Tag: Cannabinoids

Metabolic fingerprinting of Cannabis sativa L. cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes

Fischedick JT, Hazekamp A, Erkelens T, Choi YH, Verpoorte R (2010)

Phytochemistry 71:(17-18) 2058–2073

doi: 10.1016/j.phytochem.2010.10.001

Cannabis sativa L. is an important medicinal plant. In order to develop cannabis plant material as a medicinal product quality control and clear chemotaxonomic discrimination between varieties is a necessity. Therefore in this study 11 cannabis varieties were grown under the same environmental conditions. Chemical analysis of cannabis plant material used a gas chromatography flame ionization detection method that was validated for quantitative analysis of cannabis monoterpenoids, sesquiterpenoids, and cannabinoids. Quantitative data was analyzed using principal component analysis to determine which compounds are most important in discriminating cannabis varieties. In total 36 compounds were identified and quantified in the 11 varieties. Using principal component analysis each cannabis variety could be chemically discriminated. This methodology is useful for both chemotaxonomic discrimination of cannabis varieties and quality control of plant material.
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Membrane associated antitumor effects of crocine-, ginsenoside- and cannabinoid derivates

Joseph Molnar, D Szabó, Rozalia Pusztai Yukihiro Shoyama

Anticancer research 20(2A):861-7 March 2000

PMID:  10810367

In the present work a systematic study was initiated with crocine, ginsenoside and cannabinoid derivatives on multidrug resistant mouse lymphoma cells, viral tumor antigen expression and some human leukocyte functions. Among saffron derivatives, crocin and picrocrocin, triglucosyl and diglucosyl crocetin were ineffective on the reversal of multidrug resistance of lymphoma cells. Ginsenoside increased drug accumulation and tumor antigen expression at 2.0-20.0 micrograms/mL. Some cannabinoid derivatives such as cannabinol, cannabispirol and cannabidiol increased drug accumulation, while cannabidiolic acid, delta-9-THC and tetrahydro-cannabidiolic acid reduced drug accumulation of the human mdr1-gene transfected mouse lymphoma cells. The reversal of multidrug resistance is the result of the inhibition of the efflux pump function in the tumor cells. Crocetin esters were less potent than crocin itself in the inhibition of EBV early antigen expression. However crocin and diglucosylcrocetin inhibited early tumor antigen expression of adenovirus infected cells, but triglucosylcrocetin was less effective at 0.01-1.0 microgram/mL. The crocin had no antiviral effect [on HSV-2 infected vero cells] up to 25 micrograms/mL concentration. Ginsenosides had a moderate inhibitory effect except ginsenoside Rb1 (was the less effective) on the drug efflux pump. Among the cannabinoid derivatives the cannabinol and cannabispirol increased drug accumulation, while cannabidiolic acid and delta-8-THC, delta-9-THC and tetrahydro-cannabinol reduced drug accumulation in multidrug resistant mouse lymphoma cells. It is interesting that ginsenosides had a chemical structure-dependent immunomodulating effect by enhancing the activity of NK-cells and ADCC activities.
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Measuring the bioactivity of phytocannabinoid cannabidiol from cannabis sources, and a novel non-cannabis source

D. Cushing, S. Kristipati, R. Shastri, & B. Joseph

Journal of Medical Phyto Research Vol. 1, Article 2, (8-23). May, 2018

Doi: 10.31013/1002b

Phytocannabinoid Cannabidiol (CBD) has been shown to elicit a great many immunological benefits. It acts on the endocannabinoid system, namely through interactions with cannabinoid receptor 2 (CB2). CBD-CB2 affinity, which we refer to as bioactivity, is rarely tested for clinical samples. We believe that uncontrolled variation in bioactivity levels have been silently confounding many CBD experiments. In our four-part study, we validate an efficient bioactivity test that can enable greater scientific control over CBD studies. We use it to compare the bioactivity of CBD obtained from different plant organs, and we also studied whether processing methods play a role in determining bioactivity. We also examine the bioactivity and processing factors of a novel non-cannabis plant capable of producing CBD in commercial quantities, named Humulus Kriya (H. Kriya, U.S. Patent No. 15/932,529, 2018). We also test the bioactivity of some CBD isolates/extracts currently sold in the market, and compare them with a CBD product called ImmunAG, which was extracted from the inflorescence of H. Kriya. We find that the CBD from the inflorescence of the plant produces the highest bioactivity, followed by the apical buds/leaves, the petioles, and finally the stalk. We find that H. Kriya has a bioactivity profile similar to Cannabis Sativa. We find that the bioactivity levels among cannabis-based commercial CBD products are quite low, and variable. We find significantly higher bioactivity levels in ImmunAG.
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Low dose oral cannabinoid therapy reduces progression of atherosclerosis in mice

Sabine Steffens, Niels R. Veillard, Claire Arnaud, Graziano Pelli, Fabienne Burger, Christian Staub, Andreas Zimmer, Jean-Louis Frossard & François Mach 

Nature volume 434, pages782–786 (2005)

DOI: 10.1038/nature03389

Atherosclerosis is a chronic inflammatory disease, and is the primary cause of heart disease and stroke in Western countries1. Derivatives of cannabinoids such as delta-9-tetrahydrocannabinol (THC) modulate immune functions2 and therefore have potential for the treatment of inflammatory diseases. We investigated the effects of THC in a murine model of established atherosclerosis. Oral administration of THC (1 mg kg-1 per day) resulted in significant inhibition of disease progression. This effective dose is lower than the dose usually associated with psychotropic effects of THC. Furthermore, we detected the CB2 receptor (the main cannabinoid receptor expressed on immune cells2,3) in both human and mouse atherosclerotic plaques. Lymphoid cells isolated from THC-treated mice showed diminished proliferation capacity and decreased interferon-γ secretion. Macrophage chemotaxis, which is a crucial step for the development of atherosclerosis1, was also inhibited in vitro by THC. All these effects were completely blocked by a specific CB2 receptor antagonist4. Our data demonstrate that oral treatment with a low dose of THC inhibits atherosclerosis progression in the apolipoprotein E knockout mouse model, through pleiotropic immunomodulatory effects on lymphoid and myeloid cells. Thus, THC or cannabinoids with activity at the CB2 receptor may be valuable targets for treating atherosclerosis.
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Isolation and Pharmacological Evaluation of Minor Cannabinoids from High-Potency Cannabis sativa

Radwan MM, ElSohly MA, El-Alfy AT, Ahmed SA, Slade D, Husni AS, Manly SP, Wilson L, Seale S, Cutler SJ, Ross SA

J Nat Prod. 78(6), 2015

DOI: 10.1021/acs.jnatprod.5b00065

Seven new naturally occurring hydroxylated cannabinoids (1-7), along with the known cannabiripsol (8), have been isolated from the aerial parts of high-potency Cannabis sativa. The structures of the new compounds were determined by 1D and 2D NMR spectroscopic analysis, GC-MS, and HRESIMS as 8α-hydroxy-Δ9-tetrahydrocannabinol (1), 8β-hydroxy-Δ9-tetrahydrocannabinol (2), 10α-hydroxy-Δ8-tetrahydrocannabinol (3), 10β-hydroxy-Δ8-tetrahydrocannabinol (4), 10α-hydroxy-Δ9,11-hexahydrocannabinol (5), 9β,10β-epoxyhexahydrocannabinol (6), and 11-acetoxy-Δ9-tetrahydrocannabinolic acid A (7). The binding affinity of isolated compounds 1-8, Δ9-tetrahydrocannabinol, and Δ8-tetrahydrocannabinol toward CB1 and CB2 receptors as well as their behavioral effects in a mouse tetrad assay were studied. The results indicated that compound 3, with the highest affinity to the CB1 receptors, exerted the most potent cannabimimetic-like actions in the tetrad assay, while compound 4 showed partial cannabimimetic actions. Compound 2, on the other hand, displayed a dose-dependent hypolocomotive effect only.
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INFLUENCE OF AGROCLIMATIC CONDITIONS ON CONTENT OF MAIN CANNABINOIDS IN INDUSTRIAL HEMP (Cannabis sativa L.)

Vladimir Sikora, Janoš Berenji, Dragana Latković 

(2011) Genetika 01/2011; 43(3). 

DOI: 10.2298/GENSR1103449S

In a six-year field experiment eight industrial hemp varieties were examined for ∆ 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD) contents. The study analyzed the influence of growing degree days (GDD), soil temperature at 5 cm, air humidity, and growing season precipitation on the levels of the main cannabinoids in this crop.Agroclimatic conditions do not influence THC and CBD contents in industrial hemp in the same way. THC synthesis and accumulation are under the significant positive influence of GDD and air humidity and under the negative influence of precipitation, while soil temperature at 5 cm has no significant effect on it. Soil temperature at 5 cm has a significant positive effect on the CBD content, as do GDD. Precipitation has a negative influence on the CBD content of industrial hemp, while air humidity has no influence on it.
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Impact of N, P, K, and Humic Acid Supplementation on the Chemical Profile of Medical Cannabis (Cannabis sativa L)

Bernstein Nirit, Jonathan Gorelick, Roei Zerahia, Sraya Koch

June 2019 Frontiers in Plant Science 10

DOI: 10.3389/fpls.2019.00736

Mineral nutrition is a major factor affecting plant growth and function. Increasing evidence supports the involvement of macro and micronutrients in secondary metabolism. The use of the appropriate nutritional measures including organic fertilizers, supplements, and biostimulants is therefore a vital aspect of medicinal plant production including medical cannabis. Due to legal restriction on cannabis research, very little information is available concerning the effects of nutritional supplements on physiological and chemical properties of medical cannabis, and their potential role in standardization of the active compounds in the plant material supplied to patients. This study therefore evaluated the potential of nutritional supplementations, including humic acids (HAs) and inorganic N, P, and K to affect the cannabinoid profile throughout the plant. The plants were exposed to three enhanced nutrition treatments, compared to a commercial control treatment. The nutrition treatments were supplemented with HA, enhanced P fertilization, or enhanced NPK. The results demonstrate sensitivity of cannabinoids metabolism to mineral nutrition. The nutritional supplements affected cannabinoid content in the plants differently. These effects were location and organ specific, and varied between cannabinoids. While the P enhancement treatment did not affect THC, CBD, CBN, and CBG concentrations in the flowers from the top of the plants, a 16% reduction of THC concentration was observed in the inflorescence leaves. Enhanced NPK and HA treatments also produced organ-specific and spatially specific responses in the plant. NPK supplementation increased CBG levels in flowers by 71%, and lowered CBN levels in both flowers and inflorescence leaves by 38 and 36%, respectively. HA was found to reduce the natural spatial variability of all of the cannabinoids studied. However, the increased uniformity came at the expense of the higher levels of cannabinoids at the top of the plants, THC and CBD were reduced by 37 and 39%, respectively. Changes in mineral composition were observed in specific areas of the plants. The results demonstrate that nutritional supplements influence cannabinoid content in cannabis in an organ- and spatial-dependent manner. Most importantly, the results confirm the potential of environmental factors to regulate concentrations of individual cannabinoids in medical cannabis. The identified effects of nutrient supplementation can be further developed for chemical control and standardization in cannabis.
Abstract

Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides

Gagne SJ, Stout JM, Liu E, Boubakir Z, Clark SM, Page JE 

Proc Natl Acad Sci USA 109: 12811–12816. (2012)

doi: 10.1073/pnas.1200330109

!9-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The !rst intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis “owers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase- like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric “+# barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.
Abstract

Identification of candidate genes affecting Δ9-tetrahydrocannabinol biosynthesis in Cannabis sativa

Marks MD, Tian L, Wenger JP, Omburo SN, Soto-Fuentes W, He J, Gang DR, Weiblen GD, Dixon RA (2009)

J Exp Bot 60: 3715–3726

doi: 10.1093/jxb/erp210

RNA isolated from the glands of a D9-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.
Abstract

Identification and Determination of Cannabinoids in both Commercially Available and Cannabis Oils Stored Long Term

Mamoru Yotoriyama, Eiji Ishiharajima, Yoko Kato, Ikuo Yamamoto

Journal of health science 51(4):483-487 August 2005

DOI: 10.1248/jhs.51.483

Among cannabinoids (CNs) in two commercially available and long-term stored cannabis oils obtained from the seeds of Cannabis sativa L. in Japan, tetrahydrocannabinol (THC), canabidiol (CBD), cannabinol (CBN), and cannabichromene were identified and determined using high-performance thin-layer chromatography and capillary gas chromatography-mass speectrometry after partitioning extraction with n-hexane/acetonitrile. CNs were determined in two commercially available cannabis oils, designated “Hemp oil” (THC, 16.3 mu g/g and CBN, 5.9 mu g/g) and “Taima-yu” (CBD, 26.1 mu g/g). The concentration of CBD in a cannabis oil that had been stored for 20 years was 107 4 mu g/g. However, no other CNs including THC were detected in the oil. The CBD content in the original oil was estimated to be about 130 mu g/g from the determination values of the two periods. These results suggest that cannabis oils generally contain CNs to some extent.
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