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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5515_Библиотеки_им_академика_М_И_Перельмана.pdf
The logic of the ABC model is both simple and stunning. But like anything in
nature, if you look hard enough you will find something that is an exception, or many
things that are exceptions. Meyerowitz and colleagues explained this phenomenon in
1991, when sequencing genes and studies to elucidate the molecular nature of genetic
systems were still in their infancy. The patterns got even more complicated when
flowers that aren’t part of the known model systems were examined. Novel patterns of
floral structure in these other kinds of flowers led to the need for more explanation, and
like any good model the ABC model could accommodate additional factors to explain
novel variation. In its current form, the more inclusive model is called the ABCDE
model, which includes two more controlling factors, D and E. The D and E factors are
important for telling the floral primordium to recognize that it will sit as the terminal
structure on a stem. Because the role of the D factor is species-dependent, the general
model has been shortened and is known as the ABCE model.
The need for yet another model is suggested because there are structures other than
sepal, petal, carpel, and stamen that make up flowers. The best model would not only be
able to explain floral development in Arabidopsis but also in other plants. The ABC
model accommodates four developmental fields (the four whorls), but what if the
definition of a flower is everything that is interior to and including a sepal? This would
include the ovules of the female part of the flower, which would require an explanation
for five developmental fields. And what about flowers that have strayed from the
typical sepal–petal–stamen–carpel structure? Indeed, some flowers do away with their
petals. They do not eliminate that outer whorl but replace it with something else. This is
where flowers such as lilies are relevant; they have modified their two outer whorls
into what are called tepals. Tepals share characteristics of both sepals and petals and
are found throughout the monocots—such as lilies and tulips. Lilies have the flower
structure of tepal– tepal–stamen–carpel.
As Goethe wrote, “With this model and the key to it, one will be able to invent
plants … which, even if they do not actually exist, nevertheless might exist.” Too bad he
didn’t make a deal with the devil to live to the end of the twentieth century. He would
have been quite impressed with himself.
2000 (Floral Development)
In chapter 5 we followed a cannabis plant through the 0000 and 1000 development
stages (according to Mediavilla and colleagues’ digital scheme). It is now time to take
it through the 2000 stage: the part of the plant’s development during which flowers
form. This stage is complicated, because cannabis has two sex systems (monoecy and
dioecy), which is why the 2000 level of digital development is subdivided into 2100
(dioecious male, or a plant that makes only male flowers), 2200 (dioecious female, or a
plant that makes only female flowers), and 2300 (monoecious, or a single plant that
makes both male and female flowers). In addition, some cannabis plants produce
flowers that are hermaphroditic, or have both male and female organs in the flower (fig.
6.5). By far the most instructive way to examine the formation of flowers of one species
is by comparing them to other closely related species. Fortunately, studies comparing
cannabis to its close relatives exist. Members of the genera Trema and Celtis have been
used to understand floral development in the Cannabaceae, and this has given
researchers a good idea of the intricacies of floral development in the family in general
and cannabis specifically.
The floral morphology of cannabis has a whorled architecture, just as discussed for
Arabidopsis. Since the flowers are mostly unisexual, however, we need to discern
between male (staminate) and female (pistillate) flowers. Because cannabis flowers
have lost their petals, they only have sepals as their nonreproductive floral organs. The
sepals are green and leaflike and protect the flower when in bud. As detailed in chapter
5, the onset of floral development is highly dependent on the photoperiod that the plant
experiences. Once the photoperiod is optimum, flowers will begin to develop. The left
side of figure 6.6 illustrates a staminate (male) flower.
Figure 6.5. Flower proliferation in female, male, true hermaphrodite, and mixed-gender cannabis plants
(from left to right). The flowers grow in the axial areas of diverging branches.
Note the composition of the male flower structure. The black crescent at the bottom
is a bract, which is a modified leaf that subtends the flower itself. In terms of the ABCE
model, the bract is simply a modified leaf that is “outside” and not part of the model.
The light gray crescents on the right and left are called prophylls and may be what is
considered the first field, while the medium gray crescents are sepals (together they are
called the calyx), making up the second developmental field. This whorl is followed by
the whorl of stamens. Since this is a diagram of a male flower, there is no whorl of
carpels at the center.
The sequence of events of the development of the staminate (male) flower is as
follows: The meristem of the developing flower is rounded; all the subsequent floral
structures will emanate from this meristem as the result of genetic signals and protein
interactions. The first tissues that will develop into sepals appear in a spiral whorl, and
the sepals begin to develop. The tissue that will eventually become the stamens appears
and develops in a spiral pattern too. At the same time the sepals elongate. The stamens
take up the central position of the flower, excluding the development of carpels and
ovule and completing the components of the male flower. Important structures known as
trichomes arise on the upper and lower sides of the bract and the calyx as the flower
develops. Chapter 9 discusses the trichomes in more detail, because these tissues are
important for producing the thousand or so compounds made by the cannabis plant.
Figure 6.6. Left: Diagram of a staminate cannabis flower. The sepals together form the calyx. Right:
Diagram of a pistillate cannabis flower. Adapted from Leme et al. (2020).
The pistillate (female) cannabis flower also has a subtending bract (fig. 6.6, right
side), but it grows laterally to nearly encircle the entire inner developmental fields. The
female flower lacks prophylls but has a fused calyx (or sepal whorl) that encloses the
carpels. Two carpels fuse to form the ovary. The sequence of events of the development
of the pistillate cannabis flower is as follows: The early floral meristem is domeshaped and protected by a large bract. The first sepal primordium appears, followed
closely by the appearance of the second sepal primordium, which appears to fuse with
the first to form a ring of sepal tissue around the base of the flower. At the center of the
still developing flower, the tissue that will become carpel elongates, and two carpels
form and fuse to make up the ovule. The carpels close at their tops, and the tips of the
carpels elongate to form the styles (the tubes that allow pollen access to the ovules).
The calyx ultimately forms as a cuplike structure that covers the ovary. The subtending
bract will develop large numbers of trichomes and enlarge to eventually enclose the
entire female flower.
It appears that cannabis flowers have the typical four- or five-whorled structure of
most flowers and could therefore fit the ABCE model nicely. However, in a 2020
review of cannabis genetics and development, plant geneticist Gianni Barcaccia and
colleagues pointed out a lack of information on how the ABCE model applies to floral
development in the Cannabis genus. This is more than likely due to the lack of formal
work on the genetics of Cannabis because of now-removed legal restrictions and the
difficulties of performing genetic experiments with cannabis plants. Their generation
times are long, and they are rather big plants (unlike the tiny Arabidopsis plants that can
be grown on a petri dish). But Barcaccia and his colleagues thought of a different way
to extend the ABCE model to cannabis. To explore whether the genes involved in floral
development might fit the ABCE model for cannabis, the group scanned cannabis
genome sequences to see if they could identify some of the same genes that are involved
in the floral development in Arabidopsis. They found almost all of the MADS box
transcription factors that facilitate the ABCE model in the cannabis genome.
The structures of the cannabis flower have clear affinities or homologies with other
flowers. It is also clear that cannabis has most of the genetic toolbox to participate in an
ABCE-like developmental dance. It is likely that Cannabis uses a similar genetic toolkit
to make its flowers, with modifications to the ABCE model that specifically address
traits inherent to cannabis, such as the loss of petals and the formation of unisexual
flowers. Further genetic research will, as Goethe wrote, reveal the “inner truth and
logic” of the cannabis flower—perhaps both aesthetically and commercially.
7
Decarboxylation
Cannabis ingestion has historical and cultural contexts that are part of the cannabis story. This book
does not endorse the use of cannabis, and the discussion that follows in this chapter does not
advocate cannabis ingestion. It is well known that many health issues and problems can arise. All
cannabis users should be aware that vaping, the use of water pipes or vaporizers, and other
methods of consuming cannabis can be harmful to human health. Furthermore, some methods of
ingestion, such as shared use of water pipes or other devices, can contribute to the spread of
infectious diseases and should be avoided.
The first beings who ingested cannabis probably ate it. And the chemicals that the
cannabis plant synthesizes are many and varied. The two that are most relevant to its
recreational and medicinal use are tetrahydrocannabinolic acid (THCA) and
cannabidiolic acid (CBDA). It is their derivative activated chemicals cannabidiol and
tetrahydrocannabinol (THC and CBD) that we need to ingest for cannabis to work its
wonders. In chapter 8 we will follow a THCA and a CBDA molecule synthesized by a
Cannabis plant on their journeys into and through the human body. But before taking that
journey, we need a little chemistry and then a bit of head-shop madness to get the THC
and CBD in proper form for their effects to work.
Light Up or Leave Me Alone
Figure 7.1 depicts the chemical structures for THCA and CBDA. There is one small
part of these molecules (called a carboxyl group) on which we need to focus at this
juncture. The rest of the structure is discussed in chapter 9.
To simplify these structures, I have placed a “black box” over most of both
molecules in figure 7.1. This black box is the same in both THCA and CBDA, so not
seeing what is obscured doesn’t matter. THCA and CBDA are inactive in their acid
forms, but by altering them to remove the carboxyl group (COOH), we get active THC
and CBD. Note that the carboxyl group is in the same place in both CBDA and THCA.
This similarity suggests that we can devise a way to alter this part of both molecules to
release the COOH.
Carboxyl groups are found all over the organic and biochemical world. One of the
most common kinds of carboxyl groups are those in amino acids, which have a carboxyl
end (COOH) and an amino end (NH3). These two kinds of ends in amino acids are
highly reactive, which is why amino acids can be strung together in long chains to make
proteins. The carboxyl and amino ends create strong bonds with each other.
Carboxyl groups have many functions in nature. For instance, by simply adding a
hydrogen component (H) to a carboxyl group we would get formic acid, a compound
made by several species of ant as a defense mechanism. By releasing formic acid,
which is noxious to other animals, the ant can fend off predators.
So how do we remove the carboxyl group to activate the THCA and CBDA? The
answer is complex and, of course, chemical. We need to yank the COOH group away
from the black boxes for both THCA and CBDA, which in chemical lingo means to
decarboxylate the THCA and CBDA. If we call our black box and the substance coming
off the lower left of the THCA black box RT, then THCA can be simplified to RTCOOH. If we do the same for CBDA (that is, call the black box and the substance
coming off the lower left of the CBDA black box RC), then CBDA can be simplified to
RC-COOH. The reactions are simply:
For generating THC = RT-COOH à RT-H + CO
2
For generating CBD = RC-COOH à RC-H + CO
2
Figure 7.1. Chemical structure of THCA (left) and CBDA (right), the two major psychoactive cannabinoids
made by Cannabis. The black boxes simplify the chemical structure, as it is the same in both THCA and
CBDA. The carboxyl groups (COOH) come off the black boxes in the upper right-hand corners.
These reactions lead to the active cannabinoids (RT-H = THC, and RC-H = CBD)
and carbon dioxide (CO2). There are, however, many ways to decarboxylate, as the
names of different decarboxylating reactions indicate: Barton decarboxylation, Kolbe
electrolysis, Kochi reaction, Krapcho decarboxylation, Tsuji—Trost reaction, and
Hunsdiecker reaction. There are also many different enzymes that can decarboxylate the
carboxyl groups. Which one would transform THCA and CBDA into THC and CBD,
respectively? None of the ones mentioned above! Instead, heating the plant material (the
buds) is the desired way to decarboxylate when Cannabis is ingested. But it is tricky:
too little heat will under-decarboxylate, and too much heat will destroy the active THC
and CBD.
The dynamics of heating cannabinoids have been studied by many chemists looking
for that sweet spot where there is maximal THC and CBD activation and minimal
breakdown to molecules other than THC and CBD. The technique of high-pressure
liquid chromatography (HPLC) is used to quantify THC activation, which is simply the
ratio of the amount of THC after activation to the amount of THCA and THC before
activation. In inactivated Cannabis buds there is a little bit of activated THC because of
the drying out of buds. But that is not enough to produce a psychoactive impact, and so
chemist Franz E. Dussy and colleagues attempted to determine the “sweet spot” for
conversion of THCA to THC using a typical titration experiment. First, they took a
dried bud, lopped off a small piece, and used HPLC to measure the amount of THCA
and THC in the original dried buds. They next heated the remaining cannabis bud to 120
degrees C (248 degrees F) and then took another part of the bud for HPLC analysis for
THC and THCA. The next step involved raising the temperature to 140 degrees C (284
degrees F), and again they took a part of the bud and measured the THC and THCA
content. They kept increasing the temperature up to 180 degrees C (365 degrees F). If
heat had nothing to do with the conversion, there should be the same ratio of THCA to
THC at each step of the experiment.
At 120 degrees C (248 degrees F), the conversion of THCA to THC was only 20
percent, but by 140 degrees C (284 degrees F), there was only a bit of the THCA left in
the bud material. By 160 degrees C (320 degrees F), all the THCA had been
decarboxylated, but two new molecules started to appear along with the activated THC.
These two molecules were cannabinol (CBN) and dihydroxy cannabinol (dihydroxyCBN). Both byproducts are not psychoactive and are for the most part unwanted when
THCA is decarboxylated. Heating to 180 degrees C (365 degrees F) merely increased
the amount of CBN and dihydroxy-CBN at the expense of activated THC.
The sweet spot thus lies somewhere between 140 and 160 degrees C (284 and 320
degrees F), but probably closer to 160 degrees C (320 degrees F). When someone lights
a marijuana cigarette with a burning match or a lighter, the cannabis that is exposed to
the fire burns at about 260 to 370 degrees C (500 to 700 degrees F). This flash of heat
will cause some of the THCA in the fire exposed part of the cigarette to be lost; in fact,
a good portion of the cannabinoids in that part of the cigarette will be shot, due to the
high temperature. But as one smokes the burning cannabis from a marijuana cigarette,
the air circulating through the burning material cools it down, so the sweet spot is only
roughly attained when smoking buds via rolled cigarettes.
The vapor points of THCA and CBDA are different; if we focus on obtaining THC,
then that temperature is not optimal for CBD. In fact, the vapor point of THCA is about
8 degrees C (14 degrees F) lower than for CBDA. Equally important are the vapor
points of THC and CBD, which are the temperatures at which these cannabinoids are
converted to nonactive forms. The difference is 10 degrees C (18 degrees F) lower for
THC than CBD. These temperature differences are the result of the different molecular
structure of the acids (THCA and CBDA) and the cannabinoids (THC and CBD). By
manipulating the decarboxylation temperatures one uses, different concentrations of
cannabinoids can be produced. Length of heating time is also involved, and lower
temperatures for longer times will do the same trick as higher temperatures for shorter
times. Basically, if you heat the buds near the vapor temperature of THCA (at about 157
degrees C, or 315 degrees F), you will convert THCA to THC, and the CBDA will be
left largely unchanged. This protocol will give you more of a psychoactive effect than a
medicinal or body effect. Raising the cooking temperature to 180 degrees C (355
degrees F) eliminates most but not all THC and optimizes the concentration of CBD,
delivering more of a body effect but a reduced psychoactive one. Raising the bake
temperature to 200 degrees C will eliminate the THC and convert more of the CBDA to
CBD, resulting in an almost complete body effect.
Pipes, Bongs, and Percolators
“Rolled cigarettes” sounds a bit nerdy and technical, so let’s add some color to the
discussion by calling it what it is: a joint, a bomb, a reefer, a spliff, a bifter, a blunt, a
bomber, a stick, a zol, a doobie, a doob, or a toke tube. But even with so many names,
there are better ways to deliver cannabis smoke to the lungs than via a spliff. These
means involve pipes of some sort. There are two reasons to go to pipes for inhaling
reefer; first, to cool the burning buds so that the decarboxylation of THCA and CBDA to
THC and CBD is better optimized, and second, to soften the harshness of the smoke.
Smoking pipes is an old practice. As with any long-established human behavior, it
is difficult to trace its origins. Some pipe experts suggest that copper pipe use occurred
as early as 2000 BCE in ancient Egypt. But the evidence for this conclusion comes from
hieroglyphs, and there is some controversy regarding whether the pipes were used in
religious ceremonies or as implements for smoking. If researchers want to claim
something is a smoking pipe, then they had better prove that there was tobacco or some
other compound in the pipe. As discussed in chapter 2, burnt plant material such as
tobacco or cannabis does leave an identifiable resin where the burning occurred. A
paper published in 2018 by paleoethnobotanist Stephen Carmody and colleagues
announced the discovery of the New World’s oldest pipe from a Native American
archaeological site in Alabama. The animal bones lying near the limestone-carved
smoking tube were radiocarbon-dated to between 1685 and 1530 BCE—about 3,700
years ago. Carmody and colleagues were also able to examine the resin in the so-called
medicine tube and found that it was dominated by tobacco remnants. This discovery
pushed the oldest known pipe back 1,000 years earlier than what was known previously
and has since been considered the oldest known pipe.
According to John Edward Philips, a pipe expert, African pipes date back at least
to the sixth century AD. Since this time was well before the introduction of tobacco to
Africa, it can be assumed that the users of these pipes were not smoking tobacco. There
is some controversy over what was being smoked in these pipes, and one researcher
(Jean-Paul Lebeuf) suggests that the plant material was from Datura metel, a close
relative to the North American jimson weed (Datura stramonium), which is poisonous.
Philips believes that it was more than likely cannabis that was being smoked in these
1,500-year-old pipes.
One of the most bizarre African pipes is a more modern one called an earth pipe. It
starts with a container of some sort, usually a bottle buried in the ground. A tube extends
from the bottle and serves as the mouthpiece. Lighted embers are placed in the bottle,
marijuana is loaded into the bottle, and the resulting smoke is inhaled through the mouth
tube. The earth surrounding the tube in the ground serves to cool the smoke as it is
inhaled from the buried bottle (fig. 7.2).
Other experts focus on the elegant pipe designs that were created after the
introduction of tobacco to Europe from the New World. Water pipes have a relatively
short history. Also known as bongs, they are a smoking device that forces the drawn
smoke through water to both control the temperature for efficient decarboxylation and to
cool the smoke for the lungs. The word “bong” comes from the Thai word baung, which
is a bamboo water pipe, but Thailand is not where the bong originated. Hookahs (also
called narghile, hubble-bubble, goza, argileh, and shisha) are other good models for
how water pipes work, but they are not the earliest ancestors. These water pipes were
used to smoke flavored tobacco; they were introduced in India and the Middle East
about 500 years ago and later refined in Turkey.
The origin of the bong probably dates back to Africa at about 1400 BCE, based on
the discovery of bong-like apparatuses in Ethiopia. Again, since this period was well
before the introduction of tobacco to Africa, these water pipes were probably used for
cannabis. There is a huge gap in the history of water pipes between the African
archaeological pipes (1400 BCE) and the first hookahs (AD 1500). A more recent
revolution in water pipes involved the use of glass and plastic in the making of the
pipes. This golden age of bongs also coincided with the expansion of recreational use
of cannabis, especially from the 1960s on.
Figure 7.2. African earth pipe. The subterranean duct cools the marijuana smoke, as does the sub- mersion
of the lighting chamber buried in the ground. Adapted from Philips (1983).