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Detection of cannabinoids inCannabis sativa plants
THCA
THC
CBCA
CBC
CBDA
CBD
Thin Layer Chromatography (TLC)
Agilent
Olivetolic acid, OA
GPP
Divaric acid
Cannabigerovarinic acid, CBGVA
Cannabichromevarinic acid, CBCVA
Cannabidivaric acid, Cannabidivarinic acid, CBDVA
Δ9-Tetrahydrocannabivarinic acid,Δ1-Tetrahydrocannabivarolic acid, THCVA
(-)-Δ9-Tetrahydrocannabinol ,THC
Cannabidivarin , Cannabidivarol, CBDV
Cannabigerolic acid,CBGA
Cannabidiolic acid, CBDA
Cannabichromenic acid, CBCA
(-)-Δ9-trans-Tetrahydrocannabivarin,Δ9- tetrahydro-cannabivarol, THCV
(-)-trans-Cannabidiol- , CBD
Cannabichromene,CBC Cannabichromevarin, CBCV
(-)-Δ9-trans-Tetrahydrocannabinolic acid, THCA
TLC ofCannabinoids
Cannabichromene,CBC
(-)-Δ1-Tetrahydrocannabinol, (-)-Δ9-Tetrahydrocannabinol ,THC
Cannabigerol, CBG
Δ9- tetrahydro-cannabivarol, THCV (-)-trans-Cannabidiol- , CBD cannabinol,CBN
Extraction methods
➢ Solid-liquid extraction (plant material + solvent) (e.g.
Methanol, Methanol:H2O, 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
6224 Accurate Mass Time of Flight (TOF)1200 HPLC system
Agilent
Extracted tissues
Stigma trichomes
Flower trichomes
Calibration curves for THC for0.6-37.5 ng in single injection
y = 1E+08x + 67134R² = 0.9953
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
4500000
5000000
0.0000 0.0050 0.0100 0.0150 0.0200 0.0250 0.0300 0.0350 0.0400
THC : 0.6-37.5 ng in injection
Olivetolic acid, OA
GPP
Divaric acid
Cannabigerovarinic acid, CBGVA
Cannabichromevarinic acid, CBCVA
Cannabidivaric acid, Cannabidivarinic acid, CBDVA
Δ9-Tetrahydrocannabivarinic acid,Δ1-Tetrahydrocannabivarolic acid, THCVA
Cannabidivarin , Cannabidivarol, CBDV
Cannabigerolic acid,CBGA
Cannabidiolic acid, CBDA
Cannabichromenic acid, CBCA
(-)-Δ9-trans-Tetrahydrocannabivarin,Δ9- tetrahydro-cannabivarol, THCV
(-)-trans-Cannabidiol- , CBD
Cannabichromene,CBC Cannabichromevarin, CBCV
(-)-Δ9-trans-Tetrahydrocannabinolic acid, THCA
Cannabinoidspathway
(-)-Δ1-Tetrahydrocannabinol, (-)-Δ9-Tetrahydrocannabinol ,THC
0.0
100.0
200.0
300.0
400.0
500.0
600.0
700.0st
igm
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row
n-6w
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ks
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O tr
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eeks
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-6w
eeks
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eeks
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O tr
icho
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eeks
med
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ks
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O tr
icho
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eeks
larg
ele
aves
-RT
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larg
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aves
-3w
eeks
larg
ele
aves
-6w
eeks
tric
hom
es o
fflo
wer
s-3w
eeks
tric
hom
es o
fflo
wer
s-6w
eeks
tric
hom
es o
fsm
all l
eave
s-3w
eeks
tric
hom
es o
fsm
all l
eave
s-6w
eeks
tric
hom
es o
fmed
ium
leav
es-3
wee
ks
tric
hom
es o
fmed
ium
leav
es-6
wee
ks
CBGA (ug) in 1 gram FW
Stigma Flower Small L. Medium L. Large L. Trichomes
*
0.00
10.00
20.00
30.00
40.00
50.00
60.00st
igm
a -b
row
n-6w
eeks
stig
ma
-gre
en-R
T-tim
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ma
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en-3
wee
ks
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ks
flow
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flow
ersN
O tr
icho
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eeks
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ks
flow
ersN
O tr
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-6w
eeks
smal
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RT-ti
me0
smal
l lea
ves-
3wee
ks
smal
l lea
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O tr
icho
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eeks
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6wee
ks
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icho
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eeks
med
ium
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ks
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aves
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aves
-3w
eeks
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aves
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eeks
tric
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fflo
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s-3w
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tric
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fflo
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s-6w
eeks
tric
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fsm
all l
eave
s-3w
eeks
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all l
eave
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eeks
tric
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es o
fmed
ium
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wee
ks
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fmed
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leav
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ks
THCA(mg) in 1 gram FW
Stigma Flower Small L. Medium L. Large L. Trichomes
180
*
Whole “head” of three strains
0
50
100
150
200
250
300
350
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
Zach AD MEND1 2 3
Small leaf with trichomes Small leaf W/O trichomes
Collection of the Trichomes usingdry-ice and 300 microne mesh
Shaken trichomes (mostly)350 micron sieve
Capitate-stalked, crysolytic and capiate gland types
Trichome types in Cannabis
Glandular trichome types in Cannabis
15-30 micron 25 to 100 micron
150 to500 m
icron
*antherial glandular trichome, which has only been found on anthers
Capitate and bulbous gland
Dayanandan and Kaufman, 1976
Capitate, long and short cystolith trichomes
Dayanandan and Kaufman, 1976
Ring of cells surroundling long hair
350-100 double layers x3-- times-3
100 -50- double layers x2
Dry ice –fresh 50-100 micron
/
.,-• 501,1mt
,,.
,
0,
501,1m
Very challenging to separate alltrichome 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 beengoing 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
ThankYou
Detection of cannabinoids inCannabis sativa plantsSlide Number 2TLC ofCannabinoidsExtraction methodsACQUITY QDa DetectorAgilentExtracted tissuesSlide Number 8Slide Number 9Flower trichomesSlide Number 11Calibration curves for THCfor0.6-37.5 ng in single injectionCannabinoidspathwayCBGA (ug) in 1 gram FWTHCA(mg) in 1 gram FWWhole “head” of three strainsSmall leaf W/O trichomesSlide Number 18Slide Number 19Slide Number 20Glandular trichome types in CannabisCapitate and bulbous glandCapitate, long and short cystolith trichomes350-100 double layers x3-- times-3100 -50- double layers x2Dry ice –fresh 50-100 micronSlide Number 27Slide Number 28Very challenging to separate alltrichome typesConclusionsAlthough extensive research has beengoing in recent years, much remainsSlide Number 32