Targeting the antioxidant, antimicrobial and anti-inflammatory activity of non-psychotropic Cannabis sativa L.: a comparison with chemotype V

Abstract

Background: Non-psychotropic Cannabis sativa L. chemotypes have gained increasing interest due to their diverse profiles of bioactive compounds. While cannabinoids such as cannabidiol (CBD), cannabigerol (CBG), are known for their biological effects, the role of other cannabinoids such cannabichromene (CBC) remains underexplored as for chemotype V, which lacks in cannabinoids but is characterized by other minor phytochemicals.

Objective: This study aimed to evaluate the individual and combined contributions of cannabinoids and non-cannabinoid phenolics to the antioxidant, antimicrobial, and anti-inflammatory properties of extracts derived from four C. sativa chemotypes, including a cannabinoid-free variant as a comparison.

Methods: Ethanolic extracts were obtained from four hemp chemotypes: CBD-rich (CS1), CBG-rich (CS2), CBC-rich (CS3), and cannabinoid-free (CS4). Phytochemical profiling was conducted using UHPLC-HRMS. Antioxidant properties were assessed via DPPH, ABTS, and FRAP assays. Antimicrobial activity was tested against Gram-positive and Gram-negative bacteria through MIC, MBC, and time-kill assays. Anti-inflammatory activity was evaluated in LPS-stimulated RAW 264.7 macrophages via gene expression analysis of pro- and anti-inflammatory mediators (IL1b, IL6, Cox2, IL10, IL1Ra).

Results: Phytochemical analysis confirmed the chemotype-specific profiles, with CS3 showing the highest levels of canniprene and the early discovered 5-methoxy-dihydrodenbinobin. Antioxidant assays revealed that cannabinoids were the main contributors to radical scavenging capacity, though CS3 exhibited additional ferric ion reducing power likely due to non-cannabinoid phenolics. Antibacterial activity was confined to Gram-positive bacteria, where CS1 showed the highest efficacy, and CS4 showed no activity, highlighting the critical role of cannabinoids. All extracts reduced LPS-induced Il1b, Il6, and Cox2 gene expression, but only cannabinoid-rich extracts upregulated the anti-inflammatory cytokines IL10 and IL1Ra, indicating a cannabinoid-dependent effect.

Conclusion: Both cannabinoids and non-cannabinoid phenolics contribute to the biological activity of Cannabis sativa extracts, with cannabinoids playing a central role in antimicrobial responses and stronger anti-inflammatory effect as a pure cannabinoid or as an extract. From this point of view, the cannabinoid-free chemotype V could be a valuable functional control for isolating the effects of cannabinoids, reinforcing the need for integrative analyses in evaluating the therapeutic potential of cannabis-derived formulations.

Supplementary Information: The online version contains supplementary material available at 10.1186/s42238-025-00336-1.

Keywords: Cannabichromene; Cannabinoid extracts; Cannabis chemotypes; Chemotype v.

Fig. 1

Major cannabinoids content and the total cannabinoids content in each extract expressed as a percentage over crude extract weight

Fig. 2

Major non-cannabinoid compounds content and the total amount in the extracts expressed as a percentage over crude extract weight

Fig. 3

Time–kill analysis of CS extracts CS1, CS2, CS3 and ciprofloxacin at 4 × MIC against methicillin-resistant S. aureus (MRSA) ATCC 43,300 over a 24 h incubation period at 37 °C

Fig. 4

Dose-dependent effect of pure cannabinoids and CS extracts on viability of RAW 264.7 macrophages. Cell viability was evaluated by Alamarblue assay after a-4-hour treatment with CBD (25–100 µM) (A), CBG (25–100 µM) (B), CBC (25–100 µM) (C), (CS1 (20–100 µg/mL) (D), CS2 (20–100 µg/mL) (E), CS3 (20–100 µg/mL) (F), or CS4 (20–100 µg/mL) (G). Data are expressed as percentage of cell viability compared to control (vehicle) and shown as mean ± SD of a triplicate. (**p < 0.05, **** p < 0.01 vs. CTR; one-way ANOVA)

Fig. 5

Cannabinoid-containing CS extracts and pure cannabinoids show enhanced anti-inflammatory activity compared to cannabinoid-free extracts. RAW 264.7 cells were pretreated with (A, C) pure cannabinoids (25 µM CBD, 50 µM CBG, 50 µM CBC) or (B, D) CS extracts (20 µg/mL CS1, 60 µg/mL CS2, 40 µg/mL CS3 and 100 µg/mL CS4) for 30 min, followed by a 4-hour treatment with 100ng/mL of LPS. mRNA levels of (A, B) pro-inflammatory (I1lb, Il6, Cox2) and (C, D) anti-inflammatory (Il10, Il1ra) were evaluated by qPCR. Gapdh was used as housekeeping gene. Normalized data are shown as fold increase over untreated cells (-). Data are depicted as mean ± SD and are representative of one out of three independent experiments with similar results. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. untreated cells (-); #p < 0.05 ####p < 0.0001 vs. LPS-induced cells