Isolation, characterization, identification and quantification of 6-F oxyphenisatin dipropionate, a novel illegal additive, from a fruit-flavored jelly.

Objective: This study is aimed to screen, identify and detect illegal additives from healthcare products which claim or imply to have weight-loss effects. Method: Ultra-high performance liquid chromatography-quadruple-time-of-flight mass spectroscopy (UPLC-Q-TOF/MS) was employed to perform non-targeted screening of illegal additives from a total of 26 batches of healthcare products with weight-loss effects. A novel oxyphenisatin dipropionate analog was discovered in a fruit-flavored jelly that was not clearly labeled as containing added drugs. After being separated and purified by silica gel column chromatography, the analog was unambiguously characterized by one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopies. The molecular structure of the analog was finally identified by comparing the spectra of the analog with those of suspected candidates prepared by de novo synthesis strategy. Thereafter, a sensitive and precise reversed phase ultra performance liquid chromatography coupled with photodiode array (UPLC-PDA) detection method was developed and verified for the determination of the analog in 15 batches of real samples. Results: In the MS/MS spectra, the signal intensity of mass/charge ratios (m/z, 242 and 214) of the novel analog fragments was highly similar to that of mass/charge ratios (m/z, 224 and 196) of oxyphenisatin dipropionate fragments. Additionally, the 1D NMR spectrum of the analog was completely consistent with that of one of the suspected candidates prepared by the de novo synthesis strategy. Based on the above analysis, the structure of the analog was determined as 3,3-bis[4'-(propionyloxy)phenyl]-6-fluoro-2-oxoindoline, which was briefly named 6-F oxyphenisatin dipropionate. A developed quantitative method showed good linearity (R2 > 0.999) in a concentration range of 1.0-100 µg/mL. The limits of detection (LOD) and quantification (LOQ) for the analog was 3 mg/kg and 10 mg/kg, respectively. The average recoveries of the analog from spiked three different matrix samples in low (1 time of LOQ), medium (2 times of LOQ), and high (10 times of LOQ) concentrations were varied from 93.9 % to 107.8 % with a precision of 0.03-1.56 %. Results of quantitative analysis in 15 batches of healthcare products revealed that the content of 6-F oxyphenisatin dipropionate in a fruit-flavored jelly and a solid beverage was 118 mg/kg and 330 mg/kg, respectively. Conclusion: In terms of its structure, 6-F oxyphenisatin dipropionate replaces hydrogen atom by the fluorine atom at position 6 on the indolinone fragment in oxyphenisatin dipropionate. To our best knowledge, 6-F oxyphenisatin dipropionate has never been detected as an illegal additive in foods. Such illegal addition of the analog to foods is more concealing, thus the supervision and testing departments should attach great importance to its application in food markets.

Identification of lymph node adulteration in minced pork by comprehensive metabolomics and lipidomics approach based on UPLC/LTQ-Orbitrap MS.

The deliberate pork adulteration with lymph nodes is a common adulteration phenomenon, and it poses a serious threat to public health and food safety. An untargeted metabolomics and lipidomics approach based on ultrahigh performance liquid chromatography coupled with linear ion trap quadrupole-Orbitrap high resolution mass spectrometry (MS) was used to distinguish lymph nodes from minced pork. The principal component analysis and orthogonal projection to latent structures discriminant analysis models were established with the good of fitness and predictivity. The results showed that there were significant differences in metabolites and lipids between lymph nodes and pork. A total of 16 significantly differentiated metabolites were identified, of which 1-palmitoylglycerophosphocholine, 12,13-dihydroxy-9-octadecenoic acid, and prostaglandin E2 (PGE2) were positively correlated with lymph node content and were identified as potential markers of lymph nodes. These three markers were combined to create a binary logistic regression model, and a combined-factor exceeding 0.75 was ultimately identified as a marker for pork adulteration with lymph nodes. The desorption electrospray ionization-MS images showed that PGE2 had a higher relative abundance in the lymph node region than in adjacent non-lymph node regions, indicating that PGE2 was a marker that contributed significantly for identifying lymph nodes adulteration into pork. Our results provide a theoretical basis for identifying lymph node adulteration, which will contribute to combating fraud in the meat industry.

A novel vardenafil analog in a healthcare product: Preparation, characterization, and quantification.

OBJECTIVE: This study is aimed to develop a qualitative and quantitative method to detect a novel vardenafil analogue from a healthcare product, which is claimed to enhance sexual function. Method: The unknown compound was detected by non-targeted screening using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). MS2 spectra showed that the characteristic fragment ions of this unknown compound were highly similar to those of vardenafil. This compound was subsequently isolated by silica gel column chromatography and characterized by 1D (dimension) and 2D nuclear magnet resonance (NMR) specta, ultra-violet (UV) spectra, and fourier transform infrared (IR) spectra. A quantitative method for analyzing this identified compound in various healthcare product was developed based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS: The unknown compound was identified as 2-(5-((4-ethylpiperazin-1-yl)sulfonyl)-2-propoxyphenyl)-5-methyl-7-propylimidazo.[5,1-f] [1,2,4]triazin-4(3H)-one based on the spectroscopic data. Quantitative results revealed that the matrix calibration curves of this compound had a good linear ranges of 2～50 ng/mL in pressed candy (R2 = 0.998), energy coffee (R2 = 0.999), and health wine (R2 = 0.997), respectively. The matrix effects, recoveries, and limit of quantitation (LOQ) of this compound all met the requirements of quantitative validation. Finally, the content of this compound in 5 batches of positive samples ranged from 1.24 to 7.20 g/kg. Conclusion: This study identified a novel vardenafil analog from a healthcare product and named it O-propyl vardenafil, and this compound was distinguished from vardenafil by the replacement of the ethyl group with a propyl group at the aryl alkyl ether moiety. Our developed quantitative method could meet practical needs. The high positive rate (16.67%) in 30 samples suggested that the related regulators should be alert to O-propyl vardenafil in routine test since it has not been detected ever before.

Optimization of adsorption performance of cerium-loaded intercalated bentonite by CCD-RSM and GA-BPNN and its application in simultaneous removal of phosphorus and ammonia nitrogen.

Excessive phosphorus (P) and ammonia nitrogen (NH3-N) in water bodies can lead to eutrophication of the aquatic environment. Therefore, it is important to develop a technology that can efficiently remove P and NH3-N from water. Here, the adsorption performance of cerium-loaded intercalated bentonite (Ce-bentonite) was optimized based on single-factor experiments using central composite design-response surface methodology (CCD-RSM) and genetic algorithm-back propagation neural network (GA-BPNN) models. Based on the determination coefficient (R2), mean absolute error (MAE), mean square error (MSE), mean absolute percentage error (MAPE), and root mean square error (RMSE), the GA-BPNN model was found to be more accurate in predicting adsorption conditions than the CCD-RSM model. The validation results showed that the removal efficiency of P and NH3-N by Ce-bentonite under optimal adsorption conditions (adsorbent dosage = 1.0 g, adsorption time = 60 min, pH = 8, initial concentration = 30 mg/L) reached 95.70% and 65.93%. Furthermore, based on the application of these optimal conditions in simultaneous removal of P and NH3-N by Ce-bentonite, pseudo-second order and Freundlich models were able to better analyze adsorption kinetics and isotherms. It is concluded that the optimization of experimental conditions by GA-BPNN has some guidance and provides a new approach to explore adsorption performance after optimizing the conditions.

Development of a microecologic product from Lactobacillus rhamnosus based on silica.

BACKGROUND: Probiotics are primarily made into microecologic products for use in the food and feed industries. The freeze-drying technique is widely used in their preparation to maintain their high level of bioactivity. This causes high costs in terms of the energy and time needed. In this study, we developed a method to produce a highly active microecologic product from Lactobacillus rhamnosus using heating and silica. RESULTS: A microecologic product was made successfully from L. rhamnosus using the whole bacterial culture broth, without waste, and using food-grade silica (4.5 mL g-1 ) to absorb water before drying at 37 °C for 8 h. The activity of L. rhamnosus cells was increased significantly by adding water extracts of green tea to the culture medium. The viable amount of L. rhamnosus in the obtained microecologic product was 9.80 × 1010 cfu g-1 with a survival rate of 224.67% in simulated gastric juice for 3 h and 68.2% in simulated intestinal juice for 3 h. The microecologic product treated an intestinal infection by multi-drug-resistant Staphylococcus aureus in mice very efficiently. CONCLUSION: The study developed an economic, eco-friendly, and efficient method for preparing highly active microecologic agents using heating and without waste. © 2022 Society of Chemical Industry.

Structure identification and toxicity evaluation of one newly-discovered dechlorinated photoproducts of chlorpyrifos.

Chlorpyrifos (CPF) is an extensively used organophosphorus pesticide. Recently, it has attracted increasing attention due to environmental health problems caused by it. Although numerous studies have discovered the dechlorinated photoproduct of CPF, its structure and toxicity remain largely unknown. In this study, we systematically investigated the structure and toxicity of dechlorinated photoproduct of CPF. The CPF degradation experiment was performed, and its products were identified by ultra high performance liquid chromatography-orbitrap fusion tribid mass spectrometer (UHPLC-Orbitrap Fusion TMS). Additionally, bond dissociation energy (BDE) calculations and photoproduct chemical synthesis were employed to determine the structure of dechlorinated photoproduct of CPF. The toxicity of CPF photoproduct was evaluated through the Ecological Structure Activity Relationships (ECOSAR) Class Program, the Toxicity Estimation Software Tool (T.E.S.T.) software, and acute toxicity testing. The results indicated that the dechlorinated photoproduct of CPF was identified as O,O-Diethyl-O-(3,5-dichloro-2-pyridyl) phosphorothioate (Dechloro-CPF), which was produced in large quantity within the first 30 min of photodegradation experiment. The acute and chronic toxicity values of Dechloro-CPF were obviously higher than those for the other two photoproducts. The median lethal dose (LD50) of Dechloro-CPF was 31.6 mg/kg for female mice and 58.4 mg/kg for male mice. This study reveals the photodegradation mechanism of CPF and confirms that Dechloro-CPF was dechlorinated photoproduct of CPF with potential acute toxicity to aquatic species and mammalian (including human). Our findings will contribute to a more comprehensive risk evaluation of CPF in food and the environment.

[Rapid screening and identification of 62 kinds of illegally added traditional Chinese medicine in food by high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry].

An analytical method based on high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS) was established for the rapid screening and identification of 62 kinds of illegally added traditional Chinese medicine (TCM) in food. According to the notice of the Ministry of Health of the People's Republic of China on further regulating the management of raw materials of health food (Weifa Jianfa (2002) No. 51), the characteristic components of the 62 kinds of TCM were screened, and the corresponding characteristic component lists of different TCM were obtained. Methanolic extracts of the 62 kinds of standard medicinal materials were subjected to HPLC-Q-TOF-MS analysis. The filtrate was separated on a Thermo Accucore aQ column (150 mm×2.1 mm, 2.6 µm) using 0.1%(v/v) formic acid aqueous solution or water and acetonitrile as the mobile phases for gradient elution in the electrospray positive and negative ion scanning mode. All the data were determined on the full scan of primary mass spectrometry and secondary mass spectrometry, with mass acquisition ranges of 100-1000 Da and 50-1000 Da, respectively. A 10 mmol/L sodium formate solution was used as the mass correction solution in both the positive and negative ion modes. Library View software was used to establish the precursor ion accurate quality database and the product ion fragment mass spectrometry database of the corresponding characteristic components of the different kinds of TCM. In the Library View database software, the name of each characteristic component of the 62 kinds of TCM was input (serial number) in order to classify the screened characteristic components. The samples were processed using the same method and analyzed. Peak View software was used to rapidly analyze and screen the target components of the TCM. The high-resolution data collected from the samples to be tested were imported into the Peak View software, followed by the compound list of the established MS database of standard medicinal materials. After setting the identification method parameters and library retrieval parameters, a matching analysis was performed, and the candidate substances for each peak were automatically identified by comparing the mass spectrum, accurate molecular ion mass number, fragment ion mass number, retention time, and other related parameters. The determination conditions of compound detection were as follows: the comprehensive score was more than 70 points. The molecular formula, retention time, mass spectrum as well as the primary isotope mass spectrometry, primary mass spectrometry, and secondary mass spectrometry data were matched with the library compounds. The corresponding list of "TCM-characteristic components" was established, and a high-resolution MS library of 388 characteristic components from the 62 types of TCM was constructed. Each TCM contains 5-10 characteristic components. According to the screening analysis of the actual food samples of the prepared wine, substitute tea, and beverage, one batch of the prepared wine sample matched with seven characteristic components of epimedium, and it was inferred that epimedium was added to the prepared wine samples. This method can allow for the qualitative screening of TCM without standards and has the characteristics of high throughput, accuracy, simplicity, and rapidity. It solves the difficulty in identifying and confirming illegally added TCM in food; provides technical methods and a basis for cracking down on the illegal addition of TCM in food; and facilitates the rapid screening and identification of illegally added TCM in food.

The capability of Bacillus pseudomycoides from soil to remove Cu(II) in water and prevent it from entering plants.

AIMS: Copper ion is widespread in wastewater and threatens the condition and human health. Micro-organisms have unique advantages to remove heavy-metal ions from water, but are rarely reported in the removal of copper ion. This aims to develop micro-organisms that can remove copper ion in water, characterize their properties and analyse their potential application in practice. METHODS AND RESULTS: Sewage sludge was used as the source to isolate wild bacteria that can remove copper ion in water. The most efficient strain was screened out from 23 obtained isolates, identified as Bacillus pseudomycoides and coded as C6. The properties of C6 in the removal of copper ion in water were investigated in the aspects of reaction conditions, reaction groups, reaction dynamic and the application in oat planting. The reaction at pH 7 within 10 min yielded the highest removal rate of copper ion, 83%. The presence of lead ion in the reaction system could promote the removal rate of copper ion. Carboxyl groups and amidogen of C6 biomass were mainly involved in the removal of copper ion. The removal of copper ion was in accord with single-layer adsorption and Langmuir adsorption isotherm model. In application, C6 biomass reduced the copper content in the oat seedlings grown in copper ion containing water by more than seven times. CONCLUSIONS: B. pseudomycoides C6 can efficiently remove copper ion in water and inhibit it from entering plants. SIGNIFICANCE AND IMPACT OF STUDY: This is the first time to report the capability of B. pseudomycoides to remove copper ion in water, which is also more efficient than the currently reported chemical and biological methods.

[Screening and confirmation of 244 pesticide residues in chilli by gas chromatography-quadrupole time-of-flight mass spectrometry].

QuEChERS pretreatment combined with gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q-TOF/MS) has been investigated for application in screening 244 pesticide residues in chilli. Fresh chilli samples were extracted with acetonitrile, and dried chilli samples were extracted using an acetonitrile/acetic acid (99∶1, v/v) mixture. The two extraction solvents were stored at -20 ℃. After salting out and cleaning by dispersive solid phase extraction (dSPE), heptachlor epoxide B was added as an internal standard, and the resulting residues were dissolved in 1.00 mL acetone. The dissolved sample solution was loaded onto an HP-5MS UI column (30 m×0.25 mm, 0.25 µm) and eluted by GC-Q-TOF/MS with a programmable temperature vaporizer and splitless injection in the full-scan mode. The compensation effects of the analytical protectant (AP) and matrix-matched calibration method on the matrix effect were established. AP could be used in the fresh chilli matrix to compensate for matrix effects, but it was not effective in the dried chilli matrix. The matrix-matched calibration method was effective in both matrices, which was selected for the quantification of pesticide residues in the samples. Because of the existence of the isomers of one compound and the same characteristic ions of different compounds, analyte detection was based on a flexible retention time deviation of ±0.25 min and accurate mass deviation of ±20×10 -6. Screening was performed by the software in the automatic matching mode. Compound identification and quantitation were based on a database and calibration curve established with reference materials. Suspicious samples were subjected to manual analysis. Quantitative analysis of 244 pesticide residues in fresh chilli and 222 pesticide residues in dried chilli was performed. The results showed that the developed database and method can provide a reference for the high-throughput screening and quantitation of fresh and dried chilli. Different levels of pesticides were added to the blank chilli samples, and the addition level corresponding to a signal-to-noise ratio (S/N) of 10 was used as the limit of quantification (LOQ). The LOQs of 44 pesticides with a maximum residue limit (MRL) ≤0.05 mg/kg in fresh chilli did not exceed 0.010 mg/kg. The linear ranges of these 44 pesticides were 0.01-1.00 mg/L. At spiked levels of the LOQ and 2.5 times the LOQ, the ratios of the 44 pesticides with recoveries of 60% to 120% were 88.64% and 100%, respectively. The LOQs of 200 pesticides with MRLs ≥0.05 mg/kg or without MRLs in fresh chilli did not exceed 0.025 mg/kg. The linear ranges of these 200 pesticides were 0.05-1.00. At spiked levels of the LOQ, twice the LOQ, and 10 times the LOQ, the ratios of the 200 pesticides with recoveries of 60% to 120% were 49.50%, 87.00%, and 89.50%, respectively. The linear correlation coefficients (r 2) of the 244 pesticides in fresh chilli were greater than 0.99. The LOQs of 222 pesticides in dried chilli were less than 0.15 mg/kg, and the linear ranges were 0.04-1.00 mg/L. The ratios of these 222 pesticides with r 2 greater than 0.99 was 95.46%. At spiked levels of the LOQ, twice the LOQ and 10 times the LOQ in dried chilli, the ratio of the 222 pesticides with recoveries of 60% to 120% were 72.52%, 73.42%, and 81.53%, respectively. The established screening and confirmation method was used to analyze 12 fresh chilli samples and 14 dried chilli samples. Eight pesticides were found in nine fresh chilli samples and three dried chilli samples, all of which were confirmed to be positive after manual identification. The concentrations of these pesticides were lower than the MRLs required by GB 2763-2019: National Food Safety Standard Maximum Residue Limits for Pesticides in Food. The results demonstrate that the established method is rapid, easy to execute, efficient, and reliable. It can be used for the high-throughput screening and quantitation of pesticide residues in fresh and dried chilli.

Therapeutic effect of Lactobacillus rhamnosus SHA113 on intestinal infection by multi-drug-resistant Staphylococcus aureus and its underlying mechanisms.

Staphylococcus aureus, especially multi-drug-resistant (MDR) pathogenic S. aureus, poses a severe threat to food safety and human health. Probiotics offer promising potential for the control of MDR pathogens because of their safe and biofunctional properties. This study shows that Lactobacillus rhamnosus SHA113, a strain isolated from the milk of healthy women, could efficiently inhibit MDR S. aureus both in vitro and in vivo. In vitro, L. rhamnosus efficiently inhibited and even killed drug resistant and drug sensitive S. aureus strains. In vivo experiments showed that SHA113 could efficiently decrease the number of S. aureus cells, inhibit the expression of inflammatory factors TNF-α and IL-6, and restore the level of white cells and neutrophils in the blood. SHA113 could also efficiently repair damage of the intestinal barrier and other functions impaired by S. aureus infection. This was indicated by a change of intestinal villi length and structure, and an up-regulated expression of tight junction proteins ZO-1 and occludin. SHA113 also restored the structural damage of immune organs, such as the enlargement of the spleen and the increased level of inflammatory cytokines caused by S. aureus infection. More importantly, L. rhamnosus SHA113 showed more effective inhibitory and therapeutic effects on MDR S. aureus strain ZBQ006 than on drug sensitive S. aureus strain 29213. These results illustrated that L. rhamnosus SHA113 has great potential for the treatment of MDR S. aureus contamination as food control and for therapeutic treatment.

Novel Biomedical Functions of Surfactin A from Bacillus subtilis in Wound Healing Promotion and Scar Inhibition.

Surfactin produced by Bacillus subtilis is a powerful biosurfactant in food, cosmetics, and pesticide industries. However, its suitability in wound healing applications is uncertain. In this article, we determined the effects of surfactin A from B. subtilis on wound healing, angiogenesis, cell migration, inflammatory response, and scar formation. The results indicated that 80.65 ± 2.03% of surfactin A-treated wounds were closed, whereas 44.30 ± 4.26% of the vehicle-treated wound areas remained open on day 7 (P < 0.05). In mechanisms, it upregulated the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), accelerated keratinocyte migration through mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways, and regulated the secretion of proinflammatory cytokines and macrophage phenotypic switch. More attractive, surfactin A showed a seductive capability to inhibit scar tissue formation by affecting the expression of α-smooth muscle actin (α-SMA) and transforming growth factor (TGF-ß). Overall, the study revealed a new function and potential of surfactin A as an affordable and efficient wound healing drug.

(-)-Syringaresinol-4-O-ß-D-glucopyranoside from Cortex Albizziae inhibits corticosterone-induced PC12 cell apoptosis and relieves the associated dysfunction.

The neuroprotective effects and potential mechanisms of (-)-Syringaresinol-4-O-ß-D-glucopyranoside (SRG), a natural lignan glycoside extracted from Cortex Albizziae, were investigated using corticosterone (CORT)-induced PC12 cells as an in vitro anxiety model. PC12 cells were treated with 100 µM CORT and 5, 10, or 20 µM SRG for 48 h. Cell viability and lactate dehydrogenase (LDH) leakage were measured. Apoptosis were detected using FITC-coupled Annexin V (AV) and propidium iodide (PI) staining flow cytometric analyses and TUNEL assays. Rhodamine 123 and Fluo-3-AM staining flow cytometric analyses were used to detect mitochondrial membrane potential (ΔΨm) and intracellular calcium concentration ([Ca2+]i), respectively. Western blot was used to detect brain-derived neurotrophic factor (BDNF), Bax, Bcl-2, cAMP-response element binding protein (CREB), cytosolic cytochrome c (Cyt c), caspase-3, and cleaved caspase-3. Experimental data showed that SRG promoted cell proliferation, reduced LDH release, inhibited apoptosis, improved ΔΨm values, decreased [Ca2+]i, up-regulated CREB, BDNF, and Bcl-2, down-regulated Bax and Cyt c protein expression levels, and reduced caspase-3 activity. This suggests that SRG has neuroprotective and antiapoptotic effects in the pathogenesis of anxiety disorders, and its mechanisms are partly connecte to inhibition of the mitochondrial apoptotic pathway and activation of pathways involving CREB and BDNF.

A Review of ULK1-Mediated Autophagy in Drug Resistance of Cancer.

The difficulty of early diagnosis and the development of drug resistance are two major barriers to the successful treatment of cancer. Autophagy plays a crucial role in several cellular functions, and its dysregulation is associated with both tumorigenesis and drug resistance. Unc-51-like kinase 1 (ULK1) is a serine/threonine kinase that participates in the initiation of autophagy. Many studies have indicated that compounds that directly or indirectly target ULK1 could be used for tumor therapy. However, reports of the therapeutic effects of these compounds have come to conflicting conclusions. In this work, we reviewed recent studies related to the effects of ULK1 on the regulation of autophagy and the development of drug resistance in cancers, with the aim of clarifying the mechanistic underpinnings of this therapeutic target.

Conversion of DON to 3-epi-DON in vitro and toxicity reduction of DON in vivo by Lactobacillus rhamnosus.

Deoxynivalenol (DON) is a mycotoxin that contaminates grains and feed. Degradation and toxicity reduction of DON by probiotics benefit human and animal health. Lactobacillus rhamnosus is an FDA-approved probiotic that can be used in children. After screening seven L. rhamnosus strains isolated from human milk, SHA113 showed the highest DON degradation rate of 60% under the optimal conditions of 37 °C, pH 6.0, OD600 = 1.5, 5 mg L-1 DON, and 48 h. When dead and live SHA113 cells were used separately, only the live cells reduced the DON concentration and transformed it into 3-epi-DON. Mice feeding experiments showed that pretreatment with SHA113 for 48 h reduced the toxicity of DON to the immunological system and organs. Directly feeding SHA113 cells could also slightly reduce the DON toxicity. Both in vitro and in vivo experiments indicated that L. rhamnosus has potential to reduce DON toxicity.