Evaluación de los resultados en términos de minería de datos

3.2.1 Biogenesis

Until the mid 1960’s the only plant cannabinoid whose structure was fully elucidated was cannabinol (CBN) — a constituent which actually may represent an oxidation artifact. However, on the basis of CBN, the main cannabinoid structure skeleton became known. Thus, cannabidiol (CBD),

which had been independently isolated in pure form by Adams22,23 and

by Todd,24 was correctly assumed to be, like CBN, a terpenoid deriva-

tive attached to olivetol. But its exact structure was not elucidated. The psychoactive components of cannabis were assumed to be related tricyclic derivatives. On the basis of the tentatively elucidated constituents, Todd25 suggested that the cannabinoids may be formed initially in the plant by condensation of a menthatriene with olivetol.

Gaoni and Mechoulam26 suggested that cannabigerol (CBG) origi-

nated in nature most probably by the condensation of geranyl pyrophos- phate with olivetol. In their following papers,27–29they expanded the bio- genetic scheme by suggesting subsequent conversions of CBG to CBD, tetrahydrocannabinol (THC) and finally to CBN.

Yamauchi et al.30 demonstrated that the neutral cannabinoids and

cannabinolic acid are artifacts produced from cannabinoid acids during the harvest and storage of cannabis.

Shoyama et al.31 made use of labeled calcium malonate-[2-14C];

sodium mevalonate-[2-14C]; geraniol-[1-3H]; nerol-[1-3H] and labeled

cannabigerolic acid (CBGA)-carboxyl-14C; CBGA-[U-3H] and CBDA-

carboxyl-14C to clarify the biogenesis. The plants were nourished with the labeled substances for the period of six days applied through a cotton wick or directly to the leaves and the production of radioactive cannabi- noids was followed. On the evidence of the identified substances, a defini-

tive biogenetic scheme could be formulated.32 In a later publication,

Shoyama et al.33reported that the cannabinoid acids with a n-propyl side chain, are biosynthesized along the same biogenetic pathway as the pentyl homologues.

In their next study, Shoyama and Nishioka34 isolated new spirocom- pounds: cannabispirol and acetyl cannabispirol. This is in addition to the already known cannabispirone and cannabispirenone from a Japanese hemp variety. The two scientists included them in their biogenetic schema alongside the cannabinoid acids. In a further study, Shoyama et al.35dealt with the biosynthesis of propylcannabinoid acids by in vitro incubation with raw enzyme solution from three species of Cannabis sativa KL. A bio- genetic schema is presented illustrating the relationship between methyl, propyl and pentyl cannabinoid acids.

Crombie et al.36isolated spiranes from Thai hemp and proposed the biogenesis of their origin. Turner and El-Sohly37isolated a series of polyoxi- dized cannabinoids as minor substances in cannabis.They modified the bio-

genetic scheme by assuming that∆9-THC is converted into CBN through

epoxy and hydroxylated cannabinoids. More recently, a new cannabinoid, cannabinerolic acid was isolated from the leaves of a Mexican strain of Cannabis sativa L.38 This cannabinoid acid is assumed to be involved in the biosynthesis of∆9-THCA. The same authors presented the first direct

experimental evidence for the mechanism of∆9-THCA biosynthesis from

CBGA through oxidocyclization by a new enzyme.39This∆9-THCA syn-

thase was the first isolated and identified enzyme involved in cannabinoid biogenesis.

The absence of CBD and CBDA was reported in Cannabis of South African origin.40,41In samples of cannabis from three regions of Mexico,42 likewise, no CBD was present. The absence of CBD and its acid, alongside the presence of THC and THC acid, indicated that the two types of com- pounds presumably originate by separate pathways. Indeed, Taura et al.43 identified a unique enzyme, cannabidiolic acid synthase, that catalyzes the oxidocyclization of CBGA (predominantly) as well as cannabinerolic acid

to CBDA. One year later, Morimoto et al.44 identified in young leaves

of Cannabis sativa, an enzyme, which is involved in the biosynthesis of cannabichromenic acid. Cannabichromenic acid synthase catalyze the oxi- docyclization of CBGA and cannabinerolic acid to cannabichromenic acid (predominantly from CBGA). As THCA and CBCA originate in the plant by two independent different pathways from CBGA (and not from

OH HO COOH O OH COOH O OH COOH

Cannabidiolic acid (CBDA) _∆9_{-Tetrahydrocannabinlic acid (∆}9_-THCA)

Cannabinolic acid (CBNA) HO

OH

Cannabigerolic acid (CBGA) C5H11

C5H11

C5H11

O C5H11

OH

Cannabicyclolic acid (CBLA)

O C5H11 OH Cannabichromenic acid HO C5H11 O COOH OH Cannabielsoic acid A COOH light O2 O2 COOH COOH OP2O63- Geranyl pyrophosphate + HO C5H11 Olivetolic acid COOH OH

Fig. 1. Proposed biogenesis of cannabinoid acids in the plant Cannabis sativa L.

CBDA, as was theoretically assumed previously) with the help of appro-

priate enzymes45 and because CBDA is synthesized in the plant by an

independent pathway, it is easy to explain why some cannabis strains do not contain CBD and CBDA.

The biogenesis of cannabinoid acids in Cannabis sativa L. based on experimentally acquired knowledge is presented on Fig. 1.

3.2.2 Cannabinoid Substances from Cannabis sativa L

The first successful attempt to identify a typical cannabis constituent (today called cannabinoid) was achieved by Wood et al.46, who isolated cannabinol (CBN) from the exuded resin of Indian hemp (“charas”), which analyzed for C21H26O2. Another, big step was made by Cahn47, who advanced

the elucidation of the structure of CBN (Cahn, 1932). Several years later, Todd’s group and Adam’s group elucidated the correct structure of CBN by synthesizing and comparing various possible structures.48–50

A second cannabis constituent, cannabidiol (CBD) was also isolated, but its structure was only partially clarified.51,52 Synthetic tetrahydro- cannabinols, which showed cannabis-like activity in animal tests, were prepared, but they obviously differed from the active natural product, on the basis of their UV spectrum.53–56

In a systematic study of the antibacterial substances in hemp, Krejèí and Šantavý found that an extract containing carboxylic acids was effective against Staphylococcus aureus and other gram positive micro organisms. They isolated cannabidiololic acid (CBDA)57,58 and partially elucidated the structure.The only problems remaining being the position of the double bond in monoterpene ring and the stereochemistry.

The advances in isolation methods made possible a clarification of the chemistry of cannabis. In 1963, our group reisolated CBD and reported its correct structure and stereochemistry.59A year later we finally succeeded in isolating pure∆9-tetrahydrocannabinol (∆9-THC), elucidated its struc- ture, obtained a crystalline derivative and achieved a partial synthesis from CBD.60Several years later, a minor psychotomimetically active constituent,

∆8_{-THC, was isolated from marijuana.}61_{Whether this THC isomer is a}

natural compound, or an artifact formed during the drying of the plant, remains an open problem.

The absolute configuration of CBD, and hence also of THC, was established by the analysis of the shift of optical rotation values62and fully by correlation with known terpenoids.63

Several additional, non-psychotropic cannabinoids were also identified at that time. The best known are cannabigerol (CBG),26cannabichromene

(CBC)64,65 and cannabicyclol.66 Cannabinolic acid (CBNA) and

HO OH

C₅H₁₁ R = H, Cannabidiol(CBD) R = COOH, Cannabidiolic acid (CBDA)

O OH

C5H11

R = H, D9_{-Tetrahydrocannabinol (D}9_-THC)

R = COOH,D9_{-Tetrahydrocannabinolic acid}

(D9_-THCA)

HO OH

C5H11

R = H, Cannabigerol (CBG) R = COOH, Cannabigerolic acid (CBGA) O C5H11 OH O C₅H₁₁ OH O OH C₅H₁₁ R = H, Cannabichromene (CBC) R = COOH, Cannabichromenic acid (CBCA)

R = H, Cannabicyclol (CBL) R = COOH Cannabicyclolic acid (CBLA)

R = H, Cannabinol (CBN) R = COOH, Cannabinolic acid (CBNA) R R R R R R

Fig. 2. The main cannabinoids in Cannabis sativa L.

∆9_{-THC acids, A and B,}30,67,68 _{as well as∆}8_{-THC acid}69 _{and canna-}

bielsoic acid.70The decarboxylated product of cannabielsoic acid, canna- bielsoin, is found in mammals as a metabolite of CBD.71The syntheses of some of the cannabinoid acids have been reported.72

It is possible, that some of the natural neutral cannabinoids (if not all of them) are artifacts formed by decarboxylation, photochemical cyclization (cannabicyclol), oxidation (cannabielsoic acid) or isomerization (∆8_-THC

and∆8-THC acid) of other constituents.

For the main representative cannabinoids see Fig. 2. 3.2.3 Non Cannabinoid Constituents of Cannabis sativa L

Like all plants, Cannabis sativa L. forms a huge number of chemicals. Many of these have been identified.73–77None of them, except the cannabinoids are specific for this plant and none of these non-cannabinoids has been found to contribute to the cannabinoid effects. However, very few have been directly tested together with cannabinoids for any biological effects. Recently, ElSohly and Slade,78 published a comprehensive review. Up to date, 70 natural cannabinoid compounds were documented. Althogether, they record 419 constituents: nitrogenous compounds (27), amino acids

(18), proteins (3), enzymes (6), glycoproteins (2), sugars and related com- pounds (34), hydrocarbons (50), simple alcohols (7), simple aldehydes (12), simple ketones (13), simple acids (20), fatty acids (23), simple esters (12), lactones (1), steroids (11), terpenes (120), non cannabinoid phenols (25), flavonoids (23), vitamins (1), pigments (2) and elements (9).

3.3 CANNABINOID RECEPTORS IN LIVING ORGANISMS