Terpenoids and Phytocannabinoids Co-Produced in Cannabis Sativa Strains Show Specific Interaction for Cell Cytotoxic Activity

Abstract

Mixtures of different Cannabis sativa phytocannabinoids are more active biologically than single phytocannabinoids. However, cannabis terpenoids as potential instigators of phytocannabinoid activity have not yet been explored in detail. Terpenoid groups were statistically co-related to certain cannabis strains rich in Δ⁹-tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), and their ability to enhance the activity of decarboxylase phytocannabinoids (i.e., THC or CBD) was determined. Analytical HPLC and GC/MS were used to identify and quantify the secondary metabolites in 17 strains of C. sativa, and correlations between cannabinoids and terpenoids in each strain were determined. Column separation was used to separate and collect the compounds, and cell viability assay was used to assess biological activity. We found that in "high THC" or "high CBD" strains, phytocannabinoids are produced alongside certain sets of terpenoids. Only co-related terpenoids enhanced the cytotoxic activity of phytocannabinoids on MDA-MB-231 and HCT-116 cell lines. This was found to be most effective in natural ratios found in extracts of cannabis inflorescence. The correlation in a particular strain between THCA or CBDA and a certain set of terpenoids, and the partial specificity in interaction may have influenced the cultivation of cannabis and may have implications for therapeutic treatments.

Keywords: Cannabis sativa; cancer cell line; entourage effect; phytocannabinoids; terpenoids.

Figure 1

Principal Components Analysis (PCA) with phytocannabinoid–phytocannabinoid and phytocannabinoid–terpenoid correlations calculated for THCA and CBDA affinity. (For information regarding specific strains and chemotyping, see Supplementary Table S1). THCA co-related compounds are circled in red, while CBDA co-related compounds are circled in black. Strong correlations are indicated by larger circles, smaller correlations with smaller circles. Independent compounds, showing weak correlations to either THCA or CBDA are circled in blue. THCA: Δ⁹-tetrahydrocannabinolic acid; CBDA: cannabidiolic acid.

Figure 2

XTT cell viability assay of MDA-MB-231 cell line, treated for 48 h with (A) THC and terpene fraction; (B) CBD and terpene fraction. THC/CBD—in all treatments the phytocannabinoid concentration was 10µg/mL. The various concentrations of co-related terpenes and cross-related terpenes are detailed in the figure. Control—mixture of methanol and hexane (1:1), the solvent used for extraction and treatment. Doxo—doxorubicin 1 µg/µL. THCA-related terpenes—terpenes listed in Table 1A for Cs12 strain, fraction 6. CBDA-related terpenes—terpenes listed in Table 1B for the Arbel strain, fraction 3. THC + CBDA-related terpenes and CBD + THCA-related terpenes treatments were carried out at a ratio of 1:10 (1 µg/mL:10 µg/mL terpenoid:phytocannabinoid). Total amounts were extrapolated from the dry weight of the extracts and the relative amounts of the different molecules identified, as calculated from the area under the peak, valued by GC-personalized integration parameters (see Table 2). Percent cell viability is presented as mean ± SD. Columns with different letters are significantly different from all combination pairs, according to the Tukey–Kramer honest significant difference (HSD, P ≤ 0.05).

Figure 3

XTT cell viability assay of the HCT-116 cell line, treated for 48 h with (A) THC; and (B) CBD. THC/CBD—in all treatments the phytocannabinoid concentration was 20 µg/mL. The various concentrations of co-related terpenes and cross-related terpenes are detailed in the figure. Control—mixture of methanol and hexane (1:1), the solvent used for extraction and treatment. Doxo—doxorubicin 1 µg/µL. THCA-related terpenes—terpenes listed in Table 1A for Cs12 strain, fraction 6. CBDA-related terpenes—terpenes listed in Table 1B for the Arbel strain, fraction 3. THC + CBDA-related terpenes and CBD + THCA-related terpenes treatments were carried out at a ratio of 1:20 (1 µg/mL:20 µg/mL terpenoid:phytocannabinoid). Total amounts were extrapolated from the dry weight of the extract and the relative amounts of the different molecules identified, as calculated from the area under the peak, valued by GC-personalized integration parameters. Percent cell viability is presented as mean ± SD (uppercase letters mark statistical significance) of all treatments in replicas. Values with different letters are significantly different from all combinations of pairs, according to the Tukey–Kramer honest significant difference (HSD, P ≤ 0.05).