Rapid Discrimination of Panax quinquefolium and Panax ginseng Using the Proofman-Duplex-LMTIA Technique.

This study aims to establish a rapid identification method based on the Proofman-LMTIA technique for distinguishing between Panax quinquefolium and Panax ginseng. By targeting specific 18S rDNA sequences, suitable primers and Proofman probes labeled FAM or JOE were designed for LMTIA. Initially, single-species-primer Proofman-LMTIA assays were performed separately for each ginseng type to optimize reaction temperature, assess sensitivity and specificity, and determine the detection limit. Subsequently, both sets of primers and their corresponding probes were combined in the same reaction system to further optimize reaction conditions, evaluate sensitivity, and assess stability. Finally, the developed Proofman-duplex-LMTIA technique was employed to detect P. quinquefolium and P. ginseng slices available in the market. Single-plex Proofman-LMTIA assays revealed that the optimal reaction temperature for both P. quinquefolium and P. ginseng was 62 °C. The sensitivity was as low as 1 pg/µL, with a detection limit of 0.1%, and both showed excellent specificity. The optimal temperature for Proofman-duplex-LMTIA assays was 58 °C. This method could simultaneously identify P. quinquefolium and P. ginseng. Testing 6 samples of P. ginseng and 11 samples of P. quinquefolium from the market resulted in a 100% positive rate for all samples. This study successfully established a rapid, simple, sensitive, and specific Proofman-duplex-LMTIA identification method for P. quinquefolium and P. ginseng. It provides an effective means for quality control of P. quinquefolium, P. ginseng, and related products.

Detection of Soybean-Derived Components in Dairy Products Using Proofreading Enzyme-Mediated Probe Cleavage Coupled with Ladder-Shape Melting Temperature Isothermal Amplification (Proofman-LMTIA).

Food adulteration is a serious problem all over the world. Establishing an accurate, sensitive and fast detection method is an important part of identifying food adulteration. Herein, a sequence-specific ladder-shape melting temperature isothermal amplification (LMTIA) assay was reported to detect soybean-derived components using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. The results showed that, under the optimal temperature of 57 °C, the established Proofman-LMTIA method for the detection of soybean-derived components in dairy products was sensitive to 1 pg/µL, with strong specificity, and could distinguish soybean genes from those of beef, mutton, sunflower, corn, walnut, etc. The established Proofman-LMTIA detection method was applied to the detection of actual samples of cow milk and goat milk. The results showed that the method was accurate, stable and reliable, and the detection results were not affected by a complex matrix without false positives or false negatives. It was proved that the method could be used for the detection and identification of soybean-derived components in actual dairy products samples.

Isolation and characterization of antioxidative monoterpenes from Cynanchum atratum roots.

One novel monoterpene rhamnoside (1) and 7 known monoterpenes (2-8) were isolated from the ethanol extract of Cynanchum atratum for the first time. Their structures were identified by comprehensive spectroscopic data analysis such as nuclear magnetic resonance, high-resolution electrospray ionization mass spectra, optical rotatory dispersion, and acid hydrolysis. In the subsequent antioxidant assay, compound 8 exhibited obvious 2,2-diphenyl-2-picrylhydrazyl hydrate radical scavenging activity.

Development of a Ladder-Shape Melting Temperature Isothermal Amplification (LMTIA) Assay for the Identification of Cassava Component in Sweet Potato Starch Noodles.

Food authenticity has become increasingly important as a result of food adulteration. To identify the authenticity of sweet potato starch noodles, the ladder-shape melting temperature isothermal amplification (LMTIA) method of determining cassava (Manihot esculenta Crantz) DNA in sweet potato starch noodles was used. A set of primers targeted at the internal transcription spacer (ITS) of cassava was designed, genomic DNA was extracted, the LMTIA reaction temperature was optimized, and the specificity of the primer was verified with the genomic DNAs of cassava, sweet potato (Ipomoea batatas L.), Solanum tuberosum L., Zea mays L., Vigna radiate L., Triticum aestivum L., and Glycine max (L.) Merr. The sensitivity with the serially diluted genomic DNA of cassava and the suitability for the DNA extracted from sweet potato starch adulterated with cassava starch were tested. The LMTIA assay for identifying the cassava component in sweet potato starch noodles was established. At the optimal temperature of 52 °C, the primers could specifically distinguish a 0.01% (w/w) cassava component added to sweet potato starch. Additionally, the LMTIA method was applied to the cassava DNA detection of 31 sweet potato starch noodle samples purchased from retail markets in China. Of these, 14 samples were positive. The LMTIA assay could be a reliable method for the rapid detection of cassava components in sweet potato starch noodles, to protect the rights of consumers and to regulate the sale market order of starch noodles.

A Comparison of In-House Real-Time LAMP Assays with a Commercial Assay for the Detection of Pathogenic Bacteria.

Molecular detection of bacterial pathogens based on LAMP methods is a faster and simpler approach than conventional culture methods. Although different LAMP-based methods for pathogenic bacterial detection are available, a systematic comparison of these different LAMP assays has not been performed. In this paper, we compared 12 in-house real-time LAMP assays with a commercialized kit (Isothermal Master Mix) for the detection of Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145 and Streptococcus agalactiae. False-positive results were observed in all 12 in-house real-time LAMP assays, while all the negative controls of Isothermal Master Mix remained negative after amplification. The detection limit of Isothermal Master Mix for Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121 and Streptococcus agalactiae was 1 pg, whereas the sensitivity of the commercialized kit for E. coli O145 was 100 pg. In conclusion, the 12 in-house real-time LAMP assays were impractical to use, while the commercialized kit Isothermal Master Mix was useful for the detection of most bacterial pathogens.

Accelerating diabetic wound healing with Ramulus Mori (Sangzhi) alkaloids via NRF2/HO-1/eNOS pathway.

Diabetic foot ulcers (DFUs) represent a severe complication of diabetes mellitus. Ramulus Mori (Sangzhi) alkaloids (SZ-A), an approved oral medication for type 2 diabetes, have not been explored for their potential to enhance the processes involved in diabetic wound healing. This study aims to investigate SZ-A's role in diabetic wound healing mechanisms. The in vivo experimentation involves dividing the subjects into NC and SZ-A groups, with SZ-A dosed at 200 and 400 mg/kg, to assess the therapeutic efficacy of SZ-A. The results of the animal studies show that SZ-A intervention accelerates the processes of diabetic angiogenesis and wound healing in a manner dependent on its concentration. Additionally, a pathological model using advanced glycation end products (AGEs) in HUVECs demonstrates SZ-A's cytoprotective effect. In vitro, SZ-A intervention significantly increases cell proliferation, migration and tube formation, protecting HUVECs from oxidative stress injury induced by AGEs. Mechanistically, SZ-A exerts a protective effect on HUVECs from oxidative stress damage through the activation of the NRF2/HO-1/eNOS signaling pathway. The findings suggest that SZ-A exhibits considerable potential as a promising candidate for treating DFUs, which will aid in more effectively integrating plant-based therapies into clinical settings.

Significantly enhanced specific activity of Bacillus subtilis (2,3)-butanediol dehydrogenase through computer-aided refinement of its substrate-binding pocket.

(2,3)-Butanediol dehydrogenases (BDHs) are widely utilized for the stereoselective interconversion between α-hydroxy ketones and vicinal diols to produce various functional building blocks. In this study, to enhance the specific activity towards (R)-phenyl-1,2-ethanediol (1a) for 2-hydroxyacetophenone (1b), the substrate-binding pocket of a Bacillus subtilis BDH (BsBDHA) was refined through site-directed mutagenesis. Based on molecular docking simulations, 14 residues were identified and subjected to alanine scanning mutagenesis. After screening, two residues, His42 and Gly292, were singled out for partial site-saturation mutagenesis. The results revealed that BsBDHAH42A and BsBDHAG292A displayed high activities of 3.21 and 1.97 U/mg, respectively. Employing combinatorial mutagenesis, a superior mutant, BsBDHAI49L/V266L/G292A, was developed, exhibiting significantly enhanced specific activity and catalytic efficiency towards (R)-1a, achieving 14.81 U/mg and 4.47 mM-1 s-1, respectively, which were 27.4- and 55.9-fold higher than those of BsBDHA. Further substrate spectrum analysis revealed that the superior mutant displayed increased specific activities for (R)-2a-6a by 1.4- to 10.3-fold. The integration of BsBDHAI49L/V266L/G292A into a three-enzymatic cascade for the synthesis of 1b effectively elevated the yield from 58.1 to 82.4 %. Molecular mechanism analysis indicated that the mutation-induced changes in intermolecular forces resulted in a higher frequency of reactive conformations for (R)-1a in BsBDHAI49L/V266L/G292A compared to BsBDHA.

Study on detection of soybean components in edible oil with ladder-shape melting temperature isothermal amplification (LMTIA) assay.

A ladder-shape melting temperature isothermal amplification (LMTIA) assay was established and used to detect soybean components in edible oils. LMTIA primers were designed with the sequence of the internal transcribed spacer (ITS) gene as the target, the reaction temperatures were optimized, the sensitivity was determined, and the suitability of the DNA extraction method for edible oil was assessed, with H2O and genomic DNA (gDNA) from corn, rapeseed, cottonseed, sesame, chili, chicken, pork, beef, and mutton as negative controls to test the false positives of the LMTIA assay. The established LMTIA assay gave a sensitivity of 1 pg at an optimal temperature of 57 °C. The Edible Oil DNA Extraction Kit was suitable for the LMTIA assay to detect soybean components in refined plant oil. No false positives occurred from all negative controls. This study successfully established the LMTIA assay for the detection of soybean ITS genes in edible oils, which could be used to detect soybean components in edible oils.

Natural glycosidic antioxidants from Cynanchum atratum roots.

Two new rhamnosides, 18-O-α-l-rhamnopyranosylabietic acid (1) and (E)-3,5-dimethoxystilben-4'-O-α-l-rhamnopyranoside (2), five known glucosides (3-7) along with three others were isolated from Cynanchum atratum roots. The structures of new compounds were elucidated by physical data analyses such as NMR, UV, IR, HR-ESI-MS, as well as acid hydrolysis. All of them were assessed for their antioxidant activities through 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical ion (ABTS•+), 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH•) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•) assay, with l-ascorbic acid used as the positive control. As a result, compounds 3-5 exhibited obvious antioxidant activities. These bioactive components could be promising antioxidants.

Synthesis of cinnamoyl glucoside derivatives and their antiproliferation activities against murine melanoma B16-F10 cell line.

Twelve cinnamoyl glucoside derivatives were prepared by glycosylation of glucosyl trichloroacetimidate and cinnamic acid derivatives, followed by dechloroacetylation with a pyridine/H2O mixture. Their structures were characterized by 1H and 13C NMR, as well as mass analysis. All the products were tested for their antiproliferation activities against murine melanoma B16-F10 cell line. Compounds 4e-4j were able to inhibit the proliferation of murine melanoma B16-F10 cell line with IC50 values of 17.38 ± 0.07, 9.87 ± 0.09, 9.69 ± 0.12, 29.42 ± 0.04, 32.95 ± 0.08, 25.68 ± 0.09 µM, respectively.

Ladder-shape melting temperature isothermal amplification of nucleic acids.

A novel method, termed ladder-shape melting temperature isothermal amplification (LMTIA), was developed in this study. As a proof of concept, one pair of primers or two pairs of nested primers and a thermostable DNA polymerase were employed to amplify the internal transcribed spacer of Oryza sativa with the ladder-shape melting temperature curve. Our results demonstrated that the LMTIA assay with nested primers was 50-fold more sensitive than the LAMP assay with the same level of specificity. The LMTIA method has the potential to be used for the prevention and control of emerging epidemics caused by different types of pathogens.

Two new phenylethanoid glycosides from Ginkgo biloba leaves and their tyrosinase inhibitory activities.

Two undescribed phenylethanoid glycosides, Ginkgoside C (1) and D (2), together with ten known glycosides (3-12) were isolated from Ginkgo biloba leaves. Their structures were characterized by physical data analyses such as NMR, HRESIMS, as well as chemical hydrolysis. All compounds were tested for their tyrosinase inhibitory activities. At a concentration of 25 µM, compounds 2, 4, 5, 6, and 11 showed obvious mushroom tyrosinase inhibition activities, with %inhibition values of 19.12 ± 2.59%, 25.79 ± 1.83%, 16.07 ± 1.07%, 24.46 ± 1.10%, 18.64 ± 3.62%, respectively, with kojic acid used as the positive control (27.50 ± 2.72%).

Chemoenzymatic synthesis of feruloylated monoacyl- and diacyl-glycerols in ionic liquids.

Feruloylated monoacyl- and diacyl-glycerols (FMAGs and FDAGs) are lipophilic antioxidants and potential UV absorbers. FMAGs and FDAGs were synthesized by a novel chemoenzymatic method: firstly, ferulic acid was esterified with glycerol to synthesize glyceryl ferulate, using p-toluenesulfonic acid as chemical catalyst in 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF(4)); secondly, glyceryl ferulate was esterified with oleic acid to synthesize FMAGs and FDAGs, using Novozym 435 as biocatalyst in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF(6)). The conversion of ferulic acid and yield of glyceryl ferulate in the first reaction were both 98%. The yields of FMAGs and FDAGs in the second reaction reached 34 +/- 2% and 66 +/- 3%, respectively.

Necessity of screening water chestnuts for microcystins after cyanobacterial blooms break out.

Water chestnut is one of the most popular vegetables in Asian countries that grows in shallow water. Eighteen water chestnut samples were collected from Lake Tai and six samples were bought at markets in Wuxi, China, in October 2007. Extraction solution of water chestnut was cleaned up with a solid phase extraction column and immunoaffinity chromatography cartridges, then the microcystin (MC) level was detected by indirect competitive enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS). The results of ELISA showed that there were six samples collected from Lake Tai which contained MCs; the highest level of total MCs was 7.02 ng/g. The results of LC-MS confirmed that MC-LR and MC-RR were present in five samples. The highest level of MC-LR was 1.02 ng/g and that of MC-RR was 4.44 ng/g. Heavy cyanobacterial blooms had occurred, and MCs were detected in water at the points in Lake Tai where MCs occurred in water chestnuts collected in 2007. MCs were not detected in the six samples bought at Wuxi markets. The results suggest that MCs can accumulate in water chestnuts, which is a potential hazard for human health.

Determination of microcystin-LR in water from Lake Tai, China.

Microcystin-LR (MC-LR) is a heptapeptide hepatotoxin produced by cyanobacteria. Immunoaffinity chromatography (IAC) column was prepared with CNBr-activated Sepharose 4B and monoclonal antibody of MC-LR. Water sample was cleaned up by IAC column and the content of MC-LR in water was determined by liquid chromatography-mass spectrometry (LC-MS). The results suggested that the IAC column exhibited highly selective specificity for MC-LR and selective removed interference from complex water sample. Water sample was concentrated for 2,000-fold through once purification. Cyanobacterial blooms had broken out in 2007 in Lake Tai, the third largest freshwater lake in China. Water samples from two parts of Lake Tai had been analyzed. The highest concentration of MC-LR in water from Lake Tai was 0.522 microg/L.

Enzymatic epoxidation of soybean oil using ionic liquid as reaction media.

An ionic liquid (IL) system for the enzymatic epoxidation of soybean oil was studied. The effects of active oxygen carriers (different fatty acids) and ILs ([Bmim]PF6 and [Bmim]BF4) on the enzymatic epoxidation were investigated. Response surface methodology (RSM) was used to study and optimize the effects of variables (reaction time, reaction temperature, molar ratio of H2O2/C=C-bonds, and molar ratio of fatty acid/C=C-bonds) on the epoxy oxygen group content (EOC) of epoxidized soybean oil (ESO). Results showed that the enzymatic epoxidation of soybean oil can be enhanced using tetradecanoic acid (C13H27COOH) as active oxygen carrier and [Bmim]PF6 as reaction medium. The optimum EOC of ESO was 5.9 ± 0.3% under the following conditions: reaction temperature 46°C, reaction time 11 h, enzyme load 3% (w/w, relative to the weight of soybean oil), molar ratio of H2O2/C=C-bonds 1.8:1, and molar ratio of C13H27COOH/C=C-bonds 0.5:1.