Detection of cannabinoids in - foodsecurity.tau.ac.il · Olivetolic acid, OA. GPP. Divaricacid....
Transcript of Detection of cannabinoids in - foodsecurity.tau.ac.il · Olivetolic acid, OA. GPP. Divaricacid....
-
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
a -b
row
n-6w
eeks
stig
ma
-gre
en-R
T-tim
e0
stig
ma
-gre
en-3
wee
ks
stig
ma
-gre
en-6
wee
ks
flow
ers-
3wee
ks
flow
ersN
O tr
icho
mes
-3w
eeks
flow
ers-
6wee
ks
flow
ersN
O tr
icho
mes
-6w
eeks
smal
llea
ves-
RT-ti
me0
smal
l lea
ves-
3wee
ks
smal
l lea
vesN
O tr
icho
mes
-3w
eeks
smal
l lea
ves-
6wee
ks
smal
l lea
vesN
O tr
icho
mes
-6w
eeks
med
ium
leav
es-R
T-tim
e0
med
ium
leav
es-3
wee
ks
med
ium
leav
esN
O tr
icho
mes
-3w
eeks
med
ium
leav
es-6
wee
ks
med
ium
leav
esN
O tr
icho
mes
-6w
eeks
larg
ele
aves
-RT
-tim
e0
larg
ele
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
e0
stig
ma
-gre
en-3
wee
ks
stig
ma
-gre
en-6
wee
ks
flow
ers-
3wee
ks
flow
ersN
O tr
icho
mes
-3w
eeks
flow
ers-
6wee
ks
flow
ersN
O tr
icho
mes
-6w
eeks
smal
l lea
ves-
RT-ti
me0
smal
l lea
ves-
3wee
ks
smal
l lea
vesN
O tr
icho
mes
-3w
eeks
smal
l lea
ves-
6wee
ks
smal
l lea
vesN
O tr
icho
mes
-6w
eeks
med
ium
leav
es-R
T-tim
e0
med
ium
leav
es-3
wee
ks
med
ium
leav
esN
O tr
icho
mes
-3w
eeks
med
ium
leav
es-6
wee
ks
med
ium
leav
esN
O tr
icho
mes
-6w
eeks
larg
ele
aves
-RT
-tim
e0
larg
e le
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
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