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Citation: Lee, S.; Kim, E.J.; Kwon, E.;
Oh, S.J.; Cho, M.; Kim, C.M.; Lee, W.;
Hong, J. Identification of Terpene
Compositions in the Leaves and
Inflorescences of Hybrid Cannabis
Species Using Headspace-Gas
Chromatography/Mass
Spectrometry. Molecules 2023,28,
8082. https://doi.org/10.3390/
molecules28248082
Academic Editor: Gavino Sanna
Received: 23 October 2023
Revised: 11 December 2023
Accepted: 12 December 2023
Published: 14 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
molecules
Article
Identification of Terpene Compositions in the Leaves and
Inflorescences of Hybrid Cannabis Species Using
Headspace-Gas Chromatography/Mass Spectrometry
Sangin Lee 1, Eun Jae Kim 1, Eunjeong Kwon 1, Seo Jeong Oh 1, Mansoo Cho 2, Chul Min Kim 3,
Wonwoong Lee 4,* and Jongki Hong 1,*
1College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea; ahfqqhsi@naver.com (S.L.);
dmswo3537@naver.com (E.J.K.)
2Graduate School of Techno Design, Kookmin University, Seoul 02707, Republic of Korea;
chomansoo@gmail.com
3Department of Horticulture Industry, Wonkwang University, Iksan 54538, Republic of Korea;
chulmin21@wku.ac.kr
4Research Institute of Pharmaceutical Sciences, College of Pharmacy, Woosuk University,
Wanju 55338, Republic of Korea
*Correspondence: wwlee@woosuk.ac.kr (W.L.); jhong@khu.ac.kr (J.H.);
Tel.: +82-63-290-1573 (W.L.); +82-2-961-9255 (J.H.); Fax: +82-2-961-0357 (J.H.)
Abstract:
Although cannabidiol and tetrahydrocannabinol in Cannabis species exert their pharmaco-
logical effects via the endocannabinoid system, it is believed that other phytochemicals, particularly
terpenes, can modulate therapeutic outcomes through the entourage effect. Therefore, to gain a better
understanding of the pharmacological effects of Cannabis, obtaining information on phytochemical
compositions, including mono-, di-, and sesqui-terpenes in Cannabis species is essential. Applying a
sophisticated analytical method is indispensable. In this study, headspace-gas chromatography/mass
spectrometry (HS-GC/MS) was employed to identify major terpenes in the leaves and inflorescences
of hybrid Cannabis species. The incubation time and temperature conditions for HS-GC/MS were
optimized. This method was successfully applied to the leaves (n= 9) and inflorescences (n= 7)
of hybrid Cannabis species. A total of 26 terpenes in Cannabis species were detected, and six major
components, such as
α
-pinene (9.8–2270
µ
g/g),
β
-pinene (2.6–930
µ
g/g), myrcene (0.7–17,400
µ
g/g),
limonene (1.3–300
µ
g/g),
β
-caryophyllene (60–3300
µ
g/g), and
α
-humulene (40–870
µ
g/g), were
quantified. Each sample showed different terpene compositions, but six major terpenes among all the
terpenes detected were consistently found in both the leaves and inflorescences of hybrid Cannabis
species. In this study, the six major terpenes’ potential in hybrid Cannabis species was evaluated
as biomarkers to distinguish hybrid Cannabis species samples. This study contributes to a better
understanding of the entourage effect of Cannabis-based botanical drugs.
Keywords:
hybrid Cannabis species; terpenes; leaf; inflorescence; headspace; gas chromatography;
mass spectrometry; statistical analysis
1. Introduction
Cannabis species contain various bioactive phytochemicals, categorized as cannabi-
noids and non-cannabinoids [
1
], used for food, medicine, and even ornamental plants [
2
].
Their unique pharmacological effects have generated increased interest in many areas,
including academia, industry, and the government. Among the bioactive phytochemicals
in Cannabis, tetrahydrocannabinol (THC) and cannabidiol (CBD) have psychoactive effects
by binding to endocannabinoid receptors [
3
] and therapeutic effects for epilepsy, pain, and
drug addiction [
4
]. Although both THC and CBD are known to be the most potent bioactive
compounds, other phytochemicals in Cannabis species also exhibit characteristic effects on
the human body [
5
]. In particular, terpenes reportedly contribute to the entourage effect,
Molecules 2023,28, 8082. https://doi.org/10.3390/molecules28248082 https://www.mdpi.com/journal/molecules
Molecules 2023,28, 8082 2 of 13
which can modulate the unique psychoactive effects of cannabinoids such as THC and
CBD [
6
]. Therefore, to better understand the entourage effect, identifying and quantifying
not only cannabinoids but also terpenes in Cannabis species is crucial.
To enhance their psychoactive and therapeutic effects, crossbreeding different Cannabis
species has become an industrial and commercial preference rather than cultivating original
Cannabis species. Consequently, in the Cannabis industry and market, finding landrace
Cannabis that is not hybrid Cannabis is challenging [
7
]. Hybrid Cannabis species are known
to have distinct effects and are promoted and sold based on their unique entourage effects.
Although cannabinoid contents (THC and CBD) in individual hybrid Cannabis species
have been presented, providing the terpene contents in these strains to understand the
characteristic entourage effect of individual hybrid Cannabis is essential.
Numerous analytical methods have been developed to determine terpenes in Cannabis
plants [
8
–
12
]. Although high-performance liquid chromatography (HPLC) has been used to
determine various bioactive phytochemicals in Cannabis species [
13
–
15
], gas chromatogra-
phy (GC) has been widely employed to analyze terpenes contributing to the fragrance and
flavor of products [
16
]. In particular, flame ionization detection (FID) realizes a simple and
easy operation method combined with GC, while mass spectrometry (MS) provides reliable
qualification and quantification results [
17
]. To analyze volatile terpenes in Cannabis plants,
a delicate sample preparation method should be performed to extract volatile terpenes
without significant losses. Solid-phase microextraction (SPME) has been widely used to
extract terpenes from natural products [
18
,
19
] since it is one of the representative methods
to extract volatile compounds from various matrices. Nonetheless, optimization procedures
for SPME conditions (such as temperature, solvents, and fibers) should precede sample
application and be accompanied by intensive labor and time [
20
]. A headspace (HS)-SPME
method may serve as an alternate simple sample preparation method with automated
operation and no solvent usage [
21
]. Another excellent alternative might be an automated
HS method, allowing for direct extraction of volatile compounds from various matrices
without needing fibers and solvents [
22
]. Consequently, automated HS methods have been
widely employed to extract bioactive volatile phytochemicals from plant samples [
11
,
23
,
24
].
In this study, we developed an HS-GC/MS method to determine volatile terpenes
in the leaves (n= 9) and inflorescences (n= 7) of hybrid Cannabis. Twenty-six terpenes in
hybrid Cannabis samples were detected and identified using this method. The developed
HS-GC/MS method was optimized and validated to quantify six major and abundant ter-
penes, including
α
- and
β
-pinene, myrcene, limonene,
β
-caryophyllene, and
α
-humulene,
in the leaves and inflorescences of hybrid Cannabis species. Since individual Cannabis
samples exhibited characteristic distributions for terpenes, even in leaves or inflorescences,
they could not be categorized into similar distributions. In conclusion, this study provides
characteristic terpene distributions and quantification results for six major terpenes in
the leaves and inflorescences of hybrid Cannabis. Furthermore, this study helps to better
understand the characteristic entourage effect of terpenes in individual hybrid Cannabis.
2. Results and Discussion
2.1. Optimization of HS-GC/MS Conditions
Although GC/FID provides several advantages, such as ease, simplicity, and low cost,
GC/MS has been the ‘gold standard’ for identifying and quantifying volatile phytochem-
icals in plant samples due to its indispensable sensitivity and selectivity [
25
]. Moreover,
when combined with an automated HS system, a GC/MS method reduces the need for
labor, minimizes processing time, and decreases the use of harmful solvents. Therefore, in
this study, we employed an HS-GC/MS method to identify and quantify terpenes in the
leaves and inflorescences of hybrid Cannabis.
To efficiently extract terpenes from the leaf and inflorescence samples, the HS condi-
tions were optimized in terms of incubation time and temperature using a standard solution
of representative terpenes, including
α
-pinene, myrcene,
β
-caryophyllene, and
α
-humulene.
As shown in Figure 1, the targeted terpenes were more affected by temperature than by
Molecules 2023,28, 8082 3 of 13
incubation time. In particular, the most effective total area for terpene extraction was at
120
◦
C. However, it should be noted that terpenes with high volatility, such as
α
-pinene and
myrcene, exhibited lower levels than other investigated temperatures. This finding might
be attributed to the degradation of terpenes caused by high temperatures [
26
]. Although
the incubation time parameter had a lesser influence on terpene extraction efficiency, an
incubation time of 30 min at 100 ◦C was shown to be effective.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 13
To efficiently extract terpenes from the leaf and inflorescence samples, the HS condi-
tions were optimized in terms of incubation time and temperature using a standard solu-
tion of representative terpenes, including α-pinene, myrcene, β-caryophyllene, and α-hu-
mulene. As shown in Figure 1, the targeted terpenes were more affected by temperature
than by incubation time. In particular, the most effective total area for terpene extraction
was at 120 °C. However, it should be noted that terpenes with high volatility, such as α-
pinene and myrcene, exhibited lower levels than other investigated temperatures. This
finding might be aributed to the degradation of terpenes caused by high temperatures
[26]. Although the incubation time parameter had a lesser influence on terpene extraction
efficiency, an incubation time of 30 min at 100 °C was shown to be effective.
Figure 1. Influence of incubation time and headspace temperature on terpene extraction efficiency
in leaf samples of Victory: (a) total peak area of terpenes and individual peak area of terpenes such
as (b) α-pinene, (c) myrcene, (d) β-caryophyllene, and (e) α-humulene.
Furthermore, we preliminarily investigated total ion chromatograms of representa-
tive hybrid Cannabis leaf samples at varying incubation temperatures, where incubation
was performed for 30 min. As shown in Figure S1, peak areas for all terpenes in leaf sam-
ples increased until 100 °C, while highly volatile terpenes (early eluted) were degraded at
120 °C. Moreover, signal noise, resulting from matrix influences, increased at 120 °C.
Therefore, we selected 100 °C and 30 min as the optimal incubation temperature and time,
respectively.
2.2. Investigation of Terpenes in the Leaves and Inflorescences
The optimized HS-GC/MS method was preliminarily applied to collected leaf and
inflorescence samples of hybrid Cannabis. As shown in Figure 2, a total of 26 terpenes were
detected in leaf and inflorescence samples using HS-GC/MS. The detected terpenes were
identified based on the National Institute of Standards and Technology (NIST) database
and their mass spectral paerns. To further confirm the identified terpenes, the Kovats
index (KI) was calculated using an alkane standard solution (C
8
–C
20
) and compared to
Figure 1.
Influence of incubation time and headspace temperature on terpene extraction efficiency in
leaf samples of Victory: (
a
) total peak area of terpenes and individual peak area of terpenes such as
(b)α-pinene, (c) myrcene, (d)β-caryophyllene, and (e)α-humulene.
Furthermore, we preliminarily investigated total ion chromatograms of representative
hybrid Cannabis leaf samples at varying incubation temperatures, where incubation was
performed for 30 min. As shown in Figure S1, peak areas for all terpenes in leaf samples
increased until 100
◦
C, while highly volatile terpenes (early eluted) were degraded at
120
◦
C. Moreover, signal noise, resulting from matrix influences, increased at 120
◦
C.
Therefore, we selected 100
◦
C and 30 min as the optimal incubation temperature and
time, respectively.
2.2. Investigation of Terpenes in the Leaves and Inflorescences
The optimized HS-GC/MS method was preliminarily applied to collected leaf and
inflorescence samples of hybrid Cannabis. As shown in Figure 2, a total of 26 terpenes were
detected in leaf and inflorescence samples using HS-GC/MS. The detected terpenes were
identified based on the National Institute of Standards and Technology (NIST) database
and their mass spectral patterns. To further confirm the identified terpenes, the Kovats
index (KI) was calculated using an alkane standard solution (C
8
–C
20
) and compared to
reference KI [
27
]. The KI and characteristic ions for the 26 terpenes in hybrid Cannabis are
summarized in Table 1.
Molecules 2023,28, 8082 4 of 13
Molecules 2023, 28, x FOR PEER REVIEW 4 of 13
reference KI [27]. The KI and characteristic ions for the 26 terpenes in hybrid Cannabis are
summarized in Table 1.
Figure 2. Total ion chromatograms for (a) V1 leaf and (b) Blue Dream inflorescence samples of hy-
brid Cannabis. (Peak identities are as follows: 1. α-Pinene, 2. β-Pinene, 3. Myrcene, 4. Limonene, 5.
Eucalyptol, 6. E-β-Ocimene, 7. γ-Terpinene, 8. Z-Sabinene hydrate, 9. β-Caryophyllene, 10. α-Berga-
motene, 11. α-Guaiene, 12. E-β-Farnesene, 13. α-Humulene, 14. Alloaromadrene, 15. β-Selinene, 16.
α-Selinene, 17. Z,E-α-Farnesene, 18. β-Bisabolene, 19. β-sesquiphellandrene, 20. E-α-Bisabolene, 21.
Selina-3,7(11)-diene, 22. Caryophyllene oxide, 23. Guaiol, 24. γ-Eudesmol, 25. Bulnesol, 26. α-Bisab-
olol).
Tab le 1. Retention times, the Kovats index (KI), and characteristic ions of 26 terpenes in hybrid Can-
nabis species.
Elutio
n
Order
Compound
Name M.W RT
(Min)
KI
calc. KI Ref Characteristic Ions m/z
(Relative Abundance%)
1 α-Pinene 136 6.06 935 936 136 (10), 121 (15), 105 (10), 93 (100), 91 (40), 79 (25), 77 (30)
2 β-Pinene 136 7.13 981 978 136 (10), 121 (15), 93 (100), 91 (25), 79 (20), 77 (20), 69 (25)
3 Myrcene 136 7.32 989 989 136 (5), 121 (5), 93 (100), 91 (25), 79 (15), 77 (15), 69 (70), 41 (75)
4 Limonene 136 8.42 1031 1030 136 (25), 121 (25), 107 (25), 93 (75), 79 (35), 68 (100), 67 (70)
5 Eucalyptol 154 8.53 1035 1032
154 (70), 139 (60), 125 (15), 111 (80), 93 (60), 81 (90), 71 (70), 55
(40), 43 (100)
6 E-β-Ocimene 136 8.82 1046 1048 136 (5), 121 (20), 105 (20), 93 (100), 91 (45), 80 (35), 79 (40)
7
γ
-Terpinene 136 9.19 1060 1060 136 (40), 119 (50), 105 (15), 93 (100), 77 (35), 91 (60)
8 Z-Sabinene
hydrate 154 9.53 1072 1067 154 (5), 136 (25), 121 (25), 111 (15), 93 (100), 77 (35), 43 (25)
Figure 2.
Total ion chromatograms for (
a
) V1 leaf and (
b
) Blue Dream inflorescence samples of hybrid
Cannabis. (Peak identities are as follows: 1.
α
-Pinene, 2.
β
-Pinene, 3. Myrcene, 4. Limonene,
5. Eucalyptol, 6. E-
β
-Ocimene, 7.
γ
-Terpinene, 8. Z-Sabinene hydrate, 9.
β
-Caryophyllene,
10.
α
-Bergamotene, 11.
α
-Guaiene, 12. E-
β
-Farnesene, 13.
α
-Humulene, 14. Alloaromadrene,
15.
β
-Selinene, 16.
α
-Selinene, 17. Z,E-
α
-Farnesene, 18.
β
-Bisabolene, 19.
β
-sesquiphellandrene,
20. E-
α
-Bisabolene, 21. Selina-3,7(11)-diene, 22. Caryophyllene oxide, 23. Guaiol, 24.
γ
-Eudesmol,
25. Bulnesol, 26. α-Bisabolol).
Table 1.
Retention times, the Kovats index (KI), and characteristic ions of 26 terpenes in hybrid
Cannabis species.
Elution
Order
Compound
Name M.W RT
(Min) KI calc. KI Ref Characteristic Ions m/z
(Relative Abundance%)
1α-Pinene 136 6.06 935 936 136 (10), 121 (15), 105 (10), 93 (100), 91 (40),
79 (25), 77 (30)
2β-Pinene 136 7.13 981 978 136 (10), 121 (15), 93 (100), 91 (25), 79 (20), 77
(20), 69 (25)
3 Myrcene 136 7.32 989 989 136 (5), 121 (5), 93 (100), 91 (25), 79 (15), 77
(15), 69 (70), 41 (75)
4 Limonene 136 8.42 1031 1030 136 (25), 121 (25), 107 (25), 93 (75), 79 (35), 68
(100), 67 (70)
5 Eucalyptol 154 8.53 1035 1032
154 (70), 139 (60), 125 (15), 111 (80), 93 (60), 81
(90), 71 (70), 55 (40), 43 (100)
6E-β-Ocimene 136 8.82 1046 1048 136 (5), 121 (20), 105 (20), 93 (100), 91 (45), 80
(35), 79 (40)
7γ-Terpinene 136 9.19 1060 1060
136 (40), 119 (50), 105 (15), 93 (100), 77 (35), 91
(60)
8Z-Sabinene
hydrate 154 9.53 1072 1067
154 (5), 136 (25), 121 (25), 111 (15), 93 (100), 77
(35), 43 (25)
9β-
Caryophyllene
204 19.13 1426 1420 204 (10), 189 (25), 175 (15), 161 (45), 147 (30),
133 (95), 120 (45), 105 (60), 93 (100), 79 (75)
Molecules 2023,28, 8082 5 of 13
Table 1. Cont.
Elution
Order
Compound
Name M.W RT
(Min) KI calc. KI Ref Characteristic Ions m/z
(Relative Abundance%)
10 trans-α-
Bergamotene 204 19.37 1435 1435 204 (5), 189 (5), 161 (5), 119 (100), 107 (30), 93
(95), 79 (25), 69 (35)
11 α-Guaiene 204 19.45 1438 1440 204 (55), 189 (35), 161 (25), 147 (90), 133 (65),
119 (45), 105 (100), 93 (75), 79 (60),
12 E-β-
Farnesene 204 19.81 1453 1456
204 (5), 189 (5), 161 (15), 133 (30), 120 (25), 107
(10), 93 (65), 79 (25), 69 (100)
13 α-Humulene 204 20.02 1461 1453
204 (10), 189 (5), 161 (5), 147 (20), 121 (40), 107
(15), 93 (100), 80 (30), 67 (10)
14
Alloaromadrene
204 20.12 1465 1460
204 (45), 189 (35), 175 (10), 161 (100), 147 (50),
133 (70), 119 (60), 105 (90), 91 (100)
15 β-Selinene 204 20.85 1494 1486 204 (70), 189 (60), 175 (30), 161 (65), 147 (50),
133 (50), 121 (60), 105 (100), 93 (90)
16 α-Selinene 204 21.01 1500 1493 204 (50), 189 (100),175 (30), 161 (35), 133 (50),
121 (25), 107 (55), 93 (55)
17 Z,E-α-
Farnesene 204 21.10 1504 1504 204 (5), 161 (10),135 (10), 123 (35), 119 (50),
107 (50), 93 (100), 79 (45), 69 (50)
18 β-Bisabolene 204 21.23 1509 1508 204 (20), 189 (5), 161 (20), 133 (10), 119 (25),
109 (30), 93 (85), 79 (35), 69 (100)
19 β-
sesquiphellandrene
204 21.62 1526 1524 204 (30), 189 (5), 161 (60), 133 (40), 120 (30),
109 (30), 93 (70), 69 (100)
20 E-α-
Bisabolene 204 22.00 1542 1540 204 (20), 189 (5), 161 (5), 147 (5), 136 (10), 119
(30), 109 (25), 93 (100), 78 (25)
21 Selina-
3,7(11)-diene 204 22.11 1546 1541
204 (55), 189 (25), 161 (100), 133 (20), 122 (60),
107 (50), 91 (30), 81 (20)
22
Caryophyllene
oxide 222 23.10 1587 1581 205 (10), 202 (20), 187 (40), 161 (35), 149 (30),
133 (45), 119 (40), 105 (65), 91 (100), 79 (85)
23 Guaiol 222 23.38 1599 1597 222 (5), 204 (25), 189 (25), 161 (100), 147 (20),
133 (25), 119 (25), 105 (60), 91 (50)
24 γ-Eudesmol 222 24.04 1629 1631 222 (5), 204 (60), 189 (100), 161 (80), 147 (25),
133 (60), 119 (20), 105 (45), 91 (50)
25 Bulnesol 222 24.96 1669 1666 222 (5), 204 (30), 189 (35), 161 (55), 147 (25),
135 (75), 119 (45), 107 (100), 93 (85),
26 α-Bisabolol 222 25.38 1688 1683 204 (30), 189 (5), 161 (20), 135 (10), 119 (90),
109 (95), 93 (85), 79 (40), 69 (100)
To investigate the individual terpene compositions of leaf and inflorescence samples
in hybrid Cannabis species, the relative abundance of all peaks were calculated based
on the total ion chromatograms’ area under the curve. As shown in Table 2, several
terpenes, including
α
- and
β
-pinenes, myrcene, limonene,
β
-caryophyllene, bergamotene,
and
α
-humulene, were presented in all leaf samples of hybrid Cannabis. However, the
guaiol terpene was not detected in all leaves, and bulnesol was only detected in the Blue
Dream variety. The overall terpene compositions in inflorescence samples are shown
in Table 3. As shown in Table 3, most terpenes were detected in all inflorescences of
hybrid Cannabis species, except for the bulnesol terpene, which was only detected in Blue
Dream, similar to the leaf samples. As shown in Tables 2and 3, even though some leaf
and inflorescence samples originated from the same hybrid Cannabis species, including
White Widow, individual terpene compositions substantially differed between leaves
and inflorescences. To investigate the relationship between plant organs and/or hybrid
Cannabis species, all hybrid Cannabis plants should be grown under uniform growing
conditions since spatial differences, organs, and locations can influence individual terpene
accumulation [
28
]. Among the identified 26 terpenes, six monoterpenes (including
α
-
and
β
-pinenes, myrcene, limonene,
β
-caryophyllene, and
α
-humulene) were commonly
detected in both the leaves and inflorescences of hybrid Cannabis species. These six terpenes
were well-known as predominant terpenes [
29
] and may contribute to the “entourage
effect” of cannabinoids [30].
Molecules 2023,28, 8082 6 of 13
Table 2. Relative abundances of 26 terpenes in leaves of hybrid Cannabis.
Elution Order Compound
Name
Relative Abundance (%)
Cherry
Blossom V1 V4 White Widow Chung Sam Blue Dream Bubble Gum Purple Victory
1α-Pinene 28 ±8 50 ±30 40 ±20 2.29 ±0.04 4.8 ±0.5 20 ±10 5 ±1 38 ±5 17 ±1
2β-Pinene 6.6 ±0.3 15 ±3 13 ±5 1.75 ±0.09 2.0 ±0.5 7 ±5 7 ±1 14 ±2 6.5 ±0.8
3 Myrcene 6.5 ±0.2 1.14 ±0.02 2.5 ±0.2 4.41 ±0.06 0.08 ±0.03 10 ±5 9 ±2 17 ±2 14 ±2
4 Limonene 5 ±1 2.5 ±0.6 6.3 ±0.2 6.5 ±0.1 1.9 ±0.6 4 ±2 19 ±3 3.4 ±0.4 3.6 ±0.6
5 Eucalyptol 0.04±0.04 1.2 ±0.9 1.3 ±0.5 20 ±5 ND 3 ±2 ND ND 0.68 ±0.02
6E-β-Ocimene 0.6 ±0.3 0 ±1 0.47 ±0.08 ND ND 1.9 ±0.6 ND ND ND
7γ-Terpinene ND 0.1 ±0.2 0.05 ±0.07 0.40 ±0.09 ND 0.6 ±0.1 ND 0.02 ±0.01 0.13 ±0.03
8Z-Sabinene
hydrate ND 0 ±1 0.3 ±0.5 0 ±2 ND 1.0 ±0.8 ND 0.08 ±0.05 0.4 ±0.2
9β-
Caryophyllene 21 ±3 10 ±20 10 ±30 22 ±5 42 ±5 20 ±10 13 ±3 7 ±3 18 ±6
10 trans-α-
Bergamotene 4±7 5 ±9 4 ±9 1.6 ±0.5 6.3 ±0.7 3.7 ±0.3 5 ±3 2.6 ±0.4 5 ±2
11 α-Guaiene 0.2 ±0.3 <0.01 ND 0.31 ±0.01 ND ND ND ND ND
12 E-β-Farnesene 2 ±4 1 ±2 0.3 ±0.7 0.9 ±0.1 ND 0.62 ±0.08 0.5 ±0.5 ND 2 ±1
13 α-Humulene 16 ±27 10 ±20 10 ±20 16 ±4 33 ±4 12.0 ±0.3 18 ±8 7.0 ±0.2 15 ±2
14 Alloaromadrene 0.2 ±0.3 0 ±1 0 ±1 0.4 ±0.2 ND 0.3 ±0.3 ND 0.17 ±0.09 1.0 ±0.3
15 β-Selinene 0.3 ±0.5 ND ND 1.0 ±0.4 ND 0.22 ±0.03 0.57 ±0.06 0.1 ±0.1 0.7 ±0.8
16 α-Selinene 0.3 ±0.1 ND ND 1.2 ±0.3 ND ND ND 0.23 ±0.01 0.90 ±0.07
17 Z,E-α-
Farnesene 4±7 0.12 ±0.07 0.13 ±0.05 ND ND ND 1.4 ±0.8 2 ±1 ND
18 β-Bisabolene 4 ±7 0.3 ±0.8 0.3 ±0.8 3.0 ±0.9 3.4 ±0.4 1.2 ±0.6 ND 0.4 ±0.1 3.8 ±0.8
19 β-
sesquiphellandrene
ND ND <0.01 ND <0.01 ND ND ND 0.30 ±0.07
20 E-α-Bisabolene ND 1 ±2 1 ±2 ND 1.1 ±0.1 ND 9 ±6 ND 8 ±2
21 Selina-3,7(11)-
diene ND ND ND 17 ±3 0.6 ±0.4 5 ±3 9 ±3 6.6 ±0.2 2.7 ±0.1
22 Caryophyllene
oxide 0.43 ±0.09 1 ±3 2 ±6 0.1 ±0.1 4 ±1 ND ND <0.01 0.02 ±0.01
23 Guaiol ND ND ND ND ND ND ND ND ND
24 γ-Eudesmol ND 0 ±2 0 ±1 ND ND 0.2 ±0.1 ND ND ND
25 Bulnesol ND 0.1 ±0.3 ND ND ND ND ND ND ND
26 α-Bisabolol 1 ±3 0.1 ±0.4 ND 0.8 ±0.7 1 ±1 0.2 ±0.1 ND 0.09 ±0.01 0.5 ±0.9
ND means “not detected”.
Molecules 2023,28, 8082 7 of 13
Table 3. Relative abundance of 26 terpenes in the inflorescences of hybrid Cannabis.
Elution Order Compound Name Relative Abundance (%)
Cherry Blossom V1 V4 White Widow Chung Sam Blue Dream Bubble Gum
1α-Pinene 29 ±8 18 ±7 12 ±4 3 ±1 22.8 ±0.5 20 ±10 6 ±1
2β-Pinene 10 ±10 10 ±6 8 ±4 5 ±3 3.8 ±0.4 9 ±3 10 ±2
3 Myrcene 40 ±30 50 ±20 2 ±1 40 ±10 0.13 ±0.02 41 ±5 16 ±2
4 Limonene 6 ±3 6.6 ±0.9 10 ±6 13 ±9 0.2 ±0.2 2.3 ±0.6 35 ±1
5 Eucalyptol ND 0.06 ±0.08 ND 0.5 ±0.4 ND ND <0.01
6E-β-Ocimene 5 ±2 9.9 ±0.5 0.9 ±0.7 3 ±1 ND 9 ±2 ND
7γ-Terpinene 0.0 ±0.1 0.07 ±0.03 1.1 ±0.8 0.19 ±0.02 0.06 ±0.03 0.17 ±0.01 0.07 ±0.05
8Z-Sabinene
hydrate 0.02 ±0.01 0.08 ±0.01 0.2 ±0.2 0.11 ±0.06 <0.01 0.09 ±0.01 ND
9β-Caryophyllene 3 ±1 0.7 ±0.5 20 ±10 14.0 ±0.9 31 ±4 6 ±4 10.2 ±0.3
10 trans-α-
Bergamotene 0.9 ±0.4 0.09 ±0.06 6.4 ±0.9 0.4 ±0.2 8 ±5 0.66 ±0.03 0.47 ±0.03
11 α-Guaiene 0.21 ±0.09 0.19 ±0.03 0.0 ±0.2 2.4 ±0.3 0.9 ±0.1 <0.01 <0.01
12 E-β-Farnesene 1.3 ±0.6 0.05 ±0.03 0.77 ±0.09 0.5 ±0.3 0.22 ±0.04 0.08 ±0.01 0.12 ±0.09
13 α-Humulene 2.1 ±0.9 0.5 ±0.5 21 ±2 11 ±2 24 ±4 4 ±2 7.4 ±0.1
14 Alloaromadrene 0.13 ±0.05 <0.01 0.4 ±0.2 0.11 ±0.04 ND 0.09 ±0.01 ND
15 β-Selinene 0.04 ±0.02 0.03 ±0.01 0.3 ±0.2 0.8 ±0.2 1 ±1 0.20 ±0.03 0.75 ±0.07
16 α-Selinene 0.04 ±0.01 0.04 ±0.09 0.29 ±0.06 1 ±1 1.5 ±0.2 0.22 ±0.02 0.9 ±0.4
17 Z,E-α-Farnesene 0.38 ±0.09 0.2 ±0.3 2.6 ±0.3 2.0 ±0.6 1.40 ±0.06 0.13 ±0.03 0.7 ±0.1
18 β-Bisabolene 0.5 ±0.2 0.02 ±0.01 2.9 ±0.4 0 ±1 0.3 ±0.3 0.06 ±0.02 0.08 ±0.05
19 β-
sesquiphellandrene
0.09 ±0.04 ND ND 0.1 ±0.1 ND 0.05 ±0.03 ND
20 E-α-Bisabolene 0.6 ±0.3 0.0 ±0.2 3.9 ±0.5 2 ±2 4 ±3 1.7 ±0.5 ND
21 Selina-3,7(11)-
diene ND ND 0.3 ±0.1 3 ±2 0.1 ±0.5 1.5 ±0.3 12 ±1
22 Caryophyllene
oxide <0.01 0.0 ±0.1 0.7 ±0.4 0.06 ±0.01 0.2 ±0.2 ND ND
23 Guaiol 0.03 ±0.02 0.1 ±0.2 0.3 ±0.1 ND ND 0.12 ±0.08 ND
24 γ-Eudesmol 0.05 ±0.03 0.05 ±0.01 0.5 ±0.3 ND ND 0.2 ±0.2 ND
25 Bulnesol ND ND ND ND ND 0.07 ±0.05 ND
26 α-Bisabolol ND ND 0.4 ±0.7 0.15 ±0.06 0.03 ±0.01 ND ND
ND means “not detected”.
Molecules 2023,28, 8082 8 of 13
Based on the relative abundance of peak areas for terpenes, hierarchical cluster analy-
sis (HCA) was performed to cluster organs (leaves and inflorescences) in hybrid Cannabis
(Figure S2). Most leaf and inflorescence samples of hybrid Cannabis species could not be
clustered by individual strains except for Bubble Gum. However, we speculated that the
terpene compositions may be related to their organ types (leaves and inflorescences). Fur-
thermore, principal component analysis (PCA) was also performed on the data set without
scaling to find major terpenes, which can distinguish individual leaf and inflorescence
samples of hybrid Cannabis. Based on PCA results, 51.66% and 29.79% of the variance
was explained by PC1 and PC2, respectively. As shown in Figure 3, leaf samples could be
grouped. Furthermore, samples from the leaves and inflorescences of White Widow and
Blue Dream could be separated since individual leaves or inflorescences had characteristic
terpene compositions, respectively. Six terpenes (
α
- and
β
-pinenes, myrcene, limonene,
β
-caryophyllene, and
α
-humulene) out of 26 terpenes have greater potential to identify
individual leaves and inflorescence samples of hybrid Cannabis than other terpenes. These
six terpenes would likely contribute to variance explanations for PC1 and PC2 since they
were consistently and predominantly present in all leaves and inflorescences of hybrid
Cannabis. Although several characteristic terpenes (such as guaiol found in Blue Dream in-
florescences) may also be potential markers, they cannot separate all leaf and inflorescence
samples of hybrid Cannabis species. Therefore, in this study, we quantified the six major
and abundant terpenes as biomarkers using HS-GC/MS.
Molecules 2023, 28, x FOR PEER REVIEW 8 of 13
(Figure S2). Most leaf and inflorescence samples of hybrid Cannabis species could not be
clustered by individual strains except for Bubble Gum. However, we speculated that the
terpene compositions may be related to their organ types (leaves and inflorescences). Fur-
thermore, principal component analysis (PCA) was also performed on the data set without
scaling to find major terpenes, which can distinguish individual leaf and inflorescence
samples of hybrid Cannabis. Based on PCA results, 51.66% and 29.79% of the variance was
explained by PC1 and PC2, respectively. As shown in Figure 3, leaf samples could be
grouped. Furthermore, samples from the leaves and inflorescences of White Widow and
Blue Dream could be separated since individual leaves or inflorescences had characteristic
terpene compositions, respectively. Six terpenes (α- and β-pinenes, myrcene, limonene, β-
caryophyllene, and α-humulene) out of 26 terpenes have greater potential to identify in-
dividual leaves and inflorescence samples of hybrid Cannabis than other terpenes. These
six terpenes would likely contribute to variance explanations for PC1 and PC2 since they
were consistently and predominantly present in all leaves and inflorescences of hybrid
Cannabis. Although several characteristic terpenes (such as guaiol found in Blue Dream
inflorescences) may also be potential markers, they cannot separate all leaf and inflo-
rescence samples of hybrid Cannabis species. Therefore, in this study, we quantified the
six major and abundant terpenes as biomarkers using HS-GC/MS.
Figure 3. Principal component analysis (PCA) of hybrid Cannabis using relative abundances of peak
areas for 26 terpenes. (Marks were identified as follows: 1. Cherry Blossom leaf, 2. V1 leaf, 3. V4 leaf,
4. White Widow leaf, 5. Chung Sam leaf, 6. Blue Dream leaf, 7. Bubble Gum leaf, 8. Purple leaf, 9.
Victory leaf, 10. Cherry Blossom inflorescence, 11. V1 inflorescence, 12. V4 inflorescence, 13. White
Widow inflorescence, 14. Chung Sam inflorescence, 15. Blue Dream inflorescence, 16. Bubble Gum
inflorescence, A. α-Pinene, B. β-Pinene, C. Myrcene, D. Limonene, E. Eucalyptol, F. E-β-Ocimene, G.
γ-Terpinene, H. Z-Sabinene hydrate I. β-Caryophyllene, J. α-Bergamotene, K. α-Guaiene, L. β-Far-
nesene (E), M. α-Humulene, N. Alloaromadrene, O. β-Selinene, P. α-Selinene, Q. Z,E-α-Farnesene,
R. β-Bisabolene,S. β-sesquiphellandrene, T. E-α-Bisabolene, U. Selina-3,7(11)-diene, V. Caryo-
phyllene oxide, W. Guaiol, X. γ-Eudesmol, Y. Bulnesol, Z. α-Bisabolol).
Figure 3.
Principal component analysis (PCA) of hybrid Cannabis using relative abundances of
peak areas for 26 terpenes. (Marks were identified as follows: 1. Cherry Blossom leaf, 2. V1 leaf,
3. V4 leaf, 4. White Widow leaf, 5. Chung Sam leaf, 6. Blue Dream leaf, 7. Bubble Gum leaf,
8. Purple leaf, 9. Victory leaf, 10. Cherry Blossom inflorescence, 11. V1 inflorescence, 12. V4 inflores-
cence, 13. White Widow inflorescence, 14. Chung Sam inflorescence, 15. Blue Dream inflorescence,
16. Bubble Gum inflorescence, A.
α
-Pinene, B.
β
-Pinene, C. Myrcene, D. Limonene, E. Eucalyptol,
F. E-
β
-Ocimene, G.
γ
-Terpinene, H. Z-Sabinene hydrate I.
β
-Caryophyllene, J.
α
-Bergamotene, K.
α
-Guaiene, L.
β
-Farnesene (E), M.
α
-Humulene, N. Alloaromadrene, O.
β
-Selinene, P.
α
-Selinene, Q.
Z,E-
α
-Farnesene, R.
β
-Bisabolene, S.
β
-sesquiphellandrene, T. E-
α
-Bisabolene, U. Selina-3,7(11)-diene,
V. Caryophyllene oxide, W. Guaiol, X. γ-Eudesmol, Y. Bulnesol, Z. α-Bisabolol).
Molecules 2023,28, 8082 9 of 13
To quantify the six major terpenes in the leaves and inflorescences of hybrid Cannabis
species, commercially available authentic terpene standards were employed. The HS-
GC/MS method was validated in terms of quantification limits, calibration range, linearity,
precision, and accuracy. Table S1 summarizes the overall data and validation results for
quantifying six major terpenes using the HS-GC/MS method.
2.3. Quantification of the Six Major Terpenes in the Leaves and Inflorescences of Hybrid
Cannabis Species
In this study, the validated HS-GC/MS method was employed to determine six major
terpenes in the leaves (n= 9) and inflorescences (n= 7) of hybrid Cannabis species. As
depicted in Table 4, quantified terpenes were found to be highly presented in the inflo-
rescence sample of Cherry Blossom compared to other hybrid Cannabis. Furthermore, the
quantification results for most terpenes in inflorescences were higher than in leaves. In
the inflorescences of White Widow, Chung Sam, and V4,
β
-caryophyllene content was the
most abundant. The biochemical diversity of terpenes in Cannabis makes it challenging
to predict the pharmacological “entourage effect” of Cannabis. Therefore, the quantifica-
tion results of the six terpenes with characteristic bioactive effects may help demonstrate
their distinctive therapeutic outcomes and the “entourage effect” of individual leaf and
inflorescence samples of hybrid Cannabis. For example, since
α
-pinene has antioxidative
and anti-inflammatory effects [
31
–
33
], the inflorescence of Cherry Blossom may be more
effective at relieving pain when combined with cannabidiol in Cannabis [
34
]. Furthermore,
due to the analgesic and anti-cancer effects of
β
-caryophyllene [
35
], several hybrid Cannabis,
including inflorescences of White Widow, Chung Sam, V4, and Cherry Blossom and leaves
of Cherry Blossom and Victory, may be more suitable for cancer patients. Since both
myrcene and limonene are flavor and fragrance chemicals, the inflorescences of Cherry
Blossom, V1, White Widow, and Bubble Gum may be widely preferred by Cannabis users
for their potential to inhibit Cannabis use disorders, including vomiting and nausea. Table 4
summarizes the overall calculated quantification results for the six terpenes in all leaves
and inflorescences of hybrid Cannabis.
Table 4.
Quantification results for six major terpenes in the leaf and inflorescence samples of
hybrid Cannabis.
Strains Organ
Concentrations (µg/g)
α-Pinene β-Pinene Myrcene Limonene β-
Caryophyllene α-Humulene
Cherry Blossom leaf 144 ±8 32.9 ±0.5 187 ±7 14 ±1 1200 ±600 300 ±100
inflorescence 2270 ±70 930 ±20 17,400 ±300 260 ±20 2000 ±100 500 ±30
V1 leaf 100 ±10 80 ±60 10 ±10 8 ±6 220 ±20 66 ±4
inflorescence 500 ±100 290 ±60 4600 ±400 115 ±9 200 ±10 40 ±30
V4 leaf 84 ±5 27 ±3 20 ±10 8 ±2 240 ±60 70 ±20
inflorescence 93 ±1 60 ±30 50 ±50 47 ±4 2500 ±300 690 ±80
White Widow leaf 9.8 ±0.6 6.2 ±0.4 79 ±7 13 ±1 950 ±10 236 ±5
inflorescence 64 ±3 110 ±10 2000 ±1000 150 ±30 3300 ±400 870 ±80
Chung Sam leaf 6.9 ±0.3 2.6 ±0.2 0.7 ±0.2 1.6 ±0.2 263 ±4 73 ±1
inflorescence 183 ±5 30.1 ±0.3 6.1 ±0.1 1.3 ±0.2 2900 ±600 800 ±100
Blue Dream leaf 56.5 ±0.7 18.6 ±0.6 200 ±100 6.1 ±0.1 205 ±4 51 ±4
inflorescence 200 ±300 116 ±3 1400 ±900 19 ±1 400 ±200 100 ±60
Bubble Gum leaf 9.79 ±0.02 10.6 ±0.2 75 ±1 16.2 ±0.6 60 ±40 48 ±2
inflorescence 93.17 ±0.03 142 ±1 1310 ±30 300 ±20 500 ±100 130 ±40
Purple leaf 450 ±10 200 ±50 1190 ±60 34 ±6 400 ±70 130 ±30
Victory leaf 332 ±3 122 ±4 1550 ±60 42 ±3 1100 ±200 300 ±70
3. Experimental
3.1. Chemicals and Materials
Analytical grade methanol (MeOH) and ethyl acetate (EA) were purchased from J.
T. Baker (Phillipsburg, NJ, USA). Deionized water (DW) was obtained using a Milli-Q
purification system (Millipore Co., Bedford, MA, USA). Analytical grade standards of
α
-
and
β
-pinene, myrcene, limonene, eucalyptol,
β
-caryophyllene,
α
-humulene, and alkane
standard solutions (C
8
–C
20
) were purchased from Sigma–Aldrich (St. Louis, MO, USA). For
internal standards, nonane and tetradecane were also purchased from Sigma–Aldrich (St.
Molecules 2023,28, 8082 10 of 13
Louis, MO, USA). Leaves (n= 9) and inflorescences (n= 7) of hybrid Cannabis species were
provided by Nongboo Mind (Seoul, Republic of Korea). All Cannabis samples were hybrid
Cannabis species (combinations of indica and sativa) strictly supervised by the Korean
Government. Therefore, only a limited number of Cannabis samples were allowed to be
investigated in this study. All collected samples were sealed and stored in a freezer at
−20 ◦C until analysis.
3.2. Sample Preparation
Fresh leaves and inflorescences from hybrid Cannabis species were prepared by remov-
ing superficial moisture with natural drying at room temperature, chopped using scissors,
and weighed at 50 mg using an analytical balance (Mettler Toledo, Columbus, OH, USA).
Weighed leaf and inflorescence samples were transferred into 10 mL headspace vials.
3.3. HS-GC/MS Conditions
To optimize headspace conditions, the incubation temperature (60–120
◦
C) and time
(20–40 min) were tested. The sample was incubated at 100
◦
C for 30 min. The syringe
temperature, fill speed, and injection speed of the automated headspace were 120
◦
C,
100
µ
L/s, and 500
µ
L/s, respectively. GC-MS analysis was performed by an Agilent 6890N
gas chromatograph combined with an Agilent-5973 mass spectrometer equipped with
electron ionization (EI) and a quadrupole analyzer. Separation was achieved using an
Agilent Technologies DB-5MS column (30 m
×
0.25 mm i.d., film thickness of 0.25
µ
m,
J&W Scientific, Folsom, CA, USA). The sample (0.5 mL) in the automated headspace was
automatically injected into the injection port heated at 250
◦
C in split mode (10:1). As
a carrier gas, helium (purity: 99.999%) was set at a flow rate of 1 mL/min. The oven
temperature was programmed to hold at 60
◦
C for 1 min, ramp up to 200
◦
C at a rate of
5
◦
C/min, and then increase to 250
◦
C at a rate of 10
◦
C/min. The temperature of the
interface, ion source, and quadrupole was set at 250
◦
C, 230
◦
C, and 150
◦
C, respectively,
and the EI energy was set at 70 eV. The mass spectra were acquired in scan mode in the
range m/z40–250 since no substance was detected above m/z250 in all Cannabis samples
in preliminary experiments.
3.4. Qualitative and Quantitative Analysis
For qualitative analysis, the individual detection result was compared with the reten-
tion time, mass spectral pattern, database of NIST, mass spectra of authentic standards, GC
elution order, and KI values based on previous reports. KI was calculated using a C
8
–C
20
n-alkane solution and applied to a temperature-programming analysis [
36
]. Calculated KI
values were investigated according to the following equation:
KIx=100n+tx−tn
tn+1−tn(1)
where nis the number of n-alkane carbon atoms eluting before the compound x;t
n
and t
n+1
are the retention times that elute before and after compound x. The relative abundance of
terpenes was individually investigated for each leaf or inflorescence sample of different hy-
brid Cannabis varieties. Quantitative analysis was performed on
α
- and
β
-pinene, myrcene,
limonene,
β
-caryophyllene, and
α
-humulene, known as major terpenes in Cannabis. To
investigate terpenes and select biomarkers, statistical analyses (such as PCA and HCA)
were performed using R 4.1.3 (R Core Team, Vienna, Austria).
3.5. Method Validation
For reliable validation, a standard mixture solution of major volatile components in
Cannabis such as
α
- and
β
-pinene, myrcene, limonene,
β
-caryophyllene, and
α
-humulene
was dissolved in methanol at a concentration of 500
µ
g/mL using nonane and tetradecane
as internal standards. For linearity, standard solutions were prepared at 1–250
µ
g/mL
for
α
- and
β
-pinene, limonene,
β
-caryophyllene, and
α
-humulene, and 1–500
µ
g/mL
Molecules 2023,28, 8082 11 of 13
for myrcene. Calibration curves were constructed by comparing the peak area ratios of
individual compounds to their internal standards versus their concentrations in
µ
g/mL.
LOQ was evaluated as the concentration of a standard mixture with a signal-to-noise ratio
(S/N) > 10. To obtain repeatability and reproducibility, intra- and inter-day precision were
estimated by analyzing triplicates of Cannabis extract. To determine accuracy, Cannabis
samples were analyzed by spiking the standard solution at three different concentrations
(5, 10, and 20
µ
g/mL). The accuracy data was obtained by calculating differences before
and after spiking the standard solution to match the sample matrices.
Accuracy =Amo unt a f t er s pikin g −Amount be fore s piking
spiked amount ×100% (2)
4. Conclusions
In this study, we introduced the automated HS-GC/MS method to simply and easily
detect 26 terpenes and quantify six major terpenes, namely
α
- and
β
-pinenes, myrcene,
limonene,
β
-caryophyllene, and
α
-humulene, in the leaves (n= 9), and inflorescences
(n= 7) of hybrid Cannabis species without intensive time and labor. To enhance terpene
extraction efficiency from leaf and inflorescence samples, the HS incubation time and
temperature were optimized at 30 min and 100
◦
C, respectively. Using the established
HS-GC/MS method, 26 terpenes were identified based on EI mass spectral patterns and
retention indices. Furthermore, based on the PCA results, we investigated components
for identifying individual hybrid Cannabis samples and suggested six major terpenes as
potential biomarkers. These six terpenes were consistently and predominantly present
in all Cannabis samples. Furthermore, we quantified six major terpenes in both leaves
and inflorescences of hybrid Cannabis using HS-GC/MS and tried to demonstrate the
“entourage effect” specific to individual Cannabis based on quantification results for six
terpenes. The HS-GC/MS method used in this study directly detected 26 terpenes and
quantified six major terpenes in the leaves and inflorescences of hybrid Cannabis. Our
research contributes to a better understanding of terpenes’ “entourage effect” in Cannabis.
Supplementary Materials:
The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/molecules28248082/s1, Figure S1: Total ion chromatograms of terpenes
in leaves of Victory at different incubation temperature of (a) 60
◦
C, (b) 80
◦
C, (c) 100
◦
C, and
(d) 120 ◦C
; Figure S2: Cluster dendrogram of leaves and inflorescences of hybrid Cannabis using
relative abundances of peak areas for 26 terpenes; Table S1: Analytical characteristics of the HS-
GC/MS method for 6 major terpenes.
Author Contributions:
Conceptualization, W.L. and J.H.; Methodology, J.H.; Validation, S.L. and
E.J.K.; Investigation, S.L., E.K., S.J.O. and W.L.; Resources, M.C. and C.M.K.; Data curation, E.J.K.;
Writing—original draft, W.L.; Writing—review & editing, J.H.; Supervision, J.H. All authors have
read and agreed to the published version of the manuscript.
Funding:
This research was supported by the Bio & Medical Technology Development Program
of the National Research Foundation (NRF) funded by the Korean government (MSIT) (RS-2023-
00223559) as well as the Ministry of Agriculture, Food and Rural Affairs of Republic of Korea
(PJ017015 (RS2022RD010270)).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available from a publicly accessible repository.
Acknowledgments:
All leaves and inflorescences of hybrid Cannabis species were kindly provided
by Nongboo Mind (Seoul, Republic of Korea).
Conflicts of Interest: The authors declare no conflict of interest.
Molecules 2023,28, 8082 12 of 13
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