A Metabolomics and Big Data Approach to Cannabis Authenticity (Authentomics).

With the increasing accessibility of cannabis (Cannabis sativa L., also known as marijuana and hemp), its products are being developed as extracts for both recreational and therapeutic use. This has led to increased scrutiny by regulatory bodies, who aim to understand and regulate the complex chemistry of these products to ensure their safety and efficacy. Regulators use targeted analyses to track the concentration of key bioactive metabolites and potentially harmful contaminants, such as metals and other impurities. However, the metabolic complexity of cannabis metabolic pathways requires a more comprehensive approach. A non-targeted metabolomic analysis of cannabis products is necessary to generate data that can be used to determine their authenticity and efficacy. An authentomics approach, which involves combining the non-targeted analysis of new samples with big data comparisons to authenticated historic datasets, provides a robust method for verifying the quality of cannabis products. To meet International Organization for Standardization (ISO) standards, it is necessary to implement the authentomics platform technology and build an integrated database of cannabis analytical results. This study is the first to review the topic of the authentomics of cannabis and its potential to meet ISO standards.

Methionine epimerization in cyclic peptides.

Bioactive flax cyclic octa- and nona-peptides containing single methionine (Met) and its oxidized forms were treated under mild alkaline conditions to perform regio-selective epimerization. Cyclic peptide epimerization at the Met α-proton in a single chemical step has not been reported previously. The epimerization rate varies among Met oxidation states and ring size. These d-amino isomers along with the developed Met alkylation strategy will enable an approach to novel chemical functionalization of biomolecules. The amino acid configurations were confirmed by Marfey derivatizations, and cytotoxicity studies show the difference among the isomers. These d-amino analogs can act as a potential biomarker in plant protein processing and biomedical applications.

In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa.

The Cannabis sativa plant contains more than 120 cannabinoids. With the exceptions of ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD), comparatively little is known about the pharmacology of the less-abundant plant-derived (phyto) cannabinoids. The best-studied transducers of cannabinoid-dependent effects are type 1 and type 2 cannabinoid receptors (CB1R, CB2R). Partial agonism of CB1R by ∆9-THC is known to bring about the 'high' associated with Cannabis use, as well as the pain-, appetite-, and anxiety-modulating effects that are potentially therapeutic. CB2R activation by certain cannabinoids has been associated with anti-inflammatory activities. We assessed the activity of 8 phytocannabinoids at human CB1R, and CB2R in Chinese hamster ovary (CHO) cells stably expressing these receptors and in C57BL/6 mice in an attempt to better understand their pharmacodynamics. Specifically, ∆9-THC, ∆9-tetrahydrocannabinolic acid (∆9-THCa), ∆9-tetrahydrocannabivarin (THCV), CBD, cannabidiolic acid (CBDa), cannabidivarin (CBDV), cannabigerol (CBG), and cannabichromene (CBC) were evaluated. Compounds were assessed for their affinity to receptors, ability to inhibit cAMP accumulation, ßarrestin2 recruitment, receptor selectivity, and ligand bias in cell culture; and cataleptic, hypothermic, anti-nociceptive, hypolocomotive, and anxiolytic effects in mice. Our data reveal partial agonist activity for many phytocannabinoids tested at CB1R and/or CB2R, as well as in vivo responses often associated with activation of CB1R. These data build on the growing body of literature showing cannabinoid receptor-dependent pharmacology for these less-abundant phytocannabinoids and are critical in understanding the complex and interactive pharmacology of Cannabis-derived molecules.

Correction: Affinity binding of chicken apolipoprotein A1 to a novel flax orbitide (linusorb).

[This corrects the article DOI: 10.1039/C8RA01757C.].

Novel flax orbitide derived from genetic deletion.

BACKGROUND: Flaxseed orbitides are homodetic plant cyclic peptides arising from ribosomal synthesis and post-translation modification (N to C cyclization), and lacking cysteine double bonds (Nat Prod Rep 30:108-160, 2013). Screening for orbitide composition was conducted on the flax core collection (FCC) grown at both Saskatoon, Saskatchewan and Morden, Manitoba over three growing seasons (2009-2011). Two flax (Linum usitatissimum L.) accessions 'Hollandia' (CN 98056) and 'Z 11637' (CN 98150) produce neither [1-9-NαC]-linusorb B2 (3) nor [1-9-NαC]-linusorb B3 (1). Mass spectrometry was used to identify novel compounds and elucidate their structure. NMR spectroscopy was used to corroborate structural information. RESULTS: Experimental findings indicated that these accessions produce a novel orbitide, identified in three oxidation states having quasimolecular ion peaks at m/z 1072.6 (18), 1088.6 (19), and 1104.6 (20) [M + H]+ corresponding to molecular formulae C57H86N9O9S, C57H86N9O10S, and C57H86N9O11S, respectively. The structure of 19 was confirmed unequivocally as [1-9-NαC]-OLIPPFFLI. PCR amplification and sequencing of the gene coding for 18, using primers developed for 3 and 1, identified the putative linear precursor protein of 18 as being comprised of the first three amino acid residues of 3 (MLI), four conserved amino acid residues of 3 and/or 1 (PPFF), and the last two residues of 1 (LI). CONCLUSION: Comparison of gene sequencing data revealed that a 117 base pair deletion had occurred that resulted in truncation of both 3 and 1 to produce a sequence encoding for the novel orbitide precursor of 18. This observation suggests that repeat units of flax orbitide genes are conserved and suggests a novel mechanism for evolution of orbitide gene diversity. Orbitides 19 and 20 contain MetO and MetO2, respectively, and are not directly encoded, but are products of post-translation modification of Met present in 18 ([1-9-NαC]-MLIPPFFLI).

Affinity binding of chicken apolipoprotein A1 to a novel flax orbitide (linusorb).

Bioactive orbitides (linusorbs, LOs) from flaxseed (Linum usitatissimum L.) were ligated through methionine with resin to form an affinity column. The affinity resin was characterized using elemental analysis and the resin bound 70% of its weight in LOs. Chicken serum was passed over the column and washed to remove non-binding materials. The column was eluted with unbound orbitide to competitively release bound protein. A single 28 kDa protein was found in the affinity binding pool. The protein MW and sequence were identical to apolipoprotein A1 (Apo A1), a major serum protein. Its role includes reverse cholesterol transport and cholesterol efflux. The affinity technique allowed convenient and rapid isolation of Apo A1 with a recyclable affinity column. LO binding to a cholesterol carrier molecule might also help us to understand the mechanism of action of LOs in health and the biological activity of flaxseed products.

Synthesis and Characterization of Site-Selective Orbitide-BSA Conjugate to Produce Antibodies.

Bioactive flax cyclic peptides (orbitides and linusorbs) were site-specifically ligated through methionine with bovine serum albumin (BSA) to produce immunogenic compounds. In this study, modified flaxseed immunosuppressant orbitides (linusorbs or LOs) containing hydroxyl (OH) groups were synthesized for use as haptens. These compounds were extensively characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, high-performance liquid chromatography-tandem mass spectrometry, and Fourier transform infrared spectroscopy. The haptens were conjugated to BSA, and the extent of hapten incorporation was determined by matrix-assisted laser desorption and ionization, liquid chromatography-electrospray ionization-mass spectrometry, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The BSA hapten complexes were used to elicit polyclonal antibody (pAbs) production in rabbits. A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed that used orbitide-specific pAbs and horseradish peroxidase (HRP) conjugates. The LO assay detection limit was approximately 0.01 µg/mL (ppm), and thus, ELISA can be used for the detection of LOs in tissue and plant samples. The pAbs can be used to detect and quantify LOs in flax and flaxseed samples, to verify the presence of LOs in flaxseed containing foods, and for the detection of LOs in tissue samples, wastes, and body fluids of animals fed flaxseed.

Nomenclature of homodetic cyclic peptides produced from ribosomal precursors: An IUPAC task group interim report.

In 2015, an International Union of Pure and Applied Chemistry (IUPAC) Task Group was formed to develop nomenclature recommendations for homodetic cyclic peptides produced from ribosomal precursors. Delegates of the 2015 International Conference on Circular Proteins (ICCP) were presented with the strengths and weaknesses of four published approaches to homodetic cyclic peptide nomenclature, and a summary of the ensuing discussion is presented here. This interim report presents a potentially novel suggestion-the use of Cahn-Ingold-Prelog rules to specify amino acid priority in homodetic peptides for consistent numbering. Indeed, this might be the first extension of the Cahn-Ingold-Prelog rules in five decades. The authors invite interested parties to contact the corresponding author with suggestions for the improvement of the proposed nomenclature; these ideas will be discussed and considered for inclusion in the final report.

Site Covalent Modification of Methionyl Peptides for the Production of FRET Complexes.

Flax cyclic peptides (orbitides, linusorbs (LOs)) [1-8-NαC],[1-MetO2]-linusorb B1 ([MetO2]-LO1) and [1-9-NαC],[1-MetO2]-linusorb B2 ([MetO2]-LO2) are biologically active. These LOs lack active nuclei commonly used in peptide modification. We have developed reactions to activate methionine methyl sulphide to produce stable derivatives. In these reactions, LOs are converted to sulfonium intermediates and subsequently to derivatives containing active nuclei while preserving their fundamental structures. The reaction conditions preserved cyclic peptide fundamental structure and organic solvent solubility. [Met]-LO1 and [Met]-LO2 analogues containing activated groups (-CN, -COOEt, and -NH2 ) in the form of methionine, methionine (S)-oxide, and methionine (S,S)-dioxide amino acids were synthesized and characterized by LCMS and 1D and 2D NMR spectroscopy. Coumarin orbitide complexes produced in this manner bind Eu(3+) yielding FRET compounds that absorb energy through coumarin antennae and emit photons at lanthanide wavelengths.

Glycine-containing flaxseed orbitides.

Five new orbitides, cyclolinopeptides 21-25, were identified in flaxseed oil (Linum usitatissimum) extracts. Their HPLC-ESIMS quasimolecular ion peaks at m/z 1097.7 (21), 1115.6 (22), 1131.6 (23), 1018.6 (24), and 1034.6 (25) [M + H](+) corresponded to the molecular formulae C59H89N10O10, C58H87N10O10S, C58H87N10O11S, C53H80N9O9S, and C53H80N9O10S, respectively. Their structures were elucidated by extensive HPLC-ESIMS/MS analyses, and their presence was confirmed by precursor proteins identified in flax genomic DNA sequence data. The amino acid sequences of these orbitides were confirmed as [1-10-NαC]-GILVPPFFLI, [1-10-NαC]-GMLIPPFFVI, [1-10-NαC]-GOLIPPFFVI, [1-9-NαC]-GMLVFPLFI, and [1-9-NαC]-GOLVFPLFI for cyclolinopeptides 21-25, respectively. Previously reported orbitides, [1-9-NαC]-ILVPPFFLI (1), [1-9-NαC]-MLIPPFFVI (2), [1-9-NαC]-OLIPPFFVI (3), [1-8-NαC]-MLVFPLFI (7), and [1-8-NαC]-OLVFPLFI (8), were also present in flaxseed oil. The precursors of orbitides 21, 22, and 24 also produced orbitides 1, 2, and 7 by alternative cyclization. Cyclolinopeptides 3, 8, 23, and 25 contain MetO (O) and are not directly encoded, but are products of post-translational modification of the Met present in 2, 7, 22, and 24, respectively. Sufficient cyclolinopeptide 23 was isolated for characterization via 1D ((1)H and (13)C) and 2D (NOESY and HMBC) NMR spectroscopy. These compounds have been named as cyclolinopeptides U, V, W, X, and Y for 21, 22, 23, 24, and 25, respectively.

Evaluating the cytotoxicity of flaxseed orbitides for potential cancer treatment.

Flaxseed as well as its oil component possess antitumor activities against different types of cancer and have been used by some patients as complementary and/or alternative medicine. Linoorbitides (LOBs) are one family of flaxseed compounds that has implications for anticancer and antioxidant activity. The cytotoxicity of [1-9-NαC]-linusorb-B3 (LOB3), [1-9-NαC]-linusorb-B2 (LOB2), [1-9-NαC],[1-Rs,Ss-MetO]-linusorb-B2 ([MetO]-LOB2) and [1-8-NαC],[1-Rs,Ss-MetO]-linusorb-B1 ([MetO]-LOB1) was measured against human breast cancer Sk-Br-3 and MCF7 cell lines and melanoma A375 cell line. Overall cytotoxicity is cell-type specific. It scales as the hydrophobicity and concentration of the LOBs with the most abundant LOB3 being the most cytotoxic. Oral administration of LOB3 as a potential therapeutic agent might not be applicable as a much too high and/or frequent dose would be required to achieve a serum concentration of 400-500 µg/mL due to bioavailability and pharmacokinetic factors. However, LOB3 may be suitable for topical treatment formulations or as a lead compound in developing anticancer LOB derivatives.

Detection, isolation and characterisation of cyclolinopeptides J and K in ageing flax.

Methionine sulfone containing peptides CLs J (11) and K (12) may be produced from their reduced forms by oxidation but it is not known if these compounds occur in foods that contain flax. These compounds have been reported to possess greater immunosuppressive activity than their reduced methionine sulfoxide peptide forms 4 and 6, respectively. Since 11 and 12 have not been detected in commercial flax oil and milled flax seed, we tested for their presence in flax food products. Here we report that 11 and 12 accumulate in ground flaxseed that is exposed to air and heat (100°C) for more than 4h. Standards of 11 and 12 were prepared, isolated and extensively characterised using HPLC-MS/MS, 1D and 2D NMR methods. We also report the excellent thermal and oxidative stability of these peptides. Due to the harsh conditions required to produce 11 and 12, it is expected that their levels in flax based foods would be low and therefore their presence could serve as an indicative measure of severe oxidation of a food product.

Cyclo-linopeptide K butanol disolvate monohydrate.

The title compound, C(56)H(83)N(9)O(11)S·2C(4)H(10)O·H(2)O, is a butanol-water solvate of the cyclo-linopeptide cyclo(Metsulfone(1)-Leu(2)-Ile(3)-Pro(4)-Pro(5)-Phe(6)-Phe(7)-Val(8)-Ile(9)) (henceforth referred to as CLP-K) which was isolated from flax oil. All the amino acid residues are in an l configuration based on the CORN rule. The cyclic nona-peptide exhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α- and a ß-turn, each containing an N-H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) and the third residue (ß-turn), repectively. In the crystal, the components of the structure are linked by inter-molecular N-H⋯O and O-H⋯O hydrogen bonds into a two-dimensional network parallel to (001). The -C(H(2))OH group of one of the butanol solvent mol-ecules is disordered over two sets of sites with refined occupancies of 0.863 (4) and 0.137 (4).