INTRODUCTION: A protocol for the in vitro culture of the anxiolytic medicinal plant Souroubea sympetala (Marcgraviaceae) was developed, representing one of the first in vitro cultures for the family. This species was previously very difficult to cultivate from seed or cuttings. METHODS: Methods included (1) the improvement of seed germination by axenic culture (2) development of regenerative cultures in vitro, then cultivation under greenhouse and finally field conditions and (3) creation of cell suspensions. Phytochemical analysis was undertaken by liquid chromatography coupled to mass spectrometry (HPLC-MS). RESULTS: The percentage of seed germination was improved from 2% to 59% in axenic culture and the full development of the seedling with its apical shoot and root took twenty-four days. The best seedling development was obtained in Gamborg B5 culture medium. Most friable callus formation, (66.7%) was obtained in the Murashige and Skoog medium supplemented with naphthalene acetic acid (1 mg · L-1) and kinetin (0.5 mg · L-1) from which viable cell cultures were developed. Analysis identified 4 main triterpenes with both in vitro plants and greenhouse grown plants derived from them. The triterpenes were betulinic acid, ursolic acid, alpha-amyrin and beta-amyrin. The betulinic acid found in greenhouse plants was comparable to wild plants. The cell suspension cultures had much lower levels of betulinic acid than plants and are not at present a viable source of this anxiolytic triterpene. DISCUSSION: The improvement in seed germination of this recalcitrant tropical species was highly successful. The subsequent in vitro propagation and progression of plants through greenhouse and field conditions to provide mature plants with active principle concentrations comparable to wild plants was promising. Friable callus was achieved but phytochemical analysis showed that the level of betulinic acid in callus was much lower than that found in mature plant tissue. CONCLUSION: The method provides healthy plants for cultivation of this new medicinal plant and consequently harvesting of wild plants is not required.
INTRODUCTION: The U-Dream line of products are marketed as natural health product sleep aids in Canada and as dietary supplements in the United States. Several user reviews of the product mention concerning side effects not typically associated with the listed herbal ingredients stated on the product label. Based on these concerns an analytical study was undertaken to determine if the products contained any undeclared pharmaceuticals. METHODS: Product was screened by high resolution mass spectrometry (HRMS) for known pharmaceuticals with sedative and sleep promoting properties. Based on the mass spectral screens an unknown compound was isolated for characterization by nuclear magnetic resonance (NMR) and presence confirmed by Enzymelinked Immunosorbent Assay (ELISA). RESULTS: The mass spectral analyses indicated the presence of an undeclared analogue of the pharmaceutical drug zopiclone within the product lot tested. NMR characterization confirmed the compound to be a brominated analogue of zopiclone and a commercial zopiclone/eszopiclone ELISA kit tested positive. DISCUSSION: The undeclared compound was found to be an analogue of zopiclone whereby the chlorine atom was substituted with bromine. Given the results of the ELISA assay and the structural similarity to zopiclone it is likely the compound exhibits biological activity. Of considerable concern is not only the potential of the unknown compound to exhibit pharmacological activity, but the lack of a safety profile by which the risk can be properly assessed. CONCLUSIONS: The natural health product regulations provide a framework for high quality, safe and efficacious products to access the market. It is the responsibility of the manufacturer to assure traceability and transparency in their supply chain and establish verifiable compliance with GMP. This study illustrates the importance of careful evaluation of analytical data in order to detect undeclared adulterants and highlights the need for an active monitoring and surveillance system for potentially high-risk products.
Overexposure to ultraviolet (UV) light is associated with multiple health risks, from sunburn and prematurely aging skin to the development of skin cancers. The ingestion of photoprotective natural compounds through diet or supplementation is one method to increase the skin’s UV-resistance. This study’s primary objective was to determine the cellular photoprotective properties of an ingestible skincare supplement (trade name “Anti-Aging Formula” [AAF]) and compare them to its constituent active ingredients: fish oil-derived omega-3s eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), borage-derived omega-6 gamma-linolenic acid (GLA), paprika- and marigold-derived carotenoids, zeaxanthin and lutein, respectively, and vitamin D3. AAF, but not the separate individual ingredients, significantly increased the viability of primary human dermal fibroblasts after UVA exposure compared to the vehicle control. AAF and EPA/DHA-containing fish oil demonstrated similar UVB photoprotective properties whereas GLA, the carotenoids, and vitamin D3 had no significant effect. The second objective was to explore possible mechanisms of action of AAF’s photoprotective effects. AAF-treatment increased cellular antioxidant activity and the expression of genes in the glutathione and peroxiredoxin (PRDX)/thioredoxin (TXN) antioxidant pathways, suggesting an antioxidant mechanism of action. It also diminished cellular arachidonic acid (AA) levels and decreased the expression of the downstream pro-inflammatory prostaglandin-endoperoxide synthase 2 (PTGS2) gene, suggesting an anti-inflammatory mechanism of action. In conclusion, AAF is UVA/B photoprotective when applied directly to primary human dermal fibroblasts. In addition, its photoprotective effects are mainly due to its EPA/DHA components and may relate to its cellular antioxidant effects and inhibition of the AA/PTGS2 inflammatory pathway.
