Detection of cannabinoids in Cannabis sativa plants

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Shauna Bates
6 years ago
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1 Detection of cannabinoids in Cannabis sativa plants THCA CBCA CBDA THC CBD CBC

2 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

Cannabidiolic acid, CBDA (-)-Δ9-trans- Tetrahydrocannabinolic acid, THCA Δ9-Tetrahydrocannabivarinic acid, Δ1-Tetrahydrocannabivarolic acid, THCVA (-)-trans-cannabidiol-, CBD

3 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

step of extraction for GCMS) Liquid-liquid

4 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)

5 ACQUITY QDa Detector Alliance HPLC system Waters

6 Agilent 1200 HPLC system 6224 Accurate Mass Time of Flight(TOF)

7 Extracted tissues

9 Stigma trichomes

10 Flower trichomes

12 Calibration curves for THC for ng in single injection THC : ng in injection y = 1E+08x R² =

13 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

14 CBGA (ug) in 1 gram FW Stigma Flower Small L. MediumL. Large L. Trichomes * 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

15 60.00 THCA(mg) in 1 gram FW Stigma Flower Small L. MediumL. Large L. Trichomes * 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

16 Whole head of three strains 350 Zach AD MEND 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

17 Small leaf with trichomes Small leaf W/O trichomes

18 Collection of the Trichomes using dry-ice and 300 microne mesh

19 Shaken trichomes (mostly) 350 micron sieve

20 Trichome types in Cannabis Capitate-stalked, crysolytic and capiate gland types

21 Glandular trichome types in Cannabis 150 to 500 micron micron 25 to 100 micron *antherial glandular trichome, which has only been found on anthers

22 Capitate and bulbous gland Dayanandan and Kaufman, 1976

23 Capitate, long and short cystolith trichomes Ring of cells surroundling long hair Dayanandan and Kaufman, 1976

24 double layers x3-- times-3

25 double layers x2

26 Dry ice fresh micron

27 / 501,1m -., t,,,., 0

28 501,1m

29 Very challenging to separate all trichome types Solution enriched extracts - use your data wisely

30 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.

31 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

32 Thank You

Weeding out the myths on Medical Cannabis

Weeding out the myths on Medical Cannabis Presented by A/Prof David Allsop Lambert Initiative for Cannabinoid Therapeutics University of Sydney For Walk on the Wild Side 2016: Superstition or Evidence?