Aspergillus flavus (A. flavus). AFB1 is reported to have high thermal stability and is not decomposed by heat treatment during food processing. Therefore, in this study, knowing that AFB1 is metabolized by cytochrome P450 (CYP), our aim was to develop a method to detoxify A. flavus-contaminated maize, under normal temperature and pressure, using Escherichia coli expressing human CYP3A4. First, the metabolic activity of AFB1 by recombinant human CYP3A4 was evaluated. As a result, we confirmed that recombinant human CYP3A4 metabolizes 98% of AFB1. Next, we found that aflatoxin Q1, a metabolite of AFB1 was no longer mutagenic. Furthermore, we revealed that about 50% of the AFB1 metabolic activity can be maintained for 3 months when E. coli expressing human CYP3A4 is freeze-dried in the presence of trehalose. Finally, we found that 80% of AFB1 in A. flavus-contaminated maize was metabolized by E. coli expressing human CYP3A4 in the presence of surfactant triton X-405 at a final concentration of 10% (v/v). From these results, we conclude that AFB1 in A. flavus-contaminated maize can be detoxified under normal temperature and pressure by using E. coli expressing human CYP3A4.Aflatoxin B1 (AFB1) is a mycotoxin produced by  Aspergillus flavus (A. flavus) [Aflatoxin B1 (AFB1), a kind of mycotoxin, is mainly produced by  flavus) -4. In ad flavus) .e.g., using ozone, UV, clay minerals, etc.) have been reported [To date, various abiotic control methods have been tested against aflatoxins, and some of these methods  . P450 isStudies have shown that when AFB1 is metabolized by human CYP3A4, aflatoxin Q1 (AFQ1), which has relatively low toxicity, and AFB1-8,9-epoxide (AFBO), which is highly reactive with DNA and proteins, are produced . AFBO isA. flavus under normal temperature and pressure by using Escherichia coli (E. coli) expressing human CYP3A4. In addition, we attempted to improve the long-term storage stability of E. coli expressing human CYP3A4 as a biocatalyst at room temperature by evaluating the effects of disaccharides added during freeze-drying. Finally, we investigated the effect of nonionic detergents on the efficiency of AFB1 degradation by E. coli expressing human CYP3A4.In this study, we aimed to metabolize and detoxify AFB1 in maize contaminated with AFB1 was purchased from Toronto Research Chemicals (Canada), and AFM1 was purchased from LKT Laboratories Inc. (USA). We purchased 5-methoxypsoralen (5-MOP) from Tokyo Chemical Industry Co., Ltd. (Japan).Tryptone, dry yeast extract, Ampicillin sodium (Amp), and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Nacalai Tesque, Inc. (Japan). Sodium chloride (NaCl) and 5-aminolevulinic acid (5-ALA) were purchased from Kanto Chemical Co., Inc. (Japan) and Cosmo Bio Inc. (Japan), respectively.. A. flavus  was provided by RIKEN BioResource Center (Japan). Pressure-biased maize for feed was purchased from Iisaka Seibaku Co., Ltd. (Japan). Other reagents were purchased from Nacalai Tesque, Inc. or Wako Pure Chemical Industries, Ltd.Trehalose, sucrose, potato dextrose agar (PDA) medium, and Triton X-405 were purchased from Nacalai Tesque, Inc. (Japan). Acetonitrile, formic acid, and methanol were purchased from Wako Pure Chemical Industries, Ltd. (Japan), and syringe filters (Millex-LH) were purchased from Millipore (USA). AFLAKING was purchased from Horiba, Ltd. (Japan)E. coli (JM109 strain). The transformed E. coli was then inoculated into 3 ml of Luria-Bertani (LB) medium containing 50 μg/ml Amp, and cultured at 37°C, 170 rpm for 16 to 18 h. Thereafter, 1 ml of the culture solution was added to 100 ml of 2×YT medium containing 50 μg/ml Amp and cultured at 37°C and 170 rpm for 3 h. Once the optical density at 600 nm (OD600) of the culture reached 0.2-0.6, overexpression of the human CYP3A4 was induced by adding 100 μl of 0.5 M 5-ALA and 100 μl of 1 M IPTG. Subsequently, after culturing at 25°C and 170 rpm for 24 h, E. coli cells were harvested by centrifugation at 4°C and 7,000 ×g for 10 min. After removing the supernatant, the cell pellet was resuspended in a stock buffer (20% (v/v) glycerol, 0.1 M KPB, 1 mM EDTA) to obtain E. coli cells that express human CYP3A4. Expression of human CYP3A4 was calculated based on a previously developed method [An expression plasmid for human CYP3A4, named pCW3A4, was prepared as described previously  and tranE. coli expressing human CYP3A4 was diluted in stock buffer to an OD600 of 1.5. Next, we prepared the sample containing 1 ml of E. coli fraction diluted in stock buffer and 10 μl of 800 μM AFB1. This sample was incubated in a shaking incubator (170 rpm) at 25°C for 24 h. Thereafter, 4 ml of ethyl acetate containing 10 μM 5-MOP as an internal standard substance was added to the sample, and then this sample was vortexed and centrifuged at 25°C and 1,000 ×g for 10 min. Subsequently, 250 μl of the supernatant separated by centrifugation was dried under reduced pressure using a centrifugal concentrator (VC-36N/TAITEC). The sample was dissolved in 150 μl of acetonitrile, filtered with a syringe filter (Millex-LH 0.45 μm/MILLIPORE), and subjected to high performance liquid chromatography (HPLC) analysis using an HITACHI HPLC D7000 System (Japan) at excitation and detection wavelengths of 365 nm and 254 nm, respectively. We used an ODS-HPLC column  with A-Milli-Q water with 0.1% formic acid and B-acetonitrile with 0.1% formic acid for the eluent. The HPLC was run at 40°C with a flow rate of 1 ml/min with the following gradient conditions: 0 min (A: 75%) → 20 min (A: 0%) → 22 min (A: 0%) → 22.1 min (A: 75%) → 25min (A: 75%).E. coli cells expressing human CYP3A4 was repeated three times. Each metabolite was recovered in test tubes, and then concentrated using a rotary evaporator. After purification of the AFB1 metabolite, the structure of the target metabolite was analyzed by liquid chromatography-mass spectrometry (LC-MS). LC-MS analysis was carried out using a Paradigm MS2 HPLC system  at 35°C with a 150 mm × 0.1 mm Magic C18AQ column . The mobile phase was composed of solution C (water : acetonitrile (2 : 98) containing ammonium formate (0.1%)) and solution D (water : acetonitrile (90 : 10) containing ammonium formate (0.1%)). The gradient used was 95% solution C from 0.0 to 10.0 min; 95-45% solution C from 10.1 to 30 min; 45-10% solution C from 30.1 to 33 min; and 95% solution C from 33.1 to 45 min (equilibration). The flow rate was set to 0.5 μl/min and sample volumes of 0.5 μl were injected. MS was performed using an LTQ Orbitrap Discovery . The ESI source parameters were set as follows: ion spray voltage 1.8 kV, capillary temperature 200°C, and source heater temperature 300°C. The Orbitrap analyzer scanned the mass range from m/z 150 to 600. Data recording and processing were performed using the Xcalibur 2.1 software .The experiment investigating the metabolism of AFB1 by the Salmonella Typhimurium NM2009 was inoculated into 3 ml of TGA medium (1% (w/v) tryptone, 0.5% (w/v) NaCl, 0.2% (w/v) glucose) supplemented with Amp to a final concentration of 20 μg/ml and cultured overnight at 37°C with shaking at 140 rpm. The culture was then diluted to an OD600 value of 0.025 and 1 μl of the test compound was added in a 96-well microplate. Subsequently, 99 μl of 90:10:1 mixed solution of diluted bacterial solution, Cofactor1, and rat S9 fraction were added. Each sample was incubated at 37°C for 2 h. Next, 100 μl of color-developing solution was added and incubated at 37°C for 30 min, then 100 μl of 1 M sodium carbonate was added to each well and set aside for 10 min. The absorbance of each sample at 630 nm was measured using a microplate reader .The experiment was carried out as previously described . BrieflyE. coli expressing human CYP3A4 was cultured in 100 ml of 2×YT medium. E. coli expressing human CYP3A4 was suspended in 100 mM trehalose, 100 mM sucrose or sterile water. Thereafter, 5 ml each was dispensed, frozen with liquid nitrogen, and freeze-dried using a freeze dryer . The sample was then stored at 25°C. After storage for each tested time period, the sample was resuspended in KPB containing 1 mM EDTA and diluted to an OD600 value of 1.5 and AFB1 metabolic activity was evaluated.As described previously, A. flavus was inoculated in 30 ml of potato dextrose agar (PDA) medium and incubated at 27°C for 1 week [A. flavus-contaminated maize. Subsequently, the A. flavus-contaminated maize was sterilized by autoclaving. Next, after weighing 17 g of contaminated maize, 200 ml of E. coli expressing human CYP3A4, adjusted to OD600 1.5, was added to each sample and incubated at 25°C while shaking at 120 rpm for 24 h. A. flavus and E. coli expressing human CYP3A4 in each contaminated maize sample were sterilized by autoclaving. After sterilization, each contaminated maize sample was dried at 70°C for about 4 days. After crushing the contaminated maize with a coffee grinder, 34 ml of extraction solvent (acetonitrile : water = 9 : 1) and 50 μl of 100 μM AFM1 as an internal standard were added. Each contaminated maize sample was shaken at 200 rpm for 1 h at 25°C. The extract was filtered using filter paper , and the resulting solution was diluted 5-fold with sterilized water, and then filtered using glass fiber filter paper . The obtained filtrate was used as a sample diluted solution, and aflatoxins in the sample were concentrated and purified by using an immunoaffinity column  according to the manufacturer’s instructions. The same method was used to evaluate the effect of Triton X-405 on AFB1 metabolism by E. coli expressing human CYP3A4. The sample was dried under reduced pressure using a centrifugal concentrator and redissolved in 150 μl of acetonitrile. Then, the sample was filtered through a syringe filter before being subjected to HPLC analysis. The analysis conditions are detailed in section “AFB1 Metabolism by Human CYP3A4.”r 1 week . ThereafE. coli with a bacterial expression vector named pCW3A4. Thereafter, E. coli was cultured, and the CO difference spectrum was measured. As a result, an absorption peak at 450 nm was observed after CO treatment, suggesting that the human CYP3A4 enzyme was expressed and active in E. coli  or  control .+ ions were detected at m/z 329  Triton X-405 was added (A. flavus and E. coli resulting in the formation of micelle structures and improved water solubility of chemical substances. Previously, Schnaitman has shown that Triton X-100, which has a similar structure to Triton X-405, relaxes the membrane structure of E. coli but does not affect the normal morphology of the cell wall [E. coli cells, thereby increasing the incorporation of solubilized AFB1 into the cell and resulting in improved AFB1 metabolic efficiency by human CYP3A4. Recently, Chen et al. also reported that the addition of nonionic surfactants alters the structure of cell membranes of Monascus fungi and promotes the secretion of Monascus pigments, a natural food coloring [A previous study by  reportedas added . Since iell wall . Therefocoloring .A. flavus-contaminated maize and E. coli expressing human CYP3A4 in vitro under normal temperature and pressure. As a result, we succeeded in removing about 80% of AFB1 from A. flavus-contaminated maize. Therefore, it became clear that this method can be used for metabolism and detoxification of AFB1 in food. This is the first report of AFB1 detoxification from maize contaminated with A. flavus. In the future, it will be important to investigate the nutritional components of maize after CYP3A4 metabolism.In this study, we optimized the conditions for reacting


Terminalia chebula Retz. (TC) is a well-known Chinese herbal medicine and rich in chemical components with multiple pharmacological effects. In this study, an ultra-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) method was developed and used to determine the blood concentrations of nine active compounds  after oral administration of TC extracts in rats. Pretreatment of plasma samples with protein precipitate with methanol was carried out, and caffeic acid was used as the internal standard (IS). Compounds precisions of intra- and inter-day were less than 14.6%, and the accuracy ranged from −11.7% to 13.5%. The extraction recoveries of compounds were between 84.9% and 108.4%, while matrix effects occurred between 86.4% and 115.9%. Stability tests showed that all nine analytes had been stable under four storage conditions, and statistically significant the relative standard deviations were under 13.7%. The validated UPLC-MS/MS method was applied with great success to plasma pharmacokinetics analysis of the TC extracts, and the pharmacokinetic results showed that among the nine components, the area under the concentration-time curve (AUC(0-tn), 231112.38 ± 64555.20 h ng/mL) and maximum concentration  of chebulagic acid were relatively large, which indicated that it had a higher level of plasma exposure. The half-life of elimination (T1/2) of chebulinic acid, corilagin and chebulagic acid were 43.30, 26.39 and 19.98 h, respectively, suggesting that these analytes showed prolonged retention and metabolize more slowly in vivo. This study would deliver a theoretical foundation for the further application of TC in clinical practice. Terminalia chebula Retz. is to be derived from the well-ripened fruit of the plant in the family Combretaceae, which includes Terminalia chebula Retz. or Terminalia chebula Retz. var. tomentella Kurt  enzyme, indicating that it might represent an effective treatment to reduce the incidence of gastric ulcers (Chebulinic acid at a dose of 40 mg/kg has a good anti-gastric ulcer effect)  . Chebula effect) . Further effect) . It is wThe therapeutic effect of Chinese herbal medicine is based on two key prerequisites, that is, it has sufficient bioavailability and biological effect in the active site after administration, and has the inherent ability to produce the expected effect in its main action forms, namely invariance and metabolism. Because of the chemical complexity of herbal medicine, pharmacokinetics can be used as a screening method to examine whether the components are effectively utilized by the body after administration . Meanwhiin vivo, the material basis of TC to exert its efficacy can be fully elucidated, which provided a meaningful reference for the rational usage of TC and the development of safe and effective drugs.Among the compounds isolated and identified from TC, tannins and phenolic acids were found to be a widely studied chemical classes. Meanwhile, the pharmacokinetic profiles of tannins, chebulagic acid and chebulinic acid in rats based on LC-MS/MS methods have been reported . HoweverChengdu Desite Bio-Technology Co., Ltd.  had a large supply of the standard compounds of chebulic acid, gallic acid, 1,2,3,4,6-O-pentagalloylglucose, protocatechuic acid, corilagin, chebulagic acid, chebulinic acid, ellagic acid, ethyl gallate and caffeic acid (IS) (purity ≥98%). Fisher Scientific  made both methanol and acetonitrile (chromatographic grade) available. Formic acid (chromatographic grade) was provided by ROE . The Millipore-Q water purification system  was used to produce demineralized water. TC was collected from Anhui province in China.UPLC-MS/MS system was used for analysis and monitoring, which consisted primarily of an Agilent 1290 UPLC system  and an Agilent 6,470 series triple quad (TQ) mass spectrometer (MS)  equipped with an electrospray ionization source.® HSS T3 column , and temperature in the column was kept at 20°C. In the mobile phase, 0.1% formic acid in water and acetonitrile were used as solvents A and B, respectively. Gradient elution was performed as follows: 1%–5% B at 0–3 min; 5%–30% B at 3–4 min; 30%–40% B at 4–8 min; 40%–100% B at 8–9 min; 100%–100% B at 9–12 min. The flow rate was set at 0.3 ml/min and the injection volume was 10 μL.Separation was carried out on an ACQUITY UPLC2) temperature was 300°C; gas (N2) flow rate was 7 L/min; atomizer pressure was 35 psi; sheath gas flow was 11 L/min and capillary voltage was 3800 V. The specific mass spectrometric parameters of the precursor ion, product ion, fragmentor, collision energy, and ion modes of the nine compounds and IS are shown in Multiple reaction monitoring (MRM) was used for quantitative analysis in negative (−) ionization mode. The mass spectrometric parameters were set as follows: drying gas . Continuous dilution with methanol produced a working solution from the stock solution.A mixture of 20 μl serial concentration mixed working solutions of standards and 20 μl IS solutions  was added to 100 μl blank plasma to obtain standard curve samples. The final plasma sample concentrations were 100, 250, 500, 1,000, 2000, 5,000, 10000, 20000, 40000 ng/mL for chebulinic acid; 25, 62.5, 125, 250, 500, 1,000, 2,500, 5,000, 10000 ng/ml for chebulic acid, gallic acid, chebulagic acid and corilagin; 1, 2.5, 5, 10, 20, 50, 100, 200, 400 ng/ml for protocatechuic acid, ethyl gallate and 1,2,3,4,6-O-pentagalloylglucose; and 10, 25, 50, 100, 200, 500, 1,000, 2000, 4,000 ng/ml for ellagic acid.Preparation of quality control (QC) samples with three concentrations  of 16, 160, and 3,200 ng/ml for ellagic acid; 1.6, 16, 320 ng/ml for protocatechuic acid, ethyl gallate and 1,2,3,4,6-O-pentagalloylglucose; 40, 400 and 8,000 ng/ml for chebulic acid, gallic acid, corilagin and chebulinic acid and 160, 1,600, 32000 ng/ml for chebulagic acid. Both QC samples and stock solutions were stored in a refrigerator at 4°C.The plasma sample (100 μl) was sequentially spiked with 20 μl IS solutions ; 20 μL methanol, and 10 μl formic acid, then vortex-mixed for 1 min. The mixture was precipitated protein with 1,000 μl methanol by vortex mixing for 3 min. The supernatant was collected into a 1.5 ml clean centrifuge tube after centrifugation at for 10 min 14,000 rpm, and evaporated under a nitrogen stream until dry. After that, the residue was redissolved in 100 μl methanol, vortex-mixed for 3 min, then centrifuged for 10 min at 14,000 rpm. 10 μl analyte was injected into the UPLC-MS/MS system.The specificity was assessed by way of paralleling chromatograms of blank plasma samples, corresponding blank plasma samples spiked with analytes and IS, as well as plasma samples from the rats collected after oral administration of TC extracts.versus its concentration. The regression relationship is described by linear regression equation with weighting coefficient of 1/X. The signal-to-noise ratio (S/N) is defined as approximately 10 based on the lower limits of quantification (LLOQ) of the baseline noise assessment.A calibration curve was derived by plotting each analyte’s peak area ratio to IS Six replications of QC samples at three concentrations  were analyzed within 1 day or over three consecutive days to assess precision and accuracy. The accuracy was assessed by calculating the relative error (RE %), while intra and inter-day precision were defined as the relative standard deviation (RSD).The extraction recoveries and matrix effects were all investigated by six replicates at the three concentrations mentioned above low, medium and high. The recovery was then determined by comparison of the peak areas of the analytes in the pre-precipitated and post-precipitated samples of the spiked standards. While matrix effects were evaluated by calculating the area of the peak of the analyte in the precipitated spiked sample compared to the area of the peak in the standard solutions.Under variable treatment conditions: stored for 12 h at auto-sampler, for 4 hours at room temperature, under three freeze-thaw cycles, and kept at −80°C for 7 days, quality control samples were analyzed to assess the stability of the nine analytes in plasma.Male Sprague-Dawley rats (220 ± 10) g from the Beijing HUAFUKANG Bioscience Co., Inc.  were selected. In this study, six of male rats were used, given free access to water, and fasted for 12 h before the experimental period. The TC extracts were dissolved in 0.5% CMC-Na aqueous solution as a suspension at a concentration of 1 g/ml. A dose of 10 g/kg of the suspension was administered to the rats orally, the blood samples of approximately 300 μL were drawn from the fundus venous plexus of rats pre- and post-oral administration at 0.00, 0.08, 0.17, 0.25, 0.50, 0.75, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84, 96, 120, 144 and 168 h, respectively. For analysis, the plasma supernatants collected from the centrifuged plasma were frozen at −80°C after centrifugation for 10 min at 6,000 rpm.Data were expressed as mean ± standard deviation (mean ± SD). MassHunter Workstation software (version B.09.00) was quantitatively used to calculate the plasma concentrations of nine analytes. To evaluate the specific pharmacokinetic parameters, DAS Software  was used to process the pharmacokinetic results.® BEH C 18 column  and ACQUITY UPLC® HSS T3 column  on chromatographic peaks and elution time were studied experimentally. Meanwhile, water (0.1% formic acid)-acetonitrile and water (0.1% formic acid)-methanol were compared to determine the optimum mobile phase. The results displayed that water (0.1% formic acid)-acetonitrile flow has a better peak shape and lower background noise than water (0.1% formic acid)-methanol flow. Nine analytes and IS were purged in 12 min using ACQUITY UPLC® HSS T3 column at a flow phase flow rate of 0.3 mL/min, as shown in Suitable chromatographic conditions are important for the analysis of nine compounds. The effects of ACQUITY UPLCA comparison of electrospray ionisation sources in positive and negative ionisation modes was carried out. The results showed the target substances in negative ion mode are more responsive and stable. MS/MS ion conversion monitoring in multiple reaction monitoring (MRM) mode improves the specificity of the detection method. Therefore, the nine analytes were quantitated with electron spray ionization (ESI) in the negative ionisation mode.Protein precipitation with methanol acetonitrile and liquid-liquid extraction with ethyl acetate were used to find a suitable method for preparing plasma samples. The results showed that the methanol precipitation protein method had good extraction recoveries for the nine analytes and the matrix effects met the assay requirements. Thus, the protein precipitation method using methanol was used in the sample preparation. Furthermore, the effects of different volumes  of precipitation solvent on extraction recovery and matrix effect were investigated, and it was found that when the volume of methanol was 1,000 μl, the extraction recovery was between 84.9% and 108.4%, and the matrix effect was between 86.4% and 115.9%, indicating that protein precipitation with methanol could obtain satisfactory extraction recovery and matrix effect.r2 > 0.991) over the corresponding concentration ranges. The LLOQs of chebulic acid, chebulinic acid, protocatechuic acid, corilagin, chebulagic acid, gallic acid, 1,2,3,4,6-O-pentagalloylglucose, ellagic acid, ethyl gallate were 12.0, 17.0, 0.4, 0.1, 8.0, 0.9, 0.1, 8.5 and 0.2 ng/mL, separately.This study examined the pharmacokinetics of nine components of TC extract administered intragastrically to rats. 1/2 values of 1,2,3,4,6-O-pentagalloylglucose, chebulic acid, gallic acid, protocatechuic acid, ellagic acid and ethyl gallate were 6.60, 1.51, 2.06, 6.52, 8.23 and 2.08 h, separately, these analytes were shown to be eliminated shortly after oral administration. However, the T1/2 values of corilagin, chebulagic acid and chebulinic acid were 26.39, 19.98, 43.30 h, separately, and their mean residence time (MRT) were 53.45 ± 4.12, 49.78 ± 4.87 and 58.23 ± 5.63 h, separately, which demonstrated that these three analytical substances existed inside the body for a comparatively long term and may have a continuous treatment effect and enhance the clinical efficacy ((0-tn) and AUC (0-∞) values of chebulic acid, gallic acid, corilagin, chebulagic acid and chebulinic acid were a lot higher than the others, suggesting that these analytes had higher levels of plasma exposure. And the Cmax of chebulic acid, gallic acid and chebulinic acid were 1927.06 ± 572.23, 2,234.40 ± 1,201.26 and 852.55 ± 433.35 ng/ml, respectively, suggesting that the concentration of those compounds was high. In combination with AUC results, chebulic acid, gallic acid and chebulinic acid might be the potential active compounds leading to the effect of TC in vivo.The Tefficacy . The AUCFurthermore, it was found that ellagic acid and 1,2,3,4,6-O-pentagalloylglucose had double peaks phenomenon on the blank plasma concentration-time curves in In this study, a simple UPLC-MS/MS method was established and applied to study the pharmacokinetics of 10 g/kg TC extract in rats after a single oral administration. The method was verified, and the detection ability of a low concentration for nine compounds was demonstrated.  with protein precipitation method. The results indicated that the blood concentration and plasma exposure of chebulic acid, gallic acid, corilagin, chebulagic acid and chebulinic acid were higher than other compounds. In addition, compared to the other six components, corilagin, chebulagic acid and chebulinic acid showed prolonged retention and metabolize more slowly. The data obtained in this study can provide useful information for preclinical application, and also provide theoretical basis for the further development of TC.


Planococcus lilacinus Cockerell, is a primary surface-feeding insect pest of fruit and flowering plants and also transmits plant viruses, resulting in economic crop loss. Inappropriate and recurrent use of pesticides for mealybug control results in resistance building and deleterious effects on humans and the environment. Essential oils are the most excellent choice for insecticides. Insecticidal activities of pure compounds of essential oils against P. lilacinus are not reported. The present study aims to study the insecticidal activities of some pure active compounds and their binary mixture’s action by topical application against P. lilacinus. Results showed that the pure compounds of L-limonene, β-myrcene, and ocimene revealed toxicity (each at LD50 = 0.37 µg/insect) after 96 h. The binary mixtures of geraniol + L-menthol and L-limonene + geraniol exhibited synergistic effects (each at LD50 = 0.03 µg/insect) after 96 h. The monoterpenes of ocimene and β-myrcene at the higher concentration of 5,000 ppm substantially inhibited the detoxification enzyme activities of AChE  and GST  in P. lilacinus after 48 h. SEM analysis reported the significant anomalies on the morphology of abdominal cuticle, setae, and thoracic leg after 96-h treatment with ocimene at 1,250 ppm against P. lilacinus. Based on the results, the tested pure compounds and their combinations can be suggested for the control of mealybugs.Mealybug,  Planococcus lilacinus Cockerell (Hemiptera: Pseudococcidae), is a major surface-feeding pest of many fruits and flowering plants  for botanicals production.Several plant-derived essential oils of  studies . Additioeolariae , Planocous citri , Planocous ficus , Pseudocgispinus , Paracocrginatus , Planocous minor , Phenacolenopsis , and Pse viburni  have bee viburni . HencefoPhytochemicals such as 1-cyclohexyl-2-pyrrolidone, camphene, cinnamaldehyde, citral, geraniol, L-carvone, L-limonene, L-menthol, β-myrcene, ocimene, α-terpinene, and β-terpinene Figure 1P. lilacinus were collected from the outdoor area and consistently reared on live tobacco plant, Nicotiana tabacum L., in the insectary unit of Agrotechnology Division, CSIR-IHBT, Himachal Pradesh, under regulated temperature (25 ± 2°C), relative humidity (60 ± 5%), and a 16:8 light–dark environment for several generations. The fresh second instar crawlers were used for toxicity bioassays.P. lilacinus as per the standard topical application method  for the control of sucking pests was used as a positive control for comparison. Briefly, five different concentrations  of pure compounds and Neem Baan were prepared by blending each compound in 0.05% Tween-80. One microliter of sample was administered using a Hamilton micro-syringe with a repeating dispenser dorsally to each of 10 second instar crawlers of P. lilacinus. Treated crawlers were then transferred to tea leaf discs (3 cm2) in each Petri plate, pressed on top of the water-agar medium to preserve the greenness of leaf discs. Five replications were maintained for each treatment and kept under controlled conditions for observations. The mortality of the crawlers was documented after 24, 48, 72, and 96 h of treatment.The toxicity of pure compounds such as 1-cyclohexyl-2-pyrrolidone, camphene, cinnamaldehyde, citral, geraniol, L-carvone, L-limonene, L-menthol, β-myrcene, ocimene, α-terpinene, and β-terpinene was initially evaluated against the second instar crawlers of n method  for LD5050 values, for the bioassay and synergistic activity against P. lilacinus in the lab environment. Samples were prepared and administered against the second instar crawlers of P. lilacinus as mentioned above. Each treatment was performed using five replications, and the mortality of the crawlers was documented at 24, 48, 72, and 96 h after treatment for the combined compounds to calculate the LD50 and cotoxicity coefficient (CTC) values  values . The CTCInhibition activities of detoxification enzymes [acetylcholinesterase (AChE) and glutathione-S-transferase (GST) were carried out using standard procedures . Based oxProtein estimation was analyzed following the standard protocol  before pTwenty-five microliters of diluted mealybug homogenates (control and test) mixed with 25 µl of the reaction mixture that contains 50 µl of acetothiocholine and 50 µl of DTNB in 900 µl of assay buffer was incubated in triplicate at RT for 30 min. The AChE activity was evaluated spectrophotometrically at 410 nm in a microplate reader  and represented as milliunits per milligram of protein (mU/mg). The AChE Kit was purchased from Abcam, UK, for AChE determination.−1 for CDNB was used to calculate the glutathione S-transferase activity and represented as nanomolar per minute per milliliter of sample (nmol/min/ml). The GST Assay Kit was purchased from Cayman Chemical, USA, for GST determination.The total volume of 100 µl reaction mixtures was prepared by adding 10 µl of mealybug homogenates (control and test) to 75 µl of GST sample buffer, 10 µl of GST glutathione, and 5 µl of GST CDNB (initiator) in triplicate. These reaction mixtures were incubated at RT in a 96-well microplate. The enzyme kinetics were measured at the absorbance of 340 nm at 37°C for 20 min in a microplate reader with continuous mixing for 10 s after 60 s of lag time. The extinction coefficient of 0.0096 µMP. lilacinus after 72 and 96 h with ocimene  were fixed in 2.5% glutaraldehyde fixative solution in PB  for 2 h, followed by washing and air drying. After that, the samples were subjected to dehydration using 30%–100% of ethanol solutions for 15–20 min each and fixed on aluminum stubs using dual-sided sticky carbon tape. Samples were then subjected to gold sputter coating  for 10 s, maintaining 10 Pa vacuum pressure. SEM images were obtained  at the appropriate resolution for surface morphological characteristics studies  and other regression parameters were estimated by Probit analysis  against P. lilacinus after 24 h, followed by L-limonene (LD50 = 2.06 µg/insect), geraniol (LD50 = 3.32 µg/insect), and ocimene (LD50 = 5.08 µg/insect) as compared to other compounds tested. Similarly, after 48 h, L-limonene showed the most toxic effect (LD50 = 0.92 µg/insect), preceded by L-menthol (LD50 = 1.16 µg/insect), geraniol (LD50 = 1.30 µg/insect), and cinnamaldehyde (LD50 = 1.78 µg/insect). Subsequently, after 72 h, L-menthol was seen to be the most effective compound (LD50 = 0.44 µg/insect), followed by β-myrcene (LD50 = 0.52 µg/insect), L-limonene (LD50 = 0.55 µg/insect), and geraniol (LD50 = 0.65 µg/insect). Finally, the compounds, such as L-limonene, β-myrcene, and ocimene, exhibited their potency (each at LD50 = 0.37 µg/insect) after 96 h of treatment. Also, the positive control Neem Baan (pesticide) caused the toxicity with LD50 values of 5.99, 3.21, 0.62, and 0.27 µg/insect for 24, 48, 72, and 96 h, respectively.The toxicity effect of different compounds including 1-cyclohexyl-2-pyrrolidone, camphene, cinnamaldehyde, citral, geraniol, L-carvone, L-limonene, L-menthol, β-myrcene, ocimene, α-terpinene, and β-terpinene was scrutinized, and the nymphal mortality data of second instar crawlers of 50 concentration was tested for the toxicity and synergistic activity against the second instar crawlers of P. lilacinus between 24- and 96-h time intervals, and the results are shown in P. lilacinus revealed toxicity and synergistic effects. Among the multiple combinations, geraniol + L-menthol showed a significant effect (LD50 = 0.29 µg/insect), followed by L-limonene + geraniol (LD50 = 0.35 µg/insect), ocimene + geraniol (LD50 = 0.87 µg/insect), and β-myrcene + L-limonene (LD50 = 0.97 µg/insect) after 24 h. Subsequently, after 48 h, geraniol + L-menthol was found to be a more effective combination (LD50 = 0.10 µg/insect), preceded by L-limonene + geraniol (LD50 = 0.13 µg/insect), β-myrcene + geraniol (LD50 = 0.33 µg/insect), and ocimene + geraniol (LD50 = 0.42 µg/insect). Furthermore, 72-h treatment showed that geraniol + L-menthol had a significant level of toxicity (LD50 = 0.05 µg/insect), followed by L-limonene + geraniol (LD50 = 0.08 µg/insect), β-myrcene + geraniol (LD50 = 0.18 µg/insect), and ocimene + L-limonene (LD50 = 0.24 µg/insect). Eventually, the combinations such as geraniol + L-menthol and L-limonene + geraniol at 96 h showed remarkable toxicity efficiency (each at LD50 = 0.03 µg/insect).The combination of different compounds, viz., ocimene + β-myrcene, ocimene + L-limonene, ocimene + geraniol, ocimene + L-menthol, β-myrcene + L-limonene, β-myrcene + geraniol, β-myrcene + L-menthol, L-limonene + geraniol, and geraniol + L-menthol, at a 1:1 ratio of their 96 h LDP. lilacinus reflected in the percent mortality after 24, 48, 72, and 96 h is summarized in F11, 299 = 18.44, 12.45, 11.58, and 9.92) and concentrations  with p < 0.0001 for 24, 48, 72, and 96 h, respectively. However, the mean percent mortality was not significantly different (p > 0.05) between the interaction of compounds and concentrations. Results showed that, among different compounds, the pooled mean mortality was significantly higher in L-menthol (42.80 ± 19.48), L-limonene (54.40 ± 16.60), L-menthol (66.40 ± 17.05), and β-myrcene (74.80 ± 19.82) after 24, 48, 72, and 96 h, respectively, in comparison to other tested compounds. The pooled mean mortality was higher in the highest concentration at 5,000 ppm  as compared to other concentrations. The overall percent mortality was superior in L-menthol (70 ± 7.07), geraniol (82 ± 8.37), cinnamaldehyde (94 ± 5.48), and geraniol and β-myrcene (each at 98 ± 4.47) against P. lilacinus after 24, 48, 72, and 96 h, respectively, in contrast to the other remaining compounds.The toxicity of different compounds and concentrations against P. lilacinus after 24 and 48 h of treatment and are presented in F4,14 = 69.39; p < 0.0001 and F4,14 = 221.70; p < 0.0001) in P. lilacinus, as compared to the control. Similarly, all the concentrations of β-myrcene highly inhibited the AChE activity , when compared to the control after 24 and 48 h. Among different concentrations, ocimene at 5,000 ppm reported significantly higher inhibition of AChE after 24 and 48 h of treatment , followed by 2,500 ppm (1.31 ± 0.03 and 1.25 ± 0.01 mU/mg) as compared to other lower concentrations   as compared to the control. Similarly, all the concentrations of β-myrcene significantly inhibited the GST activity  in contrast to the control. Among concentrations, ocimene at 5,000 ppm exhibited higher inhibition of GST after 24 and 48 h  and was preceded by 2,500 ppm (6.60 ± 0.64 and 4.40 ± 0.64 mU/mg) than at lower concentrations  (Detoxifying enzyme inhibition activities of ocimene and β-myrcene (based on activity and feasibility) were carried out against P. lilacinus topically treated with the ocimene altered the motility and eventually caused mortality. The treated P. lilacinus exhibited several deformities on the abdominal cuticle, setae, and thoracic leg after 72 and 96 h of treatment. The P. lilacinus treated with ocimene after 72 and 96 h showed a color change from pale yellow to dark brown with the accumulation of body fluids and thick encrustations on the external surface. SEM experiments showed that the crawlers of P. lilacinus with ocimene resulted in multiple habitus symptoms based on the time interval given in P. lilacinus (control) with the distinct structure of cuticle, legs, and setae without any deformities after 96 h of treatment, whereas the severe leg deformities, lack of setae, and the abdominal cuticular damages were evidenced after 72 h . A previous study also reported that such pure compounds from the essential oil of Tagetes minuta showed residual toxicity, repellent, feeding deterrent, growth (feeding) inhibition, and weight reduction activities against the diamondback moth, Plutella xylostella  and sesquiterpenes (α-bisabolol and chamazulene) from various plant sources have been reported for their insecticidal activities against various pest insects . The conastaneum . β-myrceastaneum . These fP. lilacinus revealed that the compounds, namely, L-limonene, β-myrcene, and ocimene, were found to be most effective against P. lilacinus (each at LD50 = 0.37 µg/insect) after 96 h of treatment. Similar findings were reported earlier with the pure compounds of pulegone and menthofuran from Minthostachys verticillata against P. ficus  after 96 h of treatment. Our study indicated that all the tested combinations of the pure compounds resulted in the synergistic activity for topical toxicity against P. lilacinus. However, the present study attempted for the first time to study the combined actions of pure compounds of essential oils against P. lilacinus. The primary components of plant essential oils are blended to form a new formulation (binary mixtures) possessing additive or synergistic properties upon toxicity , which significantly inhibited the enzyme activity for 24 and 48 h after treatment compared to control mealybugs. AChE inhibition is generally involved in the accretion of acetylcholine (ACh) in cholinergic synapses that result in the higher modulation of the cholinergic system  composed of proteins and lipids and a lipophilic layer (epi/endocuticle) constituted by chitin . It is wP. lilacinus, also discussed here. Due to the lack of literature about the effect of pure compounds of essential oils against P. lilacinus, the SEM findings were not compared to the earlier studies. However, few studies on the structural deformities caused by T. minuta oil against A. craccivora , New Delhi, granting the proposal “CSIR Aroma Mission” HCP-0007 (Grant No. 33/Mission/Aroma/2016-MD). IHBT communication number for this article is 5130.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Paraglaciecola Antarctic GD virus 1 (PANV1), Paraglaciecola Antarctic JLT virus 2 (PANV2), Octadecabacter Antarctic BD virus 1 (OANV1), and Octadecabacter Antarctic DB virus 2 (OANV2). The phages infect common sea ice bacteria belonging to the genera Paraglaciecola or Octadecabacter. Although the phages are marine and cold-active, replicating at 0°C to 5°C, they all survived temporal incubations at ≥30°C and remained infectious without any salts or supplemented only with magnesium, suggesting a robust virion assembly maintaining integrity under a wide range of conditions. Host recognition in the cold proved to be effective, and the release of progeny viruses occurred as a result of cell lysis. The analysis of viral genome sequences showed that nearly one-half of the gene products of each virus are unique, highlighting that sea ice harbors unexplored virus diversity. Based on predicted genes typical for tailed double-stranded DNA phages, we suggest placing the four studied viruses in the class Caudoviricetes. Searching against viral sequences from metagenomic assemblies, we revealed that related viruses are not restricted to Antarctica but are also found in distant marine environments.Although we know the generally appreciated significant roles of microbes in sea ice and polar waters, detailed studies of virus-host systems from such environments have been so far limited by only a few available isolates. Here, we investigated infectivity under various conditions, infection cycles, and genetic diversity of the following Antarctic sea ice bacteriophages:  Sea ice covers a significant area of polar oceans every year, affecting ocean ecology, biogeochemical cycles, and climate , 2. Sea 3–9–12–Shewanella, Flavobacterium, Colwellia, Octadecabacter, Glaciecola, and Pseudoalteromonas strains , at 50°C, 55°C, 40°C, and 50°C for PANV1, PANV2, OANV1, and OANV2, respectively  . The stuectively .2+, even if residual Mg2+ ions were removed by the chelating agent ethylenediaminetetraacetic acid (EDTA)  buffer, the OANV1 titer dropped by 4 to 5 orders of magnitude, and the OANV2 titer dropped to 1/10th (2+ ions were removed using EDTA (4 PFU/mL) and the OANV2 titer dropped 100-fold. When both NaCl and MgSO4 were removed (4 PFU/mL.All four viruses studied here remained infectious without NaCl (2PO4 (see 4 PFU/mL).No statistically significant difference in virus titers was observed when virus stocks were incubated in SM buffer (pH 7.5) with either Tris-HCl or NaH2PO4 see  to h. PA10.1128/mbio.00651-22.6FIG S12PO4 (buffer 1) or Tris-HCl . Virus stocks were diluted 1,000-fold in the buffers and incubated 1 h (dark gray) and 5 h (light gray). Bars represent means of at least three independent replicates with standard error of the mean shown as error bars. Download FIG S1, PDF file, 0.1 MB.Stability of virus infectivity in SM buffer containing NaHCopyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the k calculated for the first 30 min p.i. (n = 3) was 3.9 × 10−9 and 9.0 × 10−12 mL/min for PANV2 and OANV2, respectively. PANV1 and OANV1 adsorbed with the rates of 5.4 × 10−10 and 4.6 × 10−13 mL/min, respectively, reaching ~70% adsorption by 12 h p.i.PANV1, PANV2, OANV1, and OANV2 adsorbed effectively to their hosts, achieving at least ~50% binding efficiency within 6 h at 4°C . During Paraglaciecola IceBac 372 and Octadecabacter IceBac 419 and 430 grew to early stationary stage in 4 to 7 days , showing typical growth curves of a bacterial culture. The cultures were infected at the logarithmic growth phase  using a multiplicity of infection (MOI) of 8 to 10 to analyze the one-step growth of the phages  reduced almost 2 orders of magnitude as a result of the virus addition (~2.3 × 107 CFU/mL) by 124 h p.i., while the number of viable cells in the uninfected culture of IceBac 419 was growing (~3.3 × 109 CFU/mL by 124 h). This result can be interpreted as lysis being caused by the virus infection. Similarly, the numbers of viable cells in the OANV2-infected IceBac 430 culture dropped noticeably (from ~1.4 × 109 at 0 h p.i. to ~5 × 106 CFU/mL at 124 h p.i.) compared with those of the uninfected culture (~3.5 × 109 CFU/mL at 124 h p.i.).Bacterial strains e phages . Uninfec4 h p.i. . The tur4 h p.i. . For PANPANV1, PANV2, OANV1, and OANV2 genomes are double-stranded DNA (dsDNA) molecules ranging from ~36 to ~151 kb, with GC content of 38% to 62% and 61 to 243 predicted protein-coding open reading frames (ORFs) . All fou10.1128/mbio.00651-22.1TABLE S1Table S1, PDF file, 0.3 MB.Putative functions assigned to PANV1 ORF products. Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The majority of predicted ORF products (63% to 71%) of the four virus genomes could not be assigned with any functions, including unique sequences (43% to 48%) that had no homologues in the NCBI nonredundant (nr) protein database . SurprisAbout 29% to 37% of the studied sequences had homologs in the database, allowing us to predict their functions. BLAST matches revealed mosaic similarities to the sequences of other phages, as well as moderate and psychrophilic bacteria. The following functional categories were assigned to the gene products (gps): (i) proteins involved in DNA replication, recombination, modification, and metabolism; (ii) transcription and translation regulation proteins; (iii) virion and tail structural components; (iv) cell lysis; and (v) other functions ; Fig. 5.Bordetella phage BPP-1 bbp10, which is a lysin containing a beta/gamma crystalline motif . In PANV1, gp232 and gp233 were annotated as baseplate subunits having lysozyme activity, being similar to the gene products in T4-like phages   . In OANVAlong with the categories typical for dsDNA phage genomes, additional functions were also predicted. PANV1 gp36 was predicted to be a phospholipase  and thus is possibly involved in lipid metabolism. PANV1 gp37 putatively belongs to the NAD and ADP-ribose (NADAR) superfamily, having a hydrolyzer activity and taking part in carbohydrate derivative metabolic processes . PANV1 gp51 had matches to mechanosensitive channel proteins, which are involved in transmembrane transport . However, a transmembrane helix was predicted in this protein with only ~0.6 posterior probability by TMHMM v. 2.0. In PANV2, gp30 is a putative transpeptidase, which is involved in peptidoglycan cross-linking .Myoviridae of Type 2,” adopting the structural organization of the myophage T4 neck. PANV2 was assigned to “Siphoviridae of Type 1 cluster 5,” adopting the structural organization of the siphophage SPP1 neck. Both OANV1 and OANV2 were predicted to belong to “Podoviridae of Type 3,” adopting the structural organization of the podophage P22 neck.  and short noncontractile tails  (see 13 and 3.7 × 1013 PFU/mg of protein) using two negative stains and all demonstrated consistent particle morphology. The protein patterns of the purified OANV1 particle samples were identical to each other  (a) or Nano-W (b). Scale bar is 100 nm in a for a and b. (c) Polyacrylamide gel electrophoresis of purified virus samples which were prepared in parallel  and used in transmission electron microscopy in a and b, respectively. M, marker (PageRuler unstained protein ladder). Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Caudovirales phage genome assembly (GenBank accession number LR798304), which was obtained from metagenomes from the Římov Reservoir (freshwater human-made pond), Czech Republic  between PANV1 and PANV2 and the highest (~52%) between OANV1 and OANV2. When the complete sequences were used as queries in blastn searches  against all virus sequences (NCBI taxonomy identifier [ID] 10239) in the nr nucleotide collection, typically only less than 3% of the whole-genome sequences could be aligned with other virus sequences available, highlighting the overall uniqueness of these Antarctic sea ice virus isolates. One exception was the match of the OANV2 genome to the uncultured Republic . The ove10.1128/mbio.00651-22.8FIG S3Caudovirales phage (GenBank accession number LR798304.1) found with blastx search against nr protein database (a) and scaffolds found with blastn search against IMG/VR database (b). A full list of scaffolds is presented in FIG S3, PDF file, 1.5 MB.OANV2 and the selection of similar virus genome sequences, as follows: uncultured Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.2TABLE S2Table S2, PDF file, 0.1 MB.Putative functions assigned to PANV2 ORF products. Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.3TABLE S3Table S3, PDF file, 0.2 MB.Putative functions assigned to OANV1 ORF products. Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.4TABLE S4Table S4, PDF file, 0.1 MB.Putative functions assigned to OANV2 ORF products. Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.5TABLE S5Table S5, PDF file, 0.1 MB.Scaffolds with several (at least three) regions recruited as hits in the blast search against IMG/VR with Antarctic virus isolates whole genomes as queries (dated 9 December 2020). Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In contrast, a similar blastn search with PANV1, PANV2, OANV1, and OANV2 whole-genome sequences as queries against the Integrated Microbial Genomes/Virus (IMG/VR)  databasePANV2 was similar to scaffolds assembled from saline water microbial communities from Ace Lake, Antarctica , and one10.1128/mbio.00651-22.9FIG S4FIG S4, PDF file, 0.2 MB.PANV2 and the selection of similar scaffolds found with blastn search against IMG/VR database. A full list of scaffolds is presented in Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.10FIG S5FIG S5, PDF file, 0.1 MB.OANV1 and the selection of similar scaffolds found with blastn search against IMG/VR database. A full list of scaffolds is presented in Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A blast search of PANV1, PANV2, OANV1, and OANV2 genome sequences against the IMG/VR spacer databases did not yield any information about potential additional hosts of these viruses. No reliable hits were detected to CRISPR spacers from isolate genome, and the hits to metagenome-derived spacers were on short metagenome contigs that could not be affiliated taxonomically.Shewanella or Flavobacterium sp.     and Pseuctively) . In the low zero . Phage alow zero , 61.Caudoviricetes. A few PANV1 proteins may take part in lipid and carbohydrate metabolism and membrane transport, thus possibly being auxiliary metabolic genes, which is seen commonly in marine phages   or Tris-HCl (pH 7.5 and 9). After all incubations, virus infectivity was assessed by plaque assay as described previously  when two groups were compared. Groups were considered statistically not different if the P value was >0.05.Viruses and bacteria  were groM MgSO4) , SM buffeviously . A singl550 of ~0.8) were infected with a multiplicity of infection (MOI) of ~0.001 and incubated aerobically at 4°C. Samples in which cells were replaced with broth were used as controls. To determine the number of unbound viruses, samples were diluted in 4°C broth (1:10 or 1:100), cells were removed , and supernatants were subjected to plaque assay. The percentage of adsorption was calculated from all (particles in broth) and unbound particles (in infected cultures) as follows: % bound particles = [/all] × 100%. Adsorption rate constant was calculated as (k) = [2.3/(B × t)] × log(p0/p), where B is cell concentration, p0 and p are free virus concentrations at time point zero and after time period t, respectively  was infected with PANV1 or PANV2 (MOI of 10) and incubated aerobically at 5°C. Uninfected culture was used as a control. The numbers of infective free viruses in culture supernatants  were determined by plaque assay. IceBac 419 and IceBac 430 cells (OD550 of ~0.8) were collected  and resuspended in OANV1 or OANV2 virus stocks, respectively, (MOI of ~8) or in broth (uninfected controls). During the growth, the numbers of free viruses and viable cells in supernatant and pellet fractions  were determined by plaque assay.For life cycle studies, IceBac 372 , and assembled with SPAdes v. 3.9.0 .https://www.geneious.com) was used for sequence handling. ORFs were predicted using Glimmer, GeneMarkS , MetaGeneAnnotator (http://metagene.nig.ac.jp/), and FGENESV (http://www.softberry.com/berry.phtml?topic=virus&group=programs&subgroup=gfindv). GC content was calculated with Genomics %G~C Content Calculator . Transmembrane helices were predicted using TMHMM server v. 2.0 . Whole-genome comparisons for overall nucleotide identity were done with EMBOSS stretcher  database  using thThe genome sequences of PANV1, PANV2, OANV1, and OANV2 were compared to the IMG/VR spacer databases (both “isolate” and “metagenome”)  using blSequences are available in the GenBank database .


Cells were divided into 5 groups—control, high-fat, 10 nmol/L LR + 0.6 mmol/L palmitic acid (PA) (10LR), 100 nmol/L LR + 0.6 mmol/L PA (100LR), and 1000 nmol/L LR + 0.6 mmol/L PA (1000LR). CCK-8 method to detect cell viability, GPO-PAP enzymatic method to detect intracellular triglyceride content, and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting methods to detect fatty acid translocase CD36 (FAT/CD36) and fatty acid binding protein 4 (FABP4) in L6 cells, glucose-regulated protein 78 (GRP78), glucose transporter 4 (GLUT4) expression at the mRNA and protein levels, respectively, were performed. P < 0.01). After PA intervention, compared with those in the high-fat group, GRP-78, FAT/CD36, FABP4 mRNA ((4.36 ± 0.32 vs. 8.15 ± 0.35); (1.00 ± 0.04 vs. 2.46 ± 0.08); (2.88 ± 0.55 vs. 8.29 ± 0.52), P < 0.01) and protein ((3338.13 ± 333.15 vs. 4963.98 ± 277.29); (1978.85 ± 124.24 vs. 2676.07 ± 100.64); (3372.00 ± 219.84 vs. 6083.20 ± 284.70), both P < 0.01) expression decreased in the LR group. The expression levels of GLUT4 mRNA ((0.75 ± 0.04 vs. 0.34 ± 0.03), P < 0.01) and protein ((3443.71 ± 191.89 vs. 2137.79 ± 118.75), P < 0.01) increased.  We found that after PA intervention for 24 h, the cell viability decreased significantly; the cell viability of the LR group was higher than that of the high-fat group ( Therefore, we conclude that LR can reverse PA-induced cell inactivation and lipid deposition, which may be related to the change in GRP-78, FAT/CD36, FABP4, GLUT4, and other factors. Insulin resistance (IR) is one of the pathologies of many metabolic diseases, including type 2 diabetes and obesity. Previous studies have shown that an increase in free fatty acids induces ectopic lipid deposition through multiple pathways and further leads to IR –3. GlucaFatty acid translocase FAT/CD36) is a fatty acid transport protein that can promote intracellular fat deposition. LR can improve cardiac diastolic dysfunction in patients with type 2 diabetes by affecting the expression of cardiac FAT/CD36 and converting fatty acids in cardiomyocytes into glucose  is a fat. GlucosePalmitic acid (PA) is a saturated fatty acid and one of the most common free fatty acids. Previous studies have found that PA-induced cellular IR is related to lipid deposition and ERS , 9. WhetRat L6 myoblast cells were donated by the Hebei General Hospital . Minimum essential medium (MEM) was purchased from Thermo Fisher ; LR was supplied by Novo Nord ; 10% fetal bovine serum (FBS) and trypsin-EDTA were purchased from Biological Industries ; PA and skimmed bovine serum albumin (BSA) were purchased from Sigma Aldrich (Shanghai) Trading Co. (China); FAT/CD36 and FABP4 antibodies were purchased from Abcam ; and GRP78, GLUT4, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from GeneTex .2 culture bottles containing MEM supplemented with 10% (v/v) FBS in a 5% CO2 atmosphere at 37°C. When the cell growth was close to covering the bottom of the bottle, the cells were digested in 0.25% trypsin-EDTA and transferred into 6-well plates. When cell density reached 60–70%, differentiation was induced by replacing the culture medium with MEM containing 1% FBS, and cells were cultured for 6 days in a 5% CO2 atmosphere at 37°C.L6 cells were cultured in 25 cmμm membrane, cooled to 37°C diluted in MEM to a final concentration of 0.6 mmol/L, and incubated for 24 h prior to commencing the experiment at this temperature [By reference to research by Huang et al., we used 0.6 nmol/L for Treatment with Palmitic Acid . The PA perature .Differentiated cells were randomly divided into five experimental groups: control group, high-fat group, and three LR-intervention groups. Interventions were conducted for 24 h. The control group was cultured in MEM. The high-fat and LR-intervention groups were cultured in MEM supplemented with PA at 0.6 mmol/L. The medium of the LR-intervention groups was additionally supplemented with LR at 10 nmol/L (10LR), 100 nmol/L (100LR), or 1000 nmol/L (1000LR) .4 cells/mL and transferred into wells of 96-well plates, 100 μL per well, and incubated at 37°C for 48 h. Following interventions, 10 μL of CCK-8 reagent was added to each well, and the plates were placed at 37°C for 1 h. The optical density (OD) was measured at 450 nm using a microplate reader . Stimulation index SI = /.Cell viability was measured using a Cell Counting Kit-8 . Cell suspensions were adjusted to a density of 9 × 10Lipid deposition was measured using an Oil Red O Staining Kit . Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min; washed twice with PBS, pretreated with 60% isopropanol for 20 s, and washed twice with PBS after 30 min shading, and separated with 60% isopropanol for 5 s, and then washed twice with PBS. After the staining area was calibrated and normalized using Image J image processing software, the data were counted. Stained cells were photographed with an inverted microscope  within 2 h of staining.Intracellular triglyceride content was quantified using a Triglyceride (TG) Content Assay Kit  according to the manufacturer's protocol.ΔΔCt to calculate the mRNA expression of the target gene.Total RNA was extracted from treated cells using kit  according to the manufacturer's protocol. The gene expression levels were measured by reverse transcription quantitative real-time PCR using Loading Buffer PCR Master Mix . Primers were synthesized by GeneCopoeia  or sheep anti-rat (GAPDH) secondary antibodies. Immunoreactive bands were detected using chemiluminescence reagent  according to the manufacturer's instructions. Protein expression was quantified using an enzyme labeling instrument (BD 680 automatic microplate reader) at 570 nm.n = 3). Statistical significance was assessed using Fisher LSD t-tests, and single factor ANOVA; correlations were determined using linear correlation. SPSS 20.0  was used for statistical analysis, and GraphPad Prism 6.01  was used for graphic analysis. Statistical significance was set at P < 0.05.All data are expressed as means ± standard deviation (P < 0.01), of the 10LR group (average SI = 2.63) did not differ from the high-fat group , but both the 100LR (average SI = 2.64) and 1000LR (average SI = 2.50) groups had higher cell viability (P < 0.01) [P < 0.05).Cell viability was reduced in the high fatgroup (average SI = 2.70) relative to the control group (average SI = 2.17)  . Cell viP < 0.01). The 10LR group did not differ from the high-fat group, but both the 100LR (0.09 ± 0.01) and 1000LR (0.06 ± 0.02) groups had a lower TG content than the high-fat group (P < 0.01).The TG content was higher in the high-fat group relative to the control group . Levels of FAT/CD36 and GRP78 mRNA were lower and GLUT4 mRNA higher in all three LR-intervention groups relative to the high-fat group. FABP4 mRNA in the 100LR and 1000LR groups were also lower compared with the high-fat group  cell viability assays have been performed but apoptosis assays like Annexin V assay or Hoechst assay can be performed to visualize the percent of cells undergoing apoptosis with palmitic acid and liraglutide treatment –33. (2)


Penicillium piceum W6 from the atmospheric and room-temperature plasma-mutated library of P. piceum. β-xylosidase hyperproducers filamentous fungi, P. piceum W6, exhibited an increase in β-xylosidase activity by 7.1-fold. A novel β-D-xylosidase was purified from the extracellular proteins of P. piceum W6 and designated as PpBXL. The optimal pH and temperature of PpBXL were 4.0 and 70 °C, respectively. PpBXL had high stability an acidic pH range of 3.0–5.0 and exhibited good thermostability with a thermal denaturation half-life of 10 days at 70 °C. Moreover, PpBXL showed the bifunctional activities of α-L-arabinofuranosidase and β-xylosidase. Supplementation with low-dose PpBXL (100 μg/g substrate) improved the yields of glucose and xylose generated from delignified biomass by 36–45%. The synergism between PpBXL and lignocellulolytic enzymes enhanced delignified biomass saccharification, increased the Xyl/Ara ratio, and decreased the strength of hydrogen bonds.A droplet-based microfluidic ultrahigh-throughput screening technology has been developed for the selection of high-β-xylosidase-producing  Lignocellulosic biomass is the most abundant natural polysaccharide available on Earth, which can be converted into biofuels to decrease greenhouse gas emission and strengthen energy security ,2. Lignohttp://www.cazy.org, accessed on March 2022) [A. fumigatus XC6 β-xylosidase can promote ethanol production to 80% of the theoretical value [Aspergillus, including A. fumigatus, A. brasiliensis and A. niger, exhibit optimal activities at high temperatures (55–75 °C) [β-xylosidases are categorized into different glycoside hydrolase families  . β-xylosch 2022) ,11. A vaal value . Some β-5–75 °C) . HoweverPenicillium piceum is challenging given the filamentous nature of this fungus and the secretion of β-xylosidase extracellularly. The development of the ultrahigh-throughput screening system for β-xylosidase considerably increased the chance of obtaining desired properties, which would improve strain screening efficiency and shorten strain screening cycle.Microbial β-xylosidases occur in bacteria and fungi, with those of the latter group being the most studied . At presIn this paper, a new high-throughput screening method for β-xylosidase producing filamentous fungi is proposed. The characteristics and exact role of this novel β-xylosidase in biomass degradation are demonstrated. Subsequent characterization and application reveal the novel β-xylosidase’s unique properties, making it valuable in the potential industrial applications of biomass degradation.P. piceum  was a β-glucosidase hyperproducing strain obtained by dimethyl sulphate mutagenesis [4)2SO4, 0.6% KH2PO4, 0.1% MgSO4, 0.2% Tween-80 and 0.25% CaCO3. After inoculation into 50 mL of the medium in a 300-mL conical flask, the spore suspension was grown at 28 °C for 5 days under rotatory shaking at 180 rpm [agenesis . The medT. reesei A2H  was a cellulase-hyperproducing strain generated from ARTP mutagenesis. For strain recovery, mycelia agar disks were inoculated on fresh PDA and cultivated at 30 °C for 7 days until formation of conidia. Spore suspension (1 mL) was added into 30 mL preculture  in a 250-mL flask and incubated at 28 °C for 24 h at 180 rpm. After transferring 5% precultured inoculum size to 30 mL of production medium in a 250-mL flask at 26 °C for 5 days at 180 rpm. The medium (g/L) consisted of 1.7% corn steep powder, 0.6% KH2PO4, 0.5%(NH4)2SO4, 0.25% CaCO3, 0.1% MgSO4∙7H2O, 0.2% Tween 80, and 3% inducer and had an initial pH of 5.0.7 spores/mL, 10 μL of diluted suspension was spread onto sterilized steel dishes, followed by exposure to a helium gas flow for 180 s. The plasma reactor was kept at a distance of 2 mm. Subsequently, the suspension was placed in 1 mL distilled water. After ARTP treatment, the spores were precultured at 30 °C before droplet encapsulation.After culturing for 5 days, the spores were harvested with sterile distilled/deionized water and filtered through 0.22 µm. After diluting the spore suspension to 106 spores/mL in medium containing 100 µM β-xylosidase fluorogenic substrate 4-methylumbelliferyl-β-xylobioside (4MUX) and a surfactant-containing fluorinated oil were used as the aqueous and oil phases, respectively [To produce droplets, a suspension of 10ectively . The floThe content of β-xylosidase in droplets was assessed by using the fluorogenic substrate 4MUX  at excitation and emission wavelengths of 365 and 450 nm, respectively. A focused laser was used to excite the droplets, and the obtained fluorescence intensities were determined using the microfluidic system as described previously . The higMr 10,000 cut-off Millipore membrane , the supernatant of P. piceum was loaded onto an anion exchange SP column . Based on the three peaks, the fractions were collected and subjected to further separation. After loading onto an anion exchange DEAE column , the combined fractions were equilibrated with sodium acetate buffer . Then, elution was performed in the same buffer by a linear NaCl gradient (0–1 M) at 2 mL/min, and 1 mL fraction was collected.After ultrafiltration with a https://swissmodel.expasy.org/, accessed on January 2022).Internal amino acid sequence analysis of proteins was conducted via the in-gel digestion. Mass spectrometry was used to sequence different peptides at the Tianjin Institute of Industrial Biotechnology, CAS. The simulated three-dimensional structure of the β-xylosidase was obtained by SWISS-MODEL  .2SO4, 0.6 mL/min and 60 °C, respectively [Xylose and arabinose levels were detected on an Aminex HPX-87H column  by high-performance liquid chromatography (HPLC) with a refractive index detector . The mobile phase, flow rate and temperature were 5 mM Hectively .p-nitrophenol released from p-nitrophenyl-β-D-xylopyranoside (pNPX). The assay mixture contained 20 µL diluted enzyme and 80 µL pNPX in a working volume of 100 µL. The reaction was carried out at 70 °C for 10 min and then stopped by 100 µL sodium carbonate. The p-nitrophenol released from pNPX was determined by the absorbance at 410 nm. The amount of enzyme that produced 1 µmol of p-nitrophenol per minute was denoted as 1 unit of the enzyme activity. The assay was carried out as described previously [p-nitrophenyl-α-L-arabinofuranoside (pNPAF), p-nitrophenyl-β-D-p-nitrophenyl-β-D-glucose (pNPG), o-nitrophenyl-β-D-xylopyranoside (oNPX) and birch wood xylan. In certain assays, pNPX was substituted with oNPX, pNPG or pNPAF.The β-xylosidase activity was routinely measured through the amount of eviously . The speThe optimal temperature for purified β-xylosidase was determined in phosphate buffer (pH 4.0) and 50 mM citrate at 30–70 °C for 30 min. Thermostability of the enzymes was assessed after incubation at different temperature (30–70 °C) by using the same buffer with no substrate. Sampling was conducted at different time points, and the residual activities were evaluated.The optimal pH for purified β-xylosidase was determined in phosphate buffer and 50 mM sodium citrate at 70 °C and pH 2.0–8.0 for 30 min. The pH stability of the enzymes was evaluated by detecting the residual activities at 24-h intervals.The effect of metal ions on enzyme activity was assessed with 1 mM final concentration of the substances. The activity of the enzymes with no metal ions was regarded as 100%.w/v) delignified biomass (corn stover/corn cob) and 10 FPU cellulase per gram substrate in a working volume of 50 mL. Delignified biomass were acquired after alkali pretreatment. Lignocellulolytic enzymatic preparation from T. reesei A2H was a cocktail of cellulase and hemicellulase. Lignocellulolytic enzymes from Trichoderma reesei were cultivated with filter paper for enzyme activity of 30 IU/mL and xylanase activity of 400 IU/mL. Then, 100 μg purified protein/g substrate was used as the supplementation dosage. Saccharification was carried out at 50 °C for 3 days. Glucose and xylose in enzymatic hydrolysate were measured according to the method [Synergism assays were conducted in duplicate trials in a 100-mL centrifuge tube. The reaction mixture consisted of 5%  was used to dry the biomass specimens at −20 °C for 24 h. IR spectral data were obtained using an IR spectrophotometer . The HBI was calculated in accordance with the method .p < 0.01 [To compare DDGS composition with or without lignocellulolytic enzymes, ANOVA was employed, followed by Tukey’s multiple comparison post-test. Statistical tests were conducted with SigmaPlot V11 . Level op < 0.01 .A. niger with no β-xylosidase activity was detected using this droplet-based microfluidic method as a negative control. There was no fluorescence signal released from β-xylosidase fluorogenic substrate (4MUX) in the A. niger droplets . Therefore, the second peak was concentrated and loaded onto the DEAE column and eluted with 0–1 M NaCl (The concentrated culture filtrate from  and III A. The mo1 M NaCl B. The pu1 M NaCl C.P. piceum-sequenced genome (unpublished data). The purified protein was identified as PpBXL. The protein sequence of PpBXL was compared against with publicly available sequence data through BLAST. The protein belonged to the glycoside hydrolase family 39, having 57.25% similarity with the 1.4-β-xylosidase from Rhizodiscina lignyota, 57.40% similarity with β-xylosidase from Rasamsonia emersonii, 56.20% similarity with β-xylosidase from Oidiodendron maius and 55.16% similarity with β-xylosidase from Viridothelium virens. The sequences of PpBXL have been uploaded to the NCBI database (GenBank accession number: OM055664).The single band from SDS-PAGE was hydrolyzed by trypsin digestion to generate six major peptide fragments. HPLC-ESI-MS/MS results showed that KVSVVVDASSK (peptide 1), KELGSLGKAQTYFR (peptide 2), KSYERWGK (peptide 3), KGVPLDLISFHAK (peptide 4), RVLSTQGIDK (peptide 5) and RVDNKHSNSYAK (peptide 6)  were obtThermoanaerobacterium saccharolyticum (PDB ID code: 1UHV) [10-helices a’ to f’   as temple: 1UHV) . A few we: 1UHV) . Our resa’ to f’ . The catpNPX and oNPX but more favorable towards pNPX. PpBXL showed specific activities against pNPX and oNPX, i.e., 320.5 and 79.3 IU/mg, which were higher than that of β-xylosidase from Caulobacter crescentus (215 IU/mg) or Geobacillus sp. (133 IU/mg) against pNPX [pNPAF (107.8 IU/mg), suggesting that PpBXL had arabinofuranosidase activity. PpBXL showed the bifunctional activities of α-L-arabinofuranosidase and β-xylosidase. Most of β-xylosidase shows no arabinofuranosidase activity [WSUCF1 β-xylosidase was only active against pNPX [Butyrivibrio fibrisolvens, Thermoanaerobacter ethanolicus and Sporotrichum thermophile [pNPAF and pNPX were relatively lower than that of PpBXL in this paper.As shown in nst pNPX ,13. Intermophile ,30,31 ha2+, Hg2+, Ag2+ and Ni2+ exerted strong inhibitory effects on the purified enzymes even at 1 mmol/L , Geobacillus, and Caulobacter strains present optimal activity at 55–75 °C [A. fumigatus XC6 was thermally stable within the range of 60–75 °C, and retained maximum activity at 75 °C for 24 h [Geobacillus strain (9 days at 70 °C) [The optimal temperature of PpBXL was 70 °C. PpBXL maintained 55.2% and 87.6% of its maximum activities at 80 °C and 60 °C, respectively B. Some β55–75 °C ,14,32. Tfor 24 h . HoweverT. reesei lignocellulolytic enzymes at a low dose of 100 μg/g substrate in the saccharification to elucidate the role of PpBXL in improving the enzymatic degradation of delignified biomass (delignified corn stover and delignified corn cob). PpBXL had remarkable synergism with lignocellulytic enzymes from T. reesei during the delignified biomass saccharification. Although the supplemental dose of PpBXL was low, the glucose and xylose yields released from delignified corn stover by the T. reesei lignocellulolytic enzymes increased by 41.4% and 39.3%, respectively. The glucose and xylose yields released from delignified corn cob after PpBXL supplementation increased by 36.3% and 44.6%, respectively.PpBXL was added to WSUCF1 β-xylosidase and xylanase were added to increase xylose yields to 90% during birchwoodxylan hydrolysis, which further enhanced the fermentation product yields [p < 0.01) due to PpBXL supplementation. When PpBXL could enhance the hemicellulose degradation, the hydrogen bonds between hemicellulose and cellulose were simultaneously disrupted. It is found that HBI of delignified biomass decreased (p < 0.01) after PpBXL supplementation. Hence, PpBXL supplementation evidently improved the saccharification of biomass.The delignified biomass could increase the yields of hemicellulose after alkali pretreatment. However, it remains a challenge to achieve the complete enzymatic degradation of xylan to xylose. Xylanases could hydrolyze xylan into xylose and various xylo-oligosaccharides. As these xylo-oligosaccharides were not able to undergo fermentation, they could be detected in the hydrolysates of lignocellulosic materials, thus resulting in lower yields of final product. Xylaolytic enzymes in most commercial enzyme preparations were not capable to hydrolyze xylan completely, leading to high xylo-oligomer contents retaining in the hydrolysis broth. The accumulation of xylo-oligomer led to decreased initial hydrolysis efficiency of cellulase . β-xylost yields . Similart yields . DeligniT. reesei. PpBXL might improve the hydrolysis efficiency of lignocellulose degradation by decreasing the inhibitory effect of xylo-oligomer. The development of enzyme cocktails by using novel enzyme components with unique characteristics might provide a new way for improving lignocellulose hydrolysis.A droplet-based microfluidic ultrahigh-throughput screening technology has been developed for the screening of high-β-xylosidase-producing strain, which effectively improve the quality of strain breeding. PpBXL can serve as a promising bifunctional enzyme with high β-xylosidase and α-L-arabinofuranosidase activities. Moreover, PpBXL had excellent thermostability with a thermal denaturation half-life of 10 days at 70 °C. At low concentrations, PpBXL remarkably synergized with lignocellulolytic enzymes from


Berberis nummularia and B. atrocarpa fruits, the biochemical properties , total phenolic and flavonoid content and antioxidant  capacity of ethanol extracts of B. nummularia and B. atrocarpa fruits wild-grown in Xinjiang were analyzed. The results indicated that there were no meaningful differences (p > 0.05) between the ash (1 ± 0.1 and 1 ± 0.0 g/100 g), fiber (16 ± 1.0 and 18 ± 1.4) and carbohydrate (57 ± 1.8 and 56 ± 1.8 g/100 g) content, respectively, in the dry fruits of B. nummularia and B. atrocarpa. The total fat (7 ± 0.4 and 5 ± 0.1 mg/100 g), soluble sugar (23 ± 0.6 and 12 ± 1.4 g/100 g), titratable acidity (18 ± 2.5% and 14 ± 1.3%) content, and energy value  of B. nummularia was significantly higher than that of B. atrocarpa fruits. Both species contain malic acid, acetic acid, tartaric acid, citric acid and fumaric acid, in which, malic acid is the dominant organic acid. The organic acid and mineral components of B. nummularia fruits were significantly higher than that of B. atrocarpa (p < 0.05). The total phenolic and flavonoid content of B. nummularia were 2 ± 0.0 mg GA/g DW and 2 ± 0.0 mg RE/g DW, respectively, which were significantly lower than the total phenolic and flavonoid content of B. atrocarpa (12 ± 0.1 mg GA/g DW and 9 ± 0.0 mg RE/g DW). The antioxidant capacity of B. nummularia . B. atrocarpa fruits showed significantly higher antioxidant capacity than that of B. nummularia. The fruits of the two species can be used in food coloring and nutritional supplements, and consumption of the fruits can aid in weight control and reduce blood glucose or cholesterol.To evaluate the potential health-promoting benefits of  Ugni molinae Turcz) and calafate (Berberis buxifolia Lam.), both of which grow natively in Patagonia, can be sources of anthocyanins, and bear an ascorbic acid content of 210 mg/100 g F.W. . B. nummularia and B. atrocarpa fruits were mixed with 50 mL of 80% ethanol extract. Subsequently, they were sonicated for 60 min at 70 °C at a frequency of 40 kHz. Then, the solutions were centrifuged at 5000× g, 20 °C for 15 min. The supernatants were filtered, and dried using a rotary evaporator at 50 °C followed by freeze-drying at −52 °C for 48 h. The dried extracts were reconstituted in 10 mL 80% ethanol and kept at 4 °C prior to analysis of the total phenolic and flavonoids content, and antioxidant capacity.The 5 g samples of Total Phenolic Content (TPC) was determined according to the modified Folin–Ciocalteu colorimetric method at 760 nm , by usinThe aluminum chloride colorimetric method described by Pu et al. (2018)  was modiThe DPPH assay was performed using the method of Tian et al. (2016)  with somThe FRAP assay was performed using the method of Tian et al. (2016) , at 593 The ABTS assay was performed using the method of Fang et al. (2009)  with somThe ORAC assay was performed using the method of Liu et al. (2016)  with somThe result was expressed as mg Trolox/g DW.p < 0.05. Figures were drawn using Sigma plot version 12.0.Statistical analysis of this study was performed using the SPSS (version 18.0) software . All results were expressed as means ± standard error. One-way ANOVA was used to analyze the difference between nutritional components, mineral contents, organic acids, total phenolic and flavonoid contents and antioxidant  capacity of ethanol extracts of samples at The composition including the fat, protein, fiber, ash, soluble sugar (fructose and glucose), titratable acid, carbohydrates, and energy value of berberis is shown in B. nummularia and B. atrocarpa fruits (p < 0.05), except for fiber and ash. The protein content represents one of the important indicators in evaluating fruit quality and nutritional value [B. nummularia (3 ± 0.1 g/100 g) was significantly lower than that of B. atrocarpa (4 ± 0.1 g/100 g) (p < 0.05). The soluble protein content of the two berberis was higher than that of the fruits of B. microphylla [Hippophaë rhamnoides (sea buckthorn), in which, the protein content was 17 ± 0.4% DWP [B. nummularia were significantly higher than those in B. atrocarpa (p < 0.05). The total amount of soluble sugar contained in B. nummularia (23 ± 0.6 g/100 g) is twice as high than that of B. atrocarpa (12 ± 1.5 g/100 g). The HPLC-RID chromatograms showed that the B. nummularia appears to have a significantly higher content of fructose and glucose, sharing this result with B. vulgaris [The nutrient contents of the analyzed samples  were significantly different between the al value . The sols p < 0.0, except vulgaris .B. nummularia and B. atrocarpa, which is in line with previous reports of other berberis fruit [B. nummularia and 314.41 kcal/100 g for B. atrocarpa.Dietary fiber is an essential aspect of good nutrition. The fiber content measured up to 16% for both is fruit . Researcis fruit  due to iis fruit . The conp < 0.05). Among the minerals, potassium was found to comprise the highest concentration in the fruits of the two species, measured at 1380 ± 13.6 mg/100 g for B. nummularia and 1225 ± 10.6 for B. atrocarpa, which constitutes 70.24% and 79.56% of the total mineral content, respectively. This is line with a previous study that reported potassium as the most abundant mineral element in wild fruits [B. nummularia (160 ± 16.7 mg/100 g) was higher than that of B. atrocarpa (132 ± 11.7 mg/100 g). The portion of calcium would cover up to 21% of NRV for this nutrient. Magnesium was also an abundant mineral in B. nummularia and B. atrocarpa, respectively, delivering 109 ± 1.5 mg/100 g and 58 ± 0.9 mg/100 g, which corresponds to up to 13% of NRV. The mineral contents in the two fruits of the present study were comparable to the mineral composition of three Iranian berberis species published by Rahimi-Madiseh et al. (2016) [Berberis vulgaris L. and B.crataegina DC was slightly lower, ranging from 85.07 to 97.95 mg/100 g [B. atrocarpa fruit has low levels (10 ± 0.3 mg/100 g) of iron (Fe) and Cu (1 ± 0.0 mg/100 g) content, but not present in B. nummularia. Noticeably, the content of Na in B. nummularia was significantly higher than in B. atrocarpa and other berberis [B. nummularia bears resistance to high salinity conditions in its living environment [The mineral contents and their recommended daily allowances at nutrient reference values (NRVs), are presented in d fruits . The con. 2016) . The conmg/100 g . Zn and 016 . Theberberis . This isberberis  reportedMespilus germanica L.) contain around 4 g/kg F.W. of citric acid and malic acid. Our results showed the presence of tartaric acid, malic acid, citric acid, acetic acid and fumaric acid both in B. nummularia and B. atrocarpa fruits (p < 0.001) in the content of total organic acid between the two fruits (B. nummularia (119 ± 0.1 mg/g) than in B. atrocarpa (84 ± 0.1 mg/g). The content of citric acid and tartaric acid varied from 0.48 to 4.23 mg/g. The fumaric acid was present at low concentrations in both two fruits. The difference in the organic acid contents of the two species is likely due to genetic effects.Organic acids are used as preservatives, antioxidants and acidulants , which ia fruits . The orgo fruits . Malic aB. nummularia and B. atrocarpa fruits were 2 ± 0.0 mg GA/g and 12 ± 0.1 mg GA/g DW, respectively, which is significantly different from each other (p < 0.05)  [B. microphylla fruits were 1035.03 mg GA/100 g FW. The previous studies showed that the environmental and nutrient conditions of the plant’s habitat, and the genotype and ripening steps of the fruits affect the formation and accumulation of secondary metabolites [B. nummularia and B. atrocarpa fruits in this work were collected from a waste gravel and ravine in Aksu and Yili with semi-arid climates. This could explain the higher TPC determined in this work with respect to Ruiz et al. (2014) [The TPC of the crude extracts of  < 0.05) . The tot. (2021)  showed t. (2021)  obtained. (2021)  reportedabolites ,42. B. n. (2014)  who usedB. atrocarpa fruits (9 ± 0.0 mg RE/g DW) accounted for 72% of the total content of phenols. The results of TFC content in B. atrocarpa fruits are in line with the literature reporting flavonoids as the predominant phenolic group in plant foods accounting for nearly 75% of the dietary phenols [B. nummularia, it was revealed that the TFC exceeded the content of phenols.Different amounts of TFC were revealed in the fruits of two berberis . Hassanp phenols . For theMorinda Citrifolia L.) fruit extracts (NFEs) and the results showed that the total phenolic content in Bet-Gly (Betaine: Glycerol) extracts was highest at 11.89 mg GAE/g DW, but the ethanol extracts exhibited the strongest antioxidant activities, with the results from DPPH, ABTS+, FRAP and reducing power values measuring 61.31, 123.46, 347.37 and 312.42 μmol TE/g DW, respectively. In the present study, the total antioxidant activity of the ethanol extract of B. nummularia and B. atrocarpa fruits ranged from 4 ± 0.1 to 12 ± 0.0 mg ascorbic acid/g DW for DPPH, from 32 ± 0.1 to 645 ± 1.1 mg Trolox/g DW for FRAP, from 80 ± 3.0 to 304 ± 3.0 mg Trolox/g DW for ABTS and from 61 ± 3.6 to 155 ± 2.8 mg Trolox/g DW for ORAC assays, respectively. This is consistent with the results demonstrating significantly higher values in TFC and TPC of the four assays of B. atrocarpa than that of B. nummularia (p < 0.05)  [B. vulgaris and B. crataegina had higher antioxidant capacities than B. integerrima, which is in agreement with our present results.Evaluating the antioxidant capacity of natural resources is important in the modern health lifestyle context. The highest antioxidant capacity of fruits stemmed mainly from phenolic compounds . However < 0.05) . AccordiB. nummularia and B. atrocarpa fruits with the scientific research on both species for the first time. Although the nutritional content of B. nummularia was higher than B. atrocarpa, the fruits of both species appear to be a good source of potassium and calcium, malic acid and crude fiber. It was found that the TPC, TFC, as well as the potential antioxidant properties of B. atrocarpa demonstrate significant predominance.Wild and medicinal plant species have been used for many purposes. It is important to determine the biochemical properties and antioxidant capacity of these plants when intended for consumption. In this paper, we approximate the traditional knowledge of B. nummularia and B. atrocarpa are also a promising source of natural antioxidants, with nutraceutical potential. Despite their high nutritional properties, the fruits of B. nummularia and B. atrocarpa are not considered enough and not properly recognized. When the important biochemical components are considered, one can deduce that the fruits of B. nummularia and B. atrocarpa are suitable for use in the food industry as food coloring, nutritional supplements, in addition to health and medical applications regarding weight control and blood glucose or cholesterol reduction.The biological activity of phytochemicals indicate that