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Sodium saccharin is a common artificial sweetener. However, due to its possible carcinogenic effects and causing metabolic disorders, many countries have strictly regulated its use in food. In the study, we prepared a specific monoclonal antibody (mAb 2H11) using the new hapten (6-carboxylsaccharin) and developed a direct competitive enzyme-linked immunosorbent assay (dcELISA) for the screening of sodium saccharin residue in food. The half-maximum inhibition concentration (IC50 ) and working range (IC20 -IC80 , the concentrations causing 20% and 80% inhibition by sodium saccharin) were 32.5 and 6.47 to 164 ng/mL, which was 6.5 times more sensitive than the previously reported immunoassay. The average recoveries of sodium saccharin in spiked food samples detected by dcELISA ranged from 82.1% to 117%. Among 70 food samples bought in the physical stores and online, sodium saccharin residues were only detected in four samples purchased online (one canned pineapple, two winter jujube, and one kimchi). The content measured by dcELISA agreed well with those determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The developed dcELISA was proved to be a sensitive and accurate method for determining sodium saccharin in food. PRACTICAL APPLICATION: Quantitation of sodium saccharin residue in food is very necessary and important for consumers and regulatory agencies.

Anthracnose caused by Colletotrichum gloeosporioides critically threatens the growth and commercial cultivation of Sarcandra glabra. However, the defence responses and underlying mechanisms remain unclear. Herein, we aimed to investigate the molecular reprogramming in S. glabra leaves infected with C. gloeosporioides. Leaf tissues at 0, 24 and 48 h post-inoculation (hpi) were analysed by combining RNA sequencing and Tandem Mass Tag-based liquid chromatography with tandem mass spectrometry. In total, 18 441 and 25 691 differentially expressed genes were identified at 24 and 48 hpi compared to 0 hpi (uninoculated control), respectively. In addition, 1240 and 1570 differentially abundant proteins were discovered at 24 and 48 hpi compared to 0 hpi, respectively. Correlation analysis revealed that transcription and translation levels were highly consistent regarding repeatability and expression. Analyses using databases KEGG and iPATH revealed tricitric acid cycle, glycolysis/gluconeogenesis and phenylpropanoid biosynthesis were induced, whereas photosynthesis and tryptophan were suppressed. Enzymatic activity assay results were consistent with the upregulation of defence-related enzymes including superoxide dismutases, catalases, peroxidases and chitinases. The transcriptome expression results were additionally validated by quantitative real-time polymerase chain reaction analyses. This study provides insights into the molecular reprogramming in S. glabra leaves during infection, which lay a foundation for investigating the mechanisms of host- Colletotrichum interactions and breeding disease-resistant plants. Anthracnose caused by Colletotrichum gloeosporioides critically threatens the growth and commercial cultivation of the medicinal plant Sarcandra glabra. We explored the molecular changes during the early stages of this infection through systems biology. This study provides insights into the molecular reprogramming in S. glabra leaves during infection, which lay a foundation for investigating the mechanisms of host -Colletotrichum interactions and breeding disease-resistant plants. [ABSTRACT FROM AUTHOR]

The safety of medicinal herbs is critical to human health, and the monitoring of pesticide residues in medicinal herbs is necessary. In the present study, a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for the determination of imidacloprid in medicinal herbs. Using the optimal conditions, the 50% inhibiting concentration and detection range (20–80% inhibiting concentration) of the established dc-ELISA were 0.08 and 0.03–0.29 ng/mL, respectively. Except for clothianidin (4.64%) and acetamiprid (2.52%), the cross-reactivity with analyte analogs was negligible. In purple perilla, cordate houttuynia, water lily, bitter melon, and mung bean, the average imidacloprid recoveries were from 62.97 to 134.09%, with relative standard deviations between 0.21 and 14.58%. Furthermore, the immunoassay results correlated well with spiked samples analyzed by high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS). The established immunoassay is rapid, sensitive, and specific and suitable to monitor imidacloprid residues in medicinal herbs. [ABSTRACT FROM AUTHOR]

Centella asiatica (L.) Urban is a well-known medicinal plant which has multiple pharmacological properties. Notably, the leaves of C. asiatica contain large amounts of triterpenoid saponins. However, there have only been a few studies systematically elucidating the metabolic dynamics and transcriptional differences regarding triterpenoid saponin biosynthesis during the leaf development stages of C. asiatica. Here, we performed a comprehensive analysis of the metabolome and transcriptome to reveal the dynamic patterns of triterpenoid saponin accumulation and identified the key candidate genes associated with their biosynthesis in C. asiatica leaves. In this study, we found that the key precursors in the synthesis of terpenoids, including DMAPP, IPP and β-amyrin, as well as 22 triterpenes and eight triterpenoid saponins were considered as differentially accumulated metabolites. The concentrations of DMAPP, IPP and β-amyrin showed significant increases during the entire stage of leaf development. The levels of 12 triterpenes decreased only during the later stages of leaf development, but five triterpenoid saponins rapidly accumulated at the early stages, and later decreased to a constant level. Furthermore, 48 genes involved in the MVA, MEP and 2, 3-oxidosqualene biosynthetic pathways were selected following gene annotation. Then, 17 CYP450s and 26 UGTs, which are respectively responsible for backbone modifications, were used for phylogenetic-tree construction and time-specific expression analysis. From these data, by integrating metabolomics and transcriptomics analyses, we identified CaHDR1 and CaIDI2 as the candidate genes associated with DMAPP and IPP synthesis, respectively, and CaβAS1 as the one regulating β-amyrin synthesis. Two genes from the CYP716 family were confirmed as CaCYP716A83 and CaCYP716C11 . We also selected two UGT73 families as candidate genes, associated with glycosylation of the terpenoid backbone at C-3 in C. asiatica . These findings will pave the way for further research on the molecular mechanisms associated with triterpenoid saponin biosynthesis in C. asiatica ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wan, Huang, Li, Yu, Tan, Wei, El-Sappah, Sooranna, Zhang, Pan, Zhang and Lei.)

The root of Sophora tonkinensis Gagnep is an important medicinal material in China. An unknown foliar disease, first observed In July 2018, occurred over 240 ha of S. tonkinensis (totally cultured 600 ha) in Guangxi, China, in December 2019. The initial symptoms on leaf were seen as small, tawny spots (0.5 to 1.5 mm in diam.). As the disease progressed, the lesions enlarged into grayish and dark brown concentric rings (5 to 10.0 mm in diam.) resulting in black protuberances in the center of the spots. Severe infections would adversely affect plant growth, and cut the production by 30-40%. Symptomatic leaves were sampled and the surface was sterilized with 75% ethanol for 30 s and then soaked in 0.1% HgCl2 for 2 min. After three washes with sterile distilled water, the samples were dried, placed aseptically onto potato dextrose agar (PDA) plates, and incubated at 28°C. Three days later, the isolates were placed on new PDA medium for subsequent purification and sporulation. The fungus, SDG-1, was recovered from 85% of the total 40 isolates. Its colonies were whitish initially and then became olive green 7 days after incubation at 28 °C. The pycnidias were globose to subglobose, initially brown and darken at maturity, 85 to 300 × 70 to 280 μm in size. The conidia were colorless, single-celled, rounded to ellipsoidal and 3.5 to 6.6 × 1.5 to 3.8 μm. The chlamydospores were multicellular and brick trellised, usually forming branched or unbranched chains, light to dark brown in color and measured 28.5 to 44.5 × 8.2 to 16.5 μm. The morphological characteristics were consistent with Didymella glomerata. The rDNA-ITS, β-tubulin and actin of strain SDG-1 were PCR amplified, and the DNA sequencing results were almost 100% identical to those of D. glomerata, respectively (GenBank database accession numbers MN 435377, MN447333 and MN447334, respectively). The multi-locus phylogenetic analysis was carried out with the obtained sequences, which revealed that the isolates clustered within D. glomerata with the similarity of 100% (Fig.3). Therefore, based on the morphology and phylogenetic tree, strain SDG-1 was identified as D. glomerata. For pathogenicity tests, the S. tonkinensis was cultured for two years, and the conidial suspension of SDG-1 (1 × 106 conidia /mL) was prepared by harvesting conidia from a 10-day-old culture on PDA. Conidia were sprayed onto the healthy leaves of S. tonkinensis for co-culture, while the control group was sprayed with sterile distilled water. Each experiment was performed three times. All plants were covered with plastic bags for 3 days in order to maintain high humidity and cultured in a greenhouse at 28 °C with a 12-h/12-h light/dark cycle. The symptoms appeared 7 days after the leaves were inoculated with spores, which were identical to those observed in the field. The pathogenic fungus was re-isolated from the infected leaves and identified as previously described. The control leaves remained symptomless during the pathogenicity tests. According to the previous literature, D. glomerata is a plant pathogenic fungus with a wide host range, which can damage up to 100 species of woody and herbaceous plants (Aghapour et al. 2009, Lahoz et al. 2007). To our knowledge, this is the first report of D. glomerata causing round leaf spot on S. tonkinensis in China.