Detection of cannabinoids in Cannabis sativa plants THCA CBCA CBDA THC CBD CBC
Thin Layer Chromatography (TLC) Agilent
Cannabichromene,CBC Cannabigerol, CBG (-)-Δ9-Tetrahydrocannabinol,THC Δ9- tetrahydro-cannabivarol, THCV (-)-trans-cannabidiol-, CBD cannabinol,cbn TLC of Cannabinoids Olivetolic acid, OA Divaric acid Cannabigerolic acid, CBGA GPP Cannabigerovarinic acid, CBGVA Cannabichromenic acid, CBCA Cannabidivaric acid, Cannabidivarinic acid, CBDVA Cannabichromevarinic acid, CBCVA Cannabidiolic acid, CBDA (-)-Δ9-trans- Tetrahydrocannabinolic acid, THCA Δ9-Tetrahydrocannabivarinic acid, Δ1-Tetrahydrocannabivarolic acid, THCVA (-)-trans-cannabidiol-, CBD Cannabichromene,CBC Cannabidivarin, Cannabidivarol, CBDV Cannabichromevarin, CBCV (-)-Δ9-trans-Tetrahydrocannabivarin, Δ9- tetrahydro-cannabivarol, THCV (-)-Δ1-Tetrahydrocannabinol, (-)- Δ9-Tetrahydrocannabinol,THC
Extraction methods Solid-liquid extraction (plant material + solvent) (e.g. Methanol, Methanol:H 2 O, ethanol, benzene, petrolium ether, n-hexane ) (For LCMS or first step of extraction for GCMS) Liquid-liquid extraction (extract partitioning) (e.g. n-hexane, n-hexane: ethyl acetate, (usually for GCMS)
ACQUITY QDa Detector Alliance HPLC system Waters
Agilent 1200 HPLC system 6224 Accurate Mass Time of Flight(TOF)
Extracted tissues
Stigma trichomes
Flower trichomes
Calibration curves for THC for 0.6-37.5 ng in single injection 5000000 4500000 4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 THC : 0.6-37.5 ng in injection y = 1E+08x + 67134 R² = 0.9953 0.0000 0 0.0050 0.0100 0.0150 0.0200 0.0250 0.0300 0.0350 0.0400
Cannabinoids pathway Olivetolic acid, OA Divaric acid Cannabigerolic acid, CBGA GPP Cannabigerovarinic acid, CBGVA Cannabichromenic acid, CBCA Cannabidivaric acid, Cannabidivarinic acid, CBDVA Cannabichromevarinic acid, CBCVA Cannabidiolic acid, CBDA (-)-Δ9-trans- Tetrahydrocannabinolic acid, THCA Δ9-Tetrahydrocannabivarinic acid, Δ1-Tetrahydrocannabivarolic acid, THCVA (-)-trans-cannabidiol-, CBD Cannabichromene,CBC Cannabidivarin, Cannabidivarol, CBDV Cannabichromevarin, CBCV (-)-Δ9-trans-Tetrahydrocannabivarin, Δ9- tetrahydro-cannabivarol, THCV (-)-Δ1-Tetrahydrocannabinol, (-)- Δ9-Tetrahydrocannabinol,THC
CBGA (ug) in 1 gram FW 700.0 600.0 Stigma Flower Small L. MediumL. Large L. Trichomes 500.0 400.0 300.0 * 200.0 100.0 0.0 stigma -brown-6weeks stigma -green -RT-time0 stigma -green-3weeks stigma -green-6weeks flowers-3weeks flowers NO trichomes-3weeks flowers-6weeks flowers NO trichomes-6weeks small leaves -RT-time0 small leaves-3weeks small leaves NO trichomes-3weeks small leaves-6weeks small leaves NO trichomes-6weeks medium leaves-rt-time0 medium leaves-3weeks medium leaves NO trichomes-3weeks medium leaves-6weeks medium leaves NO trichomes-6weeks large leaves - RT-time0 large leaves-3weeks large leaves-6weeks trichomes of flowers-3weeks trichomes of flowers-6weeks trichomes of small leaves-3weeks trichomes of small leaves-6weeks trichomes of medium leaves-3weeks trichomes of medium leaves-6weeks
60.00 THCA(mg) in 1 gram FW 180 50.00 Stigma Flower Small L. MediumL. Large L. Trichomes 40.00 30.00 20.00 10.00 * 0.00 stigma -brown-6weeks stigma -green -RT-time0 stigma -green-3weeks stigma -green-6weeks flowers-3weeks flowers NO trichomes-3weeks flowers-6weeks flowers NO trichomes-6weeks small leaves -RT-time0 small leaves-3weeks small leaves NO trichomes-3weeks small leaves-6weeks small leaves NO trichomes-6weeks medium leaves-rt-time0 medium leaves-3weeks medium leaves NO trichomes-3weeks medium leaves-6weeks medium leaves NO trichomes-6weeks large leaves - RT-time0 large leaves-3weeks large leaves-6weeks trichomes of flowers-3weeks trichomes of flowers-6weeks trichomes of small leaves-3weeks trichomes of small leaves-6weeks trichomes of medium leaves-3weeks trichomes of medium leaves-6weeks
Whole head of three strains 350 Zach AD MEND 1 2 3 300 250 200 150 100 50 0 CBG (ug) in 1 gram FW THC (ug) in 1 gram FW CBDA (ug) in 1 gram FW CBGA (ug) in 1 gram FW THCA(mg) in 1 gram FW CBD (ug) in 1 gram FW
Small leaf with trichomes Small leaf W/O trichomes
Collection of the Trichomes using dry-ice and 300 microne mesh
Shaken trichomes (mostly) 350 micron sieve
Trichome types in Cannabis Capitate-stalked, crysolytic and capiate gland types
Glandular trichome types in Cannabis 150 to 500 micron 15-30 micron 25 to 100 micron *antherial glandular trichome, which has only been found on anthers
Capitate and bulbous gland Dayanandan and Kaufman, 1976
Capitate, long and short cystolith trichomes Ring of cells surroundling long hair Dayanandan and Kaufman, 1976
350-100 double layers x3-- times-3
100-50- double layers x2
Dry ice fresh 50-100 micron
/ 501,1m -., t,,,., 0
501,1m
Very challenging to separate all trichome types Solution enriched extracts - use your data wisely
Conclusions Using RNA-seq data with metabolic data across multiple tissues and developmental stages could lead to discovery of new genes from the pathway Regulators of cannabinoid pathway a yet to be discovered, when approach above could contribute Developing proper tissue extraction techniques might refine the metabolic profile of specific tissues.
Although extensive research has been going in recent years, much remains to be discovered Many thanks: Ari Schaffer Oded Sagee Efraim Lewinsohn Shahar Cohen Rachel Davidovitz- Rikanati Einat Bar Shahar Sroussi Lena Esselson
Thank You