Structural analysis of a ligand-triggered intermolecular disulfide switch in a major latex protein from opium poppy.

Several proteins from plant pathogenesis-related family 10 (PR10) are highly abundant in the latex of opium poppy and have recently been shown to play diverse and important roles in the biosynthesis of benzylisoquinoline alkaloids (BIAs). The recent determination of the first crystal structures of PR10-10 showed how large conformational changes in a surface loop and adjacent ß-strand are coupled to the binding of BIA compounds to the central hydrophobic binding pocket. A more detailed analysis of these conformational changes is now reported to further clarify how ligand binding is coupled to the formation and cleavage of an intermolecular disulfide bond that is only sterically allowed when the BIA binding pocket is empty. To decouple ligand binding from disulfide-bond formation, each of the two highly conserved cysteine residues (Cys59 and Cys155) in PR10-10 was replaced with serine using site-directed mutagenesis. Crystal structures of the Cys59Ser mutant were determined in the presence of papaverine and in the absence of exogenous BIA compounds. A crystal structure of the Cys155Ser mutant was also determined in the absence of exogenous BIA compounds. All three of these crystal structures reveal conformations similar to that of wild-type PR10-10 with bound BIA compounds. In the absence of exogenous BIA compounds, the Cys59Ser and Cys155Ser mutants appear to bind an unidentified ligand or mixture of ligands that was presumably introduced during expression of the proteins in Escherichia coli. The analysis of conformational changes triggered by the binding of BIA compounds suggests a molecular mechanism coupling ligand binding to the disruption of an intermolecular disulfide bond. This mechanism may be involved in the regulation of biosynthetic reactions in plants and possibly other organisms.

Pollen identification of three notorious illicit drug plants and its potential applications in forensic practice.

Opium poppy, coca and cannabis are raw materials for three notorious illicit drugs. For a long time, drug lords have been growing and smuggling these drugs in a variety of ways and channels and are continually finding new ways of trafficking their wares, which has led to the increasing difficulty of global drug enforcement. In the present paper, we propose an innovative pollen identification system for these important drug plants, which provides a tool for screening and detection of the drugs to aid in drug enforcement. By utilizing the characteristics of these fine particles, their abundant production, and high resistance to decay, we believe this tool could be applied in the following scenarios: detecting and dynamically monitoring drug cultivation activities; determining whether a suspect has been to fields of drug plants and determining whether the site has ever been planted with a drug plant and/or was involved in drug production. In the future, combined with microscope automatic image acquisition technology and intelligent image recognition technology, this pollen identification system is expected to be used to screen three notorious illicit drug plants, thus enhancing the efficiency of drug related crime investigations.

Comparison of direct analysis in real-time mass spectrometry, atmospheric solids analysis probe-mass spectrometry, and ion mobility spectrometry for ensuring food safety by rapid screening of poppy seeds.

The opium poppy (Papaver somniferum) is a global commercial crop that has been historically valued for both medicinal and culinary purposes. Naturally occurring opium alkaloids including morphine, codeine, thebaine, noscapine, and papaverine are found primarily in the latex produced by the plant. If the plant is allowed to fully mature, poppy seeds that do not contain the opium alkaloids will form within the pods and may be used in the food industry. It is possible for the seeds to become contaminated with alkaloids by the latex during harvesting, posing a potential health risk for consumers. In the USA, there have been more than 600 reported adverse events including 19 fatalities that may be linked to the consumption of a contaminated poppy-containing product such as home-brewed poppy seed tea. Unwashed poppy seeds and pods may be purchased over the Internet and shipped worldwide. The Forensic Chemistry Center, US Food and Drug Administration (FDA) has evaluated several mass spectrometers (MS) capable of rapid screening to be used for high-throughput analysis of samples such as poppy seeds. These include a direct analysis in real-time (DART) ambient ionization source coupled to a single-quadrupole MS, an atmospheric solids analysis probe (ASAP) ionization source coupled to the same MS, and ion mobility spectrometers (IMS). These instruments have been used to analyze 17 poppy seed samples for the presence of alkaloids, and the results were compared to data obtained using liquid chromatography with mass spectral detection (LC-MS/MS). Results from the 17 poppy seed samples indicate that the DART-MS, ASAP-MS, and IMS devices detect many of the same alkaloids confirmed during the LC-MS/MS analyses, although both the false-positive and false-negative rates are higher, possibly due to the non-homogeneity of the samples and the lack of chromatographic separation.

Design and Optimisation of Sustainable Sample Treatments Based on Ultrasound-Assisted Extraction and Strong Cation-Exchange Purification with Functionalised SBA-15 for Opium Alkaloids in Ground Poppy Seeds.

An analysis methodology was optimised and validated for the quantification of opium alkaloids (OAs) in ground poppy seeds. This involved ultrasound-assisted extraction (UAE) and solid-phase extraction (SPE) purification before analysis using a high-performance liquid chromatography mass spectrometry detector (HPLC-MS/MS). UAE was optimised through the design of experiments with three factors and a three-level full factorial design. For SPE optimisation, a commercial material was compared with a previously synthesised material of SBA-15 silica functionalised with sulfonic groups (SBA-15-SO3-). The synthesised material demonstrated superior efficiency with only 25 mg and proved to be reusable for up to four cycles. The methodology was properly validated in terms of linearity, limits of detection and quantification, and selectivity. Matrix effects were negligible; adequate recovery values (85-100%) and inter-day and intra-day precision (≤15%) were obtained. The greenness of the method was evaluated with the AGREEprep metric scale, being more environmentally friendly compared to OA analysis methods. Finally, the method was applied to different samples of ground poppy seeds and revealed a concentration of 140 mg/kg of morphine equivalents in one of the samples, surpassing the legislatively established limits by sevenfold. This highlights the need to analyse these types of samples to mitigate potential public health issues.

Toxicokinetics of thebaine in those consuming non-food grade poppy seeds as a tea.

INTRODUCTION: Thebaine is an alkaloid in poppy seeds that is neurotoxic to animals. Data on its clinical effects and toxicokinetics in people are minimal. In 2022, poppy seeds high in thebaine entered the Australian food market, and people consuming tea made from these poppy seeds developed poisoning. METHODS: Three patients who drank poppy seed tea and developed neuromuscular toxicity consented for thebaine to be quantitated in serial blood samples. Blood samples were analyzed by liquid chromatography with high-resolution mass spectrometry. RESULTS: Case 1: A man in his 60s presented with drowsiness, vomiting, malaise and myoclonus. He developed metabolic acidosis with hyperlactataemia, acute kidney injury requiring haemodialysis, convulsions, rhabdomyolysis, and was in the hospital for 18 days. The admission thebaine blood concentration was 2.1 mg/L, and the apparent elimination half-life was 14.8 h. Case 2: A man in his 30s presented with myoclonus, rigidity, vomiting, and dizziness. He developed metabolic acidosis with hyperlactataemia, acute kidney injury, and myalgias. The admission thebaine blood concentration was 4.1 mg/L, and the apparent elimination half-life was 11.6 h. Case 3: A man in his 30s presented with myoclonus, rigidity, clonus, diaphoresis, and abdominal pain. The admission thebaine blood concentration was 2.2 mg/L, and the apparent elimination half-life was 8.3 h. DISCUSSION: Neuromuscular toxicity, metabolic acidosis with hyperlactataemia, acute kidney injury, and gastrointestinal symptoms were prominent clinical features in these patients after drinking poppy seed tea. Effects persisted for days, and all survived, despite thebaine concentrations far exceeding those in published forensic reports, although human data are sparse. Compared to rats, the thebaine apparent elimination half-life is much longer in humans who develop symptoms at lower concentrations. CONCLUSIONS: Despite relatively high thebaine blood concentrations and moderate to severe poisoning, outcomes were favourable with early presentations. It is possible that acute kidney injury prolongs the apparent elimination half-life of thebaine.

Structural Characterization and Molecular Dynamics Study of the REPI Fusion Protein from Papaver somniferum L.

REPI is a pivotal point enzyme in plant benzylisoquinoline alkaloid metabolism as it promotes the evolution of the biosynthetic branch of morphinan alkaloids. Experimental studies of its activity led to the identification of two modules (DRS and DRR) that catalyze two sequential steps of the epimerization of (S)- to (R)-reticuline. Recently, special attention has been paid to its genetic characterization and evolutionary history, but no structural analyses of the REPI protein have been conducted to date. We present here a computational structural characterization of REPI with heme and NADP cofactors in the apo state and in three complexes with substrate (S)-reticuline in DRS and intermediate 1,2-dehydroreticuline in DRS and in DRR. Since no experimental structure exists for REPI, we used its AlphaFold model as a scaffold to build up these four systems, which were submitted to all-atom molecular dynamics (MD) simulations. A comparison of MD results for the four systems revealed key dynamic changes associated with cofactor and ligand binding and provided a dynamic picture of the evolution of their structures and interactions. We also explored the possible dynamic occurrence of tunnels and electrostatic highways potentially involved in alternative mechanisms for channeling the intermediate from DRS to DRR.

New Validated Method for the Determination of Six Opium Alkaloids in Poppy Seed-Containing Bakery Products by High-Performance Liquid Chromatography-Tandem Mass Spectrometry after Magnetic Solid-Phase Extraction.

Bakery products containing poppy seeds are increasingly being commercialized. These seeds may be contaminated with latex from the Papaver somniferum L. plant rich in opium alkaloids (OAs). Therefore, health authorities demand the development of analytical methods to control them. In this study, an efficient and simple method was developed and validated for the first time to analyze six OAs in bakery products by high-performance liquid chromatography-tandem mass spectrometry. For this purpose, a solid-liquid extraction was optimized, and then a magnetic material [magnetite surface-modified with Fe(III) terephthalate, denoted as Fe3O4@TPA-Fe] was used for a fast magnetic solid-phase extraction. The method has been validated with adequate recoveries (70-110%) and relative standard deviations (<20%) and without matrix effects. Nine bakery samples (five breadsticks and four sliced bread) were analyzed; breadsticks showed low amounts of OAs, but two sliced bread showed higher amounts of OAs than the new amount (1.5 mg/kg) set by the Commission Regulation (EU) 2021/2142.

Opium, Street Opium, and Cancer Risk.

Opium is defined as the air-dried latex obtained by incision from the unripe capsules of Papaver somniferum L. Opium is a complex mixture that contains approximately 10% morphine and 2% codeine. It is commonly used to prepare opium tinctures for people with chronic diarrhea. Morphine and related opioids are powerful but highly addictive analgesics; designing less addictive opioids is an active area of pharmaceutical research that may lead to significant improvements in chronic pain management. Recently, the International Agency for Research on Cancer (IARC) has classified opium consumption as carcinogenic to humans (Group 1) based on sufficient evidence of carcinogenicity in human studies. However, all human studies analyzed by the IARC Working Group included participants who consumed opium that was mixed, adulterated, and/or contaminated with known and probable human carcinogens (e.g., tarry residues of combusted opium, arsenic, lead, and chromium). The working group considered that these carcinogens were part of the complex mixture that opium is, rather than co-exposure or confounders. No evidence of carcinogenicity was available for pure opium in human, animal, or mechanistic studies. To avoid confusion and concern among health professionals and patients using medicinal opium preparations and in scientists involved in the design and development of new opium derivatives, opium should be classified in Group 3 (not classifiable as to its carcinogenicity to humans). The term 'street opium' could be used to refer to opium that probably contains human carcinogens not present in pure opium and should remain in Group 1 (carcinogenic to humans).

A novel self-aggregated gold nanoparticles based on sensitive immunochromatographic assays for highly detection of opium poppy in herbal teas.

Opium poppy abused in food has aroused public concerns due to its serious side effects. Effective monitoring is essential to fight the abuse crisis. Herein, we synthesized an easily prepared, affordable, accessible highly aggregated gold nanoparticles (AGNPs) performing in lateral flow immunoassay (LFIA) for detection opium poppy in herbal teas. Simultaneously, a LFIA based ontime-resolved fluorescent microspheres (TRFMs) was developed as contrastive method. In this study, morphine (MOR), codeine (COD) and thebaine (THE) were as the specific recognition markers of opium poppy. Results demonstrated the quantitative limits of detection were 0.0049/0.0053/0.084, 0.034/0.037/0.37 ng mL-1 for AGNPs/TRFMs-LFIA, respectively. The recoveries were 95%-107.5%/91%-106.7% with coefficient of variation was 1.6%-6.6%/1.8%-7.2%, indicating excellent accuracy and precision. Parallel experiments among AGNPs/TRFMs-LFIA and LC-MS/MS analysis showed good correlation. Overall, AGNPs-LFIA executed quantitative analysis within 15 min on the basis of simple treatment while providing a rapid and sensitive analysis strategy for illegal drugs abused.

Urine thebaine determination by liquid chromatography tandem mass spectrometry after poppy seed consumption.

Laboratories are challenged to distinguish whether a positive urine morphine result is due to heroin use or possible poppy seed consumption. Thebaine is an opium alkaloid that has been shown to be present in the urine of individuals who have consumed poppy seeds, as well as those who have used opium. It is not present in heroin. We present a sensitive, specific liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for thebaine. We show that thebaine is detectable after consumption of two different poppy seed-containing products for up to 72 h in urine. We discuss limitations of the assay and suggest how the test might best be used.

Total Phenolic Content and Antioxidant and Antimicrobial Activities of Papaver rhoeas L. Organ Extracts Growing in Taounate Region, Morocco.

The objective of this study is to valorize Papaver rhoeas L. from the Taounate region of Morocco by determining the total polyphenol content (TPC), the total flavonoid content (TFC) and the antioxidant and antimicrobial activities of four organs. The quantification of TPC and TFC in root, stem, leaf and flower extracts (RE, SE, LE and FE, respectively) was estimated by the Folin-Ciocalteu reaction and the aluminum trichloride method, respectively. Two tests were used to assess antioxidant power: the DPPH test and TAC assay. The antimicrobial activity was studied against five pathogenic bacteria and yeast, using two methods: disk diffusion and microdilution. The TPC in LE and LF was twice as high as that in RE and SE (24.24 and 22.10 mg GAE/g, respectively). The TFC values in the four extracts were very close and varied between 4.50 mg QE/g in the FE and 4.38 mg QE/g in the RE. The LE and FE showed low DPPH values with IC50 = 0.50 and 0.52 mg/mL, respectively. The TAC measurement revealed the presence of a significant amount of antioxidants in the studied extracts, mainly in LE and FE (6.60 and 5.53 mg AAE/g, respectively). The antimicrobial activity results revealed significant activity on almost all of the tested strains. The MIC of FE and SE against E. coli 57 was 1.56 and 0.78 mg/mL, respectively, while against the S. aureus it was 50 and 25 mg/mL, respectively. The low MLC value (1.56 mg/mL) was recorded against E. coli 57 by RE and SE.

Papaver Plants: Current Insights on Phytochemical and Nutritional Composition Along with Biotechnological Applications.

The genus Papaver is highly esteemed in the pharmacy industry, in the culinary field, and as ornamental plants. These plants are also valued in traditional medicine. Among all Papaver species, Papaver somniferum L. (opium poppy) is the most important species in supplying phytochemicals for the formulation of drugs, mainly alkaloids like morphine, codeine, rhoeadine, thebaine, and papaverine. In addition, Papaver plants present other types of phytochemicals, which altogether are responsible for its biological activities. Therefore, this review covers the phytochemical composition of Papaver plants, including alkaloids, phenolic compounds, and essential oils. The traditional uses are reviewed along with their pharmacological activities. Moreover, safety aspects are reported to provide a deep overview of the pharmacology potential of this genus. An updated search was carried out in databases such as Google Scholar, ScienceDirect, and PubMed to retrieve the information. Overall, this genus is a rich source of alkaloids of different types and also contains interesting phenolic compounds, such as anthocyanins, flavonols, and the characteristic indole derivatives nudicaulins. Among other pharmacological properties, numerous preclinical studies have been published about the analgesic, anticancer, antimicrobial, antioxidant, and antidiabetic activities of Papaver plants. Although it highlights the significant impact of this genus for the treatment of a variety of diseases and conditions, as a future prospect, characterization works accompanying preclinical studies are required along with clinical and toxicology studies to establish a correlation between the scientific and traditional knowledge.

Spatial Distribution and Stability of Cholinesterase Inhibitory Protoberberine Alkaloids from Papaver setiferum.

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehydroprotoberberine alkaloids (1 and 2) and nine known compounds (3-11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1-4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

In vitro evaluation of chromosomal damage and DNA strand breaks after treatment with the poppy seed alkaloid thebaine.

Thebaine is an alkaloid and can be found in poppy seeds in relatively high concentrations. Acute toxicity of thebaine is fairly high, but not much is known about chronic toxicity. To investigate the genotoxicity of thebaine, cytokinesis-block micronucleus test and comet assay were conducted in TK6 cells. In addition, effects of putative thebaine metabolites were analysed using metabolically active HepG2 cells and TK6 cells with S9 mix. FDA test and trypan blue test were used together with the frequency of mitotic and apoptotic cells to assess potential cytotoxicity of thebaine treatment. Micronucleus induction was observed after high doses (150 and 500 µM) of thebaine without metabolic activation in the presence of slight to moderate cytotoxicity. No effects were observed in the comet assay or after metabolic activation up to the highest dose of 500 µM. A potential protective effect on micronucleus induction after thebaine treatment was investigated via co-treatment with MMC and BaP in TK6 cells. Only after co-treatment with MMC, a reduction of micronucleus frequency was found. Overall, this study shows a potential of thebaine to induce genotoxic effects at high concentrations. The observation of cytotoxicity at these concentrations supports the hypothesis that genotoxicity may be caused by cytotoxic effects. Further studies will need to elucidate whether these effects are directly genotoxic or indeed result from cytotoxicity.

Identification of novel anti-cancer agents by the synthesis and cellular screening of a noscapine-based library.

Noscapine is a natural product first isolated from the opium poppy (Papaver somniferum L.) with anticancer properties. In this work, we report the synthesis and cellular screening of a noscapine-based library. A library of novel noscapine derivatives was synthesized with modifications in the isoquinoline and phthalide scaffolds. The so generated library, consisting of fifty-seven derivatives of the natural product noscapine, was tested against MDA-MB-231 breast cancer cells in a cellular proliferation assay (with a Z' > 0.7). The screening resulted in the identification of two novel noscapine derivatives as inhibitors of MDA cell growth with IC50 values of 5 µM and 1.5 µM, respectively. Both hit molecules have a five-fold and seventeen-fold higher potency, compared with that of lead compound noscapine (IC50 26 µM). The identified active derivatives retain the tubulin-binding ability of noscapine. Further testing of both hit molecules, alongside the natural product against additional cancer cell lines (HepG2, HeLa and PC3 cells) confirmed our initial findings. Both molecules have improved anti-proliferative properties when compared to the initial natural product, noscapine.

Comparative Effects of Dietary Hemp and Poppy Seed Oil on Lipid Metabolism and the Antioxidant Status in Lean and Obese Zucker Rats.

The objective of this study was to compare the effects of the dietary inclusion of hemp seed oil (HO) and poppy seed oil (PO) on the lipid metabolism and antioxidant status of lean and genetically obese Zucker rats. The rats were fed a control diet for laboratory rodents or a modification with HO or PO. Both oils reduced body and epididymal fat and liver cholesterol levels and promoted oxidative stress in the liver of obese rats. The HO reduced plasma triglycerides and had a stronger liver cholesterol-lowering effect in obese rats than PO. In the lean rats, HO and PO had no effects on the body fat content, plasma lipid profile, or lipid metabolism in the liver. HO considerably elevated the content of α-linolenic acid in the liver and increased the liver ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG) in the lean rats. In conclusion, the regular consumption of both oils increases the accumulation of essential fatty acids in the liver of healthy animals, whilst not having any adverse effects on the body, whereas in genetically obese rats, the effects of both dietary oils on the lipid metabolism and antioxidant status are unequivocal and only partially beneficial.

The curious yellow colouring matter of the Iceland poppy.

The pigment responsible for the yellow colour of the Iceland poppy (Papaver nudicaule) has fascinated chemists for decades. Named nudicaulin by Sir Robert Robinson in 1939, this flavoalkaloid has been the subject of several structural elucidation, biosynthesis, total synthesis and chemical ecology investigations. This account provides a chronological overview of the fascinating history of this natural product.

Structural insights into thebaine synthase 2 catalysis.

Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4-OH from the substrate under the assistance of H89. The resulting C4-O- phenolate anion then attacks the nearby C5, and triggers intramolecular SN2' syn displacement with the elimination of O-acetyl group. Moreover, the latter SN2' reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering.