The developed and validated technique was used for the evaluation of triterpenic compounds in samples of Vaccinium macrocarpon and Vaccinium oxycoccos fruits, and their peels, pulp and seeds. The studied chromatogram profiles of Vaccinium macrocarpon and Vaccinium oxycoccos were identical but differed in the areas of the analytical peaks. Ursolic acid was the dominant compound in fruit samples of Vaccinium macrocarpon and Vaccinium oxycoccos. The highest amounts of triterpenic compounds were detected in the cranberry peels samples. The developed method for the detection of triterpene compounds can be applied in further studies for routine testing on the qualitative and quantitative composition of fruit samples of Vaccinium macrocarpon and Vaccinium oxycoccos species and cultivars.Cranberries are used in the production of medicinal preparations and food supplements, which highlights the importance of triterpene compounds determination in cranberry fruit raw material. The aim of our study was to develop and validate for routine testing suitable UPLC-DAD methodology for the evaluation of triterpene acids, neutral triterpenoids, phytosterols, and squalene content in cranberry samples. The developed and optimized UPLC-DAD methodology was validated according to the guidelines of the International Council for Harmonization (ICH), evaluating the following parameters: range, specificity, linearity (R Vaccinium oxycoccos L.), fruit raw materials are prepared in natural habitats, while American cranberry, or large cranberry (Vaccinium macrocarpon Aiton), is grown in private gardens and industrial plantations  − 144.98 and [M] − 163.00 could be identified as coumaric acid [2O] − 437.32 could be identified as ursolic acid or its isomer [Not all of the detected analytes were identified in the chromatogram of cranberry sample extracts. Some of these analytes have characteristic UV absorption maxima a–f, whicectively . The fol − 601.4 . In our ric acid , and fra, 144.98 .87%, 4372 were more than 0.999, which proves the linearity of the optimized methodology [The linear range of the identified compounds, calibration Equations, their coefficients of determination, and limits of qualitative and quantitative determination are given in hodology . The calThe accuracy parameter helps to evaluate the correspondence between the result obtained and the true value by expressing the closeness between the obtained results in percentage . The recVaccinium oxycoccos and Vaccinium macrocarpon fruit samples  of HorRat values set in the single-laboratory validation studies [The precision of the methodology was assessed based on repeatability and intermediate precision in determining the variation of the identified compounds in  samples . In the  studies . The resVaccinium macrocarpon and Vaccinium oxycoccos cranberry fruits and their parts. The test samples of large cranberries (Vaccinium macrocarpon) consisted of the whole cranberry berry, the peel, the juicy pulp of the berry, and the seeds. In addition, three test samples of small cranberries (Vaccinium oxycoccos) were prepared: the whole cranberry berry, the peel, and the juicy berry pulp with seeds. The juicy pulp of Vaccinium oxycoccos cranberry fruit contains a higher amount of water (84.2–98.8%) than the pulp of Vaccinium macrocarpon fruit (84.8–88.0%) does [The developed and validated UPLC-DAD methodology was applied to the qualitative and quantitative analysis of triterpenoids and phytosterols in 0%) does ; therefoVaccinium macrocarpon and Vaccinium oxycoccos were found to contain the highest total amount of the identified triterpene compounds and phytosterols  (Vaccinium macrocarpon) cultivar ‘Stevens’ had higher total levels of triterpenoids and phytosterols than samples of the small cranberry (Vaccinium oxycoccos) fruit did  . The conruit did . During ruit did , the totThe ursolic acid content in fruit samples of large cranberries and small cranberries was, respectively, 62.64% and 64.80% of the amount of the identified triterpenoids and phytosterols. The maximum levels of ursolic acid found in the fruit peel samples of small cranberries and large cranberries were 5809.40 ± 87.14 µg/g and 6612.79 ± 99.19 µg/g, respectively . KlavinsThe amount of oleanolic acid in fruit samples of large cranberries and small cranberries was, respectively, 17.42% and 18.82% of the identified triterpenoids and phytosterols. The largest amount of oleanolic acid found in samples of large cranberry fruit peels was 2562.29 ± 38.43 µg/g. A similar amount of oleanolic acid (2046.18 ± 30.69 µg/g) was found in samples of small cranberry fruit peels. The amount of oleanolic acid in cranberry fruit samples detected by Oszmianski et al. (894–1137 µg/g) was similar to that found in our study . MeanwhiThe largest amounts of maslinic acid found in peel samples of large cranberries and small cranberries were, accordingly, 112.45 ± 2.69 µg/g and 106.73 ± 1.60 µg/g. No maslinic acid was detected in cranberry pulp and seed samples. The maximum levels of corosolic acid found in fruit peel samples of large cranberry and small cranberry were, respectively, 241.38 ± 3.69 µg/g and 281.62 ± 4.22 µg/g. No corosolic acid was detected in cranberry pulp and seed samples. In a study by Xue et al., the content of maslinic acid in cranberry fruit extract varied from 320 µg/g to 900 µg/g, and the content of corosolic acid ranged from 1190 µg/g to 3810 µg/g .Vaccinium oxycoccos samples. Klavins et al. found the cuticle wax extract of large cranberry fruits to contain 2500 µg/g of α-Amyrin and 3000 µ/g of β-Amyrin [The largest amounts of α-Amyrin found in fruit peel samples of large cranberries and small cranberries, were, respectively, 249.53 ± 3.74 µg/g and 106.91 ± 1.60 µg/g. No α-Amyrin was detected in cranberry pulp and seed samples. The highest β-Amyrin content found in large cranberry seeds was 336.74 ± 5.05 µg/g. No β-Amyrin was detected in the β-Amyrin . In anotβ-Amyrin . In largβ-Amyrin .Vaccinium oxycoccos peel and seed pulp samples  water purification system.Acetone , methanol , formic acid , acetonitrile , reference standards for maslinic acid, corosolic acid, oleanolic acid, ursolic acid, β-Amyrin, campesterol, α-Amyrin, and β-Sitosterol were obtained from Sigma-Aldrich , and reference standards for squalene were obtained from Carl Roth . The purified deionized water used in the experiments was prepared using a Milli-QVaccinium macrocarpon and Vaccinium oxycoccos. Samples of the Vaccinium macrocarpon cultivar ‘Red Star’ and Vaccinium oxycoccos cranberry fruit used for the development and validation of the methodology were obtained from, respectively, the Botanical Garden of Vilnius University, Kairėnai, Vilnius (54°43′47.1″ N 25°24′34.9″ E) and Dubravai, Kaunas distr. (54°50’54.5” N 24°04′35.2″ E). Parts of cranberries were obtained by mechanical separation of peel, pulp, and seeds of Vaccinium macrocarpon cultivar of ‘Stevens’ collected in Giedručiai, Šakiai district. (54°57′50.8″ N 23°01′34.7″E) and samples of Vaccinium oxycoccos collected in Čepkeliai (53°59′07.3″ N 24°26′48.6″ E). The collection time was September 2021. Cranberry fruits were ground and frozen at −60 °C in an ultra-low-temperature freezer . Cranberry fruits were freeze-dried according to the methodology described by Gudžinskaitė et al. [The object of the study was samples of mature and ripe fruit of ė et al. .About 1 g (precise weight) of the lyophilized cranberry powder was extracted with 10 mL of acetone in an Elmasonic P ultrasonic bath  at 80 kHz and 1130 W at 22 ± 1 °C to 60 ± 1 °C temperature for 60 min. After the extraction, the acetonic extracts were filtered into a 10-mL volumetric flask. The prepared extracts were stored in dark glass vials at 5–8 °C. Prior to the chromatographic analysis, the acetonic extracts were filtered through membrane filters with 0.22 µm pore size .v/v) (A) and 100% methanol (B) at a flow rate of 0.2 mL/min and the following gradient change: 0 min, 8% A; 8 min, 3% A; 9 min, 2% A; 29.5 min, 2% A; and 30 min, 8% A, delaying the subsequent injection by 10 min. Mass spectrometer conditions were used as follows: a negative electrospray ionization mode with 3 kV capillary voltage and 50 V cone voltage, the source temperature was set to 150° C, the desolvation gas temperature to 600° C, flow to 1000 L/h, and cone gas flow to 20 L/h. MS analysis was performed in negative scan mode in m/z range from 110 to 1000. MS/MS analysis was performed in product ion scan at 40 eV collision energy.The analysis of the qualitative and quantitative composition of triterpenic compounds, sterols, and squalene in cranberry fruit was performed using a Waters ACQUITY Ultra High-Performance LC system  with a photodiode array detector and Xevo TQD tandem mass spectrometer . An Avantor ACE Excel UHPLC column  was used for the separation of the compounds at 25 °C. The injection volume was 1 µL, and the distribution was performed using 0.1% formic acid , the acceptance criteria of the validation were selected considering the recommendations of Eurochem, the Guidelines for Standard Method Performance Requirements, and the Guidelines for Dietary Supplements and Botanicals ,43,56,572) was set (suitable when R2 > 0.999). The limit of detection (LOD) and the limit of quantitation (LOQ) were determined at a signal-to-noise (S/N) ratio of 3:1 and 10:1, respectively. Precision was determined by evaluating the relative standard deviation (RSD%) of the retention time and the amount of 6 Vaccinium macrocarpon extracts and 6 Vaccinium oxycoccos extracts (Formula 1). The RSD% of repeatability was calculated from measurements taken on the same day (n = 6), and the intermediate precision was determined by analyzing six cranberry extracts for three consecutive days (n = 18). The acceptable precision value was determined by calculating Horwitz Ratio (HorRat) (Formula 2) [Specificity was determined by comparing the analyte retention time and the MS/MS spectrum between the reference standard and the cranberry matrix. Linearity was determined by constructing calibration regression equations consisting of 6 to 10 points and measuring standard solutions of known concentration. To evaluate linearity, the coefficient of determination (Rrmula 2) .RSD% = Recovery was determined by adding a known amount of reference standards to a blank matrix at three levels: level 1—low concentration, level 2—medium concentration, and level 3—high concentration. Concentrations of level 1 were 10 µg/mL for all standards. Concentrations of level 2 were 250 µg/mL for oleanolic acid, 800 µg/mL for ursolic acid and 50 µg/mL for maslinic acid, corosolic acid, β-amyrin, campesterol, α-amyrin, β-sitosterol, and squalene. Concentrations of level 3 were 500 µg/mL for oleanolic acid, 1600 µg/mL for ursolic acid, and 150 µg/mL for maslinic acid, corosolic acid, β-amyrin, campesterol, α-amyrin, β-sitosterol, and squalene. Recovery was calculated according to Formula (3) . Recovery was regarded as adequate when the Recovery% was in the range of 80–110% .Recover2 and to construct calibration equations for the calculation of the amounts of the identified compounds. To evaluate the difference in the amounts of triterpenic compounds between samples of cranberry, one-way analysis of variance ANOVA with Tukey’s test for multiple comparisons (the significance level set at 0.05) was used.Data analysis was performed using computer software programs Microsoft Excel 2016  and SPSS Statistics 21 . During the study, means, standard deviations (SD), and relative standard deviations (RSD) of the three independent evaluations were calculated. Linear regression analysis was performed to calculate the coefficient of determination R2 > 0.999), precision, LOD (0.27–1.86 µg/mL), LOQ (0.90–6.18 µg/mL), and accuracy (95.47–107.91%)—met the requirements of the normative documents and confirm the suitability of the methodology for application.A UPLC-DAD method was developed and validated to detect triterpene acids, neutral triterpenoids, phytosterols, and squalene in a single assay. The validation parameters of the methodology—specificity, linearity (RVaccinium macrocarpon and Vaccinium oxycoccos were identical but differed in the areas of the analytical peaks. The determination of triterpene compounds and phytosterols in different parts of cranberry fruit provides knowledge about the distribution of these compounds in cranberry organs and allows for a better understanding and prediction of how technological processes of cranberry fruit preparation and processing determine the content of triterpene compounds and phytosterols in cranberry foods and food supplements.The studied chromatogram profiles of


The results demonstrated that, despite BF decreasing the biomass and N distribution into grains, BF performed better on improved soybean yield (5.2–8.5%) by increasing the accumulation of soybean biomass (7.2 ~ 11.6%) and N (7.7%). Even though BC and BF have a detrimental effect on soybean nitrogen fixation by reducing nodule number and weight, the values of soybean nitrogenase activity and nitrogen fixation potential in BF were higher than those in BC. Additionally, BF performs better at boosting the soil’s nitrogen content and nitrate reductase and urease activity. BF increased the concentration of total N, soil organic matter, Olsen-phosphorus, and alkaline hydrolyzable N in the soil by 13.0, 17.1, 22.0, and 7.4%, respectively, compared to CK. Above all, applying BF combination with N2 (45 kg ha − 1 N) is a feasible strategy to raise crop grain output and keep soil productivity over the long term in IS.Applying Biochar (BC) or biofertilizers (BF) are potential approaches to reduce the nitrogen input and mitigate soil degradation in the maize soybean relay strip intercropping system (IS). In 2019 and 2020, a two-factor experiment was carried out to examine the effects of BC and BF on soil productivity and yield production in IS. 4 N input levels (8.4, 22.5, 45 kg, and 67.5 kg ha  Population growth widens the supply and demand gap, severely compromising food security . As a re− 1 N for IS might maximize N usage efficiency and achieve the balance between lower N input and higher yield output [The intercropping system, particularly the legume-cereal intercropping system, could provide high yield advantages , highly d output . Meanwhid output  and rhizd output  are two Applying OA in the intensive agriculture system is an age-old but gaining popular approach for boosting yields and preserving soil fertility . CommonlThus, this experiment examines the impacts of BC and BF under various N inputs on soybean biological nitrogen fixation, yield formation, and soil characteristics in the maize soybean relay strip intercropping system. 1) How do organic materials affect the development of soybean nodules and biological nitrogen fixation under various N inputs? 2) Whether the combination of BC or BF with less N input could maintain soil characteristics and soybean yield production.− 1 organic matter, 1.37 g kg − 1 total N, 22.46 g kg − 1, Olsen- phosphorus (Olsen-P), and 138 g kg − 1 exchangeable potassium (K). In this experiment, shade-tolerant soybean ‘Nandou 25’ and compact maize ‘Denghai 605’ were employed.A field experiment was carried out in 2019 and 2020 at Chong’zhou Modern Agricultural Research and Development Station , Sichuan Province, China. The field soil was a light loam with pH 6.99, 32.44 g kg − 1, 16.0 g kg − 1 N, 3.6 g kg − 1 P, 4.0 g kg − 1 K, a pH of 9.90, and an electrical conductivity of 17.9 mS/cm. The fermentation of maize straw with microbial inoculants such as B.licheniformis, B.gelatinous, and S. flavus led to the production of BF. BF has 450 g kg − 1 of organic matter, 2 billion kg − 1 of active bacteria, 28 g kg − 1 N, 53 g kg − 1 P, and 6 g kg − 1 K.The companies Liaoning Golden Future Agricultural Technology Co., Ltd. and Sowish Biotech Co., Ltd., respectively, supplied the BC and BF used in this investigation. Maize straw was pyrolyzed at a high temperature of 400–450 °C and then dried and put through a 2 mm standard sieve to create granular BC of the same size. BC had a carbon content of 749.3 g kg − 1) referred to as N0, N1, N2, and N3 were paired with various organic treatments, including BC (150 kg ha − 1), BF (300 kg ha − 1), and without organic amendments (CK). Furthermore, all the fertilizers were applied as base fertilizers for soybean by the hole-apply method. This experiment adjusted chemical fertilizer application as described in , beginning seed stage (R5), and full maturity stage (R8), three comparable soybean plants from each field were sampled. Retrieve the aboveground portion of the soybean above the cotyledon scar, then separate it into the stem, leaf, pod husk, and grains. After 4 days of drying the samples at 80 °C, weigh the organs. Next, employ an automatic Kjeldahl device to measure the nitrogen concentration using the Kjeldahl analytical method . Nitrogen accumulation in different organs is obtained by multiplying the organ’s dry weight and nitrogen content.At the R2 and R5 stages, soybean roots and nodules in the soil were collected to investigate the nodule number and fresh nodule weight. The sampled areas were 17 cm in width, 80 cm in length, and 20 cm in depth and contained four adjacent soybean plants. Peeled the nodules on the roots and soil, then washed them in tap water. After wiping the water off the nodules with absorbent paper, the number of nodules was counted, and the weight of the fresh nodules was measured. A simultaneous sample of soybean nodules was taken to assess the activity of the nitrogenase enzyme.− 1·g− 1 h− 1). The nitrogen fixation potential  is obtained by multiplying the NA and fresh nodule weight per ha.The nitrogenase (NA) enzyme activity was assessed using the acetylene reduction assay (ARA) technique . The gasAt the R5 and R8 stages, the soil samples were collected using a 2.5 cm diameter auger in a plow layer (0–20 cm). Drill holes in the center of the soybean plants, the soybean rows, and the maize-soybean row to collect soil samples. Soil samples were air-dried, ground, and sieved with 2 mm mesh before being placed in tiny bags.2Cr2O7-H2SO4 oxidation) [The enzyme assay kit evaluated the enzyme activity of soil urease and nitrate reductase . Calculate soil organic material (SOM) by determining the amount of soil organic carbon using the potassium dichromate oxidation method (Kidation) . The totidation) .At the R8 stage, 6 m non-sampling strips were harvested to record the soybean grain yield, and the water content was about 13.5%.The two-factor analysis of variance (ANOVA) and Fisher’s Least Significant Difference (LSD) tests was used to assess the data. Using Origin pro-2022 (Learning version), data analysis and figure drawing were performed .In 2 years, compared with CK, BC and BF increased soybean yield by 5.2–8.5% and 2.6–2.9%, respectively Fig. . And in p < 0.01) increased the nitrogen buildup by 5.1 and 7.7%, respectively. However, the nitrogen distribution analysis revealed that BC and BF reduced the nitrogen distribution ratio in grains compared to CK. Between each treatment, there was no difference in the soybean N accumulation in grains.After the R2 stage, soybean N accumulation quickly increased and peaked at the R8 stage Fig. . While sAt the R5 stage, soybean nodule number and weight were highest in N2 Fig. . The infp < 0.01) stronger soil urease and nitrate reductase activity than CK  higher in BF than in CK, increasing by 13.0, 17.1, 22.20, and 7.4%, respectively. The difference in total N and SOM between BC and BF was negligible. The application of BC and BF did not significantly affect the exchangeable K of the soil.At the R5 and R8 stages, BF had considerably  higher than in BC may help to explain why BF’s yield increase was greater than BC’s.Additionally, this study discovered that BC and BF boosted soybean total N accumulation while decreasing the distribution ratio in grains, indicating that the increase in soybean yield was caused by the increase in total biomass rather than the transfer efficiency of biomass to grains Figs.  and 3. PUnder low N input or low soil fertilizer circumstances, the N from the BNF plays a crucial role in increasing the ability of soybean to generate yield . The finThe previous researchers’ findings were different from this experiment. They have demonstrated that the BF greatly encouraged soybean nodulation and BNF in different legume crops , 28. MeaPrevious research has demonstrated that the soybean has two primary sources of nitrogen: inorganic nitrogen absorbed by the roots and organic nitrogen produced by the BNF of root nodules . The conThe concentrations of total N, SOM, AH-N, Olsen-P, and exchangeable K in soil were enhanced by BC and BF, particularly BF, in this study Table . ChangesFurthermore, the availability of soil nutrients not only affects soybean nodulation but is a crucial factor in determining whether an agricultural system can sustain long-term high-yield output . In orde− 1 N) boosted soybean production favorably and kept soil productivity in IS. Additionally, more research is still needed to determine how BC and BF control soybean nodulation and N accumulation under varied planting patterns and soil fertilizer conditions.Applying BC or BF in conjunction with the proper fertilizers is a great way to increase crop yields quickly while preserving sustainability over the long term in IS. Although both BC and BF suppressed soybean nodulation and BNF, BF significantly increased biomass and N accumulation, leading to the maximum grain yield in N2 under BF. When it comes to enhancing the availability of soil nutrients, including total N, SOM, Olsen-P, and Akali-hydrolysable N, BF outperforms BC. In conclusion, applying BF with N2 (45 kg ha


Ctenopharyngodon idella scales. The optimal hydrolysis conditions were investigated, and chromatography-based isolation was conducted using thermal hysteresis activity (THA) as an index. Molecular dynamic simulation (MDs) was explored to reveal the antifreeze mechanism of the AFPs. The results showed that the optimal hydrolysis conditions were 4000 U/g papain enzyme for 60 °C at pH 5.0 and substrate concentration (1:10) for 3 h, as unveiled by single-factor experiment results. The AFPs documented a THA of 2.7 °C when the Th was 1.3 °C. Hydrophilic peptide, named GCFSC-AFPs, with a THA of 5.09 °C when the Th was 1.1 °C was obtained after a series isolation of gel filtration, ion exchange, and reversed-phase HPLC chromatography. The AFPs had a molecular weight of 1107.54~1554.72 Da with three main peptides in the amino acid sequence of VGPAGPSGPSGPQ, RGSPGERGESGPAGPSG, and VGPAGPSGPSGPQG, respectively. The survival rate of yeast with GCFSC-AFPs reached 84.4% following one week of exposure at −20 °C. MDs indicated that GCFSC-AFPs interfered with the ice-water interaction and thus inhibited the ice crystallization process. Our data suggested that the GCFSC-AFPs were a novel and potential antifreeze agent in the food industry.The purpose of this study is to obtain new antifreeze peptides (AFPs) that are natural, safe, and high activity from  With the development of modernization and industrialization, the low-temperature cold chain technique has become increasingly important for preserving foods. As the most commonly used technology, frozen storage is widely used in many foods. However, the food quality and taste are extremely reduced during freeze–thawing due to ice crystal growth and recrystallization. Although commercial antifreeze agents such as sucrose and sorbitol are used in food such as surimi products and other frozen foods, they contain high sugar and calories, which may bring not only undesired flavor and taste but also excess food energy. In addition, common biological systems used cryoprotectants such as ethylene glycol, glycerol, and dimethyl sulfoxide that are not fit for food products due to the concerns regarding toxicity and changes to the natural taste and texture of the food. Therefore, developing alternative antifreeze peptides (AFPs) is necessary.Due to consumer needs for nutrition and health, foodborne antifreeze glycoproteins and polypeptides (AFPs) have attracted the increasing interest of food scientists worldwide. AFPs can reduce the freezing point of solutions, modify the morphology of ice crystals, inhibit recrystallization, and improve the survival rate of microorganisms in frozen storage. AFPs have been considered natural ice crystal growth inhibitors for use in frozen foods  and have been studied widely ,2. As crScomberomorus niphonius skin was proved to be 1.7 °C by Fu et al. [Because collagen from different species varies significantly in its amino acid composition/sequence and hydroxyproline content, the structure of peptides obtained from different species of fish collagen might be different. Therefore, their antifreeze potential also might vary considerably. For example, Chen et al.  preparedu et al. . Cao et u et al.  isolatedBecause the thermal denaturation temperature of fish collagen is lower than that of mammalian collagens  and fishCtenopharyngodon idella, commonly known as grass carp, is an essential freshwater cultured fish species in China. In 2018, grass carp generated over 5.5 million tons of freshwater food. Incomplete statistics suggested that about 100 kilotons of grass carp scales are produced annually . These by-wastes are excellent raw materials for high-value-added products such as antifreeze peptides. However, reports on antifreeze polypeptides from fish scales are scarce. Therefore, this paper aimed to obtain highly active antifreeze peptides using grass carp scales as raw materials by controlling enzymatic hydrolysis. We believed that our work might help reveal new antifreeze peptides and realize the high utilization of fish scales by-products.Ctenopharyngodon idella scales were purchased from a local supermarket , washed, dried, and stored at room temperature until use. Novozymes  Investment Co., Ltd. was the source of the alcalase, papain, trypsin, neutral protease, and pepsin protease and bovine serum albumin (BSA). Sephadex G-50 and ion exchange (sulfopropyl-Sephadex C-25) were provided by Yuanye Chemical Scientific Co. . All chemicals employed were reagent grade or higher.Ctenopharyngodon idella scales were crushed using a high-speed pulverizer. The powder was mixed with deionized water at a substrate concentration. The mixture was stirred ultrasonically  for 50 min at 45 °C and then decalcified in 1 mol/L of the citric acid solution for 24 h. After cleaning, the mixture was hydrolyzed under the following conditions. Ctenopharyngodon idella scales was mixed with distilled water at a ratio of 1:20 (w/v), and the samples were hydrolyzed with five proteases  under optimum enzymolysis conditions. The optimal conditions for the proteases were as follows: pH 9 and 50 °C for alcalase; pH 7 and 55 °C for papain; pH 8 and 50 °C for trypsin; pH 2 and 45 °C for pepsin; and pH 7 and 55 °C for papain. Microbial cryogenic protection was used as the index for comparison. THA was used as the index for further comparison.The amount of 5 g of pretreated Single-factor tests were based on enzyme dosage, reaction temperature, substrate concentration, and pH. A preliminary study was performed before finding out the data range of the study and optimizing the experiment. Preliminary experiments were conducted to optimize the enzyme and reaction parameters: enzyme dosage , temperature , substrate concentration , and pH . THA was used as the index for comparison.w/w) in distilled water to activate the hair yeast and placement on the shaking table  at 10 °C for 20 min. Then, 0.5% of the yeast slurry was added to the experimental group and mixed in a vortex mixer  to be frozen at −20 °C. The frozen yeast suspension was thawed at 4 °C for 2 h and placed on the shaking bed to score the activity. Post-thawing, the yeast suspension was diluted: 0.1 mL of yeast suspension was absorbed and coated on a YPD plate, incubated for two days at 28 °C at a constant temperature, and the survival rate of yeast was calculated .The approach of Jia et al.  was emplAs elucidated above, yeast suspensions for the test and blank groups were prepared, separated, and kept at −20 °C. The suspensions were frozen for 7 d. After the suspensions were taken out, they were cultured, and the survival rate of the yeast was calculated:(1).h, hold temperature) for 2 min followed by cooling to −25 °C. THA was defined as Th − To with To as the crystallization temperature. This `scheme was repeated at several Th values to gauge the To values.This entailed the use of differential scanning calorimetry (DSC) on a DSC200 F2 instrument  as elucidated earlier ,15 with The THA and ice crystal content (φ) were computed according to Equations (2) and (3):ΔHr is the crystallization enthalpy, and ΔHm is the melting enthalpy (both in J/g).The hydrolysate was first fractionated using a Sephadex G-50 gel filtration column (2.6 cm diameter × 100 cm length) followed by elution (5 mL/tube) with PBS water at pH 7.0 at a flow rate of 0.8 mL/min. The absorbance at 225 nm was scored, followed by THA analyses. Fractions that demonstrated higher antifreeze activity were pooled, followed by lyophilization for the ensuing step.The “best performers” were loaded in an SP-Sephadex C-25 strong cation exchange column (1.6 cm diameter × 20 cm length) and equilibrated with 20 mM HAc-NaAc buffer (pH 5.0), followed by resolution employing 0–1.4 M NaCl in this buffer at 0.5 mL/min and probing of the fractions at 225 nm. The antifreeze activity of the pooled unadsorbed and adsorbed portions was scrutinized with the pooling and lyophilization of those that demonstrated high activity.After ion-exchange chromatography, fractions with the highest THA activity were loaded onto a C18 column . Following column equilibration employing 4% acetonitrile for 4 min post-sample injection, elution ensued for 15 min with a linear gradient of 4–100% acetonitrile solution and 100% acetonitrile solution for 5 min. Those that demonstrated significant absorbance were scored. The scrutiny of THA activity entailed the same conditions listed here , with those fractions that demonstrated high absorbance scored.m/z; ESI source temperature, 100 °C; desolvation temperature, 250 °C; capillary voltage, 3.8 kV; photomultiplier tube voltage, 1600 V. Control was exerted at: scan range, 100 to 2000 m/z, full scan resolution, 70,000; source temperature, 100 °C; MS/MS scan resolution, 17,500.This entailed employing an Ultimate 3000 HPLC-MS/MS system  to probe the fractions that demonstrated evident antifreeze activity. The scheme was elution from the column into the mass spectrometer, followed by peptide sequencing retrieval and comparison analyses. The raw mass spectrometry test files were retrieved by the Uniport database with Mascot software for retrieval and comparison. LC analysis entailed loading 1 µL of the sample in an Accucore 18-MS  column in a UPLC system and PDA detector  with a linear gradient of 1–30% acetonitrile solution for 45 min for elution, 30–90% acetonitrile solution for further 5 min for elution, 90% acetonitrile solution for additional 2 min for elution, 90–1% acetonitrile solution for further 5 min for elution. MS analysis was performed using a Q Exactive QTF MS system (Thermo Fisher). The parameters for the analysis were as follows: reflector, positive; mass range, 50–2000 The 3D structure of AFPs can be constructed based on homology modeling . First, After that, the best model constructed was evaluated through the Ramachandran plot , which iwww.ks.uiuc.edu and is shown in Figure 7. Then, the peptide was placed in the center of the box and solvated with water molecules. The initialized system is shown in Figure 7. After that, the system was minimized using the steepest descent algorithm, and the periodic boundary conditions were used for all systems. Prior to the production simulations, the systems were equilibrated with the V-rescale temperature thermostat and Parrinello-Rahamn pressure thermostat. The temperature was maintained at 260 K, and the pressure was maintained at 1 bar. Then, the production simulations were carried out at least 200 ns using the GROMACS 5.1.4 software package, and snapshots were saved every 2 ps. The simulation detail was analyzed through the built-in module, and snapshots were visualized using VMD 1.9.2 software) [First, the ice model was obtained from L, USA)) .p < 0.05. Data were expressed as the mean ± SD.Statistical significance was determined by one-way analysis of variance with Dunnett’s post hoc test using SPSS 24.0 software . Significance was set at p < 0.05) and neutral protease (35.33%) (p < 0.05), but close to those for pepsin (52.10%) (p > 0.05) and alkaline (54.47%) (p > 0.05). Many previous studies showed that alkaline generally had better enzymatic hydrolysis effects. In order to accurately assess their antifreeze activity, the thermal hysteresis activity of peptides obtained with papain and alkaline was analyzed using DSC. As shown in h) was 0.8 °C, the THA of peptides obtained with papain was 1.8 °C (p < 0.05), which was higher than that of alkaline protease hydrolysate with THA of 0.9 °C at Th of 1.0 °C. Our result was consistent with the research results in [The antifreeze activity of antifreeze proteins or peptides depends primarily on the domain of the local specific peptide chain, rather than the holoprotein ,26. EnzyBy controlling the cutting conditions of endonuclease protease, the active polypeptide with specific peptide chain length and structure composition could be produced, and the peptide with potent antifreeze activity could thereby be obtained efficiently. Therefore, we optimized other conditions, including the enzyme dosage, enzymolysis temperature, pH, and substrate concentration. Thermal hysteresis is defined as the difference between the melting point and the nonequilibrium freezing point of a solution, and it is used as an indicator of AFPs activity. At present, THA is commonly measured by the direct microscopic observation of crystal growth . In thisp < 0.05). The THA of hydrolysate with papain concentration of 4000 U/g was 2.70 °C, which was higher than other concentrations. However, a further increase in papain concentration from 4000 U/g to 4500 U/g caused an evident lowering of THA (0.80 °C) (p < 0.05).As shown in The effect of temperature on THA was also measured. As shown in The THA of the hydrolysates strongly depended on papain concentration and pH . The valIncreasing the substrate level from 1:10 to 1:20 induced a minor alteration in THA . The THATaken together, the optimized conditions for AFPs preparation were as follows: papain level of 4000 U/g, temperature, 60 °C, solid–liquid ratio 1:10, pH 5.0, 3 h. Under the above optimum conditions, the THA of the hydrolysates could reach 2.7 °C.p < 0.05). In contrast, as the temperature fell, the re-crystallization of partially melted F1 was only delayed slightly. Therefore, the F2 fraction was pooled, lyophilized, and then loaded onto an SP-Sephadex C-25 strong cation exchange chromatographer for further fractioning. The elution profile is shown in p < 0.05). The amount of the S3 fraction was minimal, so we did not collect it.The hydrolysates were first separated on a Sephadex G-50 gel filtration column. Two fractions  were obtained A. As shop < 0.05), significantly higher than the THA of the other fractions  was observed for GCAFP, with an increase from −0.8 to −1.4 °C as the Th increased from 1.0 °C to 1.3 °C (o) was observed for F2, with an increase from 0 to −1.72 °C as the Th increased from 0.80 to 1.0 °C (o) was observed for S2, with an increase from −1.89 to −5.25 °C as the Th increased from −1.0 to −0.7 °C (o) was observed for P7, with an increase from −1.22 to −3.99 °C as the Th increased from 0.70 to 1.1 °C  from the  5.28 °C , but hig 5.28 °C  as well  5.28 °C . Accordi 5.28 °C . Thus, PThe peptide sequence of GCFSC-AFPs was investigated by Q EXACTIVE LC-MS/MS. As shown in The peptides sequences of GCFSC-AFPs demonstrated Gly-x1-x2 tripeptide repeats (x1 predominantly proline) with no long-chain aliphatic or aromatic amino acids, which was in good agreement with collagen’s structural properties. AFPs generally have the structural characteristics of a tripeptide repeat sequence  or Pro-Ala-Gly-Tyr as well as being rich in Ala .Scomberomorus niphonius skin (THA1.7 °C), Tenebrio Molicor (THA3.5 °C).The survival rate of yeast with GCFSC-AFPs reached 84.4% following one week of exposure at −20 ℃  was higher than that of other antifreeze polypeptides from fish such as salmon skin (THA0.53 °C), at −20 ℃ , which wThis may be due to the larger number of repeated sequences of -Gly-Pro-X tripeptide and the higher contents of characteristic amino acids Gly and Pro than the above antifreeze peptides.Previous studies revealed that molecular dynamic simulation (MDs) has great advantages in revealing the mechanism of the antifreeze activity of peptides by exploring the process of ice growth  and the p < 0.05). Furthermore, the GCFSC-AFPs predominantly formed hydrogen bonds with water rather than ice along with the simulation (The snapshots are shown in mulation F. These This work documented the preparation and isolation of antifreeze peptides with the ice-water interaction and the subsequent inhibition of the ice crystallization process.


In addition, active compounds  had good development prospects, fine affinity, and stable conformations with the target protein. In summary, this study suggested that HSBDF and its key active components in plasma  can decrease levels of pro-inflammatory factors (IL-6 and TNF-α), decrease expression of MDA, increase expression of T-SOD, and decrease cell apoptosis in an inflammatory environment. These data suggest that HSBDF has significant effect on anti-inflammation and anti-oxidative stress and also can decrease cell apoptosis in treating ALI. These findings provided an important strategy for developing new agents and facilitated clinical use of HSBDF against ALI.The Huashi Baidu Formula (HSBDF), a key Chinese medical drug, has a remarkable clinical efficacy in treating acute lung injury (ALI), and it has been officially approved by the National Medical Products Administration of China for drug clinical trials. Nevertheless, the regulated mechanisms of HSBDF and its active compounds in plasma against ALI were rarely studied. Based on these considerations, the key anti-inflammatory compounds of HSBDF were screened by molecular docking and binding free energy. The key compounds were further identified in plasma by LC/MS. Network pharmacology was employed to identify the potential regulatory mechanism of the key compounds in plasma. Next, the network pharmacological prediction was validated by a series of experimental assays, including CCK-8, EdU staining, test of TNF-α, IL-6, MDA, and T-SOD, and flow cytometry, to identify active compounds. Molecular dynamic simulation and binding interaction patterns were used to evaluate the stability and affinity between active compounds and target. Finally, the active compounds were subjected to predict pharmacokinetic properties. Molecular docking revealed that HSBDF had potential effects of inhibiting inflammation by acting on IL-6R and TNF-α. Piceatannol, emodin, aloe-emodin, rhein, physcion, luteolin, and quercetin were key compounds that may ameliorate ALI, and among which, there were five compounds  in plasma. Network pharmacology results suggested that five key compounds in plasma likely inhibited ALI by regulating inflammation and oxidative damage. Test performed  Acute lung injury (ALI), an early stage of acute respiratory distress syndrome (ARDS) , can be Inflammatory cytokine storm is a characteristic of ALI. Previous studies indicated that interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) were key cytokines involved in the pathological process of ALI . IL-6 anin vitro approaches, which provide the basic data for further scientific research and clinical use of HSBDF in treating ALI.Molecular docking and network pharmacology are widely used in researching the material basis and mechanism of TCM in the prevention and treatment of disease . HoweverEphedra sinica , Agastache rugosus , Gypsum Fibrosum , Semen armeniacae amarum , Rhizoma Pinelliae Preparata , Magnolia officinalis , Atractylodes lancea , Fructus tsaoko , Poria cocos , Astragalus membranaceus , Radix Paeoniae Rubra , Lepidium apetalum , Rheum officinale , and Glycyrrhiza uralensis  were obtained from The Affiliated Hospital of Gansu University of Chinese Medicine  at an accurate prescription dose, and their equality control adhered to the specifications and test procedures as described in the Pharmacopoeia of the People’s Republic of China (2020). HSBDF was decocted with water  for 60 min. The aqueous solutions were obtained by merging, filtrating, and concentrating to 1.23 g/ml. The extracts were kept at 4°C for preparation of a lyophilized powder for further oral administration to rats and preparation of lyophilized powder.Methanol and acetonitrile (HPLC pure grade) were purchased from MREDA , and formic acid of the same grade was provided by Thermo Fisher . Ultrapure water was obtained from Wahaha  and used throughout the study. Quercetin, luteolin, piceatannol, emodin, rhein, aloe-emodin, and physcion were all purchased from Baoji Herbest Bio-Tech Co., Ltd. . The purity of all reference standards was more than 98%. Other reagents of analytical grade were obtained from commercial sources.LPS, cell counting kit-8, and Click-iT EdU-488 cell proliferation kit were purchased from Solarbio , Yeasen , and Servicebio , respectively. DME/F-12, 1,640, and PBS were obtained from HyClone . Annexin V−FITC/PI apoptosis detection kit was provided by Meilunbio . MDA (20211202) and T-SOD (20211201) were purchased from Nanjing Jiancheng Bioengineering Institute . ELISA kits including TNF-α (2111H27) and IL-6 (2111H35) were purchased from Jiangsu Feiya Biotechnology Co., Ltd. .https://www.tcmspw.com/tcmsp.php) (http://119.3.41.228: 8000/tcmid/) (https://pubmed.ncbi.nlm.nih.gov/). A total of 1,613 compounds were retrieved for 13 herbs, among which there were E. sinica (363), S. armeniacae amarum (113), L. apetalum (68), R. paeoniae rubra (119), A. rugosus (94), M. officinalis (139), A. lancea (49), F. tsaoko (59), Astragalus membranaceus (87), Glycyrrhiza uralensis (280), R. officinale (92), P. cocos (34), and R. Pinelliae Preparata (116). It was worth noting that Gypsum Fibrosum is a mineral drug, and its main composition was CaSO4•2H2O. Therefore, its mechanism of action was not included in this study.The compounds were collected from the traditional Chinese medicine systems pharmacology   database/tcmid/) . Meanwhihttps://www.rcsb.org/).The 3D structures of IL-6R (PDB ID: 1P9M) , TNF-α , PIK3R1 3D structures of protein in 2.2.2 were preprocessed by Protein Preparation Wizard. The compounds in 2.2.1 were prepared by the LigPrep module . A totalThe binding free energy was used to evaluate the affinity between the ligand and receptor. Lower binding free energy indicates a higher affinity or catalytic activity . In this+ and Cl−. The molecular system was solvated with crystallographic water (TIP3P) molecules under orthorhombic periodic boundary conditions for a 10 Å buffer region. The system charges were neutralized by adding Na+ and Cl−. The OPLS_2005 force field was used for energy calculation. After this, an ensemble (NPT) of Nose–Hoover thermostat and barostat was applied to maintain the constant temperature (300°K) and pressure (1 bar) of the systems, respectively. The complex structural dynamic simulation for the best complex was carried out with an NPT ensemble for 50°ns, and the trajectory was set at an interval of 10°ps. The RMSD (root mean square distance) was used to compare the prediction error in disparate models of specific data sets and protein–ligand contacts with the timeline. In addition, the 3D binding interaction pattern of active compounds with protein was visualized by PyMOL.The initial structures of the protein complex with active compounds  were obtained from molecular docking. To further determine the stability of rhein and quercetin with IL-6R and rhein and emodin with the TNF-α complex in a simulated physiological solvent system, we used the Desmond module of Schrödinger to carry out molecular dynamic simulation. The molecular system was solvated with crystallographic water (TIP3P) for a 10-Å buffer region under orthorhombic periodic boundary conditions. The system charges were neutralized by adding NaThe parameters of absorption, distribution, metabolism, excretion, and toxicity (ADMET) of emodin, rhein, and quercetin were calculated by using the ProTox-II data platform. In addition, active compounds that conformed to Lipinski’s rule were predicted by the QikProp module of Schrödinger.ad libitum in a house with a 12 h light/dark cycle  for 10 days to adapt to the experimental environment. The experimental protocol, approved by the Committee for Ethics in the Laboratory Animal Center of Gansu University of Chinese Medicine (number: 2021-182), was conducted in strict accordance with the ethical principles in animal research.A total of six male Sprague-Dawley rats  were purchased from the Laboratory Animal Center of Gansu University of Chinese Medicine  with license number: SCXK (gan) 2020-0001. All animals were housed in the Laboratory Animal Center of Gansu University of Chinese Medicine [animal license number: SCXK (gan) 2020-0009]. The rats were fed standard rodent chow and water The triple quadrupole LC/MS system consisted of an ExionLC  and a SCIEX Triple Quad 5,500+  equipped with an ESI source. The chromatographic separation was achieved on a Kinetex C18 column . The water  acetonitrile, and methanol  system was used as the mobile phase. Analysis was achieved by gradient elution at a flow rate of 0.3 ml/min. The gradient elution program was: 0–2 min, 10% B; 2–5 min, 10%–40% B; 5–15 min, 40%–50% B; 15–30 min, 50%–55% B; 30–50 min, 55%–66% B; 50–63 min, 66%–80% B; and 63–73 min, 80%–100% B. The column and auto-sampler tray temperatures were kept at 30°C and 4°C, respectively. The injection volume was 2 μl. All analytes were quantified using multiple reaction monitoring (MRM) detection in the negative ion mode. The parameters of MRM are shown in In this study, standard working solutions were made up of mixed standard and single standard. The single standard was made up of quercetin (100 ng/ml). Piceatannol , emodin (90 ng/ml), rhein , aloe-emodin , physcion , and luteolin (300 ng/ml) were taken 10 µl each to form the mixed standard. Standard working solutions (100 μl) were added to blank plasma (100 μl) to form the calibration standards and stored at 4°C until use.HSBDF  was filtered, concentrated, dried, and dissolved in 50 ml water  to collect HSBDF extracts (stored at 4°C). Also, the HSBDF extracts were filtered through a 0.22-μm syringe filter when used.In previous studies, rats fasted overnight with free access to water. The plasma samples (∼0.2 ml) were collected into heparinized microcentrifuge tubes through the caudal vein at 2 h after a single oral administration of HSBDF water decocted. The plasmas were centrifuged at 4,000 rpm for 10 min immediately after collection to obtain plasma. The plasma samples were stored at −80°C until analysis.The calibration standards and plasma samples were treated with the protein precipitation method using methanol. The procedure was as follows: 100 µl samples were mixed with 800 µl methanol, then vortexed for 5 min, and centrifuged at 13,000 rpm for 10 min. The supernatant was transferred into a clean tube and evaporated with a stream of nitrogen. The residue was dissolved with methanol and was filtered using a 0.22-μm syringe filter to test.https://sea.bkslab.org/) (https://string-db.org/) (http://www.bioinformatics.com.cn/) was used to visualize the key targets (top 20 targets).The similarity ensemble approach (SEA) (ab.org/)  was useddb.org/) , and thehttps://david.ncifcrf.gov/) databases. A total of 151 targets of key compounds in plasma were imported using the DAVID database. The select identifier was set to official gene, list type was set to gene list, species was set to “Homo sapiens,” and the threshold was set at p < 0.05. Bioinformatics and GOChord of R language were used to construct the GO and pathway-target diagram, respectively.GO and KEGG enrichment was carried out using DAVID  and maintained at 37°C under 5% CO2 in DME/F-12 and 1,640 medium supplemented with 10% fetal bovine serum, respectively.HSBDF condensed extract of 1.23 g/ml was lyophilized using a freeze dryer . Finally, 15.38 g of lyophilized powder was obtained . The dry powder was stored in an 80°C refrigerator until use.3 cells/well in 200 μl of culture media and grown for 4 h. Second, they were treated with different concentrations of LPS, quercetin, luteolin, emodin, rhein, aloe-emodin, and HSBDF. Subsequently, 10 μl of CCK-8 solution was added to each well, and the plates were incubated for 1 h. The optical density of each well was determined at 450 nm using a microplate reader .Cell viability of A549 and MLE-12 cells were measured with the CCK-8 assay after compound intervention. The cells were seeded in 96-well plates at a density of 5 × 10EdU proliferation kit was employed for the determination of DNA synthetic capacity in proliferating cells. A549 cells were cultured in 96-well plates and grown for 4 h. After being treated for 24 h with compounds, the cells were strictly conducted following the instructions of the kit. Live cell imaging system  and ImageJ software were employed for calculating the number of EdU-positive cells (green cells). The percentage of EdU-positive cells to total Hoechst 33342-positive cells (blue cells) was represented as the EdU-positive rate.The A549 cells culture medium was gathered and centrifuged  at 4°C. The supernatant liquid was taken out for detection of MDA, T-SOD, TNF-α, and IL-6. Enzyme-Linked Immunosorbent Assay (ELISA) (TNF-α and IL-6), MDA, and T-SOD steps were followed as per the instructions of the reagent kit.5 cells/well) for 4 h before treatment. After adding LPS, quercetin, emodin, rhein, and HSBDF, the cells were incubated for 24 h. Next, the cells were gathered and stained using the annexin V−FITC/PI apoptosis detection kit following the manufacturer’s instructions. The resulting fluorescence signal was detected by flow cytometry .FITC-conjugated annexin V was used to detect apoptosis. A549 cells were cultured in six-well plates  of at least three independent experiments. The statistical differences between the two groups were compared by Student’s t-test. To explore the anti-inflammatory effect of HSBDF, 1,613 compounds were docked with IL-6R and TNF-α by Schrődinger. The compound with a docking score of ≤ −5 Kcal/mol was known as the potentially effective compound . It can Glycyrrhiza uralensis, Ephedra sinica, and Rheum officinale were the top three docking herbs with two targets, respectively. Previous studies also demonstrated that Glycyrrhiza polysaccharide (50 mg/kg) had a certain therapeutic effect on lung inflammation and oxidative damage in COPD mice  < 5, and rotor ≤10) and docking score ≤−5 Kcal/mol, to analyze further. Based on the Pharmacopoeia of the People’s Republic of China (2020) and the literature of every herb’s main components, seven key compounds were selected from the top 50 compounds with high docking scores . These cGlycyrrhiza uralensis, E. sinica, and R. officinale, which further provides support that Glycyrrhiza uralensis, E. sinica, and R. officinale were key herbs to treat ALI.The results indicated that piceatannol, emodin, aloe-emodin, rhein, physcion, luteolin, and quercetin had potential effects on inhibiting inflammation. Therefore, these compounds were considered key compounds to explore further. Also, we found that these compounds were mainly distributed in In order to further analyze whether the seven key compounds  were present in plasma, LC/MS was used. The results of preliminary experiments showed that the number of chromatographic peaks and intensity of peaks were the most strongly detected in 2 h plasma after HSBDF administration. Therefore, 2 h was selected to collect plasma after drug administration. Ion chromatograms of standards (S1) of key compounds, calibration standards (S2), HSBDF extracts (S3), and plasma sample (S4) are displayed in Therefore, LC/MS analysis showed that there were five key compounds in plasma of HSBDF, including emodin, aloe-emodin, rhein, luteolin, and quercetin. The structures of these compounds are shown in It was well known that network pharmacology contributes to exploring the potential mechanism of TCM in prevention and treatment of disease . TherefoIn order to further validate the prediction of network pharmacology, five nodes, namely, TP53, SRC, PIK3R1, PIK3CA, and AKT1 with the highest degree were taken as examples to be analyzed. Their crystal structure was subjected and docked with corresponding compounds. Finally, constituent with docking score ≤ −5  was selected to analyze. The total proportion of TP53 (5/5), SRC (5/5), PIK3R1 (5/5), PIK3CA (5/5), and AKT1 (4/5) was 96%, and the results are shown in G. baccata extract can upregulate AKT1 gene expressions to protect the cells against oxidative damage and inflammation , and the top five of GO enrichment were visualized, as shown in To explore the regulatory mechanisms of key compounds in plasma, a total of 267 terms were enriched (p < 0.05), and the top 20 targets and top 20 pathways are shown in Moreover, we had used 151 targets to enrich 107 pathways  in 24 h, indicating that 6.25 μg/ml of LPS is deleterious to the epithelial cell. LPS of 12.5, 25, 50, or 100 μg/ml decreased cell viability more prominently than 6.25 μg/ml LPS did, which demonstrated that LPS induced cell viability loss in a dose-dependent manner and that the lowest effective injury dose of 24 h LPS treatment was 6.25 μg/ml , emodin (3.125 μM), rhein (1.5625 μM), and HSBDF (0.03125 mg/ml) .Simultaneously, the anti-oxidative stress of HSBDF and active compounds was further researched in an inflammatory environment. We found that the oxidative stress injury was weakened after treatment of HSBDF and active compounds. The SOD was enhanced significantly by active compounds, while the MDA was decreased .These results further verify that network pharmacology results indicate that emodin, rhein, and quercetin have the function of anti-inflammation and act against oxidative damage.LPS has been known to cause alveolar epithelial cell apoptosis that characterizes ALI . TherefoTo further confirm the molecular docking insights and analyze the conformation stability of active compounds and target proteins, molecular dynamic simulations were conducted for rhein and quercetin with IL-6R and rhein and emodin with the TNF-α complex in 50 ns by the Desmond module. The root mean square deviation (RMSD) plot in The interactions of active compounds and target proteins were further elaborated based on the hydrogen bonds, hydrophobic, ionic, and water bridges during 50-ns simulation. As shown in These results suggested that binding of ligand molecules  stabilizes the protein structure without noticeable structural changes in native conformation. That is the conformation of the complex was stable, and the residues are derived from stable complexes.The ADMET property prediction is fundamental for the selection of the most promising compounds for further development. The active compounds  were subjected to predictions of pharmacokinetic properties using ProTox-II and QikProp. As shown in E. sinica, S. armeniacae amarum, and Glycyrrhiza uralensis) and HXZQP  was recommended to treat ALI at the later stages of COVID-19 by the NHC. Molecular docking results showed that the total number of compounds with potential inhibitory activities of MXSGD (120) against IL-6R and TNF-α was higher than that of HXZQP (110). The results indicated that MXSGD was more outstanding in anti-inflammation than HXZQP, which up to the TCM theory that MXSGD can regulate lung mechanisms such as diffuse the lung qi. Also, previous studies confirmed that MXSGD played a role in suppressing cytokine storm and immune regulation and protecting the pulmonary alveolar–capillary barrier  have significant effects on anti-inflammation, anti-oxidative stress, and decreasing cell apoptosis in treating ALI. Moreover, quercetin, emodin, and rhein, key active components in plasma, had a stable affinity with IL-6R and TNF-α, which are worthy of further development and utilization for ALI.In summary, this study made good use of chemical bioinformatics analysis, including molecular docking, LC/MS, network pharmacology, test


Aspergillus niger KMS on an optimized culture medium. The concentration of the six medium components in shake flasks was optimized by the Box and Wilson factor gradient method. Naringinase’s substrate, naringin, powdered albedo, flavedo, and red grapefruit segment membranes were used to stimulate naringinase biosynthesis. Rhamnose was chosen as the carbon source, while the nitrogen source was yeast extract and sodium nitrate. Naringinase biosynthesis was most favorable in the culture medium with the following composition (g 100 mL): 3.332—NaNO3; 3.427—yeast extract; 0.184—KH2PO4; 0.855—red grapefruit albedo; 0.168—naringin; 2.789—rhamnose. The obtained Aspergillus niger KMS culture fluid was concentrated, thereby precipitating the protein. As a result, a naringinase preparation with high activity, equal to 816 µmol × min−1 × g−1, was obtained.This study aimed to develop a method of naringinase biosynthesis by  Naringinase is an enzyme with the dual activity of α-L-rhamnosidase EC 3.2.1.40) and β-D-glucosidase (EC 3.2.1.21). Naringinase is used in the deglycosylation of compounds with high application potential in the food and pharmaceutical industries .40 and β,6,7. NarPenicillium [Rhizopus [Staphylococcus [Streptomyces [Bacillus [Aspergillus [Aspergillus can synthesize naringinase: A. niger [A. oryzae [A. aculetaus [A. brasiliensis [A. sojae [A. terreus [A. foetidus [A. flavus [A. tubingensis [Ferreira et al.  observedicillium ,12,13,14Rhizopus , Staphyllococcus , Streptoptomyces , and BacBacillus ; howeverergillus ,24,25,26A. niger ,33,34,35. oryzae ,20,33, Aculetaus , A. brasiliensis , A. sojaA. sojae , A. terr terreus , A. foet. flavus ,40,41, aingensis .A. niger strains [A. niger is a microorganism classified as GRAS . According to the Food and Drug Administration (FDA), it also belongs to the category of microorganisms safe for food and medical uses. It is essential due to the potential use of naringinase preparation in the food industry. A. niger is also used to produce other enzymes, food additives, and traditional fermented foods (fermented Chinese soybean paste) [A. niger species can be considered the most important source in naringinase preparation. The biosynthesis of naringinase by A. niger is influenced by many factors, e.g., the strain molds, the culture medium’s composition, and the process’s conditions. One of the most important factors influencing the culture of Aspergillus molds is the composition of the substrate. The presence of an inducer, carbon source, nitrogen source, and the content of mineral salts are the main factors determining the growth and activity of microorganisms [Most often, the authors researched naringinase biosynthesis in submerged cultivation of  strains ,30,31,43n paste) . The effn paste) ,44. For rganisms ,45. Narirganisms ,46 and trganisms ,27. Diffrganisms ,48,49. A. niger on a medium containing dried orange peel powder. Furthermore, Borkar et al. [A. niger van Tieghem MTCC 2425 submerged culture on a medium containing orange solid waste. Chen et al. [A. oryzae JMU316, and, as the only carbon source, they used pomelo peel powder. Puri et al. [Staphylococcus xylosus MAK2; for this purpose, they used dried waste from orange processing as a culture medium component. In order to produce naringinase, molds of the Aspergillus genus  were also cultured on a solid medium containing orange and grapefruit peels [A. niger, P. nalgiovense, A. flavus, and A. terreus on a solid medium containing orange peels [A. niger in a medium containing powder from dried orange peels as a carbon source. It can be assumed that other citrus peels can also be used as a carbon source for naringinase production. Puri et al. [A. niger KMS on a medium containing natural stimulants for this enzyme’s production, which were derived from waste or by-products from grapefruit juice—i.e., albedo skins and citrus seed nests.In recent years, natural particles of citrus fruit have been used as a rich source of naringin and thus as inducers of naringinase biosynthesis ,33,44,50n et al.  worked oi et al.  obtainedge peels . The typge peels ,32,34,43ge peels ,40,41,43ge peels ,29,37, sge peels ,29. Natuge peels . In 2012ge peels  patentedi et al. , for thei et al. ,52, but The goal of this work required determining the influence of temperature and optimizing the culture medium composition to maximize naringinase production.A. niger KMS on a medium containing rhamnose, glucose, sucrose, starch, maltose, glycerol, or molasses  was present in the culture medium. The analysis of variance did not show a statistically significant difference (significance level α = 0.05) between the naringinase activity obtained in the cultures on glucose and rhamnose. Despite this, rhamnose was selected for further research. It was taken into account because of the results of other authors who believe that A. niger naringinase biosynthesis is inhibited by the presence of glucose in the medium [A. niger KMS strain could result from the influence of metal ions on this process, especially calcium and magnesium. Gonzalez-Vazquez et al. [A. niger ATCC1015 strain.The data analysis presented in e medium ,29,34. Tz et al.  found th−1 [A. niger MTCC 1344 culture medium. Mateles et al. [A. niger BCC 25166 was obtained by Thammawat and Pongtanya [Rhamnose, which was selected for further research as a carbon source, was used by other authors most often at a concentration of 3.5–10 g L−1 ,32,34,44−1  by addins et al.  also rep3), ammonium sulfate ((NH4)2SO4), and ammonium dihydrogen phosphate (NH4H2PO4) was investigated. Among the organic compounds, the following were tested: amino acid, enriched broth, dry meat broth, plain broth, and yeast extract. The concentration of each nitrogen source was set at a certain level to ensure the same amount of total nitrogen, which was equal to 1.0 g L−1. A comparison of the final naringinase activities in the culture fluid obtained in submerged cultures containing the tested carbon sources is shown in Among inorganic compounds, the influence of sodium nitrate (NaNO−1 × mL−1. The highest activity of naringinase in the post-culture fluid was obtained on the medium with sodium nitrate (V).It was found  that yeaA. niger BCC 25166 with the highest activity by using a culture medium containing NaNO3 as the nitrogen source. Sheheta et al. [A. niger strain can be increased by supplementing the medium in solid culture with NaNO3 and yeast extract.Compared to organic nitrogen sources, inorganic nitrogen sources made it possible to obtain naringinase with relatively low activity. These results are in line with the results obtained by Chen et al. . It has a et al.  found thAspergillus genus [Aspergillus molds, Chen et al. [3 is a component of the Czapek substrate, and is much cheaper than yeast extract, the optimized composition of the substrate took into account the presence of both yeast extract and NaNO3.Furthermore, other authors noticed that the addition of yeast extract, to the greatest extent, stimulates the process of naringinase production by the us genus ,23,27,41n et al.  found thA. niger KMS were selected. The influence of naringin and dried, powdered citrus peel on naringinase production was investigated. Among citrus fruit particles, albedo, flavedo, and segmental membranes: red grapefruit, white grapefruit, orange, and pomelo were investigated as naringinase inducers. The effect of the biosynthesis stimulator on the activity of naringinase in the post-culture fluid in batch cultures of A. niger KMS is shown in The components of the culture medium stimulating the process of naringinase biosynthesis by A. niger KMS. Of all the components tested, the most effective inducer of naringinase biosynthesis by A. niger KMS was powdered red grapefruit albedo (AFRG). We used a natural source of naringin in the form of red grapefruit albedo as an inducer of naringinase biosynthesis, which allowed us to obtain an enzyme with the activity of 0.186 µmol × min−1 × mL−1. A slightly lower activity of naringinase was also obtained when the albedo of white grapefruit (0.166 µmol × min−1 × mL−1) or orange (0.156 µmol × min−1 × mL−1) was present in the culture medium.The data analysis presented in The team of Mendoza-Cal  comparedBased on the presented results, it was found that the composition of the culture medium should include albedo, flavedo, and segmental membranes of red grapefruit due to the highest naringinase activity in the post-culture fluid. In further studies, the composition of the Czapek medium was also supplemented with naringin as a distinguishing factor for inducing naringinase synthesis.A. terreus. As Bram et al. [Many authors believe naringin is the best stimulator of naringinase biosynthesis ,34. The m et al.  reportedm et al. ,20,33.A. oryzae JMU316 culture. The dried waste from pomelo juice production contains naringin, rhamnose, and other nutrients such as amino acids, proteins, and vitamins, which contribute to the high activity of naringinase in the culture medium [Pomelo peel powder stimulated the production of naringinase in e medium .The production of bacterial naringinase was increased by adding dried orange waste, i.e., peel, membranes, and vesicles, that were produced in the citrus processing industry to the substrate ,20. PuriA. niger KMS cultures grown in shaker flasks significantly impacts the naringinase activity in the post-culture fluid. The highest activity of the tested enzyme, amounting to 1.307 µmol × min−1 × mL−1, was obtained at the temperature of 30 °C.A comparison of the final naringinase activities in the post-culture fluid obtained in submerged cultures conducted at different temperatures is shown in A. niger KMS strain was 30 °C, which is similar to those reported in the studies of other authors. Puri et al. [A. niger MTCC1433 naringinase activity, while the enzyme that had a higher temperature (37 °C) activity was significantly reduced. Kumar et al. [A. niger VB07. Borkar et al. [A. niger van Tieghem MTCC 2425 at 29.8 °C, pH 4.7, and an inducer concentration of 14.9 g L−1 would provide the highest naringinase activity.The optimal temperature for naringinase biosynthesis by the i et al.  reportedr et al.  reportedr et al.  calculatA. niger KMS strain might have five components of the medium: NaNO3, yeast extract, rhamnose, red grapefruit peel, segment membranes, and naringin. Potassium dihydrogen phosphate (KH2PO4) was used to assess its influence on the course of naringinase biosynthesis.The results of preliminary studies showed that the most significant influence on the synthesis of naringinase by the 3 (×1), yeast extract (×2), KH2PO4 (×3), albedo, flavedo, and red grapefruit segment membranes (×4), naringin (×5), and rhamnose (×6)  .−1 × mL−1. The highest activity of naringinase, equal to 0.9 µmol × min−1 × mL−3, was obtained in the cultivation variant carried out in a culture medium with the following composition (in g·100 mL−1): 1.28—NaNO3; 2.5—yeast extract; 0.257—KH2PO4; 0.8—red grapefruit peel; 0.16—naringin; 0.5—rhamnose.As a result of the experiments based on the Box and Wilson factor plan, the naringinase activity in the post-culture fluid obtained in the individual cultivation variants varied from 0.062 to 0.9 µmol × min−1 were assumed. The calculated increments for the less significant factors were doubled. Due to the poor solubility of naringin in aqueous solutions, an increase of 10 mg 100 mL−1 was assumed.The data from factor optimization were used to determine the values of the regression coefficients, which define the linear influence of the tested concentrations of the medium components on the culture results. Based on the obtained slope regression coefficients for individual factors, the jump sizes in the gradient experiment were determined for each. For the main factors, increases in the value of 0.5 g 100 mLSubsequently, the factorial experiment was performed again with new levels of all six optimized substrate components. This procedure continued until the regression function’s local maximum was achieved (considering the limitations of the examined factors).−1 × mL−1. The highest activity of naringinase, equal to 1.965 µmol × min−1 × mL−1, was obtained in the culture medium with the following composition (in g·100 mL−1): 3.28—NaNO3; 3.35—yeast extract; 0.182—KH2PO4; 0.80—red grapefruit albedo; 0.17—naringin; 2.75—rhamnose. This variant was defined as a new central point used to develop a five-valued composition plan with seven repetitions in the central point  was obtained as a result of cultivation in a medium with the following composition (in g·100 mL−1): 3.78—NaNO3; 3.62—yeast extract; 0.187—KH2PO4; 0.86—albedo, the flavedo, and red grapefruit segmental membranes; 0.185—naringin; 3.25—rhamnose.The data analysis presented in The results obtained in this stage of the study were subjected to the ANOVA statistical analysis of variance to determine the significant effects and the values of the coefficients of the quadratic regression model using the response surface of the determined model as described. After the reduction of the most significant factors, the test results presented in −1) of the following factors: 3.3317—NaNO3; 3.4274—yeast extract; 0.1841—KH2PO4; 0.8548—red grapefruit albedo; 0.1678—naringin; 2.7891—rhamnose. The cultivation of the A. niger KMS strain on such a medium allows for obtaining naringinase with an activity of 2.2235 µmol × min−1 × mL−1.The determined equation describing the response surface was used to determine the predictable activity of naringinase in the analyzed area of the variability of the concentration of substrate components. It was also the basis for determining, using the MATLAB program, the optimal composition of the medium, thereby allowing for the maximum activity of naringinase in the post-culture fluid. The conducted research allowed us to determine the optimal concentrations  was obtained by Ni et al. [−1 × mL−1).Naringinase from i et al. . The preA. niger VB07 culture fluid, which amounted to 17.28 µmol × min−1 × mL−1. The optimized culture medium contained naringin (0.1%), rhamnose (0.5%), peptone (0.25%), and glycine (10 mM). In another study, Kumar [A. niger MTCC 344 with an activity of 9.68 µmol × min−1 × mL−1 on a medium containing naringin (0.1%), molasses (1.5%), peptone (0.5%), and mineral salts; however, it is not easy to compare the results of these actors because they measured naringinase activity at a higher temperature (60 °C).Kumar et al.  receivedy, Kumar  obtainedA. niger strains. Igbonekwu et al. [A. niger naringinase activity equal to 157.7 µmol × min−1 × mL−1. After thorough purification, they obtained a naringinase activity of 917.2 µmol×min−1 × mL−1. However, the activity results may raise doubts because the authors reported that they used a 5% naringin solution to determine the activity of naringinase. Nevertheless, the solubility of this compound is much lower (it is 1 mg per 1 mL at 40 °C).Some authors obtained significantly higher naringinase activity in the culture fluid of various u et al.  obtainedA. niger BCC 25166, amounting to the activity of 117.77 µmol × min−1 × mg−1 of protein, was obtained on the medium containing rhamnose (0.375%), naringin (0.1%), sodium nitrate (0.25%), and other components of the substrate Czapek [A. niger KMS strain with these results because the composition of the medium contained yeast extract, which resulted in a high protein content in the post-culture fluid.The maximum specific activity of naringinase from e Czapek ; howeverA. niger van Tieghem MTCC 2425 under optimized conditions on a medium containing orange particles, the activity of the naringinase preparation was about 550 µmol × min−1 × g−1 [A. niger KMS naringinase preparation was obtained using similar operations and its activity was over 800 U g−1.In turn, as a result of culturing −1 × g−1 . The enzA. oryzae JMU316 strain on a medium containing pomelo particles, peptone, and mineral salts allowed them to obtain naringinase with an activity of 0.7 µmol × min−1 × mL−1 [A. aculeatus JMUdb058 strain on an optimized medium containing, among other things, yeast extract, naringin, and soy flour, a naringinase activity of 1.16 µmol × min−1 × mL−1 was obtained [The authors who used other species and types of microorganisms obtained a relatively low activity of naringinase. The cultivation of the 1 × mL−1 . As a reobtained .In order to illustrate the effectiveness of the post-culture fluid purification, the distribution of proteins differing in molecular weight was examined using gel permeation chromatography on a YarraTM 3 μm SEC-3000 column. The post-culture fluid and the precipitated enzyme preparation were tested. The results, in the form of chromatograms, are shown in A. niger KMS in shake-flask cultures. Environmental factors (pH and dissolved oxygen) can also influence the production of naringinase by the A. niger KMS. Further studies on naringinase biosynthesis should be carried out in a bioreactor.Using waste in the form of grapefruit skins as components of the culture medium for the biosynthesis of naringinase, which can remove the bitter taste of grapefruit juices, is an effective method of managing this citrus fruit. Developing a method of naringinase biosynthesis on a substrate containing natural stimulators of the production of this enzyme, derived from waste materials or by-products from grapefruit juice, e.g., citrus peels, will enable their better management. These natural citrus fruit fragments are a rich source of carbon and substances that stimulate naringinase biosynthesis. The Box–Wilson method was confirmed to optimize naringinase biosynthesis by A. niger KMS strain was used in the research, selected from among six A. niger strains  from the microbial collection of the Department of Biotechnology and Food Analysis of the Institute of Food Chemistry and Technology of the University of Economics in Wrocław [ Wrocław .4 · 7 × H2O, p.a., KH2PO4, part a.a., FeSO4 7H2O p.a., KCl, p.a. (POCH S.A. Gliwice) NaNO3, p.a. , -naringin (naringenin-7-rhamnosidoglucoside) from citrus fruit with a purity above 90%, , yeast extract ; L-rhamnose, 99% pure . Albedo, flavedo, and segment membranes were obtained from Star Ruby red grapefruit from Turkey. The obtained fruit particles were dried at 30 °C and ground to a powder.The following substances were used to prepare the culture media: MgSO−1, carbon sources were added to the medium of Czapek with naringin (0.5 g L−1). The substrates were supplemented with water.When selecting the carbon source in the culture medium, the following saccharides were used: rhamnose, glucose, sucrose, starch, maltose, or raw materials or by-products of processing processes, such as glycerol and molasses. In a concentration of 10 g L−1. The Czapek medium supplemented with the inducer of naringinase biosynthesis was used at this research stage. The substrates were supplemented with water. Naringin and dried powdered albedo, flavedo, and segment membranes of red grapefruit, white grapefruit, orange, and pomelo were used to select ingredients stimulating naringinase biosynthesis in the culture medium. The concentration of components inducing naringinase production was 10 g LA. niger mold cultivation was carried out with the use of the Czapek medium supplemented with (in g·L−1): sodium nitrate (V)—7.8; yeast extract—20.0; potassium dihydrogen phosphate (V)—1.57, albedo, the flavedo, and red grapefruit segment membranes—5.0; naringin—1.2; rhamnose—2.5. The substrate was replenished with water.The research on the selection of the temperature of −1, yeast extract from 1 to 47.8 g·L−1, potassium dihydrogen phosphate 0.5 to 1. 97 g·L−1, red grapefruit albedo 5 to 9.8 g·L−1, naringin 0.1 to 2 g·L−1, and rhamnose from 0 to 42.5 g·L−1. The values of the concentrations of the individual components of the substrate resulted from the optimization plan applied using the Box and Wilson factor-gradient method. The substrates were supplemented with water.In the research on the optimization of the concentration of the components of the culture medium, the following were used: sodium nitrate in the range from 0 to 47.8 g·L−3. The naringinase activity is expressed in μmoles of naringin hydrolyzed per 1 min by 1 mL of culture medium.The Davis colorimetric method determined naringinase activity in the culture fluid . Sample A total of 5 mg of the naringinase preparation was dissolved in 10 mL of 0.9% sodium chloride. A total of 0.2 mL of the enzyme solution was combined with 0.3 mL of 0.1 M acetate buffer pH 4.0 and 1 mL of 0.1% naringin solution. Everything was incubated in a thermostat at 50 °C for 30 min. The Davis method  was used3; flow rate 1 mL·min−1; wavelength 280 nm. The LP-Chrom ver.1.54 software was used for the analysis of the chromatograms.Proteins were separated by size exclusion chromatography on a YarraTM 3 µm SEC-3000 column (300 × 7.8 mm). The analysis was performed using a Parkin Elmer high-pressure chromatograph with UV-Vis and RI detectors. The following separation conditions were used: phase 0.1 M phosphate buffer, pH 6.8; 0.025% NaNA. niger spore suspension (3 × 107 spores in 1 cm3 of water). The cultures were carried out in 500 mL Duran–Schott septum flasks with 100 mL of medium each. The flasks were placed on a GLF 3031 shaker with a frequency of 150 min−1 movements for seven days. During the shaking flasks’ cultivation of agitated molds of A. niger, 2 mL of samples were collected every 24 h to determine the activity of naringinase in the post-culture fluid. At the end of the culture, no increase in naringinase activity in the culture fluid was assumed.Prepared, sterile media were inoculated by adding 0.2 mL of The Box and Wilson factorial gradient method was used to optimize the culture medium composition. The concentrations of sodium nitrate, yeast extract, potassium dihydrogen phosphate (V), albedo, and flavedo of red grapefruit, naringin, and rhamnose were considered. In factor optimization, the values of the lower (−1) and upper (1) levels of the optimized parameter were determined for each of the six given culture medium components. Based on the results of factor optimization, linear regression coefficients were calculated, which were used to determine the direction and magnitude of the change in concentration of each of the optimized components (gradient optimization). The obtained results of the gradient optimization were used to determine a new central point and prepare a composition plan. Based on the results of the conducted experiments, the regression equation describing the response surface was determined, taking into account only statistically significant coefficients. The obtained function was used to calculate the maximum naringinase activity in the analyzed area of the variability of substrate components concentrations. The optimal point was determined using the hybrid method  using the MATLAB optimization package.


Alphavirus of the family Togaviridae. Natural infections of GETV have been identified in a variety of vertebrate species, with pathogenicity mainly in swine, horses, bovines, and foxes. The increasing spectrum of infection and the characteristic causing abortions in pregnant animals pose a serious threat to public health and the livestock economy. Therefore, there is an urgent need to establish a method that can be used for epidemiological investigation in multiple animals. In this study, a real-time reverse transcription fluorescent quantitative PCR (RT-qPCR) method combined with plaque assay was established for GETV with specific primers designed for the highly conserved region of GETV Nsp1 gene. The results showed that after optimizing the condition of RT-qPCR reaction, the minimum detection limit of the assay established in this study was 7.73 PFU/mL, and there was a good linear relationship between viral load and Cq value with a correlation coefficient (R2) of 0.998. Moreover, the method has good specificity, sensitivity, and repeatability. The established RT-qPCR is 100-fold more sensitive than the conventional RT-PCR. The best cutoff value for the method was determined to be 37.59 by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.956. Meanwhile, we collected 2,847 serum specimens from swine, horses, bovines, sheep, and 17,080 mosquito specimens in Shandong Province in 2022. The positive detection rates by RT-qPCR were 1%, 1%, 0.2%, 0%, and 3%, respectively. In conclusion, the method was used for epidemiological investigation, which has extensive application prospects.Getah virus (GETV) is a mosquito-borne, single-stranded, positive-sense RNA virus belonging to the genus  Culex and Aedes mosquitoes and belongs to the genus Alphavirus of the family Togaviridae (NSP)1 to NSP4 and structural proteins, including Cap, E3, E2, 6K, and E1, respectively . Chikungunya virus (CHIKV), Japanese encephalitis virus (JEV), porcine reproductive and respiratory syndrome virus (PRRSV), Akabane virus (AKAV), equine influenza virus (EIV), Sindbis virus (SINV), and Zika virus (ZIKV) kept in the laboratory were used to detect the specificity of the RT-qPCR method. One hundred and forty-three serum samples from horses serologically identified as strongly positive for GETV, stored in the laboratory, were used for ROC curve analysis. A total of 2,847 clinical serums derived from swine (1106), horses (597), bovines (667), and sheep (477) with about 17,080 mosquito specimens in 171 pools were collected in 2022 from Heze, Jining, Yantai, Qingdao, and Binzhou in Shandong Province, China.2. Cytopathic effects (CPE) were observed daily from day 1 to day 3 after inoculation. When at least 80% of the cells produce CPE, freeze–thaw 3 times and centrifuge. The supernatant was dispensed into new RNase-free tubes.Vero-E6 cells were cultured in DMEM medium containing 10% fetal bovine serum. GETV JL17/08 was inoculated into Vero-E6 monolayer cells at 0.1 MOI and incubated at 37°C with 5% CO−2, 10−3, 10−4, 10−5, and 10−6) on Vero-E6 monolayers. Incubate at 37°C with 5% CO2 for 3 h, washing off the supernatant, and covering the monolayer of cells with 1% carboxymethyl cellulose (CMC). After 3–5 days of incubation, when these lesions were observed to develop into plaques, the cells were fixed using 4% formaldehyde solution and crystal violet staining solution was added to enhance plaque visualization. Record the number of empty plaques per dilution and calculate the titer of GETV (PFU/mL) referring to Supernatant containing unknown amounts of GETV were serially diluted 10-fold and dispensed 1 mL of diluted specimens . The supernatant was collected and RNA was extracted using the QIAamp Viral RNA Mini Kit  according to the instructions. Meanwhile, the same method was applied to extract RNA from GETV , CHIKV, JEV, PRRSV, AKAV, EIV, SINV, and ZIKV viral fluids and animal serum samples. The above-extracted RNA samples were stored at −80°C until use.−7 in RNase-free centrifuge tubes, and stored at −80°C. The primers were designed based on the GETV Nsp1. All published sequences of GETV Nsp1 were retrieved from the National Center for Biotechnology Information (NCBI) (May 2022). Sixty-one sets of GETV Nsp1 gene sequences were aligned using MEGA X . Then, DNASTAR  software was used for multiple sequence analysis to select a conserved region. A set of primers based on the sequences was designed (forward: 5′-AGCATTTTCGCATCTGGCTAC-3′) (reverse: 5′-TCTGGGTCTTCCGCACTTTT-3′), and the expected length based on this primer was 150 bp. The conserved specificity of the primers was further verified by Primer-Blast (NCBI). The primers were synthesized by Sangon Biotech .The whole-genomic RNA of GETV JL17/08 was serially diluted 10-fold from the stock solution to 10® Prime Script™ PLUS RT-PCR Kit: 2 × One-Step TB Green RT-PCR Buffer 4 12.5 μl, TaKaRa Ex Taq HS Mix 1.5 μl, Prime Script PLUS RTase Mix 0.5 μl, PCR Forward Primer 1 μl, PCR Reverse Primer 1 μl, total RNA 2 μl, RNase-Free ddH2O 6.5 μl were added to prepare a total volume of 25 μl of RT-qPCR mix. The reaction conditions were Stage 1: 42°C for 5 min and 95°C for 10 s, 1 cycle, Stage 2: 95°C for 5 s and 60°C for 30 s, for each cycle, and Stage 3: 65–95°C, increment 5°C for 5 s. The RT-qPCR reaction conditions with the minimum Cq value, the highest fluorescence value, and the specific melting curve were selected as the most suitable reaction conditions to perform RT-qPCR amplification with 10-fold gradient diluted RNA as templates. The logarithm of viral PFUs (x-axis) was plotted against the corresponding Cq values (y-axis) to form the standard curve.Using the extracted GETV RNA as a template, the synthesized primers were used for the detection and quantification of GETV RNA. Primer concentration (0.1–0.5 μM/l), annealing temperature (58–65°C), and number of cycles (38–42cycels) were optimized by checkerboard method. Refer to the instructions for the One-Step TB Green−1–10−7) were used as templates for RT-qPCR and conventional RT-PCR amplification, respectively. The detection limit of qPCR was analyzed by amplification curves. Conventional PCR was first performed by reverse transcription to obtain cDNA in a total volume of 5 μl: 5 × PrimeScript RT Master Mix  1 μl, Total RNA 2 μl, and RNase-Free ddH2O 2 μl. The reaction conditions were 37°C for 15 min and 85°C for 5 s. Then, PCR Forward Primer 1 μl, PCR Reverse Primer 1 μl, 2 × Taq PCR Mix 12.5 μl, cDNA 5 μl, and ddH2O 5.5 μl were added to prepare a total volume of 25 μl of PCR mix, 94°C for 3 min, 94°C for 30 s, 55°C for 30s, 72°C for 1 min, 30 cycles, and 72°C for 5 min, and stored at 4°C. The detection limit of conventional PCR was obtained by agarose gel electrophoresis.Ten-fold gradient dilutions of RNA , and coefficient of variation (CV).Using 1 × 10Cq values of the tested methods and the qualitative test results of the gold standard were recorded. The ROC curve analysis was done using Graph Pad Prism9 software  and SPSS 16.0 for Windows . The best cutoff value that maximized the Youden index (J = Sensitivity + Specificity–1) was determined  was selected as the gold standard. Primers and probes were synthesized according to the sequences provided in the document. One hundred and forty-three laboratory-preserved serum samples verified as strongly positive by ELISA were tested by SYBR Green I RT-qPCR (established in this study) and Taqman RT-qPCR to analyze positive rate and the coincidence rate with consistency. The termined . The AUCA total of 3,018 animal serum samples from swine, bovine, horse, sheep, and mosquito samples were collected in Heze, Jining, Yantai, Qingdao, and Binzhou in Shandong Province, China, with 1,106, 597, 667, 477, and 171 specimens per species, respectively. SYBR Green I RT-qPCR was applied to the above specimens, and the positive rate was calculated for each animal in each city.6 PFU/mL (6 PFU/mL–7.73 × 10−1 PFU/mL). The linear regression equation for the standard curve was y = −3.324x + 34.924. The correlation coefficient R2) was 0.998 and the amplification efficiency (E) was 99.9% , which proved that this method has high diagnostic value. The best Cq cutoff value was calculated to be 37.59  and a decent consistency  with Taqman RT-qPCR . The ROCbe 37.59 , with a Nsp1 gene sequences. The SYBR Green I RT-qPCR and sequencing results were in 100% agreement.The results showed that the positive rates of GETV nucleic acid in Shandong  were 1%, 1%, 0.2%, 0%, and 3% in swine, horses, bovine, sheep, and mosquitoes, respectively . The posAs a mosquito-borne disease, GETV mainly causes abortion in pregnant sows and generalized rash and leg edema in horses. Natural infection of GETV has been found to occur in a variety of vertebrates, including humans. Moreover, as an effective tool for disease prevention and control, the commercialized vaccine of GETV was only successfully developed and applied in livestock industry in Japan . With itAt present, RT-qPCR methods are currently available for detecting GETV, including TaqMan and SYBR Green I. The probe approach in TaqMan has shown higher sensitivity than the SYBR-Green method. However, studies have shown that virus escape has recently been observed in various strains of the Taqman-type method . In addiR2) of 0.998. The amplification efficiency (E) was 99.9%, complied with the technical requirement of within 105%. The minimum detection limit was 7.73 PFU/mL, which was 100 times more sensitive than that of the common RT-PCR method, and meanwhile, slightly more sensitive than that of the similar method by Dong, with 1 × 101 PFU/mL , and the RNA copy number is larger than the viral titer determined by the plaque assay. The ratios of RNA copy number to viral titer range from 2 to 3 log units or more  and CAMS Innovation Fund for Medical Sciences (2020-12M-5-001).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.