Nicotinate-curcumin improves NASH by inhibiting the AKR1B10/ACCα-mediated triglyceride synthesis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a prevalent chronic liver condition. However, the potential therapeutic benefits and underlying mechanism of nicotinate-curcumin (NC) in the treatment of NASH remain uncertain. METHODS: A rat model of NASH induced by a high-fat and high-fructose diet was treated with nicotinate-curcumin (NC, 20, 40 mg·kg- 1), curcumin (Cur, 40 mg·kg- 1) and metformin (Met, 50 mg·kg- 1) for a duration of 4 weeks. The interaction between NASH, Cur and Aldo-Keto reductase family 1 member B10 (AKR1B10) was filter and analyzed using network pharmacology. The interaction of Cur, NC and AKR1B10 was analyzed using molecular docking techniques, and the binding energy of Cur and NC with AKR1B10 was compared. HepG2 cells were induced by Ox-LDL (25 µg·ml- 1, 24 h) in high glucose medium. NC (20µM, 40µM), Cur (40µM) Met (150µM) and epalrestat (Epa, 75µM) were administered individually. The activities of ALT, AST, ALP and the levels of LDL, HDL, TG, TC and FFA in serum were quantified using a chemiluminescence assay. Based on the changes in the above indicators, score according to NAS standards. The activities of Acetyl-CoA and Malonyl-CoA were measured using an ELISA assay. And the expression and cellular localization of AKR1B10 and Acetyl-CoA carboxylase (ACCα) in HepG2 cells were detected by Western blotting and immunofluorescence. RESULTS: The results of the animal experiments demonstrated that NASH rat model induced by a high-fat and high-fructose diet exhibited pronounced dysfunction in liver function and lipid metabolism. Additionally, there was a significant increase in serum levels of FFA and TG, as well as elevated expression of AKR1B10 and ACCα, and heightened activity of Acetyl-CoA and Malonyl-CoA in liver tissue. The administration of NC showed to enhance liver function in rats with NASH, leading to reductions in ALT, AST and ALP levels, and decrease in blood lipid and significant inhibition of FFA and TG synthesis in the liver. Network pharmacological analysis identified AKR1B10 and ACCα as potential targets for NASH treatment. Molecular docking studies revealed that both Cur and NC are capable of binding to AKR1B10, with NC exhibiting a stronger binding energy to AKR1B10. Western blot analysis demonstrated an upregulation in the expression of AKR1B10 and ACCα in the liver tissue of NASH rats, accompanied by elevated Acetyl-CoA and Malonyl-CoA activity, and increased levels of FFA and TG. The results of the HepG2 cell experiments induced by Ox-LDL suggest that NC significantly inhibited the expression and co-localization of AKR1B10 and ACCα, while also reduced levels of TC and LDL-C and increased level of HDL-C. These effects are accompanied by a decrease in the activities of ACCα and Malonyl-CoA, and levels of FFA and TG. Furthermore, the impact of NC appears to be more pronounced compared to Cur. CONCLUSION: NC could effectively treat NASH and improve liver function and lipid metabolism disorder. The mechanism of NC is related to the inhibition of AKR1B10/ACCα pathway and FFA/TG synthesis of liver.

Modulation of Acute Intestinal Inflammation by Dandelion Polysaccharides: An In-Depth Analysis of Antioxidative, Anti-Inflammatory Effects and Gut Microbiota Regulation.

Acute colitis is a complex disease that can lead to dysregulation of the gut flora, inducing more complex parenteral diseases. Dandelion polysaccharides (DPSs) may have potential preventive and therapeutic effects on enteritis. In this study, LPS was used to induce enteritis and VC was used as a positive drug control to explore the preventive and therapeutic effects of DPS on enteritis. The results showed that DPS could repair the intestinal barrier, down-regulate the expression of TNF-α, IL-6, IL-1ß, and other pro-inflammatory factors, up-regulate the expression of IL-22 anti-inflammatory factor, improve the antioxidant capacity of the body, and improve the structure of intestinal flora. It is proved that DPS can effectively prevent and treat LPS-induced acute enteritis and play a positive role in promoting intestinal health.

Changes in the Volatile Flavor Substances, the Non-Volatile Components, and the Antioxidant Activity of Poria cocos during Different Drying Processes.

Poria cocos (Schw.) wolf (P. cocos) is an important medicinal material with both therapeutic and edible properties. This study investigated volatile constituents, amino acids, proteins, polysaccharides, triterpenoid ingredients, and alcohol-soluble extracts on P. cocos during eight drying processes. A total of 47 volatile components were found and identified; the main volatile components of shade drying (SD) and hot-air drying at 50 °C (HD50) were esters and alcohols, while for drying in hot air at 60 °C~100 °C (△ = 10 °C) and infrared drying (ID), the main compounds were aldehydes and hydrocarbons. The amino acids in P. cocos remained the same when dried with various methods. Compared with SD samples, with the temperature increase, the content of amino acids showed a trend of first decreasing and then increasing, while the content trend of proteins was the opposite. The HD70 samples had the highest content of polysaccharide, triterpenoid ingredients, alcohol-soluble extracts, and antioxidant activity. Furthermore, volatile compounds showed a correlation between non-volatile constituents. This research provides evidence that the aroma, active components, and activity of P. cocos were affected by the drying method.

Correction: Dong et al. Identification of the Trace Components in BopuzongJian and Macleaya cordata Extract Using LC-MS Combined with a Screening Method. Molecules 2021, 26, 3851.

The article in question [...].

Sporophytic control of tapetal development and pollen fertility by a mitogen-activated protein kinase cascade in rice.

Tapetum, the innermost layer of the anther wall, provides essential nutrients and materials for pollen development. Timely degradation of anther tapetal cells is a prerequisite for normal pollen development in flowering plants. Tapetal cells facilitate male gametogenesis by providing cellular contents after highly coordinated programmed cell death (PCD). Tapetal development is regulated by a transcriptional network. However, the signaling pathway(s) involved in this process are poorly understood. In this study, we report that a mitogen-activated protein kinase (MAPK) cascade composed of OsYDA1/OsYDA2-OsMKK4-OsMPK6 plays an important role in tapetal development and male gametophyte fertility. Loss of function of this MAPK cascade leads to anther indehiscence, enlarged tapetum, and aborted pollen grains. Tapetal cells in osmkk4 and osmpk6 mutants exhibit an increased presence of lipid body-like structures within the cytoplasm, which is accompanied by a delayed occurrence of PCD. Expression of a constitutively active version of OsMPK6 (CA-OsMPK6) can rescue the pollen defects in osmkk4 mutants, confirming that OsMPK6 functions downstream of OsMKK4 in this pathway. Genetic crosses also demonstrated that the MAPK cascade sporophyticly regulates pollen development. Our study reveals a novel function of rice MAPK cascade in plant male reproductive biology.

Studies on pharmacokinetic properties and intestinal absorption mechanism of sanguinarine chloride: in vivo and in situ.

Sanguinarine (SAN) is an alkaloid with multiple biological activities, mainly extracted from Sanguinaria canadensis or Macleaya cordata. The low bioavailability of SAN limits its utilization. At present, the nature and mechanism of SAN intestinal absorption are still unclear. The pharmacokinetics, single-pass intestinal perfusion test (SPIP), and equilibrium solubility test of SAN in rats were studied. The absorption of SAN at 20, 40, and 80 mg/L in different intestinal segments was investigated, and verapamil hydrochloride (P-gp inhibitor), celecoxib (MPR2 inhibitor), and ko143 (BCRP inhibitor) were further used to determine the effect of efflux transporter proteins on SAN absorption. The equilibrium solubility of SAN in three buffer solutions (pH 1.2, 4.5 and 6.8) was investigated. The oral pharmacokinetic results in rats showed that SAN was rapidly absorbed (Tmax=0.5 h), widely distributed (Vz/F = 134 L/kg), rapidly metabolized (CL = 30 L/h/kg), and had bimodal phenomena. SPIP experiments showed that P-gp protein could significantly affect the effective permeability coefficient (Peff) and apparent absorption rate constant (Ka) of SAN. Equilibrium solubility test results show that SAN has the best solubility at pH 4.5. In conclusion, SAN is a substrate of P-gp, and its transport modes include efflux protein transport, passive transport and active transport.

Visible-Light-Promoted α-Benzylation of N-Phenyl α-Amino Acids to α-Amino Phenylpropanoids.

A new method for the synthesis of α-amino phenylpropanoids under blue light-emitting diode irradiation has been developed through α-C-H benzylation of readily available N-phenyl glycine ester with benzyl oxalates as a coupling partner under mild conditions. A range of N-phenyl glycine esters were successfully converted to α-amino phenylpropanoid products in moderate to good yields. The utility of this methodology is underlined by its application to the late-state modification of natural products.

Leaf Spot caused by Alternaria alternata on Macleaya cordata in China.

Macleaya cordata is a perennial herb that belongs to the Papaveraceae and is typically prescribed as a traditional antibacterial medicine in China (Kosina et al. 2010). The extract from M. cordata has been widely used in the manufacturing of natural growth promoters as an alternative to antibiotic growth promoters in the livestock industry (Liu et al. 2017), and the products are marketed in 70 countries such as Germany, China, etc (Ikezawa et al. 2009). During the summer of 2019, symptoms of leaf spot were observed on M. cordata (cv. HNXN-001) in two commercial fields (approximately 1, 300 m2 and 2, 100 m2) of Xinning county, Shaoyang City, Hunan Province, China, where approximately 2 to 3% of the plants were affected. The initial symptoms were irregular black and brown spots on the leaves. The lesions expanded and coalesced, eventually leading to leaf blight. Six symptomatic basal leaf sections from six plants from two fields were surface disinfested in 0.5% NaClO for 1 min, then 75% ethanol for 20 s, rinsed in sterile water three times, air dried, and placed onto potato dextrose agar (PDA), one dish for samples from a single leaf. Plates were incubated at 26°C in darkness. Nine strains with similar morphological characters were isolated, and one representative isolate ( BLH-YB-08) was used for morphological and molecular characterization. The colonies on PDA were grayish-green with white round margins. Conidia were typically obclavate to obpyriform, brown to dark brown, and 12.0 to 35.0 × 6.0 to 15.0 µm, and with 1 to 5 transverse septa and 0 to 2 longitudinal septa (n=50). Isolates were identified as Alternaria sp. on the basis of mycelial characteristics, color, and conidial morphology. To confirm identity of the pathogen, DNA was extracted from isolate BLH-YB-08 with the DNAsecure Plant Kit (TIANGEN, Biotech, China). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), actin (ACT), 28S nrDNA (LSU), 18S nuclear ribosomal DNA (SSU), histone 3 (HIS3), internal transcribed spacer (ITS) region of ribosomal DNA, and translation elongation factor 1-α (TEF) genes ( Berbee et al. 1999; Carbone and Kohn. 1999; Glass and Donaldson. 1995; White et al. 1990.) were amplified and sequenced. Sequences were deposited into the GenBank database. They were 100% sequence identity of GAPDH (OQ224996) with A. alternata strain AA2-8 (MH65578; 578/578bp), 100% sequence identity of RPB2 (OQ190460) with A. alternata strain SAX-WN-30-2 ( MK605877; 933/933bp), 100% sequence identity of ACT (OQ923292) with A. alternata strain FCBP0352 (OL830257; 939/939 bp), 100% sequence identity of LSU (OQ891167) with A. alternata XL14 (MG839509 ; 908/908 bp), 100% sequence identity of SSU (OQ139544) with A. alternata strain BJ19.4.1(OM736063; 1,067/1,067 bp), 100% sequence identity of HIS3 (MT454856) with A. alternata YJ-CYC-HC2 (OQ116440 ; 442/442 bp), 100% sequence identity of ITS (MT212225) with A. alternata CS-1-3 (OQ947366; 543/543bp), and 100% sequence identity of TEF (OQ190461) with A. alternata strain YZU 221185 (OQ512730; 252/252 bp). To test pathogenicity, the isolate BLH-YB-08 was cultured on PDA for 7 days to prepare conidial suspensions and the spore concentration adjusted to a final concentration of 1×106 spores/ml. The leaves of five potted 45-day-old M. cordata (cv. HNXN-001) plants were sprayed with conidial suspensions, and five control potted plants were wiped with 75% alcohol and washed five times with sterile distilled water. They were then sprayed with sterile distilled water. Plants were placed in a greenhouse at 25 to 30°C with 90% relative humidity. Pathogenicity tests were conducted twice. Fifteen days after inoculation, lesions were found on inoculated leaves, and the symptoms were the same as those in the field, whereas the controls were healthy. A fungus was consistently isolated from the inoculated leaves and identified as A. alternata by DNA sequencing of the GAPDH, ITS, and HIS3 genes, fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on M. cordata caused by A. alternata in China. Understanding its etiology may help to control this fungal pathogen, thus reducing economic losses. Funding: Hunan Provincial Natural Science Foundation General Project (2023JJ30341) Hunan Provincial Natural Science Foundation Youth Fund (2023JJ40367) Seed Industry Innovation Project of Hunan Provincial Science and Technology Department Special project for the construction of Chinese herbal medicine industry technology system in Hunan Province "Xiangjiuwei" Industrial Cluster Project of the Ministry of Agriculture and Rural Affairs.

Protopine Alleviates Dextran Sodium Sulfate-Induced Ulcerative Colitis by Improving Intestinal Barrier Function and Regulating Intestinal Microbiota.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD), and its pathogenesis is related to intestinal mucosal barrier damage and gut microbiota imbalance. Protopine (PRO), an isoquinoline alkaloid, is one of the main anti-inflammatory ingredients of traditional Chinese medicine Macleaya cordata(Willd.) R. Br. This study investigated the effects of PRO on the intestinal mucosal barrier and gut microbiota in dextran sodium sulfate (DSS)-induced colitis mice. C57BL/6J mice were treated with 3% DSS in drinking water to induce acute colitis, while PRO was administered orally once daily for 7 days. The results showed that PRO administration significantly alleviated the symptoms of DSS-induced colitis in mice and inhibited the expression of inflammation-related genes. In addition, PRO restored the integrity of the intestinal barrier in colitis mice by restoring colonic mucin secretion and promoting the expression of tight junction proteins. Furthermore, PRO alleviated the DSS-induced gut microbiota dysbiosis by decreasing the abundance of Proteobacteria, Escherichia-Shigella and Enterococcus, as well as enhancing the abundance of beneficial bacteria, such as Firmicutes and Akkermansia. These findings suggested that PRO effectively alleviated DSS-induced ulcerative colitis by suppressing the expression of inflammation-related genes, maintaining the intestinal mucosal barrier and regulating the intestinal microbiota.

Protective Application of Chinese Herbal Compounds and Formulae in Intestinal Inflammation in Humans and Animals.

Intestinal inflammation is a chronic gastrointestinal disorder with uncertain pathophysiology and causation that has significantly impacted both the physical and mental health of both people and animals. An increasing body of research has demonstrated the critical role of cellular signaling pathways in initiating and managing intestinal inflammation. This review focuses on the interactions of three cellular signaling pathways (TLR4/NF-κB, PI3K-AKT, MAPKs) with immunity and gut microbiota to explain the possible pathogenesis of intestinal inflammation. Traditional medicinal drugs frequently have drawbacks and negative side effects. This paper also summarizes the pharmacological mechanism and application of Chinese herbal compounds (Berberine, Sanguinarine, Astragalus polysaccharide, Curcumin, and Cannabinoids) and formulae (Wumei Wan, Gegen-Qinlian decoction, Banxia xiexin decoction) against intestinal inflammation. We show that the herbal compounds and formulae may influence the interactions among cell signaling pathways, immune function, and gut microbiota in humans and animals, exerting their immunomodulatory capacity and anti-inflammatory and antimicrobial effects. This demonstrates their strong potential to improve gut inflammation. We aim to promote herbal medicine and apply it to multispecies animals to achieve better health.

Identification of Sanguinarine Metabolites in Rats Using UPLC-Q-TOF-MS/MS.

Sanguinarine (SAN), as the main active component of a traditional Chinese veterinary medicine, has been widely used in the animal husbandry and breeding industry. However, the metabolites of SA are still uncertain. Therefore, this research aimed to investigate the metabolites of SA based on rats in vivo. The blood, feces, and urine of rats were collected after the oral administration of 40 mg/kg SAN. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) was employed to identify the metabolites of SAN. The elemental composition of sanguinarine metabolites was inferred by analyzing their exact molecular weight, and the structures of the metabolites were predicted based on their fragment ions and cleavage pathways. A total of 12 metabolites were identified, including three metabolites in the plasma, four in the urine, and nine in the feces. According to the possible metabolic pathways deduced in this study, SAN was mainly metabolized through reduction, oxidation, demethylation, hydroxylation, and glucuronidation. This present research has summarized the metabolism of SAN in rats, which is helpful for further studying the metabolic mechanism of SAN in vivo and in vitro.

Effects of sulfated ß-glucan from Saccharomyces cerevisiae on growth performance, antioxidant ability, nonspecific immunity, and intestinal flora of the red swamp crayfish (Procambarus clarkii).

The aim of this study was to examine the combined effects of sulfated ß-Glucan from Saccharomyces cerevisiae (sGSC) on growth performance, antioxidant ability, nonspecific immunity, and intestinal flora of the red swamp crayfish (Procambarus clarkii). Four experimental diets (sGSC25, sGSC50, sGSC100 and sGSC200) with different levels of sGSC (0.025%, 0.05%, 0.1% and 0.2% in diet, respectively) were fed to juvenile crayfish (average weight: 2.5 ± 0.5 g) for 8 weeks. The control diet was given with 2000 mg/kg GSC (GSC200 group). The based control diet was given without sGSC or GSC (blank group). Each group had 3 parallel test pools, 20 crayfish were reared in each pool. At the end of the growth trial, adding dietary 0.025%-0.1% sGSC could significantly improve the growth performance, antioxidant capacity and immunity of crayfish. Compared with GSC, sGSC had a better effect at lower concentration. Higher concentration of sGSC (>0.1%) would cause some side effects. sGSC also could improve the structure of the intestinal flora and optimize the function of the flora. sGSC would increase the abundances of probiotics such as Hafnia and Acinetobacter, and decreases the abundances of maleficent bacteria such as Enterobacteriaceae. Higher concentration of sGSC (>0.1%) would increase the abundance of Aeromonas. To conclude, 0.025%-0.1% sGSC can be used as a supplement in crayfish feed to increase growth, immunity, and antioxidant capacity and improve the structure of intestinal flora. These results provided a theoretical basis for the application of sGSC instead of GSC in crayfish breeding. It will be necessary to further study the optimal concentration of sGSC in feed additives in different growth stages of crayfish in the future.

A home-made sampling system coupled to hectowatt-MPT mass spectrometry in positive ion mode to confirm target ions of copper and zinc from Poyang Lake, China.

A novel home-made H2SO4-Nafion (HN) tube sampling system coupled to a line ion trap mass spectrometer (LTQ-MS) with a versatile ambient ionization source, hectowatt microwave plasma torch (HMPT), has manifested unique advantages for picking directly metal elements in aqueous samples and acquiring the fully characteristic MPT mass spectra of copper and zinc composite ions. Here, we report the development of a novel HN-HMPT-LTQ-MS for metal elements assay based on environmental water to analyze samples of Poyang Lake, China. Detailed multi-stage tandem mass spectra show that the general structural form of target ions is [M(NO3)x(H2O)y(OH)z]+ for the positive ion mode. Under the optimized conditions, the proposed method provided low limits of detection (LODs) of 0.23 µg.L-1 for 63Cu+ and 1.1 µg.L-1 for 66Zn+, with relative standard deviations (RSDs) of less than 12.7% by MPT-LTQ-MS. This new result has met the requirements of national standards (GB 5750.6-2006) and is only about one magnitude order larger than the LOD of ICP-MS method. A wide linear response range of about 4 orders of magnitude for the method with linear coefficients (R2) of 0.99709 - 0.99962 for copper and zinc tested was in accordance with that of ICP-MS. Except for the recovery of 79% for the third sample and 123.8% for the seventh sample, the present method also provided good recoveries (84 - 119.3%) in spiked 10 batches of drinking water samples. Furthermore, it is envisioned that the developed approach might build a powerful hectowatt-MPT-MS platform for food security detection, drug analysis, and origin traceability.

Safety assessment of MPTA: An oral acute and 90-day sub-chronic toxicity study in Sprague-Dawley rats.

MPTA is a novel extract product derived from Macleaya cordata (Willd.) R. Br., which has good anti-inflammatory and antioxidant activity. The aim of this study was to investigate the acute oral toxicity and 90-day sub-chronic oral toxicity of MPTA. In the acute toxicity study, 50 SD rats of both sexes were randomly divided into 5 groups and dosed in a gradient from 197.53 mg/kg to 1000.00 mg/kg bw. Toxic effects were observed up to 14 days and LD50 was calculated. In a subchronic toxicity test, male and female SD rats were orally dosed repeatedly with 96.40, 19.28, 3.86 mg/kg bw of MPTA for 90 days. In addition, a control group was set up in the subchronic study. The acute toxicity test showed that the oral LD50 of MPTA was 481.99 mg/kg with a 95% confidence interval of 404.24-574.70 mg/kg. MPTA did not appear to induce toxic effects in the longer term in terms of food and water consumption, weight gain, haematological and clinical biochemical parameters and pathological examination. The first data on the potential toxicity of MPTA was provided to highlight the safety of short-term to longer-term oral administration of MPTA, and the experimental results yield and establish a NOEAL of 96.40 mg/kg/d for MPTA.

Effect of Natural Antioxidants from Marigolds (Tagetes erecta L.) on the Oxidative Stability of Soybean Oil.

In recent years, synthetic antioxidants that are widely used in foods have been shown to cause detrimental health effects, and there has been growing interest in antioxidants realised from natural plant extracts. In this study, we investigate the potential effects of natural antioxidant components extracted from the forage plant marigold on the oxidative stability of soybean oil. First, HPLC-Q-TOF-MS/MS was used with 1,1-diphenyl-2-picrylhydrazyl (DPPH) to screen and identify potential antioxidant components in marigold. Four main antioxidant components were identified, including quercetagetin-7-O-glucoside (1), quercetagetin (2), quercetin (3) and patuletin (4). Among them, quercetagetin (QG) exhibited the highest content and the strongest DPPH radical scavenging activity and effectively inhibited the production of oxidation products in soybean oil during accelerated oxidation, as indicated by reductions in the peroxide value (PV) and acid value (AV). Then, the fatty acids and volatile compounds of soybean oil were determined with gas chromatography-mass spectrometry (GC-MS) and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). A total of 108 volatile components, including 16 alcohols, 23 aldehydes, 25 ketones, 4 acids, 15 esters, 18 hydrocarbons, and 7 other compounds, were identified. QG significantly reduced the content and number of aldehydes and ketones, whereas the formation of acids and hydrocarbons was completely prevented. In addition, the fatty acid analysis demonstrated that QG significantly inhibited oxidation of unsaturated fatty acids. Consequently, QG was identified as a potential, new natural antioxidant that is believed to be safe, effective and economical, and it may have potential for use in plant extracts feed additives.

Plant-microbe hybrid synthesis provides new insights for the efficient use of Macleaya cordata.

Sanguinarine and chelerytrine have antibacterial and anti-inflammatory effects and is the main active ingredients of growth promoters in animals. Currently, Sanguinarine and chelerytrine were extracted from the capsules of the medicinal plant Macleaya cordata. However, the biomass of M. cordata nonmedicinal parts (leaves) accounted for a large proportion and contained a rich presentation of protopine and allocryptopine which are the precursor compounds of sanguinarine and chelerytrine. The aim of this study was to develop a new method for producing sanguinarine and chelerytrine through yeast transformation of protopine and allocryptopine in M. cordata leaves. First, we isolated different genes from Papaver somniferum (PsP6H, PsCPR, PsDBOX), Eschscholtzia californica (EcP6H), Cucumis sativus (CuCPR), Arabidopsis thaliana (AtCPR) and M. cordata (Mc11229, Mc11218, Mc6408, Mc6407, Mc19967, Mc13802). Additionally, some of the gene sequences were codon optimized. Then, we transformed these genes into yeast cells to compare the catalytic efficiency. Second, we used the most efficient strains to biotransform the leaves of M. cordata. Finally, we obtained 85.415 ± 11.887 ng mL-1 sanguinarine and 4.288 ± 1.395 ng mL-1 chelerytrine, which was more than 2-3 times the content in leaves of M. cordata. Overall, we using the nonmedicinal parts of M. cordata and successfully obtained sanguinarine and chelerytrine by the plant-microbial hybrid synthesis method.

Effects of codon optimization, N-terminal truncation and gene dose on the heterologous expression of berberine bridge enzyme.

Morphine, sanguinarine and chelerythrine are benzylisoquinoline alkaloids (BIAs), and these compounds possess strong biological activities. (S)-scoulerine is a commonly shared precursor of these compounds, and berberine bridge enzyme (BBE) is a key rate-limiting enzyme in the synthesis of (S)-scoulerine. We isolated the BBE gene from Macleaya cordata (McBBE) and used CEN.PK2-1C as a chassis strain. We compared the catalytic efficiency of five codon-optimized McBBE genes in Saccharomyces cerevisiae and finally obtained a yeast strain (YH03) that exhibited a 58-fold increase in yield (1.12 mg/L). Then, we truncated the N-terminus of McBBE by 8 and 22 amino acids and found that with the increase in the number of N-terminal truncated amino acids, the production of (S)-scoulerine gradually decreased. Additionally, we used CRISPR-Cas9 to integrate the McBBE gene at the delta site of the S. cerevisiae genome to achieve stable genetic inheritance and found that the yield of (S)-scoulerine was not significantly increased in the integrated strain. In conclusion, our work achieved high-efficiency expression of McBBE in S. cerevisiae, explored the influence of N-terminal truncation on the yield of (S)-scoulerine, and obtained a genetically stable S. cerevisiae strain with high McBBE expression. This study provides a reference for further complex metabolic engineering optimization and lays a foundation for the efficient biosynthesis of BIAs.

Rapid Distinction and Semiquantitative Analysis of THC and CBD by Silver-Impregnated Paper Spray Mass Spectrometry.

The control over the amount of psychoactive THC (Δ-9-tetrahydrocannabinol) in commercial cannabidiol (CBD) products has to be strict. A fast and simple semiquantitative Ag(I)-impregnated paper spray mass spectrometric method for differentiating between THC and CBD, which show no difference in standard single-stage or tandem MS, was established. Because of a different binding affinity to Ag(I) ions, quasi-molecular Ag(I) adducts [THC + Ag]+ and [CBD + Ag]+ at m/z 421 and 423 give different fragmentation patterns. The product ions at m/z 313 for THC and m/z 353 and 355 for CBD can be used to distinguish THC and CBD and to determine their ratio. Quantification of THC/CBD ratios in commercial CBD oils was accomplished with a low matrix effect (-2.2 ± 0.4% for THC and -2.0 ± 0.3% for CBD). After simple methanol extraction (recovery of 87.3 ± 1.2% for THC and 92.3 ± 1.4% for CBD), Ag(I)-impregnated paper spray analysis was employed to determine this ratio. A single run can be completed in a few minutes. This method was benchmarked against the UHPLC-UV method. Ag(I)-impregnated paper spray MS had the same working range (THC/CBD = 0.001-1) as UHPLC-UV analysis (R2 = 0.9896 and R2 = 0.9998, respectively), as well as comparable accuracy (-2.7 to 14%) and precision (RSD 1.7-11%). The method was further validated by the analysis of 10 commercial oils by Ag(I)-impregnated paper spray MS and UHPLC-UV analysis. Based on the determined relative concentration ratios of THC/CBD and the declared CBD concentration, 6 out of 10 CBD oils appear to contain more THC than the Dutch legal limit of 0.05%.

Visible-Light-Promoted Biomimetic Reductive Functionalization of Quaternary Benzophenanthridine Alkaloids.

Reduction of an iminium C═N double bond is the most important phase I metabolism process associated with the cytotoxic property of quaternary benzophenanthridine alkaloids (QBAs). Inspired by the light-mediated reduction of QBAs with nicotinamide adenine dinucleotide, a visible-light-promoted reductive functionalization reaction of QBAs is reported in this study. C4-Alkyl-1,4-dihydropyridines (DHPs) enable the direct reductive alkylation of QBA independently, serving as both single-electron-transfer reductant reagents under irradiation with 455 nm blue light in the absence of photocatalysts and additional additives. Our protocol can be further applied to the semisynthesis of natural 6-substituted dihydrobenzophenanthridine derivatives such as O-acetyl maclekarpine E.

Cryptochrome 2 from Lilium × formolongi Regulates Photoperiodic Flowering in Transgenic Arabidopsis thaliana.

The photoperiodic flowering pathway is essential for plant reproduction. As blue and ultraviolet-A light receptors, cryptochromes play an important role in the photoperiodic regulation of flowering. Lilium × formolongi is an important cut flower that flowers within a year after seed propagation. Floral induction is highly sensitive to photoperiod. In this study, we isolated the CRYPTOCHROME2 gene (LfCRY2) from L. × formolongi. The predicted LfCRY2 protein was highly homologous to other CRY2 proteins. The transcription of LfCRY2 was induced by blue light. LfCRY2 exhibits its highest diurnal expression during the floral induction stage under both long-day and short-day photoperiods. Overexpression of LfCRY2 in Arabidopsis thaliana promoted flowering under long days but not short days, and inhibited hypocotyl elongation under blue light. Furthermore, LfCRY2 was located in the nucleus and could interact with L. × formolongi CONSTANS-like 9 (LfCOL9) and A. thaliana CRY-interacting basic-helix-loop-helix 1 (AtCIB1) in both yeast and onion cells, which supports the hypothesis that LfCRY2 hastens the floral transition via the CIB1-CO pathway in a manner similar to AtCRY2. These results provide evidence that LfCRY2 plays a vital role in promoting flowering under long days in L. × formolongi.