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The Botany of Cannabis sativa L.


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Cannabis sativa L. is among the oldest known cultivated plants, with a long history of medical use. Cannabis produces a unique class of terpenophenolic compounds called cannabinoids, 104 of which have been isolated, the major biologically active one being Δ9-tetrahydrocannabinol. Cannabidiol, an antiepileptic, is also important. Cannabis is an annual, normally dioecious and occasionally monoecious, wind-pollinated species and is highly allogamous (cross-fertilization) in nature. Therefore, maintaining the efficacy of selected high Δ9-tetrahydrocannabinol-yielding elite varieties grown from seeds under field or greenhouse conditions is very difficult. Thus, a careful screening of elite mother clones using GC-FID and their propagation using vegetative cuttings or advanced biotechnological approaches, including micropropagation, is the most suitable way to maintain quality. This chapter describes the botany, species debate, phenotype, screening of high-yielding clones using GC-FID, indoor and outdoor cultivation, micropropagation, quality assurance of propagated plants, harvesting, processing, and storage.
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The Analytical Chemistry of Cannabis
Emerging Issues in Analytical Chemistry
Series Editor
Brian F. Thomas
The Analytical Chemistry
of Cannabis
Quality Assessment, Assurance, and
Regulation of Medicinal Marijuana
and Cannabinoid Preparations
Brian F. Thomas
Analytical Chemistry and Pharmaceutics, RTI International, Research
Triangle Park, NC, United States
Mahmoud A. ElSohly
National Center for Natural Products Research, Research Institute of
Pharmaceutical Sciences and Department of Pharmaceutics, School of
Pharmacy, University of Mississippi, MS, United States
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This work is dedicated to my wife Cathy, and my mentors Billy Martin,
Ed Cook, Bob Jeffcoat, and Ken Davis.
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List of Contributors............................................................................. ix
Foreword............................................................................................. xi
Preface.............................................................................................. xiii
Acknowledgments................................................................................ xv
Chapter 1 The Botany of Cannabis sativa L. ........................................1
Botanical Description ...........................................................................2
Chemical Constituents and Phenotypes of C. sativa L. ........................5
Cannabis Biosynthesis ..........................................................................6
Selection of Elite Clones for Plant Propagation....................................8
Plant Growth and Cultivation ..............................................................9
Indoor Cultivation.............................................................................. 15
Outdoor Cultivation ...........................................................................16
Harvesting and Processing.................................................................. 18
Conclusion.......................................................................................... 19
Chapter 2 Biosynthesis and Pharmacology of Phytocannabinoids
and Related Chemical Constituents .................................... 27
Phytocannabinoid Constituents in Cannabis ...................................... 27
Monoterpenoid, Sesquiterpenoid, and Diterpenoid Constituents
of Cannabis ........................................................................................31
Phenylpropanoid Constituents of Cannabis........................................32
Therapeutic Indications for Medicinal Cannabis
and Cannabis-Derived Dosage Formulations.....................................32
Pharmacological Effects of Cannabis Constituents.............................34
Chapter 3 Medical Cannabis Formulations .........................................43
Cannabis Inflorescence and Hashish...................................................43
Teas, Tinctures, Oils, and Extracts .....................................................44
Consumables....................................................................................... 47
Formulations for Parenteral Administration ...................................... 50
Smoking and Vaporizing ....................................................................53
Bioavailability for Enteral and Transmucosal Administration ...........55
Bioavailability for Other Parenteral Routes of Administration ..........57
References........................................................................................... 59
Chapter 4 Analytical Methods in Formulation Development and
Manufacturing ...................................................................63
General Considerations in Sample Preparation for Analytical
Characterization .................................................................................64
Direct Analysis of Cannabis Inflorescence and Its Extracts................65
Separation and Analysis of Cannabis Using Gas
Chromatography ................................................................................67
Separation and Analysis of Cannabis by Thin Layer, Liquid,
and Convergence Chromatography ....................................................68
Broad-Spectrum Chemical Profiling ...................................................68
Targeted Quantitative Analytical Approaches and
Compendial Methods .........................................................................72
References........................................................................................... 78
Chapter 5 Quality Control and Stability Assessment .......................... 83
Challenges in Quality Control and Safety of Cannabis
and Cannabis-Derived Drugs .............................................................83
Variability in Composition and Strength............................................ 84
Content and Labeling Inaccuracies and Violations ............................85
Foods and Pharmaceuticals ................................................................ 86
Best Practices and Quality Control.....................................................87
Release Testing and Characterization of Chemical Delivery ..............90
Stability Assessment ........................................................................... 91
Additional Considerations ..................................................................94
References........................................................................................... 97
Chapter 6 The Roles of Research and Regulation ............................. 101
From Herbal Medicines to Controlled Substances ........................... 101
Implications to Consumers ............................................................... 102
Implications to Suppliers .................................................................. 103
Implications to Researchers.............................................................. 104
Implications to Regulators................................................................ 107
References......................................................................................... 110
Chapter 7 The Future of Cannabinoid Therapeutics.......................... 111
References......................................................................................... 114
viii Contents
Suman Chandra
National Center for Natural Products Research, Research Institute of Pharmaceutical
Sciences, School of Pharmacy, University of Mississippi, MS, United States
Mahmoud A. ElSohly
National Center for Natural Products Research, Research Institute of Pharmaceutical
Sciences and Department of Pharmaceutics, School of Pharmacy, University of
Mississippi, MS, United States
Michelle Glass
Department of Pharmacology, University of Auckland, Auckland, New Zealand
Hemant Lata
National Center for Natural Products Research, Research Institute of Pharmaceutical
Sciences, School of Pharmacy, University of Mississippi, MS, United States
Raphael Mechoulam
Institute for Drug Research, The Hebrew University of Jerusalem, Jerusalem, Israel
Roger G. Pertwee
Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, United
Brian F. Thomas
Analytical Chemistry and Pharmaceutics, RTI International, Research Triangle Park,
NC, United States
Ryan G. Vandrey
Behavioral Biology Research Unit, Johns Hopkins University School of Medicine,
Baltimore, MD, United States
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Cannabis has been used for thousands of years for recreational,
medicinal, or religious purposes. However, the determination of the
chemical structures of its cannabinoids, terpenes, and many other con-
stituents, and of the pharmacological actions and possible therapeutic
uses of some of these compounds, began less than 100 years ago. This
book begins by describing the cultivation, harvesting, and botanical
classification of cannabis plants, and then goes on to specify how these
plants produce some of their chemical constituents. Subsequent chap-
ters focus on medical formulations of cannabis and cannabis-derived
drugs, on the routes of administration of these formulations, and on
analytical methods that are used in the formulation development and
for the quality control or stability assessment of cannabis constituents.
The penultimate chapter deals with regulatory and additional
formulation-related issues for medical cannabis and cannabinoids,
while the final chapter identifies ways in which analytical chemistry
will most likely contribute to the development of cannabinoid thera-
peutics in the future. This book provides much needed insights into the
important roles that analytical chemistry has already played and is
likely to continue to play in the development of cannabis and its con-
stituents as medicines.
Roger G. Pertwee MA, DPhil, DSc, HonFBPhS
Institute of Medical Sciences
University of Aberdeen
Aberdeen, Scotland, United Kingdom
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Pharmacology began with natural products and, over some years on
either side of 1900, evolved into a rigorous scientific discipline domi-
nated, at least in the West, by well-defined chemical entities, either
extracted and processed or synthesized. The two traditions evolved
together, each informing the other, the natural strain by long experi-
ence pointing the way toward how a drug development program might
be structured, the synthetic strain contributing molecular specificity,
with analytical chemistry a common element. The resultant contribu-
tion to modern medicine, with all its caveats and controversies, must
be accounted as one of the great advancements in science.
Natural products pharmacology is very much alive. However, that
naturalis one cause of the popular misconception that herbs are in
some way better or safer than pills. Though some herbal remedies do
appear to be safe and effective, the opposite is closer to the truth.
Cannabis is a good example. The number of parameters on which can-
nabis products can vary is enormous, from strain, growing conditions,
harvesting methods, and handling to storage and processing of the raw
material to combination with a wide variety of foods and other excipi-
ents in manufacturing to methods of administration (eating, smoking,
vaping,applying to mucous membranes). At every step, from plant-
ing through consumption, myriad influences can alter dose, absorption
rate, interactions among constituents, exposure to toxins, and a host of
other factors that can result in underdosing, overdosing, and various
types and levels of acute and chronic poisoning, not excepting an
increase in the probability of lung cancer. Even if quality were well
controlled, which on the whole is very much not the case, this
complexity means that governmental oversight of cannabis products
cannot be as close and complete as that for prescription and over-the-
counter pharmaceuticals. Caveat emptor.
Governments around the world are coming slowly to the conclusion
that, in the absence of draconian enforcement, and to a nontrivial
extent in its presence, people are going to use cannabis for medicine
and recreation. The Internet spreads knowledge of genetic sequencing,
metabolomics, proteomics, and other disciplines such that people are
going to manipulate cannabis, as they have long done by selective
breeding, to maximize its mental and physical effects and tailor the
quality of those effects. The present legal status in the United States
and elsewhere cannot stop these activities by amateurs, but it does
inhibit research by professionals to investigate the basic science of can-
nabis, and to use this information to better understand neurophysio-
logical function, develop new medicines for people and animals, and
find ways to deal with cannabis addiction. Tight control of marijuana
and inhibition of legal research has arguably led to another paradoxi-
cal effect: driving the chemistry underground, which has resulted in the
proliferation of new and more dangerous synthetic cannabinoids.
There needs to be more involvement by elements of the US Food and
Drug Administration rather than the Drug Enforcement Agency.
Clearly, the policy, regulatory, and research challenges that accom-
pany the study and understanding of cannabis are unique. Despite all
the issues, research continues, understanding of cannabis and its effects
is evolving, policies are in flux, and the literature is ever-changing. The
aim of this book is to provide the reader with a detailed understanding
of the analytical chemistry of cannabis and cannabinoids as the foun-
dation for quality, safety, and utility of cannabis-derived therapeutics,
and offer direction for future advancements.
xiv Preface
The authors thank RTI Press and RTI International for their support
of this project, as well as the continued support of RTI research on
cannabis over the years by the National Institute on Drug Abuse.
We appreciate the opportunity to work with the editorial and
production team at ElsevierKaty Morrissey, Amy Clark, Vijayaraj
Purushothaman, and the many who go unmentionedin bringing this
first volume in the series Emerging Issues in Analytical Chemistry
to fruition.
Chapter 1, The Botany of Cannabis sativa L.,was prepared col-
lectively by Dr Suman Chandra, Dr Hemant Lata, and Dr Mahmoud
A. ElSohly at the University of Mississippi, whose work was supported
in part with federal funds from the National Institute on Drug Abuse,
National Institutes of Health, Department of Health and Human
Services, USA, under contract No. N01DA-10-7773.
Thanks to Dayle G. Johnson of RTI International for the cover
We are especially indebted to Dr Gerald T. Pollard for his editorial
assistance. His attention to detail and overall project management are
greatly appreciated.
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The Botany of Cannabis sativa L.
Cannabis sativa L. is a widespread species in nature. It is found in
various habitats ranging from sea level to the temperate and alpine
foothills of the Himalayas, from where it was probably spread over the
last 10,000 years.
The age-old cultivation makes its original distribu-
tion difficult to pinpoint.
Cannabis has a long history of medicinal
use in the Middle East and Asia, with references as far back as the 6th
century BCE, and it was introduced in Western Europe as a medicine
in the early 19th century to treat epilepsy, tetanus, rheumatism,
migraine, asthma, trigeminal neuralgia, fatigue, and insomnia.
As a plant, it is valued for its hallucinogenic and medicinal prop-
erties, more recently being used for pain, glaucoma, nausea, asthma,
depression, insomnia, and neuralgia.
Derivatives are used in
and multiple sclerosis.
The pharmacology and
therapeutic efficacy of cannabis preparations and its main active
constituent Δ
-tetrahydrocannabinol (Δ
-THC) have been extensively
The other important cannabinoid constituent of
current interest is cannabidiol (CBD). There has been a significant
interest in CBD over the last few years because of its reported
activity as an antiepileptic agent, particularly its promise for the
treatment of intractable pediatric epilepsy.
Other than Δ
and CBD, tetrahydrocannabivarin (THCV), cannabinol (CBN), can-
nabigerol (CBG), and cannabichromene (CBC) are major isolates.
Fig. 1.1 shows chemical structures.
Cannabis is also one of the oldest sources of food and textile
Hemp grown for fiber was introduced in Western Asia and
Egypt and subsequently in Europe between 1000 and 2000 BCE.
Cultivation of hemp in Europe became widespread after 500 CE.
The crop was first brought to South America (Chile) in 1545, and to
North America (Port Royal, Acadia) in 1606.
Now its cultivation
is prohibited or highly regulated in the United States.
The Analytical Chemistry of Cannabis. DOI:
Copyright ©2016 Elsevier Inc. All rights reserved.
Table 1.1 describes the botanical nomenclature of C. sativa L. Cannabis
is a highly variable species in terms of botany, genetics, and chemical
constituents. The number of species in the Cannabis genus has long
been controversial. Some reports proposed Cannabis as a polytypic
However, based on morphological, anatomical, phyto-
chemical, and genetic studies, it is generally treated as having a single,
highly polymorphic species, C. sativa L.
Other reported species
Figure 1.1 Chemical structures of major cannabinoids present in Cannabis sativa.Δ
nabinol; THCV, tetrahydrocannabivarin; CBN, cannabinol; CBG, cannabigerol; CBC, cannabichromene; CBD,
2The Analytical Chemistry of Cannabis
are Cannabis indica Lam. and Cannabis ruderalis Janisch, but plants
considered to have belonged to these species are now recognized as
varieties of C. sativa L. (var. indica and var. ruderalis, respectively).
Cannabis sativa and indica are widely cultivated and economically
important; Cannabis ruderalis is hardier and grows in the northern
Himalayas and the southern states of the former Soviet Union but is
rarely cultivated for drug content.
The main morphological difference between indica and sativa is in
their leaves. The leaves of sativa are much smaller and thinner,
whereas those of indica have wide fingers and are deep green, often
tinged with purple; at maturity, they turn dark purple. Indica plants
are shorter and bushier, usually under 6 ft tall and rarely over 8 ft
Indica has short branches laden with thick, dense buds, which mature
early, usually at the beginning of September in the Northern
Hemisphere. Indica buds also vary in color from dark green to purple,
with cooler conditions inducing more intense coloration. Indica flowers
earlier. The natural distribution of indica is Afghanistan, Pakistan,
India, and surrounding areas. The plants of sativa have long branches,
with the lower ones spreading 4 ft or more from the central stalk, as
on a conical Christmas tree. Height varies from 6 ft to more than 20 ft,
with 812 ft being the most common. Buds are long and thin and far
less densely populated than in indica, but longer, sometimes 3 ft or
more. Maturation time varies considerably depending on the variety
and environmental conditions. Low Δ
-THC Midwestern sativa varie-
ties (ditchweed) mature in August and September, while equatorial
Table 1.1 Botanical Nomenclature of Cannabis sativa L.
Category Botanical Nomenclature
Kingdom PlantaePlants
Subkingdom TracheobiontaVascular plants
Superdivision SpermatophytaSeed plants
Division MagnoliophytaFlowering plants
Class MagnoliopsidaDicotyledons
Subclass Hamamelididae
Order Urticales
Family Cannabaceae
Genus Cannabis
Species Cannabis sativa L.
3The Botany of Cannabis sativa L.
varieties mature from October to December. Buds of sativa require
intense light to thicken and swell; indica does not. Sativa tends to be
higher in Δ
-THC and lower in CBD than indica.Sativa is found
all over the world and comprises most of the drug type equatorial
varieties such as Colombian, Mexican, Nigerian, and South African,
where marijuana plants can be very potent. Cannabis has many local
common names, some of which are given in Table 1.2.
Normally, cannabis exhibits a dioecious (male and female flowers
develop on separate plants) and occasionally a monoecious (hermaphro-
dite) phenotype. It flowers in the shorter days (below 12-h photoperiod)
and continues growing vegetatively in the longer photoperiod. Sex is
determined by heteromorphic chromosomes (males being heterogametic
XY, females homogametic XX). Male flowers can be differentiated from
female by their different morphological appearance. At the vegetative
stage, differentiation is difficult because of morphological similarities.
Molecular techniques, however, can differentiate at an early stage.
Cannabis is wind pollinated. For the production of cannabinoids (or
phytocannabinoids), female plants are preferred for several reasons.
First, they produce higher amounts of cannabinoids. Second, once
pollinated, female plants produce seeds at maturity, whereas seed-free
Table 1.2 Common Cannabis Names in Different Languages
Language Common Names
Arabic Bhang, hashish qinnib, hasheesh kenneb, qinnib, tîl
Chinese Xian ma, ye ma
Danish Hemp
Dutch Hennep
English Hemp, marihuana
Finnish Hamppu
French Chanvre, chanvre dInde, chanvre indien, chanvrier
German Hanf, haschisch, indischer hanf
Hindi Bhang, charas, ganja
Japanese Mashinin
Nepalese Charas, gajiimaa, gaanjaa
Portuguese Cânhamo, maconha
Russian Kannabis sativa
Spanish Cáñamo, grifa, hachís, mariguana, marijuana
Swedish Porkanchaa
4The Analytical Chemistry of Cannabis
plants (sinsemilla, a Spanish word) are preferred for their higher yield of
secondary metabolites. Third, if several cannabis varieties are being
grown together, cross-pollination would affect the quality (chemical
profile) of the final product. To avoid this, removing male plants as they
appear, screening female clones for higher metabolite content, and
conservation and multiplication using biotechnological tools ensures the
consistency in chemical profile that is desirable for pharmaceuticals.
CBN was the first cannabinoid to be isolated
and identified
from C. sativa. The elucidation of CBN led to speculation that the
psychotropically active constituents of cannabis could be THCs. The
nonpsychotropic compound CBD was subsequently isolated from
Mexican marijuana
and the structure was determined.
Gaoni and
Mechoulam, two pioneers of cannabis research, determined the struc-
ture of Δ
-THC after finally succeeding in isolating and purifying this
elusive compound (see Mechoulam Close-up: How to Pamper an
Since then, the number of cannabinoids and other compounds
isolated from cannabis has increased continually, with 545 now
reported. Of these, 104 are phytocannabinoids (Table 1.3). From the
isolation and structural elucidation of Δ
-THC in 1964 until 1980,
Table 1.3 Constituents of Cannabis sativa L.
No. Groups Number of Known Compounds
1 CBG type 17
2 CBC type 8
3 CBD type 8
-THC type 18
-THC type 2
6 CBL type 3
7 CBE type 5
8 CBN type 10
9 CBND type 2
10 CBT type 9
11 Miscellaneous 22
12 Total cannabinoids 104
13 Total noncannabinoids 441
Total 545
5The Botany of Cannabis sativa L.
61 phytocannabinoids were isolated and reported.
Only nine new
ones were characterized between 1981 and 2005,
but 31 were
reported between 2006 and 2010. The 13 chemical constituent type
groups shown in Table 1.3 suggests the chemical complexity of the
cannabis plant.
The concentration of Δ
-THC and CBD, the most abundant canna-
binoids, can be characterized qualitatively and quantitatively.
Qualitative characterization is based on the Δ
-THC/CBD ratio and
assigning the plant to a discrete chemical phenotype (chemotype). In
1971, cannabis was initially characterized in two phenotypes, drug
type and fiber type, by Fetterman et al.
-THC/CBD ratio .1
was drug type, a lesser ratio was fiber type. In 1973, Small and
Beckstead proposed three categories based on the ratio: drug type if
.1, intermediate if close to 1, and fiber if ,1.
In 1987, Fournier
et al. added a rare chemotype that was characterized by a very low
content of Δ
-THC and CBD with CBG as the predominant
Quantitatively, the plant is characterized by potency through mea-
suring the level of its most abundant cannabinoids, Δ
-THC and CBD,
in its tissues (Fig. 1.2). The levels of cannabinoids are controlled by
the interaction of several genes and also influenced by the growth
environment of the plant.
Numerous biotic and abiotic factors
affect cannabinoid production, including the sex, growth stage, environ-
mental parameters, and fertilization.
Fig. 1.3 is a schematic of cannabinoid biosynthesis. In the plant,
-THC, CBD, and CBC are in their acid forms.
Two indepen-
dent pathways, cytosolic mevalonate and plastidial methylerythritol
phosphate (MEP), are responsible for terpenoid biosynthesis. The
MEP pathway is reported to be responsible for the biosynthesis of the
terpenoid moiety.
Olivetolic acid (OLA) and geranyl diphosphate
(GPP) are derived from the polyketide and the deoxyxylulose phos-
phate (DOXP)/MEP pathways, respectively, followed by condensation
under the influence of the prenylase olivetolate geranyltransferase,
yielding cannabigerolic acid (CBGA). CBGA, in turn, is oxidocyclized
6The Analytical Chemistry of Cannabis
by flavin adenine dinucleotide-dependent oxidases, namely, cannabi-
chromenic acid (CBCA) synthase, cannabidiolic acid (CBDA)
synthase, and Δ
-THCA synthase, producing CBCA, CBDA, and Δ
THCA, respectively.
THCV (6.990)
CBD (7.865)
CBD (7.899)
CBC (7.974)
THCV (6.983)
CBC (7.973)
D-8 THC (8.298)
D-8 THC (8.433)
D-9 THC (8.634)
D-9 THC (8.685)
Internal standard (9.474)
Internal standard (9.478)
CBG (9.110)
CBN (9.175)
CBG (9.109)
CBN (9.182)
6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
7.0 7.5 8.0 8.5 9.0 9.5 10.0
Figure 1.2 Gas chromatography-flame ionization detector (GC-FID) analysis of (A) a high CBD type and
(B) a high Δ
-THC type cannabis plant.
7The Botany of Cannabis sativa L.
The quality, safety, and efficacy of starting material are basic prerequi-
sites in the pharmaceutical industry. Cannabis as a feedstock is more
challenging because it is a chemically complex and highly variable
plant due to its allogamous nature. The chemical composition of can-
nabis biomass is affected by a range of factors such as genetics, envi-
ronment, growth conditions, and harvesting stage. Therefore, selection
of elite starting material (female clone) based on chemical composition,
conservation, and mass multiplication using advanced biotechnological
tools is a suitable way to ensure the consistency in chemical profile of
a crop for pharmaceuticals.
In our laboratory, we developed a GC-FID method for screening
and selection of elite biomass based on major cannabinoid content.
Geranyl pyrophosphate Olivetolic acid
Cannabigerolic acid (CBGA)
Tetrahydrocannabinolic acid
Cannabidiolic acid
Cannabichromenic acid
acid (THCA)
acid (CBDA)
acid (CBCA)
Figure 1.3 Biosynthetic pathway of tetrahydrocannabinolic acid, cannabidiolic acid, and cannabichromenic acid.
8The Analytical Chemistry of Cannabis
Briefly, quantitative analysis of seven major cannabinoids (THCV,
-THC, CBG, and CBN) is done by solvent
extraction followed by analysis using capillary gas chromatography, a
method offering short analysis time and resolution of all cannabinoids
on a single column. Three samples (100 mg each) are used for analysis
from each manicured biomass sample. A 3-mL internal standard (IS)
extraction solvent (100 mg of 4-androstene-3,17-dione 110 mL
chloroform 190 mL methanol) is added to the sample and allowed to
rest at room temperature for 1 h. The extract is then filtered through
a cotton plug and the clear filtered material is transferred to an
autosampler vial. Samples are placed onto the GC instrument along
with vials of ethanol, internal standard/Δ
-THC mixture (unextracted
standard), and controls. The results are calculated by obtaining an
average percentage of each cannabinoid from the two chromatograms
of each sample. It must be noted that the response factor for the can-
nabinoids relative to IS is 1. Therefore, the area of each cannabinoid
divided by that of the IS multiplied by the amount of IS added (3 mg)
gives the percentage of each cannabinoid in the sample, as 100 mg of
sample is used for analysis. For example, a cannabinoid with the
same peak area as that of the IS represents a 3% concentration in the
sample. The method has been validated to meet FDA-GMP
Once a female cannabis plant is screened and selected based on its
cannabinoids profile, it can be used as a mother stalk for future
Cannabis is an annual species and can be grown from seed or vegeta-
tive cuttings under indoor and outdoor conditions. Indoor cultivation
under controlled environmental conditions can generate three or four
crops per year, depending upon required per-plant biomass yield;
outdoor cultivation is limited to one crop per year. Selection of starting
material or variety depends upon the composition of active ingredients
required in the end product.
Propagation Through Seed
Fig. 1.4 shows the typical seeds of a high-Δ
-THC-yielding Mexican
variety of C. sativa L. Cultivation through seed is an ancient and
9The Botany of Cannabis sativa L.
traditional method. Seeds can be sown in small biodegradable jiffy
pots containing soil with good aeration and should be kept moist by
watering with a light spray when the upper surface begins to feel dry.
During winter, a heat mat can be used below the pots to increase the
soil temperature and enhance germination.
Normally seeds start sprouting by the fourth day of plantation, and
most of the viable seeds germinate within 15 days. Variation in the rate
of seed germination depends on the variety, seed age, storage conditions,
and soil and water temperatures. Germinated seedlings can be kept under
cool fluorescent light with an 18-h photoperiod until the seedlings are big
enough to transplant to bigger pots. Once transplanted, they can be kept
under full-spectrum grow light (1000 W high-pressure sodium or metal-
halide bulbs) with an 18-h photoperiod for further vegetative growth.
Upon completion of desired vegetative growth, plants may be
exposed to a 12-h photoperiod for flowering. (At this stage, cuttings of
selected healthy plants can be made and maintained at vegetative stage
for screening purposes.) Onset of flowering normally occurs in 2 weeks,
depending upon variety. Being a dioceous species, seed raised plants nor-
mally turn 50% male and 50% female, depending on the variety. Onset
of male flowers normally occurs a week earlier than female flowers.
For the production of useful secondary metabolites, female plants are
preferred as they produce higher cannabinoid content. At this early
flowering stage, male plants can be identified and separated from female
Figure 1.4 Cannabis sativa seeds (high-yielding Mexican variety).
10 The Analytical Chemistry of Cannabis
plants. Once all the male plants are removed, female plants can be
grown to full maturity for the production of sinsemilla (seedless) buds.
Mature buds can be analyzed for cannabinoid content using GC-FID.
Based on this analysis, elite high-yielding clones can be identified and
their vegetative backup cuttings can be used as mother plants for future
Vegetative Propagation in Soil
The pharmaceutical industry requires consistency in the active ingredi-
ents of source material. Using cannabis as a source raw material
remains especially challenging. In spite of being grown from seeds
derived from a single cannabis mother plant, a considerable degree of
variation in chemical composition of juvenile plants may be observed.
Therefore, screening of high-yielding female plants and mass propaga-
tion of vegetative clones is the most suitable way to meet the demand
for uniformity of the final product.
Once a best candidate female clone with a specific chemical profile
is screened and selected, a fresh nodal segment about 6 to 10 cm long
containing at least two nodes can be used for vegetative propagation.
A soft apical branch is cut at a 45-degree angle just below a node
and immediately dipped in distilled water. The base of the cutting is
subsequently dipped in rooting hormone and planted in biodegradable
jiffy pots (2 32 in) containing coco natural growth medium and a
mixture (1:1) of sterile potting mix and fertilome. At least one node is
covered by soil for efficient rooting. Plants are regularly irrigated and
kept under controlled environmental conditions. Rooting initiates in
2 to 3 weeks, followed by transplantation to bigger pots after 5 to 6
weeks. The cuttings can be maintained at constant vegetative state
under 18-h photoperiod (Fig. 1.5).
Vegetative Propagation in Hydroponics
Vegetative cuttings can also be grown in a hydroponics system.
A small branch consisting of a growing tip with two or three leaves is
cut and immediately dipped in distilled water. The base of the cutting
is dipped in rooting hormone and inserted (B1 in) deep into a rock-
wool cube or a hydrotone clay ball supporting medium. Plants are
supplied with vegetative fertilizer formula and exposed to fluorescent
light under 18-h photoperiod. Rooting initiates in 2 to 3 weeks.
11The Botany of Cannabis sativa L.
Figure 1.5 Indoor vegetative propagation of Cannabis sativa. (A) Vegetative cuttings under fluorescent lights,
(B) plant growing under full-spectrum metal-halide lamps.
12 The Analytical Chemistry of Cannabis
In Vitro Plant Regeneration
Tissue culture methods offer an alternative means of vegetative
propagation. Clonal propagation through tissue culture, commonly
called micropropagation, can be achieved in a short time and a
small space. It is possible to produce plants in large numbers
starting from a single clone. However, the process involves several
stages, from initiation and establishment of aseptic cultures to
multiplication, rooting of regenerated shoots, and hardening in soil.
Direct organogenesis is the most reliable method for clonal propa-
gation because it upholds genetic uniformity among progenies.
An efficient micropropagation protocol for mass growing of drug
type varieties using apical nodal segments containing axillary
(Fig. 1.6), as well as the micropropagation of a hemp
variety using shoot tips,
have been reported. Recently, our group
developed an improved one-step micropropagation protocol using
Plant tissue culture is also considered the most efficient
technology for crop improvement by the production of somaclonal
Figure 1.6 Micropropagation of Cannabis sativa. (A) A representative mother plant, (B and C) fully rooted
cannabis plants, (D) micropropagated plants under the acclimatization condition, and (EG) well-established
micropropagated plants in soil.
13The Botany of Cannabis sativa L.
and gametoclonal variants. The callus-mediated cultures have
inheritable characteristics different from those of parent plants due
to the possibility of somaclonal variability,
which may lead to
the development of commercially important improved varieties.
Micropropagation through callus production has been reported,
including production of roots through cannabis calli,
shoot regeneration,
and high-frequency plant regeneration from
leaf tissuederived calli.
Quality Control of In Vitro Regenerated Plants
The sustainability of the regeneration systems depends upon the
maintenance of the genetic integrity of micropropagated plants.
Despite its potential, in vitro techniques are known to induce soma-
clonal variations. Further, the frequency of these variations varies
with the source of explants and their regeneration pattern, media
composition, and cultural conditions. Tissue cultureinduced varia-
tions can be determined at the morphological, cytological, bio-
chemical, and molecular levels with several techniques. At present,
molecular markers are powerful tools used in the analysis of genetic
fidelity of in vitro propagated plantlets. These are not influenced by
environmental factors and generate reliable and reproducible
In our laboratory, DNA-based intersimple sequence repeat (ISSR)
markers have been successfully used to monitor the genetic stability
of the micropropagated plants of C. sativa.
Fully mature in vitro
propagated plants were also analyzed for their chemical profile and
cannabinoid content, and compared with mother plants and vegeta-
tively grown plants from the same mother plant using GC-FID for
quality assurance.
Our results showed that micropropagated plants were highly
comparable to the mother plant and vegetatively grown plants in terms
of genetics, chemical profile, and cannabinoid content. These results
confirm the clonal fidelity of in vitro propagated plants and suggest
that the biochemical mechanism used to produce the micropropagated
plants does not affect genetics or metabolic content. So these protocols
can be used for mass propagation of true to type plants of C. sativa for
commercial pharmaceutical use.
14 The Analytical Chemistry of Cannabis
Conservation of Elite Germplasm
The conservation of plant genetic resources is vital for the maintenance
and improvement of existing gene pool and plant breeding programs.
In the last few decades, in situ conservation methods have played an
important role in the conservation of elite plant germplasm. We have
developed protocols for the conservation of elite C. sativa clones using
vegetative propagation, slow growth conservation techniques, and algi-
nate encapsulation.
Indoor cultivation under controlled environmental conditions allows
total control of the plant life cycle and the quality and quantity of
the biomass as starting material for the production of a desirable
cannabinoid profile for pharmaceutical use. Parameters such as light
(intensity and photoperiod), temperature, carbon dioxide level, air cir-
culation, irrigation, relative humidity, and plant nutrition are the most
important factors.
Light is a vital component for the photosynthesis in plants.
Suitable light quality, optimum light intensity, and desirable photope-
riod are important in the indoor cultivation of cannabis. Our study
shows that cannabis plants can use high photosynthetic photon flux
density (B1500 μmol/m
/s) for efficient gas and water vapor exchange
between leaves and the environment.
Different light sources can be
used: fluorescent (for cuttings), metal-halide bulbs, high-pressure
sodium lamps (for well-established plants), conventional bulbs, and
light emitting diodes. However, with indoor lighting, it is difficult
to match photosynthetically active radiation received in the bright
outdoor sunlight. An 18-h photoperiod is optimum for vegetative
growth; 12-h is recommended for the initiation of flowering.
Irrigation and Humidity
The amount of water and the frequency of watering vary with the
growth stage, size of the plants and pots, growth temperature, humidity,
and many other factors. During the early seedling or vegetative
stage, keeping the soil moist is recommended. Once the plants are
established, the top layer of soil must be allowed to dry out
before the plants are watered again. Overall, the soil should not be kept
15The Botany of Cannabis sativa L.
constantly wet and the plant should not be allowed to wilt. In general,
watering should be done based on the requirement of the plant
depending on its growth stage and the size of the container. Vegetative
cuttings require regular moisture on the leaves to maintain a high
humidity in its microclimate until the plants are well rooted. Humidity
around 75% is recommended during the juvenile stage and about 55%
to 60% during the active vegetative and flowering stages.
Depending upon the original growth habitat and the genetic
makeup, the temperature response of photosynthesis varies with the
cannabis variety. Growth temperature of 25Cto30
most varieties.
Level of Carbon Dioxide
Air circulation in the growing room is another important factor and
is necessary for indoor cultivation of healthy plants. An elevated CO
level enhances photosynthetic carbon assimilation and so may acceler-
ate growth and improve productivity. A doubling of CO
concentration has been reported to increase the crop yield by 30% or
more in experiments conducted under close environmental conditions
such as in greenhouses and growth chambers.
Doubling of CO
concentration (B750 μmol mol
) was reported to stimulate the rate of
photosynthesis and water use efficiency by 50% and 111%, respec-
tively, as compared to ambient CO
supplementing CO
and proper air circulation in the growing room are
recommended and will scale up the rate of photosynthesis and overall
Plant Nutrition
Cannabis requires a minimum amount of nitrogen and a high level of
phosphorus to promote early root growth. For vegetative growth, a
higher level of nitrogen is required. For flowering, more potassium and
phosphorous for the production of buds is needed.
Cannabis is an annual. In the natural environment, it flowers at the
end of summer (shortening days) irrespective of planting date or plant
age. Seed is set before the arrival of winter and plant dies, if not
16 The Analytical Chemistry of Cannabis
harvested. Outdoor planting normally starts during late March or early
April, depending on weather conditions, and could last into November
or early December for some varieties. Starting from seed, plants may
be raised in small jiffy pots and the selected healthy seedlings trans-
planted to the field (seeds also may be directly planted in the field).
Male flowers start appearing within 2 to 3 months, around the middle
of July, followed by female flowers. Male plants are generally removed
from the fields for reasons stated above. Vegetative propagation of
selected elite female clones and their field plantation is generally pre-
ferred over seedlings for the consistency in the chemical profile of
the end product. Similar to seedlings, propagation of cuttings can be
done in small jiffy pots and rooted cuttings then planted in the field
by hand or by automated planters. Fig. 1.7 shows a typical outdoor
cultivation through vegetative cuttings.
Throughout the growing season, a few randomly selected plants
from different plots are periodically analyzed for cannabinoid content.
We found that the amount of Δ
-THC increases with the age of the
plant, reaching the highest level at the budding stage and plateauing
before the onset of senescence. The maturity of the crop is determined
Figure 1.7 Outdoor cultivation of cannabis crop. (A and B) plants at vegetative stage, (C and D) plants at bud-
ding stage.
17The Botany of Cannabis sativa L.
visually and confirmed using GC-FID based on the Δ
-THC and other
cannabinoid content in samples collected at different growth stages.
Since the whole plant does not mature at the same time, mature upper
buds are harvested first and other branches are given more time to
achieve maturity.
Outdoor cultivation has a few advantages and disadvantages in
comparison to indoor. Field-cultivated plants are normally bigger
and have higher biomass. Growing in the natural environment does
not require the intensive investment in equipment and maintenance
that indoor does. The primary disadvantage is less control over grow-
ing conditions. The weather may be unfavorable for harvest when the
plants have reached maturity, or a thunderstorm may seriously
damage plants when they are ready to harvest. Outdoor plants need a
longer growing season than indoor.
Fig. 1.8 shows harvesting, drying, and processing of field-grown cannabis
Figure 1.8 Harvesting, drying, and processing cannabis biomass. (A) Harvesting mature plants, (B) drying
biomass, (C) dried cannabis buds, and (D) processed plant material in barrel.
18 The Analytical Chemistry of Cannabis
Determining the optimum harvesting stage is a critical step in cannabis
cultivation. Too early or too late can significantly affect the yield of
-THC. Periodical monitoring of Δ
-THC level allows harvesting
material with the desired content. Harvesting should be done in the
morning because Δ
-THC level peaks before noon and then gradually
declines. Within the plant, the mature top buds may be harvested first
and the others allowed time to mature.
Handling, Drying, Processing, and Storage
During harvest, drying, and processing, gloves are recommended.
If the biomass is being used as starting material for pharmaceuticals,
contact with the ground should be avoided. Dry and large leaves may
be removed from mature buds before drying (Fig. 1.8).
The drying facility is based on the size of cultivation. For large-
scale growing, a commercial tobacco drying barn (such as BulkTobac,
Gas-Fired Products, Inc., Charlotte, NC) can be used. For small
samples, a simple laboratory oven will suffice for overnight drying
at 40C.
When the material is dried, it can be hand manicured. Big leaves
should be separated from buds. The buds can be gently rubbed
through screens of different sizes to separate small stems and seeds
(if any) from the dried biomass. Automated machines designed for
plant processing can separate big stems and seeds from the useable
Storing Biomass
Properly dried and processed biomass can be stored in FDA-approved
sealed fiber drums containing polyethylene bags at 18Cto20
C for
the short term. For the long term, 210C in a freezer is recommended.
Stability of Δ
-THC and other cannabinoids in biomass and products
has been reviewed by several authors.
Cannabis is very special in the plant kingdom in that it belongs to a family
(Cannabaceae) with a single genus (Cannabis) with only one species (sativa)
that has many varieties. The plant is very rich in constituents, the most
specific of which are the cannabinoids that have not been reported in any
19The Botany of Cannabis sativa L.
other plant, and it has broad pharmacological properties with tremendous
medical potential in the treatment of epilepsy, spasticity, inflammation,
irritable bowel syndrome, pain, and other disorders. Methods for growing,
harvesting, processing, formulation, and use continue to evolve towards an
important position in the pharmacopeia.
Close-Up: How to Pamper an Idea
R. Mechoulam
Hebrew University, Institute for Drug Research, Jerusalem, Israel
A scientific idea has not only to be pursued, but also pamperedin par-
ticular, if it is not welcome.
In the early 1960s as a newly appointed junior scientist at the
Weizmann Institute in Rehovot, I was supposed to work with the head of
my department, but could also explore some ideas of my own. I was
interested in the chemistry and biological effects of natural products. I
looked for biologically active plants that had not been previously well
investigated. To my great surprise, I noted that the chemistry and hence
the various activities of Cannabis sativa, the hashish plant, were not well
Morphine and cocaine had been isolated in the 19th century, and the
availability of these alkaloids had made possible biochemical, pharmaco-
logical and clinical work with them. Why not cannabis? Actually its
chemistry and biology had been investigated. There were numerous publi-
cations in the 19th century in mostly obscure journals. More recently, in
the late 1930s and early 1940s, Roger Adams, a prominent US chemist,
and Lord Alexander Todd, a Nobel Prize winner, had looked at the
chemistry of cannabis, but apparently the active constituents had never
been isolated in pure form and no definite structures had been put
forward. The reasons may have been technical. We know today that the
cannabinoidsa term I coined some years laterare present in the plant
as a mixture of constituents with closely related chemical structures which
presumably could not be separated by the methods available then.
Later, legal obstacles made work on cannabis almost impossible,
particularly in North America and Europe. Cannabisan illicit entity
was not readily available to most scientists, and research with it was next
to impossible for academics, as few of them could follow effectively the
security regulations required. By the mid 1940s cannabis research had
effectively been eliminated.
I was not aware of the legal problems. And neither were the adminis-
tration of the Weizmann Institute and even the police! In early 1963,
through the administrative head of the Institute, I requested hashish for
20 The Analytical Chemistry of Cannabis
research from the police and was asked to come over to their store of
confiscated smuggled material in Tel Aviv. There I drank a cup of coffee
with the elderly person in charge, he told me how the police had caught
hashish smugglers from the Lebanon and I told him what we wanted to
explore. I received 5 kg of hashish in the form of 10 hashish soles,
signed a receipt and boarded a bus to the Weizmann Institute some 15
miles away. On the bus travelers commented on the pleasant smell of the
vegetablesI was carrying.
We had all actually broken the strict laws on illicit substances. The
Ministry of Health was supposed to have approved the research, the
police should not have given me such a dangerous substance and I was
essentially a criminal. But at the Ministry some of the bureaucrats in
charge were my ex-colleagues, and after I was severely scolded for break-
ing the laws, we drank together some more coffee and I got a properly
signed and stamped document. Living and doing research in a small
country where people working in related areas generally know each other
has at times its positive aspects.
The first thing my colleague Yuval Shvo and I did with the now legal
hashish was to reisolate cannabidiol (CBD)already isolated previously
by Adams and Toddand elucidate its structure. Not surprisingly, for
over 40 years after our work on CBD in 1963, very few scientists and
clinicians became interested in CBD, although we showed, together with
Brazilian colleagues, already in 1980, that it is a good, novel antiepileptic
drug and together with Israeli and British colleagues and friends pub-
lished, in the early years of the 21st century, that it is also a potent drug
in autoimmune diseases. Gratifyingly, today CBD is widely acclaimed as
a novel antiepileptic drug in children.
In 1964 Yehiel Gaoni, a recent PhD chemist from the Sorbonne,
joined the hashish group, and together we isolated for the first time the
psychoactive constituent of hashish, which we named Δ
nabinol (Δ
-THC). The chemical nomenclature rules demanded a change,
and today Δ
-THC is known as Δ
-THC. However, the academic admin-
istration of the Weizmann Institute were not happy with our research.
Why could not we work on more respectable scientific topics? With a
heavy heart in 1966 I moved to the supposedly more conservative
Hebrew University in Jerusalem, where, surprisingly, I had full support
for our work and where we continued to pamper cannabinoid ideas for
over 50 years.
Over the next decades many other cannabinoids were isolated, their
structures were elucidated and compounds were synthesized by our
group. We looked at the metabolism of the cannabinoids and we collabo-
rated with numerous biologists in exploring the various cannabinoid
21The Botany of Cannabis sativa L.
Anandamide and 2-AG were discovered by my group in the 1990s,
and, with colleagues in many countries, we found that the newly discov-
ered endocannabinoid system is involved in a large number of biological
reactions and clinical conditions. The pampered idea has become a scien-
tific adult.
Looking ahead. Shall we have endocannabinoid drugs soon? By
modification of the endocannabinoid system, possibly through epige-
netics, can we possibly treat clinical conditions in the future? As we have
previously speculated, is the subtle chemical disparity of the many dozens
of endocannabinoid-like compounds in the brain somehow involved
in the huge variability in personalityan area in psychology that is yet to
be fully understood?
What next?
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26 The Analytical Chemistry of Cannabis
Biosynthesis and Pharmacology of
Phytocannabinoids and Related
Chemical Constituents
In addition to nucleic acids, proteins, lipids, and carbohydrates, cannabis
produces a large number of additional constituents, or secondary
metabolites, including phytocannabinoids, terpenoids, and phenylpro-
While phytocannabinoids are often referred to as the
activeingredients in cannabis, these other chemical constituents
have a broad spectrum of pharmacological properties and can
contribute to the effects seen upon cannabis ingestion or combustion
and inhalation, and may also be contained within and contribute to
the activity of extracts, tinctures, and other cannabis formulations.
This overview of cannabis constituents will focus on the phytocanna-
binoids, terpenoids, and flavonoids that make up a large percentage
of the pharmacologically active ingredients of current or emerging
Phytocannabinoids are a structurally diverse class of naturally occur-
ring chemical constituents in the genus Cannabis (Cannabaceae). This
chemical classification is broadly based on their derivation from a com-
mon C21 precursor (cannabigerolic acid,
CBGA), or its C19 analog
(cannabigerovaric acid,
CBGVA), the predominate phytocannabinoid
precursors formed through the reaction of geranyl pyrophosphate with
olivetolic and divarinic acid, respectively (Fig. 2.1).
Enzymatic conversion of cannabigerolic and cannabidivaric acid
produces a wide variety of C21 terpenophenolics,
-tetrahydrocannabinol (Δ
-THC), cannabigerol (CBG),
cannabichromene (CBC), cannabicyclol (CBL), cannabidiol (CBD),
cannabinodiol (CBND), and cannabinol (CBN), and their C19
The Analytical Chemistry of Cannabis. DOI:
Copyright ©2016 Elsevier Inc. All rights reserved.
homologs Δ
-tetrahydrocannabivarin (Δ
-THCV), cannabivarin (CBV),
and cannabidivarin (CBDV). More than 100 phytocannabinoids across
11 chemical classes have been isolated and identified to date.
In the
growing Cannabis sativa plant, most of these cannabinoids are initially
formed as carboxylic acids (eg, Δ
Fatty acid synthesisCoenzyme A
3 X
Polyketide synthase
Polyketide cyclase Polyketide synthase
Polyketide cyclase
Olivetolic acidDivarinolic acid
Geranyl pyrophosphate
Cannabigerolic acid (CBGA)Cannabigerovarinic acid (CBGVA)
R=propyl - Cannabidivarinic acid (CBDVA)
R=pentyl - Cannabidiolic acid (CBDA)
R=propyl - Δ9-Tetrahydrocannabivarinic acid (THCVA)
R=pentyl - Δ9Tetrahydrocannabinolic acid (THCA)
R=propyl - Cannabichromevarinic acid (CBCVA)
R=pentyl - Cannabichromenic acid (CBCA)
THCA synthase
CBDA synthase
CBCA synthase
THCA synthase
CBDA synthase
CBCA synthase
Figure 2.1 Biosynthesis of phytocannabinoids.
28 The Analytical Chemistry of Cannabis
-THCVA) that are decarboxylated to their corresponding neutral
forms as a consequence of drying, heating, combustion, or aging
(Fig. 2.2). There are also different isomers of phytocannabinoids result-
ing from variations or isomerization in the position of the double bond
THCA - R1= pentyl, R2 or R3 = COOH
THC - R1= pentyl, R2 and R3 = H
THCVA - R1= propyl, R2 or R3 = COOH
THCV - R1= propyl, R2 and R3 = H
CBDA - R1= pentyl, R2 = COOH
CBD - R1= pentyl, R2 = H
CBDVA - R1= propyl, R2 = COOH
CBDV - R1= propyl, R2 = H
CBNA - R1= pentyl, R2 = COOH
CBN - R1= pentyl, R2 and R3 = H
CBNV - R1= propyl, R2 and R3 = H
CBCA - R1= pentyl, R2 or R3 = COOH
CBC - R1= pentyl, R2 and R3 = H
CBCVA - R1= propyl, R2 or R3 = COOH
CBCV - R1= propyl, R2 and R3 = H
CBGA - R1= pentyl, R2 = COOH
CBG - R1= pentyl, R2 = H
CBGVA - R1= propyl, R2 = COOH
CBGV - R1= propyl, R2 = H
CBLA - R1= pentyl, R2 or R3 = COOH
CBL - R1= pentyl, R2 and R3 = H
CBLV - R1= propyl, R2 and R3 = H
Δ9-Tetrahydrocannabinol (THC)
Δ9-Tetrahydrocannabivarin (THCV)
Cannabigerol (CBG)
Cannabigerovarin (CBGV)
CBND - R = pentyl
CBNDV - R= propyl
Cannabinodiol (CBND)
Cannabinodivarin (CBNDV)
Cannabidiol (CBD)
Cannabidivarin (CBDV)
Cannabinol (CBN)
Cannabivarin (CBNV)
Cannabicyclol (CBL)
Cannabicyclovarin (CBLV)
Cannabichromene (CBC)
Cannabichromevarin (CBCV)
Cannabielsoin-C5 (CBE-C5)
Cannabielsoin-C3 (CBE-C3)
CBEA-C5 - R1 = pentyl, R2 or R3 = COOH
CBE-C5 - R1 = pentyl, R2 = H
CBEA-C3 - R1 = propyl, R3 = COOH
CBE-C3 - R1 = propyl, R2 = H
Cannabitriol-C5 (CBT-C5)
Cannabitriol-C3 (CBE-C3)
(–)-(9R,10R)-trans-CBT-C5 - R= pentyl
(+)-(9S,10S)-trans-CBT-C5 - R = pentyl
(±)-(9R,10S/9S,10R)-cis-CBT-C5 - R = pentyl
(± )-(9R,10R/9S,10S)-cis-CBT-C3 - R = propyl
Figure 2.2 Primary phytocannabinoid constituents in cannabis.
29Biosynthesis and Pharmacology of Phytocannabinoids and Related Chemical Constituents
in the alicyclic carbon ring (eg, (2)-trans-Δ
-THC). It is important
to note that CBN is not formed biosynthetically, but is an oxidative
degradant of Δ
Regulation of cannabinoid content in each plant phenotype
(chemotype) has been proposed to involve genetic control of the
expression of a variety of synthetic enzymes by four independent loci.
Qualitatively, the cannabinoid chemotype is controlled by the variation
in expression of these phytocannabinoid synthetic enzymes, resulting in
progenies and populations that have discrete distributions of chemical
composition (ie, chemical ratios of phytocannabinoids, such as the
-THC/CBD ratio). Quantitatively, the phytocannabinoid content is
controlled by polygenic mechanisms, and is strongly influenced by
environmental factors, such that a Gaussian distribution of total
cannabinoid content is typically observed. In addition, the cannabinoid
content and profile changes over time as the plant grows, matures, and
Wild-type chemotypes can therefore differ between Δ
predominance and CBDA predominance in discrete populations,
but vary dramatically in total cannabinoid content, with clones of
both types reaching total cannabinoid content levels of up to 2530%
(w/w) of the dry and trimmed inflorescences. Spontaneous mutations
and selective breeding have produced unique chemotypes that
show CBGA-,
or Δ
-THCVA predominance,
as well as
cannabinoid-free chemotypes.
Selective breeding has produced hun-
dreds of strains that differ in appearance and chemical composition,
and patients and recreational users often prefer specific strains for
their purported ability to produce specific pharmacological effects. De
Meijer speculates that future breeding might produce novel terpeno-
phenolic compounds such as those with branched alkyl or aromatic
side chains, or chemotypes with increased ratios of currently minor
constituents such as methyl, butyl, or farnesyl cannabinoids.
The current variation in phytocannabinoid content across and
within chemotypes has important implications in medicinal cannabis
and cannabis-based formulations and dosing. This has become increas-
ingly apparent and can be recognized by the plethora of varieties of
cannabis being cultivated, manufactured, and marketed as dosing
formulations in the medicinal and recreational market. Similarly, the
nonphytocannabinoid composition of cannabis is receiving increasing
pharmacological attention, particularly terpenoids and flavonoids.
30 The Analytical Chemistry of Cannabis
Geranyl pyrophosphate is the precursor in the synthesis of the more
ubiquitous terpenoids (Fig. 2.3), leading to the formation of limonene
and other monoterpenoids in secretory cell plastids,
or coupling with
isopentenyl pyrophosphate in the cytoplasm to form farnesyl pyro-
phosphate, a key intermediate in the biosynthesis of sesquiterpenoids
and triterpenoids.
Addition of another isopentenyl group to farne-
syl pyrophosphate leads to geranylgeranyl pyrophosphate, which is the
precursor of the diterpenoids.
Some of the predominate volatile
monoterpenes found in cannabis are β-myrcene, (E)-β-ocimene, terpi-
nolene, limonene, and β-pinene.
β-caryophyllene, α-caryophyllene
Geranyl pyrophosphate
Isopentenyl pyrophosphate
Farnesyl pyrophosphate O
Isopentenyl pyrophosphate
Geranylgeranyl pyrophosphate
FPP synthase GGPP synthase
Figure 2.3 Biosynthesis of terpenoids.
31Biosynthesis and Pharmacology of Phytocannabinoids and Related Chemical Constituents
(humulene), longifolene, α-zingiberene, and β-cedrene are among the
major sesquiterpenes found in prototypical cannabis samples.
Phenylpropanoids are of interest for their diverse pharmacological and
industrial applications.
Their biosynthesis begins with phenylalanine,
derived from the shikimate pathway, which is converted by phenylala-
nine ammonia lyase into cinnamic acid (Fig. 2.4). After hydroxylation
of cinnamic acid by cinnamate-4-hydroxylase to form p-coumaric acid,
it is converted in p-coumaroyl CoA by addition of a CoA thioester
by a 4-coumarate:CoA ligase enzyme. This common high energy
intermediate is used in the biosynthesis of cell wall constituents (lignins),
pigments (flavonoids, antocians), and UV protectant and pest resistance
compounds (stilbenoids, flavonoids, isoflavonoids, coumarins, and
furanocoumarins). A key enzyme in the flavonoid biosynthesis pathway
is chalcone synthase (CHS), a protein in the superfamily of polyketide
synthase that includes stilbene synthase, phlorisovalerophenone
synthase, isobutyrophenone synthase (BUS), and olivetol synthase
activities that can be detected during the development and growth
of glandular trichomes on bracts of cannabis.
The activities of
polyketide synthases and the resulting biosynthesis of cannabinoids,
stilbenoids, and flavonoids in the plant are induced in response to a
wide range of stimuli such as UV light, pathogens, hormones, elicitors,
growth substances, and wounding. Some of the naturally occurring
flavonoid constituents are orientin, vitesin, luteolin-7-O-β-D-glucuronide,
and apigenin-7-O-β-D-glucuronide.
Cannabis and cannabis-derived dosage formulations such as hashish
have a long history of medicinal use. However, the intoxicating effects
and associated abuse liability, scheduling, and control efforts have
limited the number of methodologically rigorous clinical studies. The
most widely supported indications for herbal cannabis and cannabis-
derived medicines are nausea and vomiting in cancer chemotherapy,
anorexia and cachexia in HIV/AIDS, chronic and neuropathic pain,
and spasticity in multiple sclerosis and spinal cord injury.
A recent
review of the randomized clinical trials investigating cannabis and
32 The Analytical Chemistry of Cannabis
cannabinoid therapeutics provides some support for these indications
as well as for sleep disorders and Tourettes syndrome.
was one of the more frequently cited medical indications in the late
twentieth century; however, this indication has received diminished
Stilbene synthase
Phenylalanine ammonia lyase
Cinnamic acid
p-Coumaric acid
4-Coumarate: CoA ligase enzyme
p-Coumaroyl CoA
Stilbenes, dihydrostilbenes,
prenylated stilbenes, etc.
Chalcone synthase
Flavanones, flavanonols,
flavans, anthoxanthins,
anthocyanidins, isoflavanoids
Cannflavin B
Figure 2.4 Biosynthesis of phenylpropanoids.
33Biosynthesis and Pharmacology of Phytocannabinoids and Related Chemical Constituents
support from the medical community due to the success of surgical
approaches and the availability of drugs with greater efficacy.
Cannabis also has a long history of use to control epileptic seizures, the
subject of recent reviews.
The use of cannabis or cannabinoid-derived
drugs for seizures is not fully supported by the Institute of Medicine;
however, CBD has gained considerable attention and anecdotal support
as a treatment for Dravet Syndrome and other specific seizure disor-
Other indications being investigated are irritable bowel
and posttraumatic stress disorder.
The contribution of the various chemical constituents in cannabis to
its therapeutic and organoleptic effects varies because of several
factors, including their differing concentrations (content), chemical
properties (eg, stability, volatility), pharmacological actions (eg, recep-
tor affinities, efficacies), physicochemical parameters (eg, lipophilicity,
solubility), pharmacokinetics, and pharmacodynamics. The principal
acidic forms of phytocannabinoids produced by plant biosynthesis
have generally been considered devoid of psychoactivity in man and
laboratory animals.
-THCA, CBNA, and CBGA have previously
been reported to lack affinity at the CB1 receptor.
More recently,
however, Δ
-THCA showed antinausea and antiemetic effects in
animals, with greater potency than Δ
and the effect was
reversed by rimonabant, the CB1 receptor antagonist. Δ
-THCA was
recently reported to bind to CB1 and CB2 receptors with greater
affinity than Δ
-THCA has shown immunomodulatory
actions that are CB1- and CB2 receptorindependent.
Thus, further
studies of the pharmacological activity and mechanism of action of
-THCA are needed.
The primary psychoactive phytocannabinoid is often considered to
be Δ
-THC because of its rapid formation from Δ
-THCA through
decarboxylation during combustion of plant material resulting in its
high concentration in smoke during inhalation and its potency at
cannabinoid receptors at physiologically relevant concentrations. In
support of this hypothesis, radioligand binding studies have shown
that Δ
-THC binds to the CB1 receptor with high affinity (K
B50 nM), while CBN has an approximately 10-fold lower affinity,
and CBD and CBG have K
values estimated to be greater than
34 The Analytical Chemistry of Cannabis
500 nM.
This receptor binding affinity correlates to both the inhi-
bition of adenylate cyclase in vitro and the analgesic and psychoactiv-
ity of these compounds in vivo.
-THC also binds to the CB2
receptor with similar high affinity and inhibits adenylate cyclase.
However, it is important to note that Δ
-THC acts as a partial agonist
in GTP-γ-S assays
as compared to the effects seen with more
efficacious synthetic cannabinoid agonists such as CP-55940 and
Because it acts as a partial agonist at both CB1 and
CB2, it elicits a response that is strongly influenced by the tissue-
specific expression levels, their extent of constitutive signaling, and the
ongoing endogenous cannabinoid release or tone of the receptor sys-
In addition to its production of psychoactivity in man, Δ
produces a myriad of additional cannabinoid receptormediated phar-
macological effects in laboratory animals and man.
Several other phytocannabinoids bind to and modulate cannabi-
noid receptor function. Δ
-THC, for example, binds to CB1 and CB2
with affinities approximating those of Δ
-THC and acts as an
-THCV also binds to CB1 and CB2 with nanomolar
and acts as a cannabimimetic agonist. High doses of
-THCV have been reported to produce a psychoactive effect
characterized as mild intoxication in man
and catalepsy
in laboratory animals. However, Δ
-THCV can also
antagonize cannabinoid receptor agonists in CB1-expressing tissues in
a manner that is both tissue- and ligand-dependent.
this compound is reported to be capable of behaving either as a CB1
antagonist or, at higher doses, as a CB1 agonist in vivo. CBN can
also bind to CB1
and CB2, but does so with lower affinity than
-THC. It also fails to inhibit forskolin-stimulated cAMP increase at
doses up to 1 μM in transfected cell lines expressing CB1 or CB2
and produces only mild intoxication in man at high
intravenous doses.
Other than Δ
-THC, CBN, and
-THCV, no phytocannabinoids have been reported to activate CB1
or CB2 receptors with nanomolar or low micromolar potency.
Neither CBD nor CBDA is psychoactive in man,
nor does CBD
bind to the CB1
or CB2
receptor with high affinity. However,
Pertwee has reported that CBD displays high potency as an antagonist
in CB1- and CB2-expressing cells or tissues.
The phytocannabinoids in cannabis can act via a plethora of other
noncannabinoid receptormediated systems to produce a wide variety
35Biosynthesis and Pharmacology of Phytocannabinoids and Related Chemical Constituents
of additional pharmacological effects. As reviewed by Pertwee in 2010,
-THC and other phytocannabinoids have been shown to modulate
the activity of GPR-55
and many other receptors and enzyme
For instance, CBD, CBN, and Δ
-THC produce noncom-
petitive/allosteric interactions with μand δ
_opioid receptors,
-THC allosterically modulates glycine receptor activation at nano-
molar concentrations.
-THC and CBD inhibit Ca(V)3 channels at
pharmacologically relevant concentrations, and Δ
-THC but not CBD
may increase the amount of calcium entry following T-type channel
activation by stabilizing open states of the channel.
These phytocan-
nabinoids also interact with transient receptor potential (TRP)
channels and enzymes of the endocannabinoid system.
De Petrocellis
et al. showed that both CBDV and CBD activate and desensitize
transient receptor potential vanilloid 1 (TRPV1) channels, and that
CBC disrupts transport of the endocannabinoid anandamide in vitro.
CBG, CBGV, and Δ
-THCV stimulate and desensitize human
TRPV1, while Δ
-THC, CBC, CBD, and CBN are potent rat TRPA1
agonists and desensitizers. All of these cannabinoids except CBC and
CBN also potently activate and desensitize rat TRPV2.
in three models of seizure, cannabis-derived botanical drug sub-
stancesrich in CBDV and CBD exerted significant anticonvulsant
effects that were not mediated by the CB1 receptor and were of com-
parable efficacy with purified CBDV.
Whether the anticonvulsant
activity produced by certain phytocannabinoids and cannabis-derived
drug formulations in vivo is related to these effects on calcium channels
remains to be determined; however, these actions are indicative
of potential therapeutic utility in the treatment of neuronal hyperexcit-
ability. Both Δ
-THC and CBN induce a cannabinoid receptor
independent release of calcitonin gene-related peptide (CGRP) from
capsaicin-sensitive perivascular sensory nerves.
Thus, the antinocicep-
tive actions of phytocannabinoids may rely on the activation of
inhibitory cannabinoid receptors (CB1) in the peripheral and central
nervous systems, as well as on the activation of excitatory ionotropic
TRP channels coexpressed with CB1 in primary nociceptive neurons
that contain and release CGRP upon activation.
-THC can also
act as an agonist at the peroxisome proliferator-activated receptor,
whereas Δ
-THCV does not. Thus it appears that these highly lipo-
philic phytocannabinoids
can gain access to and modulate a variety
of non-CB1, non-CB2 G proteincoupled receptors, transmitter-gated
channels, ion channels, and/or nuclear receptors.
36 The Analytical Chemistry of Cannabis
Nonphytocannabinoid terpenoids in cannabis contribute to the
organoleptic properties of the plant, but can also modulate the
activity of cannabinoid receptors and contribute to a wide variety of
noncannabinoid receptormediated pharmacological effects.
example, the sesquiterpenoid β-caryophyllene has been shown to bind to
CB2 with nanomolar affinity.
Upon binding to CB2, it acts like a
prototypical CB2 agonist and inhibits adenylate cylcase, produces
intracellular calcium transients, and activates the mitogen-activated
protein kinase Erk1/2 and p38 pathways in primary human mono-
cytes. β-Caryophyllene reduces the carrageenan-induced inflammatory
response in wild-type mice but not in mice lacking CB2 receptors,
evidence that this terpenoid exerts cannabimimetic effects in vivo. In
addition to modulation of cannabinoid receptors, terpenoids and
phenylpropanoids in cannabis have potent antioxidant,
and antiinflammatory activity.
Several monoterpenic alcohols,
including geraniol, nerol, and citronellol, have been reported to be
promiscuous TRP modulators.
It has been hypothesized that the
broad spectrum and prolonged sensory inhibition produced by phyto-
cannabinoids and terpenoids may allow them to act synergistically
as therapeutics for allodynia, itch, and other types of pain involving
superficial sensory nerves and skin.
Russo has proposed therapeutic
synergies and interactions among phytocannabinoids, terpenoids, and
phenylpropanoids. If clinically proven, this increases the likelihood of an
extensive pipeline of new therapeutic products and cannabis-derived
botanical drug products.
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