Tetrahydrocannabinolic Acid Research Articles

Marijuana extracts possess the effects like the endocrine disrupting chemicals

The progesterone 17alpha-hydroxylase activity, which is one of the steroidogenic enzymes in rat testis microsomes, was significantly inhibited by crude marijuana extracts from Delta(9)-tetrahydrocannabinolic acid (THCA)- and cannabidiolic acid (CBDA)-strains. Delta(9)-Tetrahydrocannabinol, cannabidiol and cannabinol also inhibited the enzymatic activity with relatively higher concentration (100-1000 microM). Testosterone 6beta- and 16alpha-hydroxylase activities together with androstenedione formation from testosterone in rat liver microsomes were also significantly inhibited by the crude marijuana extracts and the cannabinoids. Crude marijuana extracts (1 and 10 microg/ml) of THCA strain stimulated the proliferation of MCF-7 cells, although the purified cannabinoids (THC, CBD and CBN) did not show significant effects, such as the extract at the concentration of 0.01-1000 nM. These results indicate that there are some metabolic interactions between cannabinoid and steroid metabolism and that the constituents showing estrogen-like activity exist in marijuana.

The Gene Controlling Marijuana Psychoactivity: MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF Δ1-TETRAHYDROCANNABINOLIC ACID SYNTHASE FROM CANNABIS SATIVA L.

Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.

Biosynthesis of cannabinoids

The biosynthesis of cannabinoids was studied in cut sprouts of Cannabis sativa by incorporation experiments using mixtures of unlabeled glucose and [1‐13C]glucose or [U‐13C6]glucose. 13C‐labeling patterns of cannabichromenic acid and tetrahydrocannabinolic acid were analyzed by quantitative NMR spectroscopy. 13C enrichments and coupling patterns show that the C10‐terpenoid moiety is biosynthesized entirely or predominantly (> 98%) via the recently discovered deoxyxylulose phosphate pathway. The phenolic moiety is generated by a polyketide‐type reaction sequence. The data support geranyl diphosphate and the polyketide, olivetolic acid, as specific intermediates in the biosynthesis of cannabinoids.

Biosynthesis of cannabinoids. Incorporation experiments with (13)C-labeled glucoses.

The biosynthesis of cannabinoids was studied in cut sprouts of Cannabis sativa by incorporation experiments using mixtures of unlabeled glucose and [1-(13)C]glucose or [U-(13)C(6)]glucose. (13)C-labeling patterns of cannabichromenic acid and tetrahydrocannabinolic acid were analyzed by quantitative NMR spectroscopy. (13)C enrichments and coupling patterns show that the C(10)-terpenoid moiety is biosynthesized entirely or predominantly (> 98%) via the recently discovered deoxyxylulose phosphate pathway. The phenolic moiety is generated by a polyketide-type reaction sequence. The data support geranyl diphosphate and the polyketide, olivetolic acid, as specific intermediates in the biosynthesis of cannabinoids.

Direct determination of naturally occurring biologically active compound–serum albumin conjugate by matrix‐assisted laser desorption/ionization mass spectrometry

Opium alkaloids were conjugated with bovine serum albumin (BSA) to give individual antigen conjugates which were analyzed using BSA as an internal standard by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. It became clear that 9 molecules of thebaine were conjugated in a thebaine–BSA conjugate. Codeine and morphine contents in individual conjugates were determined to be 12 and 6 molecules, respectively. Using a relative molecular mass of BSA, 11.5 molecules of forskolin conjugated with BSA. Tetrahydrocannabinolic acid (THCA)–carrier protein conjugates using β‐alanine or without β‐alanine as a linker were analyzed. From these results it became evident that the hapten molecules of THCA–β‐alanine–BSA, THCA–BSA, THCA–β‐alanine–human serum albumin (HSA) and THCA–HSA were 17.2, 12.7, 27.8 and 54.4, respectively. Crocin–HSA conjugates were synthesized by two methods using succinate and NaIO4 cleavage of sugar moiety. A broad molecular peak of crocin–HSA conjugate appeared at around m/z 98,342 resulting in 30 molecules of crocin‐hemisuccinate conjugated with HSA. The crocin–HSA synthesized via NaIO4 oxidation showed a sharp molecular peak around 69,504 compared to that of hemisuccinate preparation. The number of crocin combined with HSA was, however, smaller 3.1 molecules.Analysis of monoclonal antibody by MALDI mass spectrometry determined the exact molecular weight and their purities.

Cross-Reactivity of Various Tetrahydrocannabinol Metabolites with a Monoclonal Antibody against Tetrahydrocannabinolic Acid.

A monoclonal antibody (MAb-4A4) against Δ9-tetrahydrocannabinolic acid (THCA) was prepared and its cross-reactivity analyzed with various cannabinoids including tetrahydrocannabinol metabolites by competitive enzyme-linked immunoassay (ELISA). Δ9-THC (1-100μg/ml) could be determined by the ELISA. Many metabolites of Δ8-THC and Δ9-THC reacted with the antibody, and their cross-reactivities were 44-157% of Δ8-THC. However, the antibody did not recognize the lipophilic compounds cholesterol, testosterone, β-carotene, androstene-3, 17-dione or an endogenous cannabinoid, anandamide.

Monoclonal antibody against tetrahydrocannabinolic acid distinguishes Cannabis sativa samples from different plant species

The cross-reaction of anti-Δ 1-THCA MAb against other cannabinoids was very wide. However, other naturally occurring and synthetic phenolics including opium alkaloids did not react to the MAb. Using this ELISA, this paper reports application of the competitive ELISA for detection of marijuana samples. The ELISA described here was very sensitive to the ether extracts of marijuana samples when compared to those of other plants. The assay provided a sensitive method useful for the judge of marijuana samples.

Monoclonal antibodies against naturally occurring bioactive compounds.

The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 mug/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 mug/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-ginsenoside Rb1 MAbs were produced. New western blotting method of determination for ginsenosides was established. Ginsenosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO(4) solution followed by BSA, resulting in a ginsenoside-BSA conjugate. Immunostaining of ginsenosides was more sensitive compared to other staining. Immunostaining of ginsenosides in the fresh ginseng root was succeeded using anti-ginsenoside Rb1 (GRb1) MAb after blotting to PVDF membrane.

Development of ELISA-analysis methods for the quantification of bioactive natural products in plants, phytomedicines and in humans or similar

In the course of a program in developing new ELISA-methods for the quantification of bioactive natural products in plants, phytomedicines and animals in a μg and ng scale, monoclonal antibodies against various natural products of medicinal and analytical importance have been developed. The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) of mass spectrometry. A hybridoma secreting monoclonal antibodies (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl-forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 μg/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 μg/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against Δ(1)-tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-Δ(1)-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA method was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-solamargine MAbs were produced. A method of determination for solasodine glycosides by using TLC-immunostaining was established. Solasodine glycosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO(4) solution followed by BSA, resulting in a solasodine glycoside-BSA conjugate. Immunostaining of solasodine glycosides was more sensitive compared to other staining. Immunostaining of ginsenosides in fresh ginseng root was successful using anti-ginsenoside Rb1 (G-Rb1) MAb after blotting to PVDF membrane.

Determination of tetrahydrocannabinolic acid—carrier protein conjugate by matrix‐assisted laser desorption/ionization mass spectrometry and antibody formation

AbstractIn order to form the monoclonal antibody against tetrahydrocannabinolic acid (THCA), THCA was coupled with bovine serum albumin (BSA) or human serum albumin (HSA) using β‐alanine as a linker or without β‐alanine to produce individual antigen conjugates. The number of haptens contained in the antigen conjugates was determined by matrix‐assisted laser desorption ionization mass spectrometry using BSA or HSA as an internal standard. The formation of monoclonal antibody is also discussed.

A new chromatographic method for the isolation of (−)‐Δ9‐(trans)‐tetrahydrocannabinolic acid A

Abstract(−)‐Δ9‐(trans)‐Tetrahydrocannabinolic acid A (THCA‐A), which is the main cannabinoid in ripe plants and the biogenetk precursor of the psychoactive (−)‐Δ9‐(trans)‐tetrahydrocannabinol, has been isolated from Cannabis sativa L. After extraction with petroleum ether and subsequent fractionation using alkali and diethyl ether, purification was performed by medium pressure liquid chromatography to yield an amorphous >99% pure powder. THCA‐A was characterized by its chromatographic and spectroscopic data.

Comparison of Pharmacological Activity in Mice of Different Cannabis Extracts from CBDA and THCA Strains.

Comparison of pharmacological activity of two cannabis extracts from cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA) strains in mice was evaluated using catalepsy, hypothermia, prolongation of pentobarbital (PB)-induced sleep and locomotor activity as indices. ED50s of catalepsy of CBDA strain extract (CBDA-E) and THCA strain extract (THCA-E) were 41.0 and 20.1 mg/kg, i. v., respectively. The most effective cannabis extract inducing hypothermia was CBDA-E at a dose of 50mg/kg, i. v.. By treatments (25mg/kg, i. v.) with CBDA- and THCA-Es, PB-induced sleeping time was prolonged 4.1 and 1.8 fold, as compared with that of 1% Tween 80-saline control. In its effect on locomotive activity CBDA-E showed a tendency to decrease and THCA-E at a low dose (5 and 25mg/kg) a tendency to increase, and at a high dose (50mg/kg) to decrease horizontal activity. CBDA- and THCA-Es tested, except for a dose of 5mg/kg, significantly decreased vertical motor activity of mice. From these results, CBDA-E containing CBDA as a major component and THCA-E containing THCA as a major component were found to have different pharmacological activity.

Gas chromatography of cannabis constituents, including cannabinoid acids, with on-column alkylation and esterification

Neutral cannabis constituents and cannabinoid acids were gas chromatographed with on-column methylation of phenolic hydroxyl groups and esterification of carboxylic acid functions. Injection of cannabis extracts as solution in 2 M dimethylformamide dimethylacetal in pyridine gave quantitative or near-quantitative conversion of cannabinol and tetrahydrocannabinol into methyl ethers, while cannabidio gave a monomethyl and a dimethyl derivative. Also cannabidiolic acid, tetrahydrocannabinolic acid and the tetrahydrocannabinol 7-acid metabolite chromatographed well as methyl esters and ethers. The mass spectra of the methyl derivatives gave more pertinent structural information than those of the corresponding TMS derivatives. This sample derivatization procedure should conceivably prove useful for the analysis of other heat-labile biological samples.

The Decrease of Tetrahydrocannabinolic Acid (THCA) in Cannabis Leaves during Storage

Tetrahydrocannabinolic acid (THCA) in fresh cannabis leaves has been already known to convert to tetrahydrocannabinol (THC) by decarboxylation, and then to cannabinol (CBN) by further dehydrogenation. In the present study, THCA and THC in cannabis leaves under three different environmental conditions were determined by the combination of preparative thin-layer chromatography and gas-liquid chromatography. Over 50% of THCA in cannabis leaves under one kind of environmental condition was found to convert to THC in 5 days, while THCA was hardly converted to CBN or cannabinolic acid.

Phenotype of Cannabinoids obtained by Crossing Cannabidiolic Acid (CBDA)-Cannabis and Tetrahydrocannabinolic Acid (THCA)-Cannabis

Phenotype of Cannabinoids obtained by Crossing Cannabidiolic Acid (CBDA)-Cannabis and Tetrahydrocannabinolic Acid (THCA)-Cannabis

Cannabinoid Content of a Stand of Cannabis Grown Clandestinely in Nova Scotia

The cannabinoid content of a stand of Cannabis found growing in central Cape Breton is reported. The value of Δ9-tetrahydrocannabinol, 3.03% compares favourably with values quoted for plants originating from areas known for their high potency. The possible use of tetrahydrocannabinolic acid as an indicator of age is discussed and the effect of packaging shown to bear a drastic effect on the rate of decarboxylation. Finally, the possibility of relating a sample of Cannabis to a country of origin and to other samples as to source is discussed.

Biosynthesis of cannabinoid acids

Malonic acid, mevalonic acid, geraniol and nerol were incorporated into tetrahydrocannabinolic acid and cannabichromenic acid in Cannabis sativa. The pathway from cannabigerolic acid to tetrahydrocannabinolic acid via cannabidiolic acid was established by feeding labelled cannabinoid acids. Cannabichromenic acid was shown to be formed on a side pathway from cannabigerolic acid.

Distribution of tetrahydrocannabinolic acid in fresh wild cannabis

In Abhangigkeit der Entwicklungszeit wird in Wildhanfpflanzen die Verteilung des Tetrahydrocannabinols gaschromatographisch bestimmt.

Defining individual cannabinoid compounds of Cannabis sativa L. by the Rf index by the method of the thin layer chromatography

Aim. Modern researches identified a significant number of individual cannabinoid compounds: neutral – KBD, THC, KBN and acids – KBDK, THCC. Schultz et al., R. Mechoulam believe that cannabidiolic acid – KBDK has an advantage over the other cannabinoids in the nature. It is the main component of the cannabinoid substances. Defining acids in the sorts by the value of Rf by the method of the thin layer chromatography (TLC), with the absence of "witnesses" for them is of interest. Methods. TLC method was applied to the samples (leaves, perianth, buds) of the domestic and French varieties of hemp. KBD, THC and KBN were defined by "witnesses" – KBDK and THCC by the value of Rf. Results. By the results of the chromatographic analysis, it was found that the compound with the value 0.059–0.060 of Rf was related to cannabidiolic acid while compound with the value 0.131 of Rf belonged to tetrahydrocannabinolic acid. In the chromatogram, the defined acids occupied the lowest border by the distribution front of the mobile phase. Conclusions. The study showed that during the plant vegetative growth and development only cannabidiolic acid was biosynthesised: Rf – 0.059–0.060; the French variety Futura 77 also had tetrahydrocannabinolic acid: Rf – 0.131. During the biological maturity of domestic hemp varieties seeds the neutral compounds were virtually absent. Biosynthesis of acids amplified. The French varieties demonstrated significant accumulation of both neutral cannabinoids and acids.Keywords: thin layer chromatography (TLC), individual cannabinoids compounds (KBN, KBD, THC), Rf value, cannabinoid acids (KBDK, THCC).

Psychopharmacological activity of the active constituents of hashish and some related cannabinoids.

1. The psychopharmacological activity of Δ1-tetrahydrocannabinol, (I); Δ1(6)-tetrahydrocannabinol (4′ hexyl), (II); Δ1(6)-tetrahydrocannabinol, (III); 1-ethoxyhexahydrocannabinol, (IV); 8-ethoxy-iso-hexahydrocannabinol, (V); Δ1(6)-tetrahydrocannabinolic acid Me ester, Isomer I, (VI); Δ1(6)-tetrahydrocannabinolic acid Me ester, Isomer II, (VII); cannabigerol, (VIII); Δ1(6)-tetrahydrocannabinol (3′ hexyl), (IX); cannabichromene, (X); has been examined in a variety of animal species. 2. Compounds (I) and (III) caused severe motor disturbances and a stuporous state in dogs and ptosis, “tameness” and peculiar postural changes in monkeys. In the latter animal, compound (II) elicited similar effects. 3. Compounds (I) and (III) after intraperitoneal but not subcutaneous administration, suppressed the gerbil digging activity; reduced the rat conditioned avoidance response and induced a cataleptoid reaction in mice, rats and gerbils. In addition, compound (I) reduced the performance of mice on the rotating-rod. Both compounds, administered subcutaneously, induced a measurable ataxic gait in rats. 4. Amphetamine reversed the behavioural changes elicited by compounds (I) and (III) in monkeys, as well as the cataleptoid reaction in rats. 5. None of the other compounds provoked observable changes in any of the species studied. 6. It is suggested that Rhesus monkeys might serve as a suitable model for assessing the psychopharmacological activity of active cannabinoids.