Developing Robust Standardised Analytical Procedures for Cannabinoid Quantification: Laying the Foundations for an Emerging Cannabis-Based Pharmaceutical Industry.

The plant genus Cannabis is a prolific producer of unique pharmaceutically relevant metabolites, commonly referred to as cannabinoids. Robust and standardised methods for the quantification of cannabinoids within botanical and drug forms is a critical step forward for an emerging Cannabis-based pharmaceutical industry, which is poised for rapid expansion. Despite a growing body of analytical methods for the quantification of cannabinoids, few have been validated using internationally accredited guidelines. Moreover, standardised methods have yet to be developed for application at various stages of manufacture as well as for different levels of processing and refinement. Validation parameters for establishing robust standardised methods for cannabinoid quantification within Cannabis-based drug forms are critically discussed. Determining an appropriate level of specificity (discrimination) among heterogeneous botanical matrices as well as evaluating accuracy (recovery) and inter-laboratory precision (reproducibility) within strict and volatile regulatory environments are potential obstacles to the establishment of robust analytical procedures. We argue that while some of these challenges remain unique to Cannabis, others are common to botanical-based drug development and manufacture. In order to address potential barriers to analytical method standardisation, a collaborative research initiative inclusive of academic and commercial stakeholders is proposed.

Detection of delta-9-tetrahydrocannabinol (THC) in oral fluid, blood and urine following oral consumption of low-content THC hemp oil.

Hemp-derivative (Cannabis sativa L.) food products containing trace levels of Δ-9-tetrahydrocannabinol (THC) are proposed for consumption in Australia and New Zealand; however, it is unclear whether use of these products will negatively affect existing drug screening protocols. This double-blind, within-subjects, cross-over trial assessed 35 adults (17 male; 18 female), aged 22-52 years [Mean=30.7, Standard Deviation (S.D)±7.6]. Low dose THC oil [5mL bearer sesame oil containing 10mg/kg THC (0.046mg THC per 5mL dose)]; high dose THC oil [5mL bearer sesame oil containing 20mg/kg THC (0.092mg THC per 5mL dose)]; and a placebo oil (THC negative) was consumed during a three-week protocol. The Securetec Drugwipe® II Twin device assessed THC presence (cut-off 20ng/mL) in oral fluid at baseline, at 5, 30, 60, 120 and 240min post-treatment. Blood was drawn at baseline, 30, 120 and 240min post-treatment, and urine at baseline and 240min post-treatment. No THC was detected in oral fluid, blood or urine samples at any time-point following consumption of the low or high THC dose. Trace concentrations of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCa) were detected in blood 4-h after consumption of the high THC treatment (M=0.0001mg/L) and in urine at 4-h post consumption of both low and high THC treatments (M=0.0001mg/L and 0.0004mg/L, respectively). Consumption of low-content THC oil does not result in positive biological assessments. It is therefore highly unlikely that ingestion of products containing these levels of THC will negatively impact existing region-specific drug driving enforcement protocols.

Ecotype Variation of Methyl Eugenol Content in Tea Tree (Melaleuca alternifolia and Melaleuca linariifolia).

Methyl eugenol is a natural phenylpropanoid compound found in a wide range of plants used for food, flavouring, cosmetics, and health-care. As a suspected rodent carcinogen, methyl eugenol may also be harmful to humans when present in significant concentrations. Consequently, its level has been restricted in some foodstuffs and cosmetics for some markets. In order to assess the potential to breed uniformly low methyl eugenol cultivars for an essential oil crop, tea tree, the source of 'Oil of Melaleuca, terpinene-4-ol type', we examine levels in individual trees (n = 30) from two geographic regions and six terpene chemotypes. Overall, methyl eugenol levels were low in this species (Mean [SD] 354 [239] ppm, n = 30), much lower than levels predicted to be of toxicological concern. Within each chemotype, there was a lack of evidence for correlations between terpenoid constituents and methyl eugenol levels. Further support for the independence of methyl eugenol and terpene biosynthesis was evident from similar mean levels in selected (Mean [SD] 586 [339] ppm, n = 12) and undomesticated Melaleuca alternifolia trees (Mean [SD] 480 [299] ppm, n = 5) with terpinen-4-ol type oils. By contrast, methyl eugenol level varied by geographic origin and chemotype. Trees from the upland region, where there is a prevalence of terpinolene type trees, had lower average methyl eugenol levels than trees from the coastal region, where there is a prevalence of terpinen-4-ol and 1,8-cineole type trees.

Investigation of nutritional properties of three species of marine turban snails for human consumption.

Turban snails (family Turbinidae) are gastropod molluscs that are harvested for human consumption yet little is known about the nutritional properties of these snails, particularly from Australian waters. This study compares the proximate composition (ash, moisture, protein, and lipid content), fatty acid profiles, mineral, and trace element content of three species of turbinid snails; Turbo militaris, Lunella undulata, and Lunella torquata from northern New South Wales, Australia. They were all found to have relatively high protein in their flesh (16.0% to 18.5% of the fresh weight). L. torquata had a significantly higher lipid content (8.5% w/w) than L. undulata (5.2% w/w), whereas T. militaris (5.6% w/w) was not significantly different to either. Analysis with gas chromatography showed there was no significant difference in monounsaturated fatty acid (MUFA) content, with an average of approximately 14% of the total fatty acids in all three species. However, saturated fatty acids (SFA) were significantly higher in T. militaris (41%), whereas polyunsaturated fatty acids (PUFA) were significantly higher in L. undulata (46%). The ratio of n-3/n-6 fatty acids ranged from 1.1 in T. militaris to 1.4 in L. torquata, which is good for human health and comparable to other high value gastropods. The results indicate that T. militaris, L. undulata, and L. torquata provide a good source of essential elements such as zinc, selenium, and iron. At the location studied, toxic metals and metalloids were below safe recommended standards for human consumption. Overall, this study confirms the suitability of turban snails as a nutritional food for human consumption.

Validation of Quantitative HPLC Method for Bacosides in KeenMind.

Brahmi (Bacopa monnieri) has been used by Ayurvedic medical practitioners in India for almost 3000 years. The pharmacological properties of Bacopa monnieri were studied extensively and the activities were attributed mainly due to the presence of characteristic saponins called "bacosides." Bacosides are complex mixture of structurally closely related compounds, glycosides of either jujubogenin or pseudojujubogenin. The popularity of herbal medicines and increasing clinical evidence to support associated health claims require standardisation of the phytochemical actives contained in these products. However, unlike allopathic medicines which typically contain a single active compound, herbal medicines are typically complex mixtures of various phytochemicals. The assay for bacosides in the British Pharmacopoeia monograph for Bacopa monnieri exemplifies that only a subset of bacosides present are included in the calculation of total bacosides. These results in calculated bacoside values are significantly lower than those attained for the same material using more inclusive techniques such as UV spectroscopy. This study illustrates some of the problems encountered when applying chemical analysis for standardisation of herbal medicines, particularly in relation to the new method development and validation of bacosides from KeenMind.

Ocean Warming and CO2-Induced Acidification Impact the Lipid Content of a Marine Predatory Gastropod.

Ocean warming and acidification are current global environmental challenges impacting aquatic organisms. A shift in conditions outside the optimal environmental range for marine species is likely to generate stress that could impact metabolic activity, with consequences for the biosynthesis of marine lipids. The aim of this study was to investigate differences in the lipid content of Dicathais orbita exposed to current and predicted future climate change scenarios. The whelks were exposed to a combination of temperature and CO2-induced acidification treatments in controlled flowthrough seawater mesocosms for 35 days. Under current conditions, D. orbita foot tissue has an average of 6 mg lipid/g tissue, but at predicted future ocean temperatures, the total lipid content dropped significantly, to almost half. The fatty acid composition is dominated by polyunsaturated fatty acids (PUFA 52%) with an n-3:6 fatty acid ratio of almost 2, which remains unchanged under future ocean conditions. However, we detected an interactive effect of temperature and pCO2 on the % PUFAs and n-3 and n-6 fatty acids were significantly reduced by elevated water temperature, while both the saturated and monounsaturated fatty acids were significantly reduced under increased pCO2 acidifying conditions. The present study indicates the potential for relatively small predicted changes in ocean conditions to reduce lipid reserves and alter the fatty acid composition of a predatory marine mollusc. This has potential implications for the growth and survivorship of whelks under future conditions, but only minimal implications for human consumption of D. orbita as nutritional seafood are predicted.

Proliferative and Inhibitory Activity of Siberian ginseng (Eleutherococcus senticosus) Extract on Cancer Cell Lines; A-549, XWLC-05, HCT-116, CNE and Beas-2b.

Siberian ginseng (Eleutherococcus senticosus) is used primarily as an adaptogen herb and also for its immune stimulant properties in Western herbal medicine. Another closely related species used in East Asian medicine systems i.e. Kampo, TCM (Manchuria, Korea, Japan and Ainu of Hokkaido) and also called Siberian ginseng (Acanthopanax senticosus) also displays immune-stimulant and anti-cancer properties. These may affect tumour growth and also provide an anti-fatigue effect for cancer patients, in particular for those suffering from lung cancer. There is some evidence that a carbohydrate in Siberian ginseng may possess not only immune stimulatory but also anti-tumour effects and also display other various anti-cancer properties. Our study aimed to determine the inhibitory and also proliferative effects of a methanol plant extract of Siberan ginseng (E. senticosus) on various cancer and normal cell lines including: A-549 (small cell lung cancer), XWLC-05 (Yunnan lung cancer cell line), CNE (human nasopharyngeal carcinoma cell line), HCT-116 (human colon cancer) and Beas-2b (human lung epithelial). These cell lines were treated with an extract from E. senticosus that was evaporated and re- constituted in DMSO. Treatment of A-549 (small cell lung cancer) cells with E. senticosus methanolic extract showed a concentration-dependent inhibitory trend from 12.5 - 50µg/mL, and then a plateau, whereas at 12.5 and 25 µg/mL, there is a slight growth suppression in QBC-939 cells, but then a steady suppression from 50, 100 and 200µg/mL. Further, in XWLC-05 (Yunnan lung cancer cell line), E. senticosus methanolic extract displayed an inhibitory effect which plateaued with increasing dosage. Next, in CNE (human nasopharyngeal carcinoma cell line) there was a dose dependent proliferative response, whereas in Beas-2 (human lung epithelial cell line), an inhibitory effect. Finally in colon cancer cell line (HCT-116) we observed an initially weak inhibitory effect and then plateau.

Adulteration of Ginkgo biloba products and a simple method to improve its detection.

Extracts of ginkgo (Ginkgo biloba) leaf are widely available worldwide in herbal medicinal products, dietary supplements, botanicals and complementary medicines, and several pharmacopoeias contain monographs for ginkgo leaf, leaf extract and finished products. Being a high-value botanical commodity, ginkgo extracts may be the subject of economically motivated adulteration. We analysed eight ginkgo leaf retail products purchased in Australia and Denmark and found compelling evidence of adulteration with flavonol aglycones in three of these. The same three products also contained genistein, an isoflavone that does not occur in ginkgo leaf. Although the United States Pharmacopeia - National Formulary (USP-NF) and the British and European Pharmacopoeias stipulate a required range for flavonol glycosides in ginkgo extract, the prescribed assays quantify flavonol aglycones. This means that these pharmacopoeial methods are not capable of detecting adulteration of ginkgo extract with free flavonol aglycones. We propose a simple modification of the USP-NF method that addresses this problem: by assaying for flavonol aglycones pre and post hydrolysis the content of flavonol glycosides can be accurately estimated via a simple calculation. We also recommend a maximum limit be set for free flavonol aglycones in ginkgo extract.

Parvifloranines A and B, two 11-carbon alkaloids from Geijera parviflora.

Two novel alkaloids (parvifloranines A and B), possessing an unusual 11-carbon skeleton linked with amino acids, were isolated from Geijera parviflora, an endemic Australian Rutaceae. Their structures were elucidated by extensive spectroscopic measurements including 2D NMR analyses. Parvifloranine A was found to be a mixture of two enantiomers, (S)-1 and (R)-1, in a ratio of 1:4, based on their separation using a chiral column. Parvifloranine B is also believed to be a mixture of enantiomers. Proposed biosynthetic pathways are discussed. Parvifloranine A inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages with an IC50 value of 23.4 µM.

Kava in the treatment of generalized anxiety disorder: a double-blind, randomized, placebo-controlled study.

Kava (Piper methysticum) is a plant-based medicine, which has been previously shown to reduce anxiety. To date, however, no placebo-controlled trial assessing kava in the treatment of generalized anxiety disorder (GAD) has been completed. A total of 75 participants with GAD and no comorbid mood disorder were enrolled in a 6-week double-blind trial of an aqueous extract of kava (120/240 mg of kavalactones per day depending on response) versus placebo. γ-Aminobutyric acid (GABA) and noradrenaline transporter polymorphisms were also analyzed as potential pharmacogenetic markers of response. Reduction in anxiety was measured using the Hamilton Anxiety Rating Scale (HAMA) as the primary outcome. Intention-to-treat analysis was performed on 58 participants who met inclusion criteria after an initial 1 week placebo run-in phase. Results revealed a significant reduction in anxiety for the kava group compared with the placebo group with a moderate effect size (P = 0.046, Cohen d = 0.62). Among participants with moderate to severe Diagnostic and Statistical Manual of Mental Disorders-diagnosed GAD, this effect was larger (P = 0.02; d = 0.82). At conclusion of the controlled phase, 26% of the kava group were classified as remitted (HAMA ≤ 7) compared with 6% of the placebo group (P = 0.04). Within the kava group, GABA transporter polymorphisms rs2601126 (P = 0.021) and rs2697153 (P = 0.046) were associated with HAMA reduction. Kava was well tolerated, and aside from more headaches reported in the kava group (P = 0.05), no other significant differences between groups occurred for any other adverse effects, nor for liver function tests. Standardized kava may be a moderately effective short-term option for the treatment of GAD. Furthermore, specific GABA transporter polymorphisms appear to potentially modify anxiolytic response to kava.