Vapor Phase Terpenes Mitigate Oxidative Degradation of Cannabis sativa Inflorescence Cannabinoid Content in an Accelerated Stability Study.

Introduction: As Cannabis sativa L. (Cannabaceae) ages, inflorescence phytochemicals are susceptible to oxidative degradation. Reduction of Δ9-tetrahydrocannabinol (Δ9-THC) content has the potential to impact the reliability and accuracy of dosing. Advances that improve cannabinoid stability during storage would have an important impact in medical cannabis markets. Reported here is the use of C. sativa terpenes with antioxidant properties that improve inflorescence cannabinoid stability. Materials and Methods: Killer Kush inflorescence samples were stored in a temperature-controlled environment, in opaque jars. To accelerate the rate of oxidate degradation, samples were stored with the oxidizing agent hydrogen peroxide. Vapor phase terpenes were added to inflorescence packaging. Two terpene blends and three different dosage amounts were evaluated. Inflorescence stability samples were prepared in triplicate for each sample type. Cannabinoid content was quantitatively assessed after 24, 81, and 127 days of storage using high-performance liquid chromatography. Terpene content was assessed using headspace gas chromatography mass spectrometry. Results from inflorescence stored with and without external terpenes were compared by analysis of variance (ANOVA) data processing. Results: After 127 days of storage, inflorescence in the accelerated study experienced a loss of 18.0% and 34.3% total Δ9-THC content for samples stored with and without external terpenes, respectively. The differences in cannabinoid content were found to be statistically significant at all timepoints using ANOVA processing. In the nonaccelerated study, only one of the six sample types investigated had a statistically significant greater total Δ9-THC content than control at all timepoints. Nevertheless, a dose-dependent relationship between the amount of external terpenes added to inflorescence and the preservation of total Δ9-THC content was observed. Discussion: In the accelerated study, exogenous terpenes reduced the degradation of inflorescence cannabinoid content by 47.4%. This represents the first reported addition of terpene antioxidants to inflorescence packaging for cannabinoid preservation. Of note, the antioxidants used in this system can be obtained from C. sativa. This is advantageous from a toxicological perspective as inhaling synthetic antioxidants presents unknown and unpredictable risks. When fully developed, the novel system has applications for inflorescence packaged for individual sale, as well as long-term storage of bulk biomass.

(-)-trans-Δ9-Tetrahydrocannabiphorol Content of Cannabis sativa Inflorescence from Various Chemotypes.

The (-)-trans-Δ9-tetrahydrocannabiphorol (Δ9-THCP, 1) content of the inflorescence from six Cannabis sativa chemotypes, including 14 plants of distinct genotypes, and two extracts was determined quantitatively via high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This represents the first comprehensive quantitative screening for 1 from various C. sativa chemotypes. Compound 1 was detected in all 13 inflorescence samples originating from "(-)-trans-Δ9-tetrahydrocannabinol (Δ9-THC, 2) dominant" C. sativa chemotypes, but was not detected in a "cannabidiol (CBD, 3) dominant" chemotype. The inflorescence content of 1 ranged approximately from 0.0023% to 0.0136% (w/w). Comprehensive inflorescence sampling was performed for each specimen investigated. A trend between inflorescence cannabinoid potency and the location of which the inflorescence was sampled on the C. sativa plant was observed for the three cannabinoids tested (1-3). The preliminary results obtained indicate Δ9-THCP (1) may have a higher degree of prevalence in C. sativa inflorescence than previously estimated.

The preservation and augmentation of volatile terpenes in cannabis inflorescence.

BACKGROUND: Terpenes contribute to the pharmacology, efficacy, aroma, and flavor of cannabis inflorescence, improving the experience for medical and recreational users. Terpenes are inherently volatile, resulting in the loss of terpene content as inflorescence ages. A method to establish and/or maintain a desired terpene content of cannabis inflorescence is needed. A novel packaging method was investigated for the preservation of native terpenes and the replenishment of terpenes to depleted inflorescence over various storage durations. METHODS: Inflorescence samples from two different chemotypes (DJ's Gold, Cream Caramel) were obtained from a state licensed medical cannabis organization. Samples from the DJ's Gold chemotype were depleted of terpenes whereas samples from the Cream Caramel chemotype had a terpene content representative of inflorescence available for medicinal or recreational purposes. Inflorescence samples were stored using the novel packaging approach, in airtight containers in the presence of external terpenes. Control samples were similarly stored without external terpenes. Terpene content of the inflorescence samples were quantitively determined by headspace gas chromatography mass spectrometry (HS GC-MS) after various storage durations. Main effects analysis was used to determine the impact of various parameters on the effectiveness of the system. RESULTS: All samples stored using the novel packaging approach had a higher terpene content than their corresponding control. 1.18% (w/w) of external terpene, relative to inflorescence weight, was the minimum amount required to maintain the initial terpene content of the inflorescence after 6 weeks of storage. Main effects analysis showed that augmentation of inflorescence terpene content was dependent upon the amount and type of external volatile utilized. The terpene profile of inflorescence samples from two separate harvests were selectively adjusted, reducing the percent difference of the two sample's terpene profiles by 39.5%. CONCLUSIONS: A successful proof of concept was achieved for preservation, augmentation, and replenishment of terpenes to cannabis inflorescence over various storage durations. Inflorescence stored using the novel packaging approach is a significant step towards providing patients with cannabis inflorescence of reproducible and reliable terpene content, an important component of inflorescence efficacy. The novel approach for replenishment of terpenes to depleted inflorescence represents an exciting development for patients and manufacturers.

Structure and properties of alpha-synuclein and other amyloids determined at the amino acid level.

The structure of alpha-synuclein (alpha-syn) amyloid was studied by hydrogen-deuterium exchange by using a fragment separation-MS analysis. The conditions used made it possible to distinguish the exchange of unprotected and protected amide hydrogens and to define the order/disorder boundaries at close to amino acid resolution. The soluble alpha-syn monomer exchanges its amide hydrogens with water hydrogens at random coil rates, consistent with its natively unstructured condition. In assembled amyloid, long N-terminal and C-terminal segments remain unprotected (residues 1- approximately 38 and 102-140), although the N-terminal segment shows some heterogeneity. A continuous middle segment (residues approximately 39-101) is strongly protected by systematically H-bonded cross-beta structure. This segment is much too long to fit the amyloid ribbon width, but non-H-bonded amides expected for direction-changing loops are not apparent. These results and other known constraints specify that alpha-syn amyloid adopts a chain fold like that suggested before for amyloid-beta [Petkova et al. (2002) Proc. Natl. Acad Sci. USA 99, 16742-16747] but with a short, H-bonded interlamina turn. More generally, we suggest that the prevalence of accidental amyloid formation derives mainly from the exceptional ability of the main chain in a structurally relaxed beta-conformation to adapt to and energy-minimize side-chain mismatching. Seeding specificity, strain variability, and species barriers then arise because newly added parallel in-register chains must faithfully reproduce the same set of adaptations.

The E46K mutation in alpha-synuclein increases amyloid fibril formation.

The identification of a novel mutation (E46K) in one of the KTKEGV-type repeats in the amino-terminal region of alpha-synuclein suggests that this region and, more specifically, Glu residues in the repeats may be important in regulating the ability of alpha-synuclein to polymerize into amyloid fibrils. It was demonstrated that the E46K mutation increased the propensity of alpha-synuclein to fibrillize, but this effect was less than that of the A53T mutation. The substitution of Glu(46) for an Ala also increased the assembly of alpha-synuclein, but the polymers formed can have different ultrastructures, further indicating that this amino acid position has a significant effect on the assembly process. The effect of residue Glu(83) in the sixth repeat of alpha-synuclein, which lies closest to the amino acid stretch critical for filament assembly, was also studied. Mutation of Glu(83) to a Lys or Ala increased polymerization but perturbed some of the properties of mature amyloid. These results demonstrated that some of the Glu residues within the repeats can have significant effects on modulating the assembly of alpha-synuclein to form amyloid fibrils. The greater effect of the A53T mutation, even when compared with what may be predicted to be a more dramatic mutation such as E46K, underscores the importance of protein microenvironment in affecting protein structure. Moreover, the relative effects of the A53T and E46K mutations are consistent with the age of onset of disease. These findings support the notion that aberrant alpha-synuclein polymerization resulting in the formation of pathological inclusions can lead to disease.

BACE overexpression alters the subcellular processing of APP and inhibits Abeta deposition in vivo.

Introducing mutations within the amyloid precursor protein (APP) that affect beta- and gamma-secretase cleavages results in amyloid plaque formation in vivo. However, the relationship between beta-amyloid deposition and the subcellular site of Abeta production is unknown. To determine the effect of increasing beta-secretase (BACE) activity on Abeta deposition, we generated transgenic mice overexpressing human BACE. Although modest overexpression enhanced amyloid deposition, high BACE overexpression inhibited amyloid formation despite increased beta-cleavage of APP. However, high BACE expression shifted the subcellular location of APP cleavage to the neuronal perikarya early in the secretory pathway. These results suggest that the production, clearance, and aggregation of Abeta peptides are highly dependent on the specific neuronal subcellular domain wherein Abeta is generated and highlight the importance of perikaryal versus axonal APP proteolysis in the development of Abeta amyloid pathology in Alzheimer's disease.