which could help to pave the way for the development of alternative uses of the leaf with potential applications in the food, cosmetic, textile, and agrochemical industries and also to enhance exploration of anticancer, analgesic, and anti-inflammatory compounds [11]. To our knowledge, this is the first attempt to comprehensively describe the metabolic capacities of C. sativa leaf (including both primary and secondary metabolism) based on a Genome-Scale Metabolic reconstruction; the contextualization of the reconstruction was carried out via LC-QTOF-MS to favor the identification of metabolites with known (anticancer, due to cannabinoids) and alternative properties (nutraceuticals, due to flavonoids and amino acids) [43].