Search

Natural health products in North America represent a $9.6 billion USD industry that has seen increased demands, particularly in products for immune support and in anti-viral supplements as a response to the COVID-19 pandemic. It has been estimated that approximately 40% of the plant raw materials used in natural health products are wild harvested each year, but exact data are missing for many species. Our data show that there are at least 1445 medicinal plant species harvested for commercial products in Canada, and 1217 of these are native species. Medicinal plants and non-timber forest crops are also increasingly impacted by climate change as suitable growing areas shift. To ensure long-term species survival in the wild, it is necessary to generate accurate information about the distribution of wild populations, the need for conservation, and the development of sustainable methods. Our objective was to provide a preliminary assessment of the conservation status of Canadian medicinal plants by analyzing the available data resources.

Light mediates plant growth through diverse mechanisms and signaling networks including plant growth regulators (PGRs). We hypothesized that a novel class of PGRs, the indoleamines, are plant signaling molecules that perceive changes in light composition and initiate a cascade of metabolic responses. We used three Scutellaria model species (skullcap): S. lateriflora, S. galericulata and S. racemosa that produce high levels of melatonin and serotonin to investigate this hypothesis. Axenic Scutellaria cultures were exposed to red, blue, green or full spectrum white light spectra provided by light emitting diode (LED) lighting systems, or daylight fluorescent bulbs. Melatonin (MEL), serotonin (5HT), abscisic acid (ABA), auxin (IAA), and jasmonic acid (JA), were quantified by liquid chromatography with tandem mass spectrometry. Melatonin was detected consistently in plants grown under blue light in all species of Scutellaria. In S. galericulata, significant quantities of ABA were detected in plants grown under white light but not detected in plants grown under other light spectra. In timeline studies of S. racemosa plants exposed to limited red or blue light spectra had significantly reduced levels of tryptamine (TRM), 5HT and MEL in the shoots initially but melatonin was detected after 12 hours and quantifiable amounts of 5HT were detected after 7 days. Supplementation of the culture medium with MEL or 5HT did not change the pattern of MEL in blue light grown cultures but did change patterns of 5HT accumulation. 5HT was highest in plants grown under red light immediately after culture and decreased over 7 days. These data indicate that the relative amounts of MEL and 5HT are responsive to light spectra and redirect metabolic resources to enable plant adaptations to changing environments.

INTRODUCTION Nuclear magnetic resonance (NMR) spectroscopy combined with multivariate statistical analysis can provide tools to help detect differences in plant chemistry when grown under varying conditions. Hypericum perforatum, or Saint John's wort, plants are a suitable model to explore methods of discrimination between early stage plants grown in different conditions. OBJECTIVES The purpose of this work was to develop a method for identifying differences in chemical profiles between young Hypericum perforatum plants grown under different lighting conditions. MATERIAL AND METHODS Cuttings were grown for 3 weeks under different light conditions. Plant extracts were prepared in MeOD-d4 and analysed by 1 H-NMR. A multivariate analysis method of the NMR data was developed in an effort to determine variations in chemical profiles. RESULTS The method identified specific metabolites as drivers of difference between the plants grown under different light conditions. STOCSY (statistical total correlation spectroscopy) and quantification of highlighted metabolites supported the findings of the multivariate analysis. Glutamine, sucrose and fructose were found to be chemical markers of light quality in this study. CONCLUSION NMR metabolomics using a medium field instrument could find differences in plant chemistry when grown in different conditions. This method could easily be extended to benchtop instruments and be used for crop monitoring and growth condition optimisation.

Wildfires are having both devastating and regenerative impacts on the ecosystems in the Pacific Northwest of North America. Balsamorhiza sagittata and B. deltoidea (balsamroot) are ecologically important species in this region, and B. sagittata populations are increasing, while B. deltoidea is critically imperiled. The aim of this research was to establish in vitro protocols for germination and regeneration of Balsamorhiza spp. to enable conservation efforts. It was hypothesized that karrikins, which are plant growth regulators released from burning plants during wildfires, would induce seed germination in Balsamorhiza spp. Three karrikins (KAR1, KAR2, and KAR11) were tested for the ability to enhance germination in these species at two levels (5 or 10 μM). KAR2 had the strongest positive effect on germination and induced 47% and 60% germination, respectively, in B. sagittata seeds compared to 14% germination obtained in the control (water agar media). In B. deltoidea, KAR2 treatment resulted in a germination rate of 73.1% and 100%, compared to 69% in the control. A germplasm collection of seedlings of both species was established for conservation and regeneration experiments. Thidiazuron treatment (10 μM) induced formation of embryo-like structures in seedlings of both B. sagittata and B. deltoidea, with regenerants originating from the crown of seedlings. The present study provides in vitro methods for conservation and mass propagation of Balsamorhiza species.

β-N-Methylamino-L-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria that can accumulate in ecosystems and food webs. Human exposure to cyanobacterial and algal blooms may be a risk factor for neurodegenerative diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis. Analytical chemists have struggled to find reliable methods for BMAA analysis in complex sample matrices. Analysis of BMAA is complicated by at least 3 naturally occurring isomers: N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB), and β-aminomethyl-L-alanine (BAMA). More than 350 publications have reported detection and quantification of BMAA and its isomers, but varying results have led to controversy in the literature. The objective of this study was to perform a single laboratory validation (SLV) of a frequently published method for BMAA analysis using a ZIC-HILIC column. We investigated the selectivity, linearity, accuracy, precision, and sensitivity of the method and our data show that this HILIC method fails many of the criteria for a validated method. The method fails the criterion for selectivity as the chromatography does not separate BMAA from its isomer BAMA. Sensitivity of the method greatly decreased over the experimental period and it demonstrated a higher limit of detection (LOD) (7.5 pg on column) and a higher lower limit of quantification (LLOQ) (30 pg on column) than other published validated methods. The method demonstrated poor precision of repeated injections of standards of BMAA with % relative standard deviation (%RSD) values that ranged from 37 to 107% while HorRat values for BMAA had a fail rate of 80% and BAMA had a fail rate of 73%. No HorRat values between 0.5 and 2 were found for repeated injections of standards of AEG and DAB. Recovery of 13C3,15N2-BMAA in a cyanobacterial matrix was

Cyanobacteria are among the earth’s oldest known living groups of organisms and can form layered accretions called microbialites, found in both the fossil record and existing lakes. Studies of cyanobacterial biochemical processes help to understand the evolution of life on earth. The conserved metabolism of cyanobacterial species includes the biosynthesis of unusual nonprotein amino acids such as N-(2-aminoethyl)glycine, hypothesized to have constituted an early form of genetic information in cells. Pavilion Lake in British Columbia, Canada, hosts a population of unique, actively growing microbialites covered in biofilms dominated by cyanobacteria. We hypothesized that the living microbial communities produce dinitrogenous nonprotein amino acids, such as N-(2-aminoethyl)glycine and its structural isomers β-N-methylamino-l-alanine, 2,4-diaminobutyric acid and β-aminomethyl-l-alanine. We analyzed samples in sediment traps collected between 2007 and 2014 in depths ranging from 11 to 46 m. N-(2-aminoethyl)glycine, 2,4-diaminobutyric acid and β-aminomethyl-l-alanine were found in highest concentration in the shallowest microbialite biofilms with a maximum of 22 ng/g, 33 ng/g and 0.4 ng/g, respectively. In contrast, the concentration of β-N-methylamino-l-alanine was highest in collections between 18 and 26 m depths and only β-N-methylamino-l-alanine was found in the deepest water collections. These data provide evidence indicating that the production of these nonprotein amino acids is highly conserved through the evolution of cyanobacteria and suggest that the nitrogen-rich metabolites may have had both an important role in ancient and modern cyanobacterial metabolism. Further research will determine the role of N-(2-aminoethyl)glycine and its isomers in early life metabolism and their current function in photosynthetic cells.

The phytochemical diversity of Cannabis chemovars is not well understood, and many chemovars were created in informal breeding programs without records of parentage or the criteria for selection. Key criteria for selection sometimes included aroma notes and visual cues, which some breeders associated with pharmacological activity. We hypothesized that the process of selection for scents believed to be related to specific tetrahydrocannabinol levels has resulted in modified terpene biosynthesis in these chemovars. Thirty-two cannabinoids, 29 monoterpenes and 38 sesquiterpenes were measured in 33 chemovars from 5 licensed producers. A classification system based on cannabinoid content was used with targeted metabolomic tools to determine relationships in the phytochemistry. Three monoterpenes, limonene, β-myrcene, and α-pinene, and two sesquiterpenes, caryophyllene and humulene, were abundant in the majority of chemovars. Nine terpenes were present in tetrahydrocannabinol-dominant chemovars. Three monoterpenes and four sesquiterpenes were predominantly found in cannabidiol-containing chemovars. Low abundance terpenes may have been the aromatic cues identified by breeders. The medicinal activity of some of the terpenes is likely to contribute to the pharmacological effect of specific chemovars. Together, these data demonstrate the synergy of compounds in Cannabis chemovars and point to the need for additional research to understand the phytochemical complexity.

Climate change is now a reality and is altering ecosystems, with Canada experiencing 2-4 times the global average rate of warming. This will have a critical impact on berry cultivation and horticulture. Enhancing our understanding of how wild and cultivated berries will perform under changing climates will be essential to mitigating impacts on ecosystems, culture and food security. Our objective was to predict the impact of climate change on habitat suitability of four berry producing Vaccinium species: two species with primarily northern distributions (V. uliginosum, V. vitis-idaea), one species with a primarily southern distribution (V. oxycoccos), and the commercially cultivated V. macrocarpon. We used the maximum entropy (Maxent) model and the CMIP6 shared socioeconomic pathways (SSPs) 126 and 585 projected to 2041-2060 and 2061-2080. Wild species showed a uniform northward progression and expansion of suitable habitat. Our modeling predicts that suitable growing regions for commercial cranberries are also likely to shift with some farms becoming unsuitable for the current varieties and other regions becoming more suitable for cranberry farms. Both V. macrocarpon and V. oxycoccos showed a high dependence on precipitation-associated variables. Vaccinium vitis-idaea and V. uliginosum had a greater number of variables with smaller contributions which may improve their resilience to individual climactic events. Future competition between commercial cranberry farms and wild berries in protected areas could lead to conflicts between agriculture and conservation priorities. New varieties of commercial berries are required to maintain current commercial berry farms.

Tart cherries (TC) are a rich source of polyphenols that elicit antioxidant and anti-inflammatory effects. As a consequence, the effects of TC derived supplements on markers of human health, exercise performance and sleep have been investigated. Supplementation protocols have been highly variable across studies and the dose of bioactive compounds used has often been poorly characterized. Specific and non-specific analytical methods were employed for measuring the total polyphenol and anthocyanin content in TC supplements. This review critically analyses the supplementation protocols and the analytical methods used for the characterization of TC supplements, culminating in recommendations for good practice in the analysis and reporting of the polyphenol content and profile of TC products. A literature search was conducted using PubMed/Medline and Web of Science up to May 4th, 2020, including studies published in all years prior. Only articles written in English that provided a TC dietary supplement as opposed to fresh whole TC were included in this review. Forty-three studies were identified as eligible and included for analysis in this review. The studies investigated the effects of TC supplementation on various aspects of human health, exercise recovery and performance and sleep. Twenty studies conducted an analysis of TC supplement and reported total polyphenol/anthocyanin content. Six studies did not report the polyphenol content of the TC supplement used. Seventeen studies reported the TC supplement polyphenol content but this was derived from previously published studies and presumably different supplement batches. The duration of the supplementation protocol ranged from acute supplementation to 84 days, meanwhile the total polyphenol and anthocyanin dose ranged from 143 to 2,140 mg/day and 15 to 547 mg/day, respectively. Due to the variety of specific and non-specific analytical methods used, the relative efficacy of different doses and polyphenol blends cannot reliably be extrapolated from critical analysis of the literature. Future studies should conduct an analysis of the study supplement batch. In addition to analysis and reporting of total polyphenol content, specific analytical methods such as HPLC UV/MS should be used to quantify total and individual anthocyanin contents.

Breadfruit is a traditional staple crop from Pacific islands with the potential to improve worldwide food security and mitigate diabetes. Flour produced from breadfruit is a gluten-free, low glycemic index, nutrient dense and complete protein option for modern foods but basic scientific knowledge of health impacts of a breadfruit-based diet in animals and humans was lacking. We designed a series of studies to provide basic and fundamental data on impacts of a breadfruit-based diet through an in vitro and in vivo model. Cooked breadfruit flour was digested through a multi-stage enzyme digestion model to estimate protein digestibility in comparison to wheat flour. Breadfruit protein was found to be easier to digest than wheat protein in the enzyme digestion model. The flour digestions were applied to Caco-2 cells to test the cytotoxicity and to measure the immunogenicity through cytokine expression. No significant differences were observed for immune factors and cytokines (IL-4, IL-10, IL-8, TNF-α, IFN-γ) on Caco-2 cells between the breadfruit and wheat groups. A breadfruit-based rodent chow was formulated by substitution of all of the wheat in the standard formulation with breadfruit. The diets were isocaloric, nutrient equivalent and used to feed male and female C57BL/6 mice for 21 days. No sign of malnutrition, discomfort, illness or death was observed among the mice because of the diet. The histology and the cytokine expression of the mice ileum from both groups were analyzed and showed similar results. The expression of major bacteria was measured in the colon and showed similar results. Mice fed the breadfruit diet had a significantly higher growth rate and body weight than standard diet fed mice. No negative health outcomes were observed in studies with in vitro or in vivo models and breadfruit flour is a healthy alternative to other starches for modern foods.

Cabbage looper (Trichoplusia ni) larvae are generalist herbivores that feed on numerous cultivated plants and weeds including crucifers, other vegetables, flowers, and field crops. Consuming plant material from a wide range of plant species exposes these larvae to a considerable variety of plant secondary metabolites involved in chemical defense against herbivory. The ability of the cabbage looper larvae to detoxify plant secondary metabolites, such as nicotine, has been attributed to the rapid induction of excretion via the Malpighian tubules. However, the role of metabolism in the detoxification of plant secondary metabolites in cabbage looper larvae is not well studied. We investigated nicotine metabolism in 4th larval instar cabbage looper using UPLC-MS/MS analysis to resolve the time course of nicotine metabolism, the kinetic distribution of nicotine, and the presence or absence of major metabolites of nicotine in larval tissue and excretions. The major metabolite found in our analysis was cotinine, with trace amounts of cotinine N-oxide and nicotine N-oxide. The nicotine metabolites detected are similar to those of the nicotine-tolerant Lepidopteran tobacco hornworm (Manduca sexta). The results of our study demonstrate that the 5′C-oxidation of nicotine to cotinine is the primary pathway for nicotine metabolism in cabbage looper larvae. This study showed that metabolism of nicotine and subsequent excretion of nicotine and its metabolites occurs in the larvae of the cabbage looper. Our results suggest that 5′C-oxidation in lepidopteran insects is a conserved metabolic pathway for the detoxification of plant secondary metabolites.

In June 2016, massive cyanobacterial blooms occurred in the St. Lucie River in Florida, caused by nutrient and cyanobacterial-laden water releases from Lake Okeechobee. We independently collected and analyzed bloom material for cyanotoxin diversity and concentrations. The concentrations of microcystins, potent hepatotoxins, present in the bloom material greatly exceeded World Health Organization Guideline Values for drinking and recreational water. We also detected the neurotoxins anatoxin-a(S) and β-N-methylamino-L-alanine (BMAA). The Florida State Governor declared a state of emergency, but many affected aquatic recreational areas in St. Lucie County remained open during the bloom event without adequate hazard notification to citizens. During the bloom event, issues with preparedness, communication, sampling, analysis, closures and contingencies were observed. We suggest better ways that cyanobacterial bloom events can be predicted, managed, and mitigated in the future throughout the world. As similar problems with cyanobacterial bloom frequency and occurrence present worldwide, understanding governmental responses to the 2016 Florida incident can help in the development of effective mitigation and management strategies for future bloom events.

Piper methysticum (Kava) is a plant whose roots are used in the preparation of traditional beverages with spiritual, medicinal, and social importance for the Pacific Islanders. Kava is also sold as a herbal supplement or recreational beverage consumed for its mild inebriating effect in Europe and North America. With an ongoing interest in the safety and quality of kava products, it is necessary to develop a validated method for determination of kava chemical composition to ensure confidence in quality assessment. Thus, an high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method was developed, optimized, and validated for determining six major kavalactones and three flavokavains in kava raw materials and finished products based on AOAC single-laboratory validation guidelines. This is the first fully validated analytical method for measuring kavalactones and flavokavains in a single run. The separation of the analytes was achieved in 10 min with an Agilent Poroshell C18 column using gradient separation. The sample was extracted with methanol first and then acetone. The signals were detected at 240 nm and 355 nm. The limit of quantification was under 1.2 µg/mL (0.3 mg/g) for kavalactones and under 0.35 µg/mL (0.01 mg/g) for flavokavains. The Horwitz ratio values described ranged from 0.3 to 1.82. The spike recovery experiments showed an accuracy between 92 and 105% for all analytes. The results of the study demonstrate that the method is fit for the purpose of determining methysticin, dihydromethysticin, kavain, dihydrokavain, yangonin, desmethoxyyangonin, flavokavain A, flavokavain B, and flavokavain C in kava raw material and finished products (dry-filled capsule, liquid phytocaps, and tincture).

Breadfruit (Artocarpus altilis and A. altilis × A. mariannensis) is a sustainable and highly nutritious staple crop that has been granted “Generally Recognised as Safe” status by the FDA. There are more than 300 cultivars of breadfruit selected by Indigenous Peoples of the Pacific Islands. To date, specific starch characteristics of individual cultivars have not been investigated. We hypothesized that starch and fiber vary in type, yield, and quality across breadfruit cultivars. The starch yield, structure, swelling power, solubility, dietary fiber, and starch-bound protein of starch isolated from 16 breadfruit cultivars were analyzed. Starch granule size ranged from 2.04 μm to 17.66 μm with the starch structures of hybrid cultivars appearing uniquely membranous. Three cultivars, Yuley, Ulu fiti, and Ma’afala, had particularly distinguishing characteristics. Ma’afala had the highest protein content. Ulu fiti had the highest starch yield but the lowest swelling power and solubility. Yuley had the highest dietary fibre (total, soluble, insoluble), swelling power, and low starch-bound protein. This variability in characteristics suggests Yuley is an ideal cultivar for various starch applications in foods, pharmaceuticals, industrial chemicals, and other applications.

Plants respond to changes in their environments through hormonal activation of a physiological cascade that redirects metabolic resources and growth. In filberts (Corylus sp.), chelated iron promotes the growth of new shoots but the mechanism(s) are not understood. To use untargeted metabolomics and hormonomics approaches to generate novel hypotheses for the morphoregulatory role of ferric ethylenediamine-N,N'-di-(ortho-hydroxyphenyl) acetic acid (Fe-EDDHA) in filbert shoot organogenesis in vitro. Data were generated using previously optimized standardized untargeted metabolomics protocols with time of flight mass spectrometry. Multivariate statistical tools (principal component and partial least squares discriminant analysis) did not detect significant differences. Discovery tools Significance Analysis of Microarrays (SAM), multiple linear regression analysis, Bayesian analysis, logical algorithms, machine learning, synthetic biotransformations, targeted hormonomics, and online resources including MetaboAnalyst were used. Starch/sucrose metabolism and shikimate pathway metabolites were increased. Dose dependent decreases were found in polyphenol metabolism, specifically ellagic acid and its methylated derivative 3,4,3′-tri-O-methylellagic acid. Hormonomics analysis revealed significant differences in phytohormones and their conjugates. FeEDDHA treatment reduced indole-3-acetic acid, abscisic acid, salicylic acid, jasmonic acid conjugates (JA-Trp, JA-Ile, OH-JA) and dihydrozeatinglucoside in regenerating explants. Serotonin (5HT) was decreased in FeEDDHA-treated regenerating tissues while the related metabolite melatonin was increased. Eight phenolic conjugates of 5HT and eight catabolites were affected by FeEDDHA indicating that metabolism to sequester, deactivate and metabolize 5HT was induced by Fe(III). Tryptophan was metabolized through kynurenine but not anthranilate. Seven novel hypotheses were generated to guide future studies to understand the regulatory control(s) of shoot organogenesis.

Nutraceuticals are foods containing medicinally active plant secondary metabolites that can be beneficial to human health, but it is hubris to think that the plants make these secondary metabolites solely for our benefit. Plants make secondary metabolites in order to adapt, survive and thrive in hostile environments. Plants cannot flee from danger or migrate to a new place in search of better opportunities for growth. Plants must sense changes in their environments and respond to those changes through the production of specialized chemicals. Plants sense their environment through a complex system of plant growth regulators and signaling molecules including melatonin and serotonin. When faced with an environmental challenge, plants produce these signaling molecules to initiate metabolic cascades that redistribute metabolic resources. Changing climates are exposing plants to more and different challenges than they have previously encountered, and these environmental cues are stimulating plants to alter secondary metabolite production. This chapter will highlight some of the plant responses to changing climates and the impact of changing environments on medicinally active plant chemicals in nutraceuticals.

Nuclear magnetic resonance (NMR) spectroscopy combined with multivariate statistical analysis can provide tools to help detect differences in plant chemistry when grown under varying conditions. Hypericum perforatum, or Saint John's wort, plants are a suitable model to explore methods of discrimination between early stage plants grown in different conditions.The purpose of this work was to develop a method for identifying differences in chemical profiles between young Hypericum perforatum plants grown under different lighting conditions.Cuttings were grown for 3 weeks under different light conditions. Plant extracts were prepared in MeOD-dThe method identified specific metabolites as drivers of difference between the plants grown under different light conditions. STOCSY (statistical total correlation spectroscopy) and quantification of highlighted metabolites supported the findings of the multivariate analysis. Glutamine, sucrose and fructose were found to be chemical markers of light quality in this study.NMR metabolomics using a medium field instrument could find differences in plant chemistry when grown in different conditions. This method could easily be extended to benchtop instruments and be used for crop monitoring and growth condition optimisation.

Neurodegenerative diseases are influenced by environmental factors such as exposure to toxins including the cyanotoxin β-N-methylamino-l-alanine (BMAA) that can bioaccumulate in common food sources such as fish, mussels and crabs. Accurate and precise analytical methods are needed to detect and quantify BMAA to minimize human health risks. The objective of this review is to provide a comprehensive overview of the methods used for BMAA analysis from 2003 to 2019 and to evaluate the reported performance characteristics for each method to determine the consensus data for each analytical approach and different sample matrices. Detailed searches of the database Web of Science™ (WoS) were performed between August 21st, 2018 and April 5th, 2019. Eligible studies included analytical methods for the detection and quantification of BMAA in cyanobacteria and bioaccumulated BMAA in higher trophic levels, in phytoplankton and zooplankton and in human tissues and fluids. This systematic review has limitations in that only the English language literature is included and it did not include standard operating protocols nor any method validation data that have not been made public. We identified 148 eligible studies, of which a positive result for BMAA in one or more samples analyzed was reported in 84% (125 out of 148) of total studies, 57% of HILIC studies, 92% of RPLC studies and 71% of other studies. The largest discrepancy between different methods arose from the analysis of cyanobacteria samples, where BMAA was detected in 95% of RPLC studies but only in 25% of HILIC studies. Without sufficient published validation of each method's performance characteristics, it is difficult to establish each method as fit for purpose for each sample matrix. The importance of establishing methods as appropriate for their intended use is evidenced by the inconsistent reporting of BMAA across environmental samples, despite its prevalence in diverse ecosystems and food webs.

There is an explosion in the number of labs analyzing cannabinoids in marijuana (Cannabis sativa L., Cannabaceae) but existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging solvents. The objective of this work was to develop and validate an accurate method for analyzing cannabinoids in cannabis raw materials and finished products that is more efficient and uses fewer toxic solvents. An HPLC-DAD method was developed for eight cannabinoids in cannabis flowers and oils using a statistically guided optimization plan based on the principles of green chemistry. A single-laboratory validation determined the linearity, selectivity, accuracy, repeatability, intermediate precision, limit of detection, and limit of quantitation of the method. Amounts of individual cannabinoids above the limit of quantitation in the flowers ranged from 0.02 to 14.9% w/w, with repeatability ranging from 0.78 to 10.08% relative standard deviation. The intermediate precision determined using HorRat ratios ranged from 0.3 to 2.0. The LOQs for individual cannabinoids in flowers ranged from 0.02 to 0.17% w/w. This is a significant improvement over previous methods and is suitable for a wide range of applications including regulatory compliance, clinical studies, direct patient medical services, and commercial suppliers. Electronic supplementary material The online version of this article (doi:10.1007/s00216-017-0256-3) contains supplementary material, which is available to authorized users.

cv. Anthos) is presented. Isotope tracer experiments were performed on plantlets regenerated from thidiazuron-induced stem explants and grown on MS basal medium for 2 months. Radiolabel from 14C-tryptophan was recovered as 14C-indoleacetic acid, 14C-tryptamine, 14C-5-hydroxytryptophan, 14C-serotonin and 14C-melatonin in the treated St. John's wort plantlets. Chromatographic peak identity was confirmed by high performance liquid chromatography-mass spectrometry-mass spectrometry and quantification of melatonin by radioimmunoassay. Significantly more radiolabel was recovered in serotonin relative to melatonin under low light conditions with this ratio being reversed under increased lighting, indicating that the rate of flow through this biosynthetic pathway is regulated, at least in part, by light.

Cannabis is an interesting domesticated crop with a long history of cultivation and use. Strains have been selected through informal breeding programs with undisclosed parentage and criteria. The term “strain” refers to minor morphological differences and grower branding rather than distinct cultivated varieties. We hypothesized that strains sold by different licensed producers are chemotaxonomically indistinguishable and that the commercial practice of identifying strains by the ratio of total THC and CBD is insufficient to account for the reported human health outcomes. We used targeted metabolomics to analyze 11 known cannabinoids and an untargeted metabolomics approach to identify 21 unknown cannabinoids. Five clusters of chemotaxonomically indistinguishable strains were identified from the 33 commercial products. Only 3 of the clusters produce CBDA in significant quantities while the other 2 clusters redirect metabolic resources toward the THCA production pathways. Six unknown metabolites were unique to CBD-rich strains and/or correlated to CBDA and 3 unknowns were found only in THC-rich strains. Together, these data indicate the domestication of the cannabis germplasm has resulted in a loss of the CBDA pathway in some strains and reallocation of resources between CBDA and THCA pathways in others. The impact of domestication is a lack of chemical diversity and loss of biodiversity in modern cannabis strains.

Melatonin and serotonin are important phytochemicals enabling plants to redirect growth in response to environmental stresses. Despite much research on their biosynthetic routes, localization of their biosynthetic enzymes and recent identification of a phytomelatonin receptor, localization of the molecules themselves has to date not been possible. Elucidation of their locations in living tissues can provide an effective tool to facilitate indolamine research across systems including both plants and animals. In this study, we employed a novel technique, quantum dot nanoparticles, to directly visualize melatonin and serotonin in axenic roots. Melatonin was absorbed through epidermal cells, travelled laterally, and accumulated in endodermal and rapidly dividing pericycle cells. Serotonin was absorbed by cells proximal to the crown with rapid polar movement toward the root tip. Thermal stress disrupted localization and dispersed melatonin and serotonin across cells. These data demonstrate the natural movement of melatonin and serotonin in roots directing cell growth and suggest that plants have a mechanism to disperse the indolamines throughout tissues as antioxidants in response to environmental stresses.

Serotonin (5-hydroxytryptamine) is an indoleamine with important physiological functions in plants, including developmental processes and stress responses. In this study, a simple and rapid bioassay was developed to investigate the inhibition of the fruit browning enzyme, polyphenol oxidase (PPO) via serotonin, employing cut apple discs as well as apple tissue homogenate treated with serotonin. The present study demonstrated that serotonin is significantly effective in inhibiting browning compared to ascorbic acid. Serotonin reduced fruit browning in tissues as well as suppressed the activity of PPO enzyme in the browning model solutions containing epicatechin (Epi) and caffeoyl tartrate (CT) as polyphenol substrates. The determination of enzyme kinetic parameters also suggested that serotonin may act as a putative, non-competitive inhibitor of PPO. Apart from its anti-browning effect, the application of serotonin significantly increased total phenolics content and antioxidant capacity when applied to the apple homogenate. Furthermore, the analysis of MdPPOs showed that five (MdPPO2, MdPPO4, MdPPO6, MdPPO8 and MdPPO10) out of the nine MdPPOs expressed in apple fruits were down-regulated in apple tissues treated with serotonin. To the best of our knowledge, this is the first report documenting a role for serotonin in the biochemical and transcriptional regulation of PPO enzymes and a simple system to study the biochemical control of browning in pome fruits.

A single-laboratory validation study was completed for the determination of β- N -methylamino-L-alanine (BMAA), N -(2-aminoethyl)glycine (AEG), and 2,4-diaminobutyric acid (DAB) in bulk natural health product supplements purchased from a health food store in Canada. BMAA and its isomers were extracted with acid hydrolysis to free analytes from protein association. Acid was removed with the residue evaporated to dryness and reconstituted with derivatization using 6-aminoquinolyl- N - hydroxysuccinimidyl carbamate (AccQ-Fluor). Chromatographic separation and detection were achieved using RP ultra-performance LC coupled to a tandem mass spectrometer operated in multiple reaction monitoring mode. Data from biological samples were evaluated for precision and accuracy across different days to ensure repeatability. Accuracy was assessed by spike recovery of biological samples using varying amino acid concentrations, with an average recovery across all samples of 108.6%. The analytical range was found to be 764–0.746 ng/mL prior to derivatization, thereby providing a linear range compatible with potentially widely varying analyte concentrations in commercial health food products. Both the U. S. Food and Drug Administration (FDA) and U. S. Pharmacopeia definitions were evaluated for determining method limits, with the FDA approach found to be most suitable having an LOD of 0.187 ng/mL and LLOQ of 0.746 ng/mL. BMAA in the collected specimens was detected at concentrations lower than 1 μg/g, while AEG and DAB were found at concentrations as high as 100 μg/g. Finding these analytes, even at low concentrations, has potential public health significance and suggests a need to screen such products prior to distribution. The method described provides a rapid, accurate, and precise method to facilitate that screening process.

More than 80% of the world's hungry live in tropical and subtropical regions where small increases in the costs of imported food, fuel and fertilizer create periods of increased food insecurity. Over time, the traditional knowledge and the traditional crops of the region are being lost and diet-based diseases such as type II diabetes are increasing in frequency. Breadfruit, Artocarpus altilis (Parkinson) Fosberg, has been a staple food and traditional crop in the Pacific for more than 3000 years and is now being cultivated for food security in the Caribbean and other tropical regions. While there is some evidence to suggest that a traditional diet based on breadfruit and other Pacific staples can prevent onset of type II diabetes, detailed scientific studies have not been conducted. One of the important issues is the wide variability in reported nutritional composition of the fruit in studies that included many different cultivars grown in widely different ecosystems. We conducted a review of the nutritional data to determine the best consensus for fruit nutrition. We identified 41 individual studies that provide some proximate, carbohydrate, vitamin and/or mineral data. A majority of the studies do not provide sufficient botanical data such as species, cultivar name, or descriptive information that would indicate the stage of maturity of the fruit or factors of the local environment such as soil composition or rainfall. Despite these shortcomings, compositional data for breadfruit suggests that it has potential to mitigate type II diabetes and obesity in Oceania and elsewhere in the tropics where breadfruit is grown. Further studies will identify specific elite cultivars recommended for this purpose.

Primulina dryas ( P. dryas ), a popular ornamental plant species has recently gained interest among hobbyists and collectors due to its attractive foliage and flowers. An efficient protocol for in vitro regeneration of P. dryas from leaf and petiole explants was developed using thidiazuron (TDZ) as the sole inductive stimulus. The objective of this study was to evaluate the potential of P. dryas for mass propagation in vitro. Orientation of the explants on the culture medium also affected regeneration with significant enhancement in the number of shoots and somatic embryos when the abaxial surface of the leaf was in contact with the culture medium. Explants also showed a capacity to regenerate on a medium devoid of growth regulators indicating active endogenous hormone metabolism sufficient to stimulate de novo differentiation. The inhibitors of auxin biosynthesis and transport, p -chlorophenoxyisobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid (TIBA) significantly reduced regeneration in an optimal growth environment suggesting the involvement of endogenous auxin in regeneration of P. dryas . Growth and development of regenerants was further improved in temporary immersion bioreactors and the resulting plants were successfully transplanted in the greenhouse. High efficiency and reproducibility of Primulina regeneration system reported here may facilitate large scale production of this ornamental species.

Hypericum perforatum L. (St. John’s wort) is an important medicinal herb and a subject of intensive research for its complex and diverse bioactive chemicals. An in vitro-grown germplasm collection of elite H. perforatum lines, established to provide easy access to physiologically uniform plants, was used for ploidy assessment studies. Germplasm lines were maintained by repeated subculture of shoot tips for over 10 yr with little change in their capacity to produce multiple shoots. Shoots of four of these lines were rooted and grown in the greenhouse to obtain plants to provide anther and filament explants. Culture of explants on a regeneration medium supplemented with 1 mg L-1 ?-naphthaleneacetic acid (NAA) and 1 mg L-1 6-benzylaminopurine (BA) induced large numbers of calluses and shoots on all explants. Flow cytometric (FCM) analysis of nuclei samples revealed that the nuclear DNA contents of calluses and shoots developed from anther and filament explants of germplasm lines were not significantly different from those of the donor plants. FCM screening of in vitro-maintained germplasm lines in the collection showed that they had similar nuclear DNA amounts and were all tetraploid (2n = 4x). Analysis of seedlings obtained from the original seed source used to derive the germplasm lines showed that ~11% of them were hexaploid (2n = 6x). Data obtained from FCM screens confirmed the preservation of tetraploidy in in vitro-maintained H. perforatum germplasm and the regenerants obtained from male floral organs. The consistent ploidy of the H. perforatum plants of in vitro origin further supports the usefulness of such technologies to ensure genetic uniformity of medicinal plants over extended periods of culture and may facilitate long-term preservation of their elite clones. © 2015, The Society for In Vitro Biology.

Metabolomics is the qualitative and quantitative analysis of all of the small molecules in a biological sample at a specific time and influence. Technologies for metabolomics analysis have developed rapidly as new analytical tools for chemical separations, mass spectrometry, and NMR spectroscopy have emerged. Plants have one of the largest metabolomes, and it is estimated that the average plant leaf can contain upward of 30 000 phytochemicals. In the past decade, over 1200 papers on plant metabolomics have been published. A standard metabolomics data set contains vast amounts of information and can either investigate or generate hypotheses. The key factors in using plant metabolomics data most effectively are the experimental design, authentic standard availability, extract standardization, and statistical analysis. Using cranberry (Vaccinium macrocarpon) as a model system, this review will discuss and demonstrate strategies and tools for analysis and interpretation of metabolomics data sets including eliminating false discoveries and determining significance, metabolite clustering, and logical algorithms for discovery of new metabolites and pathways. Together these metabolomics tools represent an entirely new pipeline for phytochemical discovery.

Protein deficiency has been observed as a leading cause of malnutrition and child death in the tropics. The current study evaluated the protein quality of 49 important breadfruit cultivars (41 Artocarpus altilis and 8 hybrids of A. altilis × A. mariannensis). While significant differences were found between cultivars, all varieties contained a full spectrum of the essential amino acids and are especially rich in phenylalanine, leucine, isoleucine, and valine. The cultivar Ma'afala contained significantly higher total essential amino acid content than other varieties and higher-quality protein than staples such as corn, wheat, rice, soybean, potato, and pea.

Arbuscular mycorrhizal (AM) fungi can affect many different micronutrients and macronutrients in plants and also influence host volatile compound synthesis. Their effect on the edible portions of plants is less clear. Two separate studies were performed to investigate whether inoculation by AM fungi (Rhizophagus irregularis, Funneliformis mosseae, or both) can affect the food quality of tomato fruits, in particular common minerals, antioxidants, carotenoids, a suite of vitamins, and flavor compounds (sugars, titratable acids, volatile compounds). It was found that AM fungal inoculation increased the nutrient quality of tomato fruits for most nutrients except vitamins. Fruit mineral concentration increased with inoculation (particularly N, P, and Cu). Similarly, inoculated plants had fruit with higher antioxidant capacity and more carotenoids. Furthermore, five volatile compounds were significantly higher in AM plants compared with non-AM controls. Taken together, these results show that AM fungi represent a promising resource for improving both sustainable food production and human nutritional needs.

Introduction. Breadfruit, Artocarpus spp., is a staple crop with the potential to alleviate hunger and increase food security in tropical regions. Guidelines and recommenda- tions for cultivar selection and production practices are now required for establishment of breadfruit in new areas. Materials and methods. To respond to this need for spreading breadfruit, our study quantified the growth, development, yield and seasonality of 24 bread- fruit cultivars (26 trees) established in Kauai, Hawaii, over a 7-year period from 2006-2012. Individual production profiles were generated for each accessioned cultivar based on major agricultural factors. Results. Across all cultivars of breadfruit (A. altilis), an average of 269 fruits per year was produced by each tree with an average fruit weight of 1.2 kg. Based on the planting density of 50 trees⋅ha -1 , this translates to an average projected yield of 5.23 t⋅ha -1 after 7 years. Hybrids (A. altilis × A. mariannensis) had a higher yield than bread- fruit. The data of our article support the previously proposed hypothesis for predicting bread- fruit seasonality. On average, the peak season occurred from July to November. Conclusions. Ma'afala, the first widely available commercial cultivar, started to bear fruit within 22 to 23 months of planting. Other cultivars with potential for commercial production include Toneno, White, Rotuma and Meinpadahk. Hawaii / Artocarpus / fruits / breadfruit / variety trials / choice of species / crop yield / seasonality / adaptation

Artemisia tridentata Nutt. and related species in the subgenus Tridentatae are ecologically important plants with a rich history of ceremonial and medicinal use by the indigenous people of North America. With the exception of antimicrobial and insecticidal bioassays, there is limited data to support the traditional uses of A. tridentata. Additionally, wild A. tridentata populations are declining and conservation of genetic resources is warranted. The current study was designed to (a) develop in vitro protocols to conserve A. tridentata in axenic culture and to provide plant tissues for phytochemical analysis, (b) to investigate the presence of neurologically-active phytochemicals in A. tridentata and (c) to develop metabolomics as a tool for understanding secondary metabolite biosynthesis under aseptic conditions. A collection of in vitro-grown germplasm lines was established from wild-harvested seeds of A. tridentata. Neurotransmitters acetylcholine, GABA, melatonin and serotonin were identified and quantified in the plant tissues. Crude extracts of A. tridentata inhibited acetylcholinesterase in a bioassay. A metabolomics analysis with chemometric statistics quantified changes in the phytochemical profiles of wild-harvested plants and plantlets in axenic culture. A total of 1,543 phytochemicals were found in all samples of A. tridentata including 52 significant ions putatively identified as monoterpene, phenolic or sesquiterpene compounds. Together, these data provide the foundation for further investigations of the phytochemical diversity and medicinal activity of A. tridentata and demonstrate a research approach for use of metabolomics as a tool for understanding secondary metabolite biosynthesis under aseptic conditions.