Accumulation of bioactive metabolites in cultivated medical Cannabis

Abstract

There has been an increased use of medical Cannabis in the United States of America as more states legalize its use. Complete chemical analyses of this material can vary considerably between producers and is often not fully provided to consumers. As phytochemists in a state with legal medical Cannabis we sought to characterize the accumulation of phytochemicals in material grown by licensed commercial producers. We report the development of a simple extraction and analysis method, amenable to use by commercial laboratories for the detection and quantification of both cannabinoids and terpenoids. Through analysis of developing flowers on plants, we can identify sources of variability of floral metabolites due to flower maturity and position on the plant. The terpenoid composition varied by accession and was used to cluster cannabis strains into specific types. Inclusion of terpenoids with cannabinoids in the analysis of medical cannabis should be encouraged, as both of these classes of compounds could play a role in the beneficial medical effects of different cannabis strains.

Fig 1. Biosynthetic schema for cannabinoids.

This schema is derived from pathways reviewed in [2].

Fig 2. GC-FID separation of terpenoids and cannabinoids.

Peaks are labeled with the Peak ID code. A. Mixture of 21 terpenoid and 6 cannabinoid chemical standards separated by GC-FID. B. Sample was extracted from C-Plus, a strain of Cannabis with approximately equal abundances of CBD (C3) and Δ9-THC (C4).

Fig 3. Accumulation of Δ9-THC in organs of Sour Willie or Bohdi Tree following floral induction.

Δ9-THC levels in samples collected from Sour Willie (A, B) or Bohdi Tree (C, D) at days post-induction are represented. Panels A and C report average (n = 3) Δ9-THC levels in floral samples (solid line) and leaf samples (dotted line). Panels B and D report Δ9-THC levels in floral samples from the top of the plant (solid) or bottom of the plant (dotted line).

Fig 4. Paired comparisons of CBD levels in leaf and floral samples of Cannabis plants.

Flowers and leaves were collected from 16 Cannabis strains. The Δ9-THC dominant strains fall within the red circle, while the CBD co-dominant strains fall within the blue circle.

Fig 5. Paired comparisons of total cannabinoid and total terpenoid levels.

Cured trimmed flowers were extracted and the terpenoid and cannabinoid composition determined by GC-FID. The total values for cannabinoids and terpenoids in each sample were plotted.

Fig 6. Hierarchical clustering of medical Cannabis strains based on floral terpenoid composition.

The relative abundance of 19 terpenoids detected by GC-FID in cured floral samples from 72 medical Cannabis strains were used to generate the tree using the agglomerative hierarchical clustering method in the XLSTAT plug-in for Excel. Pie charts of a sample terpenoid composition for individuals within each clade are shown at the branch points, terpenoid composition is indicated by numerical code (Table 1, 1 = T1, 2 = T2, etc). Terpenoid types are identified: α- and β-pinene, Pin; β-myrcene, Myr; β-caryophyllene, C; d-limonene, L; terpinolene, Terp.

Fig 7. Transcript accumulation in leaf samples of cannabinoid biosynthetic genes.

Leaf RNA from the indicated strains were assayed in triplicate by qRT-PCR, using primers for CBDAS (black), for THCAS (purple) or prenyl transferase (blue).

Fig 8. Transcript accumulation of cannabinoid biosynthetic genes during floral development.

RNA was isolated from flowers collected from the indicated strains at early (blue), middle (orange) or late (purple) after the floral induction phase. Triplicate RNA isolations were assayed by qRT-PCR, using primers for THCAS (A), CBDAS (B) or prenyl transferase (C).