Exogenous regulation of macronutrients promotes the accumulation of alkaloid yield in anisodus tanguticus (Maxim.) pascher.

BACKGROUND: Anisodus tanguticus (Maxim.) Pascher (A. tanguticus) is a valuable botanical for extracting tropane alkaloids, which are widely used in the pharmaceutical industry. Implementing appropriate cultivation methods can improve both the quality and yield of A. tanguticus. A two-year field experiment was conducted from 2021 to 2023 using a single-factor randomized complete block design replicated three times. The study examined the effects of different nutrient levels (nitrogen: 0, 75, 150, 225, 300, 375 kg/ha; phosphorus: 0, 600, 750, 900, 1050, 1200 kg/ha; potassium: 0, 75, 112.5, 150, 187.5, 225 kg/ha) on the growth, primary alkaloid contents, and alkaloid yield of A. tanguticus at different growth stages (S-Greening, S-Growing, S-Wilting; T-Greening, T-Growing, and T-Wilting) in both the roots and aboveground portions. RESULTS: Our results demonstrate that nutrient levels significantly affect the growth and alkaloid accumulation in A. tanguticus. High nitrogen levels (375 kg/ha) notably increased both root and aboveground biomass, while phosphorus had a minimal effect, especially on aboveground biomass. For alkaloid content (scopolamine, anisodamine, anisodine, atropine), a moderate nitrogen level (225 kg/ha) was most effective, followed by low potassium (75 kg/ha), with phosphorus showing a limited impact. Increased phosphorus levels led to a decrease in scopolamine content. During the T-Growing period, moderate nitrogen addition (225 kg/ha) yielded the highest alkaloid levels per unit area (205.79 kg/ha). In the T-Wilting period, low potassium (75 kg/ha) and low phosphorus (750 kg/ha) resulted in alkaloid levels of 146.91 kg/ha and 142.18 kg/ha, respectively. This indicates nitrogen has the most substantial effect on alkaloid accumulation, followed by potassium and phosphorus. The Douglas production function analysis suggests focusing on root biomass and the accumulation of scopolamine and atropine in roots to maximize alkaloid yield in A. tanguticus cultivation. CONCLUSIONS: Our findings show that the optimum harvesting period for A. tanguticus is the T-Wilting period, and that the optimal nitrogen addition is 225 kg/ha, the optimal potassium addition is 75 kg/ha, and the optimal phosphorus addition is 600 kg/ha or less.

Simultaneous Determination of 23 Pyrrolizidine and Tropane Alkaloids in Infusions from Dry Edible Flowers Using Optimized µSPEed® Microextraction Prior to Their Analysis by UHPLC-IT-MS/MS.

A miniaturized solid-phase extraction of two tropane alkaloids (TAs) and twenty-one pyrrolizidine alkaloids (PAs) from infusions of dry edible flowers using optimized µSPEed® technique was developed. The optimization of the µSPEed® methodology involved testing different cartridges and comparing various volumes and numbers of loading cycles. The final conditions allowed for a rapid extraction, taking only 3.5 min. This was achieved using a C18-ODS cartridge, conditioning with 100 µL of methanol (two cycles), loading 100 µL of the infusion sample (seven cycles), and eluting the analytes with 100 µL of methanol (two cycles). Prior to their analysis by UHPLC-IT-MS/MS, the extracts were evaporated and reconstituted in 100 µL of water (0.2% formic acid)/methanol (0.2% ammonia) 95:5 (v/v), allowing for a preconcentration factor of seven times. The methodology was successfully validated obtaining recoveries ranging between 87 and 97%, RSD of less than 12%, and MQL between 0.09 and 0.2 µg/L. The validated methodology was applied to twenty samples of edible flower infusions to evaluate the safety of these products. Two infusion samples obtained from Acmella oleracea and Viola tricolor were contaminated with 0.16 and 0.2 µg/L of scopolamine (TA), respectively, while the infusion of Citrus aurantium was contaminated with intermedine and lycopsamine (PAs) below the MQL.

Mycotoxins and pyrrolizidine alkaloids in herbal dietary supplements.

The market demand for herbal dietary supplements is rapidly growing and such products are becoming more common and accessible to consumers. However, the knowledge about their safety remains incomplete. Herbal dietary supplements are one of the food groups that can contribute significantly to human health concerns arising from chronic exposure to pyrrolizidine alkaloids and mycotoxins. This study aimed to simultaneously determine 79 natural contaminants, including mycotoxins, as well as pyrrolizidine and tropane alkaloids in herbal dietary supplements in one analytical run. Exposure assessment and human health risks were assessed for all compounds included in this study. The total concentration of naturally occurring contaminants in herbal dietary supplements reached 5.3 mg kg-1 and the most frequently detected mycotoxins were tentoxin and alternariol monomethyl ether. The latter was detected with the highest frequency, reaching concentrations up to 2.5 mg kg-1. The obtained results indicate a potential risk to public health related to herbal dietary supplement consumption.

A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna.

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6ß-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer.

Occurrence and health risk assessment of tropane alkaloids in cereal foods consumed in Korea.

Tropane alkaloids (TA) are natural toxins found in certain plants, including cereals, of which atropine and scopolamine are the main species of concern due to their acute toxicity. This study aimed to determine the occurrence of TA in cereal foods and assess the potential health risks associated with their consumption in Korea. TA levels were analyzed in 80 raw and 71 processed cereal samples, which were distributed throughout Korea in 2021, using ultra-performance liquid chromatography-tandem mass spectrometry. At least one of the six TA species, namely atropine, scopolamine, pseudotropine, tropinone, scopine, and 6-hydroxytropinone, was detected in 10 out of the 151 samples at levels ranging from 0.12 to 88.10 µg kg-1. Dietary exposure (mean, 0.23 ng kg-1 bw day-1) to atropine and scopolamine in the Korean population was estimated to be low across all age groups. This is despite considering worst-case scenarios using the total concentrations of atropine and scopolamine in a millet sample, both of which were detected, and 95th percentile consumption for consumers of millet only. Both the hazard index and margin of exposure methods indicated that the current levels of TA exposure from millet consumption were unlikely to pose significant health risks to the Korean population.

Effects of dietary exposure to plant toxins on bioaccumulation, survival, and growth of black soldier fly (Hermetia illucens) larvae and lesser mealworm (Alphitobius diaperinus).

In their natural habitat, insects may bioaccumulate toxins from plants for defence against predators. When insects are accidently raised on feed that is contaminated with toxins from co-harvested herbs, this may pose a health risk when used for human or animal consumption. Plant toxins of particular relevance are the pyrrolizidine alkaloids (PAs), which are genotoxic carcinogens produced by a wide variety of plant species and the tropane alkaloids (TAs) which are produced by a number of Solanaceae species. This study aimed to investigate the transfer of these plant toxins from substrates to black soldier fly larvae (BSFL) and lesser mealworm (LMW). PAs and the TAs atropine and scopolamine were added to insect substrate simulating the presence of different PA- or TA-containing herbs, and BSFL and LMW were grown on these substrates. Bioaccumulation from substrate to insects varied widely among the different plant toxins. Highest bioaccumulation was observed for the PAs europine, rinderine and echinatine. For most PAs and for atropine and scopolamine, bioaccumulation was very low. In the substrate, PA N-oxides were quickly converted to the corresponding tertiary amines. More research is needed to verify the findings of this study at larger scale, and to determine the potential role of the insect and/or substrate microbiome in metabolizing these toxins.

Functional divergence of two arginine decarboxylase genes in tropane alkaloid biosynthesis and root growth in Atropa belladonna.

Putrescine, produced via the arginine decarboxylase (ADC)/ornithine decarboxylase (ODC)-mediated pathway, is an initial precursor for polyamines metabolism and the root-specific biosynthesis of medicinal tropane alkaloids (TAs). These alkaloids are widely used as muscarinic acetylcholine antagonists in clinics. Although the functions of ODC in biosynthesis of polyamines and TAs have been well investigated, the role of ADC is still poorly understood. In this study, enzyme inhibitor treatment showed that ADC was involved in the biosynthesis of putrescine-derived metabolites and root growth in Atropa belladonna. Further analysis found that there were six ADC unigenes in the A. belladonna transcriptome, with two of them, AbADC1 and AbADC2, exhibiting high expression in the roots. To investigate their roles in TAs/polyamines metabolism and root growth, RNA interference (RNAi) was used to suppress either AbADC1 or AbADC2 expression in A. belladonna hairy roots. Suppression of the AbADC1 expression resulted in a significant reduction in the putrescine content and hairy root biomass. However, it had no noticeable effect on the levels of N-methylputrescine and the TAs hyoscyamine, anisodamine, and scopolamine. On the other hand, suppression of AbADC2 expression markedly reduced the levels of putrescine, N-methylputrescine, and TAs, but had no significant effect on hairy root biomass. According to ß-glucuronidase (GUS) staining assays, AbADC1 was mainly expressed in the root elongation and division region while AbADC2 was mainly expressed in the cylinder of the root maturation region. These differences in expression led to functional divergence, with AbADC1 primarily regulating root growth and AbADC2 contributing to TA biosynthesis.

A Trip Back Home: Resistance to Herbivores of Native and Non-Native Plant Populations of Datura stramonium.

When colonizing new ranges, plant populations may benefit from the absence of the checks imposed by the enemies, herbivores, and pathogens that regulated their numbers in their original range. Therefore, rates of plant damage or infestation by natural enemies are expected to be lower in the new range. Exposing both non-native and native plant populations in the native range, where native herbivores are present, can be used to test whether resistance mechanisms have diverged between populations. Datura stramonium is native to the Americas but widely distributed in Spain, where populations show lower herbivore damage than populations in the native range. We established experiments in two localities in the native range (Mexico), exposing two native and two non-native D. stramonium populations to natural herbivores. Plant performance differed between the localities, as did the abundance of the main specialist herbivore, Lema daturaphila. In Teotihuacán, where L. daturaphila is common, native plants had significantly more adult beetles and herbivore damage than non-native plants. The degree of infestation by the specialist seed predator Trichobaris soror differed among populations and between sites, but the native Ticumán population always had the lowest level of infestation. The Ticumán population also had the highest concentration of the alkaloid scopolamine. Scopolamine was negatively related to the number of eggs deposited by L. daturaphila in Teotihuacán. There was among-family variation in herbivore damage (resistance), alkaloid content (scopolamine), and infestation by L. daturaphila and T. soror, indicating genetic variation and potential for further evolution. Although native and non-native D. stramonium populations have not yet diverged in plant resistance/constitutive defense, the differences between ranges (and the two experimental sites) in the type and abundance of herbivores suggest that further research is needed on the role of resource availability and adaptive plasticity, specialized metabolites (induced, constitutive), and the relationship between genealogical origin and plant defense in both ranges.