Each fermenter consisted of 40 mL goat ruminal fluid, 160 mL warm buffer, 2 g mixed substrates, and 50 mg FO-0, FO-5, FO-10 or FO-15. Fish oil inclusion reduced (P < 0.05) digestibility and nitrogen retention in Experiment 1. Increasing doses of FO in the diet induced a strong drop (P < 0.001) in ruminal total volatile fatty acid (VFA) concentration and protozoa population at 3 h post incubation, but did not affect individual VFA proportions. Substitution of FO for LO decreased mean concentrations of C18:0 (P = 0.057), c-9,c-12 C18:2 and C18:3n-3 (P < 0.001), but increased (P < 0.001) C20:5n-3 and C22:6n-3. Feeding FO-10 enhanced formation of ruminal c-9,t-11 conjugated linoleic acid (CLA) concentration compared with FO-0. Overall, combined data suggest that to improve ruminal concentrations of C20:5n-3, C22:6n-3, and c-9,t-11 CLA for deposition in tissues or milk with minimal risk of affecting digestibility and ruminal fermentation, a dietary supplementation of 15 g/kg LO and 10 g/kg FO would be suitable.This study was performed to investigate effects of partial replacement of fish oil (FO) for linseed oil (LO) on digestibility, ruminal fermentation and biohydrogenation in growing goats. Experiment 1 was carried out in four growing male goats aged 6 months in a 4 × 4 Latin square design. Goats were fed a basal diet supplemented with 25 g/kg dry matter either LO alone or in combination with tuna FO. Treatments were developed by replacing FO for LO at ratios of 0, 5, 10 and 15 g/kg DM corresponding to FO-0, FO-5, FO-10 and FO-15, respectively. Experiment 2 was carried out in an  Linseed oil is one of the richest sources of ALA (Conjugated linoleic acids (CLA) are known to have anti-carcinogenic, anti-obesity, antioxidant and anti-inflammatory effects . Ruminan product . The t-1s of ALA . Regarditrans C18:1 available for incorporation in tissue lipids . Diets were offered in equal amounts twice daily at 07:00 and 17:00 h. Goats were assigned to treatments according to a 4 × 4 Latin square design. The treatment diet consisted of the basal diet supplemented (DM basis) with 25 g/kg DM either LO alone or in combination with tuna FO. Treatments were developed by replacing FO for LO at ratios of 0, 5, 10 and 15 g/kg DM corresponding to FO-0, FO-5, FO-10 and FO-15, respectively. Diets , aged 6 months and weighing 18.13 ± 0.25 kg, were used in this study. Goats were kept in individual wooden cages  and had free access to water and a mineral block. The basal diet consisted of concentrate fed in pelleted form and chopped fresh guinea grass offered y. Diets  were fory. Diets . Pellete2SO4 to keep the final pH below 3. After recording the weight, 10% proportions of 24 h feces were collected and dried in a forced-air oven at 60°C for 48 h, milled through a 1-mm mesh and stored at −20°C for subsequent chemical analysis. On d21, ruminal fluid samples were collected at 0 and 3 h post morning feeding using a 100-mL syringe. A portion of ruminal fluid was immediately fixed with 10% formalin solution in sterilized 0.9% normal saline  for direct counting of protozoa (2SO4 (9:1 w/w), centrifuged at 10,000 × g for 15 minutes and stored at −20°C for analyses of volatile fatty acids (VFA) and NH3-N concentrations.Dry matter intake (DMI) was determined by weighing daily feed offered and refused during the experiment and correcting for the DM content of each dietary component. Feed samples were pooled and stored at −20°C for further analysis. From d15 to d19, total feces and urine were collected to calculate apparent nutrient digestibility and nitrogen balance. Feces were collected in wire-screen baskets placed under the floor of the cages, and urine was collected through a funnel into plastic buckets containing 50 mL of 10% Hprotozoa . Anotherin vitro using an incubation system with 12 continuous fermenters. The experiment was a completely randomized design including the same four treatments as in Experiment 1. Total added oil alone or in the mixtures was 2.5% DM in each fermenter.Assessment of FA BH was carried out in vitro experiment. Oils were prepared and added into in vitro fermenters mixed with tween 80 solution . Ruminal contents were obtained before the morning feeding from four male goats, aged 6 months, fed a diet based on fresh guinea grass and 21% crude protein (CP) concentrate (C:F 35:65) twice daily at 07:00 and 17:00 h for a 1-week prior to sampling. Ruminal fluid was transported in four pre-warm thermos flasks to the laboratory within 30 min of collection. Ruminal fluid was filtered through a metal sieve with a pore size of 1-mm to retain small particles under continuous flushing with CO2 at 39°C. Fatty acid profiles of mixed substrates, oils and inoculum are presented in Feeds including guinea grass and the concentrate mix collected from Experiment 1 were used as substrates. Guinea grass and concentrate were mixed at a 65:35 ratio . Feed samples were analyzed for FA profiles before conducting the 2 gas. Samples for FA analysis (5 mL) were taken at 0, 1, 2, 4, 6, 12 and 24 h. Reactions were immediately stopped by cooling in an ice bath. Strained ruminal fluid (40 mL) from each goat was added to the fermenter containing warm buffer (160 mL) and mixed substrates (2.0 g). After 30 min, oil solutions were directly added into the fermenters. Cultures were continuously mixed in slow-shaking water bath at 39°C under continuous flushing with COTM 8000, Foss, Denmark). The concentration of N in acidified urine samples and ruminal NH3–N concentration were analyzed by the micro-Kjeldahl method  equipped with a 100 m × 0.25 mm × 0.2 μm film fused silica capillary column  and a flame ionization detector. The column temperature was kept at 70°C for 4 min, then increased at 13°C/min to 175°C and held for 27 min, then increased at 4°C/min to 215°C and held for 17 min, then increased at 4°C/min to 240°C and held for 10 min. Fatty acids were identified by comparison of retention times with external FAME standards . The CLA mixture  contained c-9,t-11 CLA, t-10,c-12 CLA, c-9,c-11 CLA, and t-9,t-11 CLA.Feed and fecal samples were analyzed for DM, organic matter (OM), CP, ether extract (EE), and ash using standard methods . Crude pl method . VFA conl method  equippedYijk = µ+ Oi + Aj + Pk+ εijk. Where Yijk observation from animal j, receiving diet i in period k; µ, the overall of mean; Oi, the effect of fish oil level ; Aj, the effect of animal ; Pk, the effect of period ; εijk, the residual effect. Mean amounts of FA in the Experiment 2 were statistically analyzed by a PROC MIXED procedure with the statistical model Yijk= µ + Oi + Tj + (O×T)ij + εijk, where Yijk = the dependent variable; µ = the overall mean; Oi = the fixed effect of added oil; Tj = the fixed effect of incubation time; (O×T)ij = the fixed effect of interaction between added oil and incubation time; εijk = the random residual error. Goat inoculum source was considered as a random factor. Orthogonal polynomial contrasts (linear and quadratic) were used to examine treatment effects on response variables. Significant differences among treatment means were statistically compared using Tukey. Statistical tests were performed using SAS University Edition 2019 . Significant effect of treatment on least squares means was declared at P < 0.05 and tendency was declared at 0.05 ≤ P < 0.1.Data in Experiment 1 were analyzed using the GLM procedure. The statistical model was Linseed oil was rich in ALA (557.50 μg/mg), whereas FO contained (µg/mg) high amounts of FA including EPA (82.61) and DHA (373.74) that were not present in feeds or LO. However, compared with LO, FO also contained high amounts (µg/mg) of some saturated FA consisting of C14:0, C16:0 and C18:0, thus, replacement of FO for LO in the diet also increased their concentrations. Ruminal fluid + buffer contained high amounts (μg/mL) of C16:0 and C18:0.P < 0.01) intakes of C14–C18, EPA and DHA, but linearly decreased (P < 0.01) intakes of LA and ALA. Quadratic decreases (P < 0.05) in apparent digestibility of DM, OM and CP were observed with increasing levels of FO in the diet. There was a remarkable drop (P < 0.01) in OM digestibility when goats were fed FO-15. Increasing dietary FO decreased linearly (P < 0.05) nitrogen retention, accounting for −12.90% in FO-15 as compared to FO-0  by feeding FO, with the lowest value observed in the FO-15 group relative to FO-0. Surprisingly, replacement of FO for LO did not cause any shifts in the proportions of individual VFA (P > 0.05). Concerning ruminal microbes, compared with those fed only LO, protozoa population decreased significantly (P < 0.01) in goats fed the blended FO and LO diets.Replacement of FO for LO in diets had no effect on ruminal pH, NH feeding . Howevert-9 C18:1, EPA and DHA (P < 0.001) mean concentration of C18:0. Further analysis of FA changes during incubation revealed that concentration of C18:0 remained unchanged until 12 h of incubation; however, a different change was detected at 24 h incubation (P < 0.001) concentrations of ALA and LA (P < 0.01) of LA among the treatments was observed only at 0, 6 and 24 h incubation  to later stages of incubation. The greatest amounts of c-9,t-11 CLA in the FO-10 treatment were measured at 6 and 24 h of incubations  and 2.18-fold (at 24 h). Increasing amount of FO in the diets resulted in an increase of t-9,t-12 C18:2 , the highest value in FO-10 (5.96 μg/mL) versus the lower value in FO-0 (3.68 μg/mL). Fish oil substitution for LO notably increased (P = 0.01) concentration of t-9 C18:1 during incubation (P < 0.05) detected with FO-10 at 6 h of incubation compared with FO-0  with FO-10 at 6 h incubation relative to FO-0 (P < 0.001) increases in concentrations of EPA and DHA (P < 0.001) in DHA concentration over time of incubation (Dietary replacement of FO for LO resulted in time-dependent shifts in ruminal FA concentrations characterized by decreases in C18:0, LA and ALA and concomitant increases in  and DHA . Comparecubation  with theA and LA . Except A and LA , the difcubation . The FO-cubation , with thith FO-0 . Concent to FO-0 . Regardl and DHA  and a licubation .Although A transient reduction in ruminal total VFA concentration after 3 h feeding FO diets indicated that this oil disturbed fermentation by ruminal microbes, which was in agreement with c-9,t-11 CLA and C18:0 likely due to BH of ALA and LA (cis and trans C18:1 isomers in the rumen (c-9,t-11 CLA synthesis by Δ9 desaturase in mammary gland (t-9 C18:1 (The shift in FA concentrations that occurred during the 24 h incubations –7 indicaA and LA . It is wA and LA , which whe rumen , and usery gland . Althoug-9 C18:1  increase-9 C18:1 .t-11 C18:1 peak from other trans isomers of similar elution time; therefore, it could not explain the changes of all FA involving the process of LA and ALA BH. The greater amounts of t-9 C18:1 with FO diets suggested there was an increase in the conversion efficiency of VA to t-9 C18:1 or that the production of VA was large enough to co-elute with the t-9 C18:1 peak. trans-18:1 isomers. t-9 C18:1 concentration in milk fat. The higher amount of c-9,t-11 CLA in FO-10 at 6 h after incubation suggested that use of FO at this level in place of LO not only inhibited the conversion of VA to C18:0, but also prevented hydrogenation of c-9,t-11 CLA to VA. However, feeding FO-15 elicited a modest improvement in c-9,t-11 CLA compared with FO-10 largely because LO-15 had a lower amount of LA. Feeding fish oil in dairy goats increased milk c-9,t-11 CLA (A limitation of the FA protocol in this study was that it could not define the t-11 CLA . A reduct-11 CLA . A lineac-9,t-11 CLA was not change by feeding FO, but the higher concentration of this FA was detected in FO-10 at 6 and 24 h incubation. To improve ruminal concentrations of EPA, DHA, c-9,t-11 CLA and reduce C18:0 concentration without or less affecting digestibility and ruminal fermentation, a dietary supplementation of 15 g/kg LO and 10 g/kg FO would be suitable.Substitution of FO for LO decreased nutrient digestibility and ruminal total VFA concentration without affecting individual VFA proportions. Replacement of FO for LO from 5 to 15 g/kg DM remarkably decreased ruminal protozoa populations. Increasing dose of FO in the diet resulted in decreased mean amounts of C18:0, LA and ALA but increased mean amounts of EPA and DHA. Mean amount of


Crataegus pinnatifida is used to treat various diseases, including indigestion, congestive heart failure, hypertension, atherosclerosis, and myocardial dysfunction. We evaluated antioxidant and anti-α-glucosidase activities of various solvent extracts and major bioactive components from the fruit of C. pinnatifida. Ethyl acetate extracts showed potent antioxidant activities with IC50 values of 23.26 ± 1.97 and 50.73 ± 8.03 μg/mL, respectively, in DPPH and ABTS radical scavenging assays. Acetone extract exhibited significant anti-α-glucosidase activity with IC50 values of 42.35 ± 2.48 μg/mL. HPLC analysis was used to examine and compare the content of active components in various solvent extracts. We isolated four active compounds and evaluated their antioxidant and anti-α-glucosidase properties. Among the isolated compounds, chlorogenic acid and hyperoside showed potential antioxidant activities in ABTS and superoxide radical scavenging assays. Moreover, hyperoside also displayed stronger anti-α-glucosidase activity than other isolates. The molecular docking model and the hydrophilic interactive mode of anti-α-glucosidase assay revealed that hyperoside might have a higher antagonistic effect than positive control acarbose. The present study suggests that C. pinnatifida and its active extracts and components are worth further investigation and might be expectantly developed as the candidates for the treatment or prevention of oxidative stress-related diseases and hyperglycemia. Free radicals are generated from normal biochemical reactions in the body and increase oxidative stress and probably damage biological molecules such as lipids, proteins, and DNA . Free ra+ to the free radicals during oxidation and become a radical themselves. These radical intermediates are stabilized by the resonance delocalization of the electron within the aromatic ring [Phenolic compounds are free radical scavengers (FRS) that delay or repress the initiation step or hinder the propagation step of lipid oxidation . The anttic ring . For theOxidative DNA damage links pathogenically to multiple aging-regarding degenerative diseases such as cancer, coronary heart disease, and diabetes . Growingα-Glucosidase is an essential enzyme in human carbohydrate metabolism. Many studies have affirmed the association between α-glucosidase inhibitors and blood glucose regulation in type II diabetic patients . α-GlucoCrataegus pinnatifida is a traditional Chinese medicinal (TCM) herb that is classified as the Rosaceae family and is widely distributed in the north of China [C. pinnatifida can be used as medicine and food for its safety [C. pinnatifida is used in prescriptions to treat indigestion with epigastric distention, abdominal pain, diarrhea, hyperlipidemia, amenorrhea, and hypertension [C. pinnatifida also possesses various biological activities, including lipid regulating, anti-atherosclerosis, antihypertensive, antibacterial, antiviral, anticancer, immunoregulating, anti-inflammatory, and antioxidant activities [C. pinnatifida. Moreover, we conducted molecular docking between α-glucosidase and hyperoside with the most potent anti-α-glucosidase activity. In addition, the amounts of isolated compounds were quantified by HPLC analysis. The antioxidant and anti-α-glucosidase effects of isolated components were also evaluated.of China . Fruits s safety . In tradrtension . Previoutivities ,16,17,183) and p-nitro-phenyl-α-D-glucopyranoside (p-NPG) were obtained from Alfa Aesar . Disodium hydrogenphosphate, potassium peroxodisulfate, sodium carbonate, and potassium dihydrogenphosphate were acquired from the SHOWA Chemical Co. Ltd. . 2,2-diphenyl-1-hydrazyl (DPPH), deoxyribose, nitroblue tetrazolium (NBT), phenazine methosulphate (PMS), and 2-thiobarbituric acid (TBA) were supplied by Tokyo Chemical Industry Co., Ltd. . Butyl hydroxytoluene (BHT), acarbose, nicotinamide adenine dinucleotide (NADH), sodium acetate, trichloroacetic acid (TCA), and potassium acetate were acquired from Acros Organics . Acetic acid was supplied by Avantor Performance Materials, LLC . Sodium hydroxide solution was acquired from Merck .Ascorbic acid, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), α-glucosidase, ethylenediaminetetraacetic acid (EDTA), gallic acid, Folin-ciocalteau’s reagent, hydrogen peroxide solution, Trolox, and 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) were purchased from Sigma-Aldrich . Ferric chloride  was added into 20 g of pieces and shaken by orbital shakers for 24 h at 25 °C. The extracts were filtered through filter paper (Whatman No. 1) and condensed by a rotary evaporator at 37 °C. All extracts were stored at −20 °C until further experiments.The fruits of C. pinnatifida were extracted and shredded with MeOH  at room temperature. The MeOH extract was concentrated under reduced pressure at 37 °C, and the MeOH extract (5.61 g) was obtained. MeOH extract (5.61 g) was purified by column chromatography (CC)  to afford 14 fractions: A1–A14. Part (153 mg) of fraction A9 was further purified by preparative TLC  to afford epicatechin (4.98 mg) (Rf = 0.12) and procyanidin B2 (3.81 mg) (Rf = 0.35). Part (215 mg) of fraction A11 was further by preparative TLC  to obtain hyperoside (1.70 mg) (Rf = 0.30) and chlorogenic acid (5.81 mg) (Rf = 0.23). The TLC graphs of isolated compounds are shown in The fruits (20 g) of ® 100 RP-18 Endcapped  purchased from Merck KGaA, Darmstadt, Germany. HPLC-PDA chromatographic fingerprints were obtained with an Agilent 1260 Infinity II HPLC instrument equipped with a 1260 Infinity II quaternary pump, a 1260 Infinity II degasser, a 1260 Infinity II vialsampler, a 1260 Infinity II column thermostat, a 1260 Infinity II diode array detector HS, and a PC with the Agilent Chemstation software. All of them are from Agilent Technologies . Gradient separation using 0.2% acetic acid in water (v/v) (solvent A) and water:methanol:acetonitrile = 3:2:5 (solvent B) as mobile phase was as follows: 0–20 min, linear gradient from 0 to 15% B; 20–65 min, 15% B with isocratic elution; 65–110 min, linear gradient from 15 to 40% B; 110–120 min, linear gradient from 40 to 100% B; 120–130 min, back to initial conditions at 0% B; and 130–135 min, at 0% B. The flow rate was 1.0 mL/min, and the injection volume was 10 μL. Peaks were detected at 280 nm. Different compounds were identified by retention time. To guarantee peak purity, DAD acquisition from 200 to 650 nm was conducted to register UV-spectra. For the quantitative analysis of four compounds in the extracts, aliquots of samples were dispersed in 10 mL of a methanol solution by sonication for 5 min. Then, the samples were centrifuged for 15 min at 3500 rpm, and the supernatant extracts were filtered through 0.45 μm PTFE syringe filters .Reversed-phase separations were performed using a LiChrospher2O. Gallic acid was diluted by MeOH (0–100 μg/mL) as standard. Each extract or gallic acid (50 μL) was added to a 96-well microplate. The mixture was incubated for 5 min. Subsequently, 20% Na2CO3 solution (100 μL) was added. After 40 min, it was incubated in darkness at room temperature before measuring the absorbance of the supernatant at 750 nm. The TPC of the extracts was determined from a standard calibration curve using gallic acid in the range of 0–100 μg/mL with R2 value of 0.9992. The concentration of TPC was expressed in milligram (mg) gallic acid equivalents (GAE) per gram of dried extract. All measurements were conducted in triplicate.Total phenolic content (TPC) of different extracts was determined by Folin-Ciocalteau’s method with a slight modification of . Brieflyw/v) aluminum chloride solution (200 µL) and 0.1 mM potassium acetate solution (200 µL). The mixture was reacted at room temperature for 30 min. Then, the absorbance of the mixture was measured at 415 nm. The TFC of the extracts was determined from a standard calibration curve using quercetin with R2 value of 0.9997. The concentration of TFC was expressed in milligram quercetin equivalents (GAE) per gram of dried extract. All measurements were conducted in triplicate.The total flavonoid content (TFC) of each extract was measured using the aluminum chloride colorimetry method described by Do et al.  with slit is the absorbance of the test sample, and Ac is the absorbance of the control (untreated group). BHT was used as the positive control. All half-maximal inhibitory concentration (IC50) values of tested activities were determined by the linear regression of the percentage of remaining DPPH radical against the sample’s concentration.The scavenging activity of DPPH radical was investigated as described with slight modification . DPPH so2O  and leaving the mixture in the dark for about 16 h at room temperature before use. The working solution was diluted by EtOH to reach the absorbance of 0.70 ± 0.02 at 740 nm for further experiments. Different concentrations  of extracts or compounds (10 µL) were mixed with the working solution (190 μL). The mixture was incubated at room temperature for 6 min, the antioxidant activity of the mixture was determined by calculating the decrease in absorbance measured at 740 nm by the following equation:ABTS radical scavenging activity of each extract was measured based on the method with slight changes . In briec and At are the absorbance of the control (untreated group) and test sample, respectively. The IC50 values of all tested activities were determined by the liner regression of the percentage of remaining ABTS radical against the sample’s concentration.Where A2•−) was evaluated using a modified method [t is the absorbance of the test sample, and Ac is the absorbance of the control (untreated group). The IC50 values of all tested activities were determined by the linear regression of the percentage of remaining superoxide radical against the sample concentration.The scavenging activity of superoxide anion radical  and 100 μL H2O2 (20 mM) in sodium phosphate buffer (20 mM pH 7.4) was added. The mixture was incubated in a water bath at 37 °C for 1 h to trigger the Fenton reaction and form a hydroxyl radical. After incubation, 2.8% TCA (200 μL) and 1% TBA (200 μL) were added and boiled in the dry bath at 100 °C for 1 h. The mixture was measured at 532 nm by the following equation:t and Ac are the absorbance of the test sample and the control (untreated group), respectively. The IC50 values of all tested activities were determined by the linear regression of the percentage of remaining hydroxyl radical against the sample’s concentration.The scavenging activity of hydroxyl radical was measured based on the method described by Mathew and Abraham with slight revision . In shor2 value of 0.998. Results are expressed in mM TE/g dry weight. Additional dilution was needed if the FRAP value measured was over the linear range of the standard curve.Ferric reducing antioxidant power was measured based on previous study with slight modification . The worp-NPG , the substrate, was added and incubated in dry baths at 37 °C for 40 min after the mixture vortexed. The reaction was terminated after adding 0.1 M Na2CO3 solution (500 μL). The absorbance of released p-nitrophenol (p-PNP) was measured by calculating the decrease in absorbance measured at 400 nm by the following equation:c is the absorbance of the control (untreated group), and At is the absorbance of the test sample. The IC50 values of all tested activities were determined by the linear regression of the percentage of remaining α-glucosidase against the sample concentration.The inhibition assay of α-glucosidase was conducted using the conditions previously reported with slight modifications . The α-GAll calculations are performed by Discovery Studio 2019  software. Primarily, the structure of hyperoside or acarbose is energy minimized until the default derivative convergence criterion of 0.01 kcal/mol is met. The crystal structure (PDB: 3A4A) is retrieved from the Protein Databank, and hydrogen atoms are added to prepare the docked receptor. This protein structure is subsequently used in the CDocker program to dock hyperoside or acarbose into the active site. Ten different docking poses are calculated and ranked by using the PLP-Score scoring function. The top-ranked docking solution is visually analyzed to determine the binding mode of hyperoside or acarbose.t-test. A probability of 0.05 or less was considered statistically significant. All experiments were performed at least 3 times.All data are expressed as mean ± SEM. Statistical analysis was carried out using Student’s C. pinnatifida were evaluated. TPCs, TFCs, and extraction yields of n-hexane, chloroform, dichloromethane, EtOAc, acetone, EtOH, and MeOH extracts from C. pinnatifida are shown in TPCs, TFCs, and yields of various solvent extracts from C. pinnatifida ranged from 2.3 ± 1.67% (n-hexane extract) to 28.0 ± 0.75% (MeOH extract). Higher polar solvent (including MeOH and EtOH) extracts had higher yields. The results indicated that C. pinnatifida is abundant in high polar components.The yields of various solvent extracts from C. pinnatifida were measured by the Folin–Ciocalteu method. TPC in the extracts was calculated from the regression equation  of the calibration curve. The TPCs of solvent extracts ranged from 12.12 ± 0.26 to 63.53 ± 0.27 mg of GAE/g. Among all different solvent extracts, the EtOAc extract contained the highest amount of TPC (63.53 ± 0.27 mg/g) followed by acetone (45.81 ± 0.61 mg/g), MeOH (44.20 ± 1.30 mg/g), EtOH (32.82 ± 1.21 mg/g), chloroform (21.09 ± 0.99 mg/g), dichloromethane (19.77 ± 0.13 mg/g), and n-hexane (12.12 ± 0.26 mg/g). These results suggested that solvents with higher relative polarity are suitable for the extraction of phenolic compounds from C. pinnatifida.Phenolic contents in different solvent extracts from C. pinnatifida were evaluated by aluminum chloride colorimetric method. TFC in the extracts was calculated from the regression equation  of the calibration curve. The TFCs of different solvent extracts ranged from 17.40 ± 3.11 to 37.93 ± 5.31 mg of QE/g. Among all different solvent extracts, chloroform extract contained highest amount of TFC (37.93 ± 5.31 mg/g) and followed by dichloromethane (34.87 ± 1.95 mg/g), n-hexane (31.74 ± 1.07 mg/g), EtOAc (25.22 ± 4.21 mg/g), EtOH (22.61 ± 0.90 mg/g), acetone (19.41 ± 3.00 mg/g), and MeOH (17.40 ± 3.11 mg/g).The TFCs of solvent extracts from n-hexane, chloroform, dichloromethane, EtOAc, acetone, EtOH, and MeOH) from the fruits of C. pinnatifida was first mentioned in this study. This can supply a guide for the option of suitable solvents in TPC and TFC extraction applications.The comparative assessment of total phenolic content (TPC) and total flavonoid content (TFC) of various solvent extracts  exhibited the strongest DPPH radical scavenging activity and was even better than BHT (IC50 = 34.28 ± 1.40 μg/mL). In addition, acetone (IC50 = 40.06 ± 0.18), chloroform (IC50 = 68.61 ± 3.14 μg/mL), dichloromethane (IC50 = 76.22 ± 4.90 μg/mL), MeOH (IC50 = 74.13 ± 5.61 μg/mL), and EtOH (IC50 = 96.21 ± 4.26 μg/mL) extracts showed moderate activities. n-Hexane (IC50 = 199.18 ± 16.82 μg/mL) extract showed relatively high IC50 value.The hydrogen atom or electron donation abilities of various solvent extracts were measured by the reduction of a purple DPPHydrazine ,28. Tabl•+ that can be reduced by antioxidants. The high-pigmented and hydrophilic antioxidants were better reflected by ABTS assay than DPPH assay [50 = 50.73 ± 8.03 μg/mL) followed by acetone (IC50 = 87.75 ± 2.70 μg/mL), MeOH (IC50 = 105.86 ± 6.25 μg/mL), n-hexane (IC50 = 131.14 ± 7.15 μg/mL), dichloromethane (IC50 = 131.44 ± 7.13 μg/mL), EtOH (IC50 = 140.29 ± 6.76), and chloroform (IC50 = 141.97 ± 3.70 μg/mL).The ABTS assay is based on the generation of a blue/green ABTSPH assay . As show50 = 122.95 ± 9.07 μg/mL) on the superoxide radical scavenging assay.This assay is measured by the PMS/NADH–NBT system. Superoxide anion radicals generated from dissolved oxygen by PMS/NADH coupling reaction reduce NBT. The decrease in absorbance at 560 nm with antioxidants indicates a reduction in superoxide anion radicals in the reaction mixture . The res3-EDTA-ascorbic acid and H2O2 [50 = 92.30 ± 7.47 μg/mL) showed relatively potent antioxidant activity in scavenging hydroxyl radical followed by chloroform (IC50 = 114.41 ± 1.80 μg/mL), n-hexane (IC50 = 124.45 ± 6.27 μg/mL), acetone (IC50 = 127.25 ± 2.26 μg/mL), MeOH (IC50 = 138.04 ± 21.91 μg/mL), and EtOH (IC50 = 139.03 ± 17.33 μg/mL)  .3+) complex to ferrous iron (Fe2+) complex by antioxidants present in the samples [n-hexane (70.11 ± 4.13 TE mM/g)  .C. pinnatifida possessed the highest phenolic content and the highest hydrogen atom or electron donation abilities in DPPH and ABTS radical scavenging assays. In addition, the EtOAc extract also showed potent superoxide and hydroxyl radical scavenging effects. Moreover, acetone extract showed the highest reducing ability in the FRAP assay.Based on the above results of the antioxidant assays, the EtOAc extract from n-hexane, chloroform, dichloromethane, EtOAc, acetone, EtOH, and MeOH) from the fruits of C. pinnatifida was first proposed in our study. This can provide an indication for the selection of appropriate solvents in antioxidant extraction applications.The comparative evaluation of antioxidant assays  of various solvent extracts (C. pinnatifida exerted the most anti-α-glucosidase activity (IC50 = 42.35 ± 2.48 μg/mL) followed by MeOH (IC50 = 52.02 ± 0.24 μg/mL), EtOH (IC50 = 58.69 ± 6.91 μg/mL), n-hexane (IC50 = 99.75 ± 4.37 μg/mL), dichloromethane (IC50 = 120.41 ± 6.44 μg/mL), EtOAc (IC50 = 126.36 ± 9.81 μg/mL), and chloroform (IC50 = 207.46 ± 9.52 μg/mL). Seven solvent extracts showed higher anti-α-glucosidase activities than antidiabetic agent, acarbose (IC50 = 317.80 ± 16.36). Among all solvent extracts, acetone, MeOH, and EtOH extracts showed relatively potent anti-α-glucosidase activity. These results indicated that the suitable relative polarity of extracting solvents for anti-α-glucosidase activity from C. pinnatifida would be ranged from 0.355 to 0.762.α-Glucosidase inhibitors decrease the rate of hydrolytic cleavage of oligosaccharide and delay carbohydrate digestion ,33. The C. pinnatifida by HPLC analysis with reverse-phase. The contents of four active compounds in each solvent extract was shown in n-hexane extract) in succeeding order of EtOH > MeOH > EtOAc > acetone > dichloromethane > chloroform > n-hexane extract. EtOH (33.62 ± 0.49 μg/kg) and MeOH (27.76 ± 0.45 μg/kg) extracts exhibited higher amounts of four active compounds compared with other extracts. Procyanidin B2 was the most abundant among the four active compounds in organic solvent extract followed by chlorogenic acid, epicatechin, and hyperoside.n-hexane, chloroform, dichloromethane, EtOAc, acetone, EtOH, and MeOH) from the fruits of C. pinnatifida by HPLC analysis is first conducted in our study.The comparative evaluation for identification and quantification of the major active components  of different solvent extracts (50 = 10.59 ± 0.22 μg/mL) exerted a higher superoxide radical scavenging activity than other isolated compounds. Epicatechin (IC50 = 20 ± 3.49 μg/mL), chlorogenic acid (IC50 = 18.38 ± 0.56 μg/mL), and procyanidin B2 (IC50 = 13.69 ± 1.88 μg/mL) significantly inhibited hydroxyl radical with a lower IC50 value than positive control, BHT (IC50 = 60.62 ± 2.58 μg/mL). The antioxidant activities of isolates were also evaluated by FRAP assay. Hyperoside, epicatechin, and procyanidin B2 show higher antioxidant power (TE ≥ 7726.67 ± 39.97 mM/g) than BHT (TE = 4257.97 ± 44.90 mM/g).The isolated compounds, epicatechin, chlorogenic acid, hyperoside, and procyanidin B2 , were meC. pinnatifida against α-glucosidase is shown in 50 = 317.80 ± 16.36 μg/mL). Hyperoside and epicatechin displayed significant activities with IC50 values of 34.98 ± 0.89 and 81.79 ± 6.94 μg/mL followed by procyanidin B2 (IC50 = 118.44 ± 7.34 μg/mL) and chlorogenic acid (IC50 = 170.37 ± 10.68 μg/mL).The inhibitory activity of four isolated compounds from According to the results of anti-α-glucosidase assay, hyperoside exhibited the most potent anti-α-glucosidase activity among all isolated compounds. Thus, the interaction between α-glucosidase and hyperoside was evaluated by molecular modeling docking. The 3D crystal structure of α-glucosidase showed that it mainly contains numerous structural domains including the N-terminal domain, the barrel domain in which the active site is located, and the C-terminal domain. The α-glucosidase active site is primarily formed by numerous β-sheets and several loops or α-helices. Importantly, the active site from different species shows certain conformational similarities. It mainly contains hydrophilic residues and can accept compounds with distinct sizes to enter. The crystal structure of α-glucosidase from beta vulgaris complexed with acarbose  CDocker modeling program. The 3D crystal structure for α-glucosidase of Saccharomyces cerevisiae is not available at this moment; thus, the crystal structure (PDB: 3A4A) of Saccharomyces cerevisiae (PDB: 3A4A) containing 72% sequence homology with α-glucosidase from Saccharomyces cerevisiae is usually used to perform the docking study and is also employed in this study. In the crystal structure (PDB: 3A4A), the configuration of its active site is quite similar to that of α-glucosidase from beta vulgaris, but it is deep and narrow. The crystal structure of this active site reveals that its co-crystallized ligand, α-D-glucopyranose, locates deeply in the ligand-binding pocket and makes three essential H-bond interactions including (1) the 4-hydroxyl group interacting with His 351 and Asp 352 by acting as the H-bond donor; (2) the 5-hydroxyl group that makes H-bond contact with Glu 277 as well as Asp 352 by acting as the H-bond donor and also serves as the H-bond acceptor to interact with Arg 213; and (3) the 6-hydroxymethyl group that acts as the H-bond donor to interact with Glu 277 and Asp 352.To further study how hyperoside b might iAs shown in As the positive control, the docking model for acarbose is also generated to compare its binding mode with hyperoside. Acarbose was bound to the catalytic site similarly as hyperoside by leaning its A ring toward the binding position of α-D-glucopyranose and located its D ring to protrude out of the entrance of the active site. Since it contains a lot of hydrophilic moieties, acarbose mainly interacted with the active site by the H-bond interactions, including the following: (1) the 5-hydroxymethyl group on the A ring interacted with Asp 215 by acting as the H-bond donor; (2) the 4-hydroxyl group on the A ring interacted with His 351 as well as Asp 352 by acting as the H-bond donor and also served as the H-bond acceptor to contact with Arg 442; (3) the 5′′-hydroxymethyl group on the C ring interacted with His 280 by acting as the H-bond donor; (4) the 1′′′-hydroxyl group on the D ring made H-bond interaction with Lys 156 as well as Ser 240 by acting as the H-bond acceptor, and it also made H-bond contact with Leu 313 by acting as the H-bond donor; (5) the 2′′′-hydroxyl group on the D ring interacted with the backbone of Pro 312 as the H-bond donor; and (6) the 3′′′-hydroxyl group on the D ring contacted with the side chain of Asp 242 by serving as the H-bond donor.Saccharomyces cerevisiae complexed with hyperoside or acarbose is needed to further establish the nature of their respective interaction.For the comparison of the binding modes of hyperoside and acarbose, hyperoside can reside in the middle of the substrate binding pocket and its D ring is located at the entrance of the pocket to block the entrance and enhance its inhibitory effect. For acarbose, its A ring can be located in a similar position as the A ring of hyperoside but its D ring partially protrudes out of the substrate-binding pocket. For their hydrophilic interactive binding mode b, the A C. pinnatifida and its isolated compounds were investigated with different antioxidant systems and anti-α-glucosidase activity assay. Ethyl acetate extract from C. pinnatifida showed significant antioxidant activities through DPPH, ABTS, superoxide, hydroxyl radical scavenging tests, and FRAP assay. TPC in the extract of ethyl acetate also showed the highest value among all solvent extracts, indicating that the ethyl acetate extract possessed the largest amounts of polyphenols, and it corresponded to the results of antioxidant activities. The acetone extract possessed relatively high ferric reducing antioxidant power among all solvent extracts. In addition, acetone extract also showed the strongest α-glucosidase inhibitory property.Various solvent extracts of C. pinnatifida were quantified by HPLC and identified as epicatechin, chlorogenic acid, hyperoside, and procyanidin B2. The bioactivity assays demonstrated that all isolates displayed antioxidant activities. In addition, hyperoside showed strong anti-α-glucosidase property. As the result of molecular modeling docking, hyperoside also exhibited the high affinity with α-glucosidase and, thus, displayed potential inhibitory activity against α-glucosidase.Four isolated compounds from C. pinnatifida is first mentioned in this study. The results are enough to support the importance of the suitable solvents to extract bioactive compounds. The bioactive extracts and their isolated compounds mentioned above can be used as natural antioxidants in the food industry and dietary supplements against oxidative damage. Furthermore, the acetone extract and hyperoside can also be used as natural α-glucosidase inhibitors.The comparative evaluation of antioxidant and anti-α-glucosidase activities in various solvent extracts and bioactive compounds from the fruits of


Facial emotional recognition is something used often in our daily lives. How does the brain process the face search? Can taste modify such a process? This study employed two tastes (sweet and acidic) to investigate the cross-modal interaction between taste and emotional face recognition. The behavior responses (reaction time and correct response ratios) and the event-related potential (ERP) were applied to analyze the interaction between taste and face processing. Behavior data showed that when detecting a negative target face with a positive face as a distractor, the participants perform the task faster with an acidic taste than with sweet. No interaction effect was observed with correct response ratio analysis. The early  and mid-stage [early posterior negativity (EPN)] components have shown that sweet and acidic tastes modified the ERP components with the affective face search process in the ERP results. No interaction effect was observed in the late-stage (LPP) component. Our data have extended the understanding of the cross-modal mechanism and provided electrophysiological evidence that affective facial processing could be influenced by sweet and acidic tastes. Facial expressions play a significant role in social situations, and they become even more intriguing during fine dining. So much so that one of the critical social skills for high table culture is detecting others’ emotions while dining. Thus, there is a subtle interplay of cross-modal integration in this matter. How does taste influence facial expression by affecting emotion? More specifically, does the chewing of food influence the affective face identification? These questions motivated our study.Emotional face detection is indeed a well-established model to study cross-modal sensory integration. Different emotional facial processing has been studied extensively . Many evIn the real environment, emotional facial recognition can be influenced by other sensory stimuli or background . The senIt is not difficult to understand that such cross-modal sensory information integration can happen at different stages. With evolution, such situations frequently occur in the natural environment, which forces human beings to develop optimal neural network processes for cross-modal information. These different successive ERP components provided the time-windows to observe the stages of cross-modal information interaction. For instance, emotional facial processing has been integrated with additional emotional cues like body gestures or voice information at an early stage. Through the four ERP components, one can observe the interaction effect and estimate the stage  of cross-modal interaction. Moreover, with taste and vision, The additional sensory information of vision and sound has provided a similar interaction effect on affective face recognition. However, is it necessary for the gustatory system to contribute to the affective face detection system in a similar manner? This question is still puzzling. Though previous studies have researched the olfactory system and have found some interaction effect between smell and face detection , the int∗Power version 3.1 (Gsion 3.1  is used via the link1). Half of them were male faces and the other half were female faces. There were three types of emotional faces: positive, neutral, and negative. Each type contains 12 face images. With the emotional face search task, two face images were displayed parallelly on the screen (left and right), which allowed around 10° × 9° of visual angle. One emotional face was taken as a target emotional face (either positive or negative); the other distracting image was a neutral or opposite emotional face. The gustatory stimuli were vitamin C tablets  and sugar crystals . The tablets or crystals were placed on the participant’s tongue during the experiment  .  in a sound-proof chamber and were fitted with a 64-channel ANT equipment (ANT Neuro) electrode cap. All electrodes were positioned according to the international 10–20 system  and refeBefore the formal experiment, the participants were trained in the practice section for as long as required. They learned to breathe through their nose, keep their bodies as still as possible, and fix their eyes on the monitor and avoid blinking or moving their eyes or mouth while performing the task.The experiment was based on the two factors within the subject design. The two variables of emotional faces and taste were manipulated. The emotional faces had four levels: positive , positive (negative), negative , and negative (positive). The tastes had two levels: sweet or acidic. Thus, there were a total eight conditions of different stimuli combination : PositivThe procedure is depicted in via the ocular correction software. Based on previous research (t-tests) with Bonferroni correction were performed. (The recorded EEG data are assessable via the link2).Brain Vision Analyzer 2.0 software  was used for data processing and EEG filtering . The data were segmented into 1,000 ms intervals , and corrected to the 200 ms pre-stimulus baseline. Trials with correct behavioral responses were selected for further ERP analysis. Artifacts due to eye movements were corrected research  and the F = 25.197, p < 0.001, ηp2 = 0.448]. Post hoc tests revealed the correct response ratios of different visual combinations: PoNu (99.02% ± 2.80%) > PoNg (97.36% ± 2.93%) > NgNu (96.06% ± 3.04%) > NgPo (94.17% ± 4.86%)  = 1.022, p = 0.320, ηp2 = 0.032]. There was no significant interaction effect between vision and taste .A 4  × 2 (taste: sweet and acidic) repeated measures ANOVA was used to test the main effects and interaction effect of correct response ratios. The main effect of emotional faces has been observed . Post hoc tests revealed the RT with different visual stimuli: PoNu (623.59 ± 172.91 ms) < PoNg (655.45 ± 164.74 ms) < NgNu (818.90 ± 170.57 ms) < NgPo (824.59 ± 170.28 ms). There was no significant main effect of taste . However, the notable interaction between vision and taste existed . A simple effect test with Bonferroni correction only revealed that the RT of NgPoA (790.78 ± 162.02 ms) was remarkably shorter than that of NgPoS . All other pairwise comparisons were not significant (ps > 0.05).The reaction time (RT) of trials with the correct response was selected and further examined  right. RF = 36.491, p < 0.001, ηp2 = 0.557]. The amplitude of the target negative faces  was significantly larger than that of the target positive faces  (p < 0.001). There was no main effect of taste  and brain areas .A 3  × 4  × 2 (taste: sweet and acidic) repeated-measures ANOVA was used to analyze P1 . The maiF = 37.635, p < 0.001, p2η = 0.565]. A simple effect test was conducted to show that PoNuS (4.56 ± 0.56) stirred larger responses than PoNuA (2.68 ± 0.62) (p < 0.001) (p < 0.001). NgNuA (5.00 ± 0.68) induced larger responses than NgNuS (3.55 ± 0.50) (p < 0.01).The interaction between taste and vision was substantial . A simple effect test showed, in all brain areas, NgNu and NgPo initiated stronger responses than PoNu and PoNg, respectively . In the mid and right brain areas, responses of PoNu are larger than those of PoNg (p < 0.001).The interaction between brain area and vision was significant , nor among area, taste, and vision .There is no interaction between area and taste . The right hemisphere (−1.53 ± 0.98) responded stronger than the left hemisphere (0.75 ± 0.40). The emotional faces have shown the main effect . PoNu (−0.86 ± 0.62) induced larger amplitudes than NgNu (−0.05 ± 0.54) (p < 0.01). The taste had a main effect as well . Sweet (−0.65 ± 0.63) caused stronger responses than Acidic (−0.13 ± 0.59).Similar analysis has been applied . The maiF = 12.279, p < 0.001, p2η = 0.297]. A simple test has demonstrated that PoNuS (−1.39 ± 0.65) has led to larger responses than PoNusA (−0.32 ± 0.62) (p < 0.01). PoNgS (−1.28 ± 0.74) induced larger responses than PoNgA (0.02 ± 0.75) (p < 0.001).The interaction was significant between vision and taste . A simple test showed that on the right hemisphere PoNu (−2.29 ± 0.97) influenced larger responses than PoNg (−0.78 ± 0.89) (p < 0.001).The interaction effect between the area and vision was commendable .No noticeable interaction effect was shown between area and taste . A simple effect test was conducted to show that PoNuS (0.16 ± 0.41) induced stronger negative response than PoNuA (0.99 ± 0.54) (p < 0.01) on the left hemisphere, whereas NgNuA (0.45 ± 0.35) induced stronger negative response than NgNuS (0.91 ± 0.39) (p < 0.01). NgNuA (0.45 ± 0.35) induced stronger negative response than NgNuS (0.91 ± 0.39) (p < 0.01). On the right hemisphere, on the other hand, PoNuS (−2.94 ± 1.12) induced a stronger negative response than PoNuA (−1.63 ± 0.86) (p < 0.05) and PoNgS (−3.19 ± 1.21) induced a stronger negative response than PoNgA (−1.03 ± 1.06) (p < 0.001).The interaction among vision, taste, and brain areas was significant . PoNu (4.49 ± 0.84) induced more negative responses than NgNu (5.31 ± 0.75) (p < 0.01), while PoNg (3.91 ± 0.88) induced more negative responses than NgPo (4.88 ± 0.77) (p < 0.05) and PoNg induced more negative responses than PoNu (p < 0.05). There was no noteworthy effect of the brain areas , nor was there any significant impact from the taste .A similar analysis has been applied and it was found that the main effect of the emotional faces was evident . A simple effect test was conducted which showed that PoNgS (4.15 ± 0.89) induced more negative responses than PoNuA (4.84 ± 0.81) (p < 0.05); PoNgS (3.50 ± 0.87) induced more negative responses than PoNgA (4.33 ± 0.91) (p < 0.01) and NgNuA (4.89 ± 0.87) induced more negative responses than NgNuS (5.73 ± 0.65) (p < 0.05). Significant interaction was observed between emotional faces and brain areas . A simple test showed that, on the right hemisphere, PoNu (4.71 ± 0.90) induced more negative responses than NgNu (6.33 ± 0.83) (p < 0.001); PoNg (3.76 ± 0.85) induced more negative responses than NgPo (5.45 ± 0.83) (p < 0.01). PoNg induced more negative responses than PoNu (p < 0.01) and NgPo induced more negative responses than NgNu (p < 0.01). No significant interaction was seen between brain areas and taste .The interaction between taste and emotional faces was significant . A simple test has shown that on the left hemisphere, PoNuS (3.90 ± 0.92) induced more negative responses than PoNuA (4.65 ± 0.92) (p < 0.01) and PoNgS (3.77 ± 1.01) induced more negative responses than PoNgA (4.35 ± 1.09) (p < 0.05). Whereas on the right hemisphere, PoNuS (3.22 ± 0.93) induced more negative responses than PoNuA (4.30 ± 0.84) (p < 0.05) and PoNgS (5.92 ± 0.92) induced more negative responses than PoNgA (6.75 ± 0.76) (p < 0.05).The interaction among vision, taste, and brain areas was noteworthy . PoNu (4.10 ± 0.37) induced more positive responses than NgNu (2.74 ± 0.28) (p < 0.001); on the other hand, PoNu induced more positive responses than PoNg (3.32 ± 0.34) (p < 0.01). There was a significant main effect of brain areas . The average responses on the right brain (4.14 ± 0.44) were more positive than those of the mid brain (3.03 ± 0.32) and the left brain (2.88 ± 0.28) (p < 0.01). There was no main effect from taste .The main effect of emotional faces was significant , or between brain areas and taste . Significant interaction was observed between emotional faces and brain areas . A simple effect test was conducted and it showed that PoNu (3.88 ± 0.39) induced more positive responses than NgNu (1.89 ± 0.19) in the whole brain . Additionally, PoNu initiated more positive responses than PoNg (3.02 ± 0.34) on the left brain (p < 0.001). The interaction among vision, taste, and brain areas was significant . This simple test also showed that on the left brain, PoNuS (4.95 ± 0.74) induced more positive responses than PoNuA (2.81 ± 0.29) (p < 0.05).There was no interaction between emotional faces and taste [Our results have shown that gustatory stimuli can influence affective facial processing at behavioral and neural levels. Behavior data has shown that while detecting a negative target face with a positive face as a distractor, the participants perform the task faster with the acidic taste than with the sweet taste. With temporal dynamic ERP analysis, the significant interaction effects between the emotional face and the taste have been observed with P1, N170, and EPN.In previous literature, multiple sensory stimuli have been applied as the congruent or incongruent emotional background to observe the cross-modal interaction for emotion detection at behavioral and physiological levels . UsuallyIn previous ERP studies, the main observation of cross-modal interaction was focused on the early, mid, and late time windows of facial processing . The emoBoth EPN and LPP have been taken as the prototypical emotional-related ERP comments. The EPN has implicated early tagging and prioritized processing . The LPPIt is important to note that the current study has some limitations. The taste stimuli were kept in the mouth throughout the task performance. The subject could not avoid taste adaptation effect, although our pilot study showed that the taste could remain within each block task. It has been shown that visual perception can be modulated by even time interval adaptation . Thus, iTaken together, our finding has provided the behavioral and electrophysiological evidence that sweet and acidic tastes could interact with the emotional face search process. We expect that the cross-modal interaction patterns observed at the electrophysiological level are more sensitive and robust than the behavior level’s response pattern. However, we are also aware of the constraints on generality  mentioneThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by Institute Ethics Committee of Liaoning Normal University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.PL and JJ designed the experiment. JJ and JC performed the experiments. JJ, LW, and PL performed the data analysis. PL wrote the manuscript. LW and JC improved the manuscript. All authors contributed to the article and approved the submitted version.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.


Salmonella outbreaks. The objective of this study was to compare effects of different kinds of natural antimicrobials on Salmonella inactivation in undiluted and diluted tahini during thermal treatment and storage. Results showed that the Weibull model was more suitable to describe the thermal inactivation behavior of S. montevideo CICC21588 in two kinds of tahini than the first-order model. Inactivation curves were concave-upward in undiluted tahini but concave-downward in diluted tahini. During storage of undiluted tahini, 3% oregano oil caused extra 1.44 or 0.80 log CFU/g reductions after 7 days at 25 °C or 4 °C compared to the control and 0.5% citric acid caused an extra reduction of 0.75 log CFU/g after 7 d at 4 °C. For diluted tahini, 2–3% oregano oil and 0.4–0.5% ε-polylysine reduced more populations compared to undiluted tahini. These antimicrobials all inhibited the growth of S. montevideo during 24 h at 25 °C and ε-polylysine had the best effect. Furthermore, these antimicrobials enhanced the Salmonella inactivation in diluted tahini during thermal treatment, and there was less of a synergistic effect of thermal and antimicrobials in undiluted tahini due to less sublethal injured cells caused by heat. This study may provide useful information for Salmonella inactivation in tahini.Tahini and tahini-based products are popular with consumers due to their special flavor and high nutritional values, but often have been linked to  Tahini (sesame seeds paste) is a well-known ready-to-eat product in Middle Eastern and Eastern Mediterranean countries due to its special flavor and high nutritional value . It has Salmonella illness outbreaks and recalls related to the tahini and tahini-based products consumption happened at 2001 [Salmonella illness outbreaks happened at 2001–2019, including Salmonella serotypes, outbreak locations, associated products, and product sources.However, a number of  at 2001 , 2002–20 at 2001 , 2011 [9 at 2001 , 2013 [1 at 2001 , and 201 at 2001 , resultiw) [Salmonella cannot grow in tahini due to its low aw (0.16–0.25), the high fat content may help them persist for a long period in low aw environment [Salmonella contamination in tahini or tahini-based products.Generally, the production of tahini involves soaking, dehulling, roasting, and milling of sesame seeds  and the w) ,17. In aironment ,17 and aironment . FurtherIn recent years, the interest in using natural antimicrobials to control pathogens in the food industry has increased due to their safety. Essential oils (EOs), ε-polylysine (ε-PL) and organic acid, have been widely studied in many food products to control the growth of pathogens, such as fresh lettuce , beef 2, milk 2, almond Salmonella in undiluted or diluted tahini, (2) to evaluate the sublethal injured cells by thermal treatments in undiluted or diluted tahini, (3) to evaluate the inhibitory effects of oregano oil, ε-PL and citric acid in undiluted or diluted tahini during storage and (4) to evaluate the synergistic effects of heating and antimicrobials in undiluted or diluted tahini.The objectives of this study were (1) to determine thermal inactivation kinetics of Salmonella enterica serotypes were used in this study. Salmonella montevideo CICC21588 was obtained from the China Center of Industrial Culture Collection. Salmonella typhimurium CMCC50115 was obtained from National Center for Medical Culture Collections. Salmonella mbandaka NYS5-8 and Salmonella enteritidi R8-1-1 were provided by the College of Food Science and Engineering, Northwest A & F University, Yangling, China. All strains were stored at −20 °C with 20% glycerol. Prior to the experiment, one loopful of frozen culture was streaked onto tryptic soy agar  plates and incubated at 37 °C for 24 h. A single colony was transferred to 30 mL of tryptic soy broth  and incubated at 37 °C for 24 h. The 3 mL of culture was transferred to 300 mL of fresh TSB and incubated at 37 °C to reach the stationary phase. The strain cultures were centrifugated (4000× g for 10 min) and the cell pellets were resuspended using 20 mL of phosphate buffer saline , then centrifugated again and finally resuspended with 300 μL of PBS to obtain a required concentration of 1010–1011 CFU/mL.Four 2 CFU/g, which did not interfere with the count of Salmonella cells grown on TSA. Diluted tahini (50% w/w) was prepared by adding sterilized water, simulating common edible methods at home or restaurant in the Chinese diet [Tahini  was used in this study and the indigenous microbiota in tahini was less than 10Essential oils  including oregano, cinnamon, thyme, tea tree, eucalyptus, peppermint, garlic and turmeric oils were directly added into undiluted and diluted tahini, obtaining the concentration of 1–3%. ε-PL , tea-polyphenol  and citric acid  were dissolved or diluted to reach appropriate concentrations using sterilized water before adding to samples, obtaining the final concentration of 0.05–0.5% for organic acid and 0.05–0.4% for ε-PL or tea-polyphenol. The pH values of samples were measured at room temperature using a pH meter . Salmonella suspension was inoculated on samples to achieve a cell density of about 108–109 CFU/mL. To study the Salmonella inactivation of EOs, ε-PL and organic acid during heating treatments, antimicrobials were added to the cells to obtain the target concentrations . The aw of control group samples was adjusted by adding the same amount of deionized water as treated samples, but without antimicrobials.The thermal treatment was conducted using a heating block system (HBS) as described in a previous study . About 0Five TDT cells filled with 0.8 g inoculated samples were heated to the target temperatures  with a heating rate of 5 °C/min using HBS, which was used to simulate the fast-heating rate in radio-frequency treated products. When the sample temperatures reached the target set-point, one cell was removed and defined as a time-zero sample. All the test cells were immediately cooled using ice-water after heat treatment.Salmonella of different serotypes rather than one specific serotype, Salmonella montevideo CICC21588, Salmonella typhimurium CMCC50115, Salmonella mbandaka NYS5-8 and Salmonella enteritidi R8-1-1 were used and compared. Antimicrobial activity of EOs, ε-PL, tea polyphenol and organic acid against Salmonella was determined by oxford cup assay (ID of 6 mm and OD of 8 mm) according to Bian et al. [w/v agar) and then TSB with 0.75% agar containing Salmonella (106 CFU/mL) was poured onto the plates and allowed to solidify. Afterwards, 100 μL EOs or 200 μL of two concentrations (5% and 10%) of ε-PL, tea polyphenol and organic acid were added into the oxford cups. The plates were incubated at 37 °C for 24 h and the inhibitory zone diameters were determined.To study antibacterial effects of these natural antimicrobials against n et al.  with somv/w). For water soluble antimicrobials, 20%  or 40%  of ε-PL and 80% CA (w/v) were added to undiluted tahini for obtaining final concentrations of 0.2% or 0.4%, and 0.3% or 0.5% (w/w), respectively. The aw of control group samples was adjusted to the same with that of the treated samples. Afterwards, samples were inoculated with Salmonella suspension, obtaining a cell concentration of 108–109 CFU/g and stored at 25 °C or 4 °C for 7 days. S. montevideo CICC21588 populations in inoculated samples were enumerated after 0, 1, 2, 3 and 7 days. Oregano oil was added to undiluted tahini to obtain the final concentrations of 1% and 3% (v/w). For water soluble antimicrobials, 20% ε-PL (w/v) and 20% CA (w/v) were added to diluted tahini to obtain final concentrations of 0.05–0.5% (w/w). Then samples were inoculated with Salmonella suspension, obtaining a cell concentration of 106 CFU/g and stored at 25 °C or 4 °C for 7 days. Bacterial enumeration was conducted at 0 h, 4 h, 7 h, 11 h, 14 h, 24 h, 36 h, 52 h and 72 h and after 7 days.Oregano oil was added to diluted tahini to obtain final concentrations of 1–3%  agar plate  and TSA ectively , becauseS(t) is the survival ratio, N (CFU/g) represents the bacteria population at the time t (min) of isothermal treatment, N0 (CFU/g) represents the bacteria population at the time reaching the target temperature and D is the decimal reduction time (min) at temperature T (°C). The first-order kinetic and Weibull distribution models were used to fit the thermal inactivation data. The first-order kinetic model was defined by the following equation:δ is scale parameter and p is shape parameter, indicating the survival curve’s concavity. p = 1, <1 and >1 represent liner, concave-upward (tailing) and concave-downward (shoulder) inactivation curves, respectively. For both the first-order kinetic and the Weibull distribution models, the coefficient of determination (R2) reflects the goodness of fit.The Weibull model can be expressed as:p = 0.05) using the SPSS statistics 17.0 software . The parameters for the first-order kinetic and Weibull distribution models were obtained using the SPSS Statistics 17.0. All treatments were conducted in triplicate. The statistical significance of differences was evaluated by a one-way analysis of variance (ANOVA) and the Duncan post-hoc test . The thermal inactivation curves of S. montevideo CICC21588 in undiluted or diluted tahini with a heating rate of 5 °C/min in the HBS under different temperatures are shown in p-values obtained from first-order kinetic and Weibull distribution models for describing the thermal inactivation behavior are listed in R2 = 0.973–0.996) and lower root mean square deviation (RMSE = 0.078–0.279) of the Weibull model compared to those  of the first-order kinetic model indicated that the Weibull model was more suitable to describe the thermal inactivation behavior of S. montevideo CICC21588 in undiluted and diluted tahini. In undiluted tahini, δ-values were 10.6, 5.21 and 3.84 at 75 °C, 85 °C and 95 °C, respectively. The high thermal resistance of Salmonella was reported in undiluted sample due to its extremely low aw (0.256) and high fat content (52.6%). He et al. [S. enterica serotypes in peanut butter  at 90 °C were 6.32–12.08 min based on the Weibull model. Krapf and Gantenbein-Demarchi [Salmonella in dark chocolate  and found that the D-value at 90 °C was 25 min. In diluted tahini, δ-values were 16.52, 4.38 and 0.92 at 56 °C, 58 °C and 60 °C, respectively. δ-value decreased with the increase of temperature and aw, similar to results obtained by Gautam, Govindan, Gänzle and Roopesh [e et al.  also fouDemarchi  studied  Roopesh .w = 0.256), p-values of the Weibull model were less than one under three thermal treatments of 75 °C, 85 °C and 95 °C. The inactivation curves were concave-upward, as shown in Salmonella in peanut butter [In undiluted tahini , indicating that cell membrane damages occurred under these treatments. However, the injured cell populations were less than 1 log CFU/g. The similar result was also obtained by Lee et al. [Salmonella enteritidis on the raw shelled almonds without causing significant sublethal injury.e et al. , who fouS. montevideo CICC21588. It has been reported that a mild thermal stress (54–60 °C) causes a large number of injured cells of Escherichia coli O157:H7 in fruit juice, however, more outer membrane damages are observed than cytoplasmic membrane damages, which is inconsistent with the results in this study possibly due to the different strain used [For diluted tahini, a large number of injured cells were produced under the thermal treatments of 56 °C, 58 °C and 60 °C, as shown in ain used ,35.S. montevideo CICC21588 populations were inactivated directly by relatively high temperatures in undiluted tahini without causing significant injured cells and curves were concave-upward. While in diluted tahini, a damage accumulation was observed at the beginning of heating and the inactivation curves were concave-downward.That more sublethal injured cells caused by thermal stress in diluted tahini than in undiluted tahini corresponds to the thermal inactivation curves as described in Salmonella of EOs, ε-PL, tea polyphenol and organic acid using the oxford cup assay. For EOs, as listed in Salmonella serotypes compared to oregano, cinnamon and thyme oils. Furthermore, oregano oil exhibited the strongest antimicrobial activity with inhibition zones of 21.38–24.00 mm. Salmonella with inhibition zones of 16.25–23.50 mm and 13.00–18.50 mm at the concentration of 10%, respectively. For water-soluble antimicrobial agents, as presented in Salmonella with inhibition zones of 25.75–27.70 mm and 24.00–27.20 mm at the concentration of 10%, respectively. The antimicrobials effect of lactic acid was slightly smaller than that of the citric acid. Therefore, oregano oil, ε-PL, tea polyphenol and CA were further selected to control S. montevideo CICC21588 in undiluted and diluted tahini according to the inhibition zones.Organic acids, such as citric acid and lactic acid, are widely used in the food industry to control pathogens or regulate acid flavor, generally regarded as safe . From TaS. montevideo CICC21588 population was significantly reduced by 3.61 log CFU/g in undiluted tahini with 3% oregano oil after 7 days at 25 °C, compared to 2.17 log CFU/g in the control (p < 0.05). After 7 days at 4 °C, 1.53 and 1.47 log CFU/g reductions were observed in undiluted tahini with 1% and 3% oregano oil, respectively, compared to 0.67 log CFU/g in the control (p < 0.05). This result indicated that 3% oregano oil could effectively inactivate Salmonella in undiluted tahini without bringing water into samples, causing no influence on its shelf life. However, 3% oregano oil influenced the flavor of tahini and might not be accepted by consumers. As shown in Salmonella population in undiluted tahini declined faster at higher temperatures during the storage period of 7 d, in accordance with previous research related to LMFs [S. Tennessee cells in peanut butter was significantly lower at 22 °C, compared to 4 °C during the storage period of 14 d. The possible reason probably is that the metabolism of bacteria at the relatively low temperature is lower than that in the high temperature, which helps Salmonella to maintain biological activity.In addition, results showed that the  to LMFs . Park, O to LMFs  found thSalmonella reduction during the storage period of 7 days at 4 °C or 25 °C (p > 0.05). A reduction of 1.42 log CFU/g was observed in undiluted tahini with 0.5% CA after 7 days at 4 °C, compared to the control of 0.73 log CFU/g (p < 0.05). This result indicated that 0.5% CA only caused less than 1 log CFU/g extra reduction compared to the control after 7 days of storage period. Although Al-Nabulsi et al. [Salmonella typhimurium in undiluted tahini by CA compared to our results, but the amount of water brought into tahini is not shown in their study, which would influence the bactericidal effect.The addition of ε-PL and CA may bring water into undiluted tahini, which might accelerate the oxidative rancidity of fat, influencing its shelf life. Therefore, it is necessary to ensure the original concentrations of ε-PL and CA high. From i et al.  have fouS. montevideo CICC21588 in diluted tahini during the storage period of 7 days at 4 °C or 25 °C are shown in w/w) diluted tahini provided a suitable environment for Salmonella growth, posing a great threat to consumer health. After 7 days at 25 °C, samples without antimicrobials suffered deterioration caused by Bacillus subtilis originally existed in tahini (characterized by 16S rRNA gene sequencing), influencing the Salmonella enumeration. During the storage period of 7 days at 4 °C, S. montevideo CICC21588 population remained stable, about 6.19–6.40 log CFU/g in diluted tahini without antimicrobials.The inhibitory effects of oregano oil, ε-PL and CA against S. montevideo CICC21588 and slightly reduced the population at 4 h. 3% oregano oil could inhibit the growth up to 7 h. After 2 or 3 days at 25 °C, the population reached levels of 8.99 or 8.67 log CFU/g with 2% and 3% oregano oil, respectively. While after 7 days at 4 °C, 2–3% oregano oil significantly reduced S. montevideo CICC 21588 population by 0.70–3.57 log CFU/g (p < 0.05) as listed in Bacillus subtilis.As shown in Results also showed that oregano oil was more effective in diluted tahini than in undiluted tahini at 4 °C. In undiluted tahini, 1.47 Log CFU/g reduction was observed with 3% oregano oil compared to 0.67 log CFU/g reduction in the control, while 3.57 log CFU/g reduction in diluted tahini compared to no reduction in the control after 7 days at 4 °C. Diluted tahini used in this study had less fat, more water content and better fluidity than undiluted tahini. It is reported that increased fat content has a negative effect on the antimicrobial efficacy of EOs in model foods, such as peanut paste , soft chS. montevideo CICC21588 for 4 h with 0.05% ε-PL and for 72 h with 0.5% ε-PL. However, after 7 days at 25 °C, the populations reached the maximum numbers of approximately 9 log CFU/g. While at 4 °C, S. montevideo CICC21588 population remained stable for 72 h, about 6 log CFU/g with the addition of 0.05–0.5% ε-PL  of S. typhimurium compared to the control (5.56 h) in pork at 24 °C [S. typhimurium population by 0.68–4.93 log CFU/g in roast beef after 7 days at 4 °C. However, some researchers found that ε-PL could exhibit excellent effect at particularly low concentrations. Geornaras and Sofos [S. typhimurium in TSAYE on day 6 at 4 °C. Furthermore, Geornaras, Yoon, Belk, Smith and Sofos [S. typhimurium populations to low levels even below the detection limit in fat-free/whole fat milk, beef, bologna, ice and vegetables at 12 °C on day 6 and the antimicrobial activity was influenced by food composition. As shown in .5% ε-PL b and aft.5% ε-PL . Furtherat 24 °C . Chang eat 24 °C  found thnd Sofos  found thnd Sofos  also demAs reported by previous studies, ε-PL has been widely used to control pathogens in high water foods but rarely in LMFs. In this study, ε-PL did not exhibit any antimicrobial activity in undiluted tahini b, possibS. montevideo CICC21588 in diluted tahini at 25 °C within 24 h due to the decrease of pH value (pH 5.53-4.95). The population reached to similar levels of 8.85–9.06 log CFU/g at 52 h in the absence or presence of CA. However, after 7 d, samples with CA suffered deterioration and influenced the Salmonella enumeration, indicating that 0.05–0.5% CA had no antibacterial effect on Bacillus subtilis. While during the period of 7 days at 4 °C, 0.3% and 0.5% CA had no antimicrobial activity against S. montevideo in diluted tahini and kept stable populations of about 6 log CFU/g . As shown in S. montevideo CICC21588 of heating combined with 1% oregano oil or 0.4% ε-PL or 0.3% CA. δ- and p-values of the Weibull model for thermal inactivation in combination with antimicrobials. The δ-values at 56 °C combined with 1% oregano oil, 0.4% ε-PL or 0.3% CA were 3.98 min, 4.20 min and 4.92 min, respectively, while the δ-value was 16.46 min by heating alone at 56 °C. Furthermore, the δ-values at 58 °C combined with these antimicrobials were also significantly smaller than that of heating alone at 58 °C (p < 0.05), indicating that oregano oil, ε-PL and CA all significantly reduced the thermal resistance of S. montevideo CICC21588 in diluted tahini.w of tahini samples, which influenced the thermal resistance of Salmonella [w increased from 0.256 of original sample to 0.283 or 0.335 of samples with 0.3% CA or 0.4% ε-PL. Therefore, the aw of control group samples was adjusted to the same with antimicrobials treated samples. Although the δ-values at 75 °C with 0.3% CA or 0.4% ε-PL significantly decreased as compared to the respective control (p < 0.05), significant differences between the population reductions were only observed at 75 °C for 20 min (p < 0.05) and no significant difference at 75 °C for 40–100 min (p > 0.05), indicating that the addition of CA and ε-PL in undiluted tahini slightly reduced the thermal resistance of S. montevideo CICC21588 but the synergistic effects were far lower than those in diluted tahini samples. From lmonella . The aw Escherichia coli O157:H7 [w paste-like foods, such as tahini, peanut paste or nut paste, due to the limited effect in these foods. Espina et al. [E. coli O157:H7 in orange juice. Sun-Ah et al. [Listeria monocytogenes in sous-vide salmon [The different synergistic effects obtained between undiluted and diluted tahini may be due to the lower sublethal injured cells caused by thermal treatments in undiluted tahini compared to that in diluted tahini . Antimic O157:H7 . For org O157:H7 . Furthera et al.  reportedh et al.  found thSalmonella inactivation in undiluted and diluted tahini during thermal treatment and storage. Oregano oil and CA had antimicrobial effects against S. montevideo CICC21588 in undiluted tahini during the storage period of 7 days at 4 °C or 25 °C. However, only high concentration of oregano oil (3%) exhibited a satisfactory effect but might cause undesirable flavor. In diluted tahini, oregano oil, ε-PL and CA all could inhibit the growth of S. montevideo CICC21588 at 25 °C to varying degrees and ε-PL had the best inhibitory effect. These antimicrobials all enhanced Salmonella inactivation effects in diluted tahini during thermal treatment. Further research may focus on developing combination of natural antimicrobials to further enhance synergistic effects and reduce the unfriendly flavor in treated products. In addition, the safety and quality including sensory evaluation of tahini samples with these antimicrobials should be considered in further studies. This study may provide some useful information for thermal inactivation of Salmonella in tahini or tahini-based products and selecting effective appropriate antimicrobials for practical pasteurization.The present study compared effects of hydrophobic antimicrobials, oregano oil and hydrophilic antimicrobials, ε-PL and CA on


I)  and its derivatives 1 , 3 , 7  showed higher anti-TMV activity in vivo at 500 mg/L than commercial ribavirin . Some compounds exhibited insecticidal activities against Plutella xylostella, Mythimna separate and Spodoptera frugiperda. Especially, compounds 7 and 27 displayed excellent insecticidal activities against Plutella xylostell  even at 0.1 mg/L. Additionally, most echinopsine derivatives exhibited high fungicidal activities against Physalospora piricola and Sclerotinia sclerotiorum.Based on the broad-spectrum biological activities of echinopsine and acylhydrazones, a series of echinopsine derivatives containing acylhydrazone moieties have been designed, synthesized and their biological activities were evaluated for the first time. The bioassay results indicated that most of the compounds showed moderate to good antiviral activities against tobacco mosaic virus (TMV), among which echinopsine ( As a well-studied plant virus, tobacco mosaic virus (TMV) belongs to single-stranded RNA virus of the family togaviridae4 and it can infect 268 species of plants in 38 families, such as tobacco, tomato, pepper, cucumber, causing their leaves to grow spots, wither and even leading to yield reduction7. Although commercially available plant virus inhibitors ningnanmycin and ribavirin are widely used to control TMV, their inhibitory effects are lower than 60%8. Thus, the development of efficient alternative TMV inhibitors is still in great request.Plant virus diseases can be caused by more than 900 viruses, which reduce grain production and lead to huge economic losses all over the worldetc10. Song et al. reported that the EC50 value of purine nucleoside derivative for the inactivating activity against TMV was 48 mg/L, which was better than that of ningnanmycin (88 mg/L)11. Li et al. first found that phenanthroindolizidine alkaloid, (R)-antofine, exhibited a good inhibitory effect against TMV12. Wang et al. found some β-carboline analogues7, hemigossypol13, dehydrobufotenine derivatives14, pityriacitrin marine alkaloids15, pulmonarin alkaloids16 and hamacanthin derivatives17 exhibited higher anti-TMV activities than ningnanmycin. Many other natural alkaloids derivatives were also developed as potential TMV inhibitors27. Although a variety of natural product derivatives have been found to exhibit high anti-TMV activity, few of them have been applied successfully in agriculture. Thus, it is necessary to discover novel natural TMV inhibitors with diverse structures.Natural products are an important source of plant virus inhibitor discovery. Compared with traditional synthetic plant virus inhibitor, plant virus inhibitor derived from natural products have many advantages, including low toxic, environmentally friendly, easy to decompose and specific to target species, Echinops sphaerocephalus L., the root of which was used as traditional Chinese medicine for treatment of deep-rooted breast carbuncles, ulcer, sodoku and breast milk stoppage. Although the bioactivity of Echinops sphaerocephalus L. extract has been widely studied28, the biological activity of echinopsine is still not clear. The anti-TMV activity of echinopsine has not been reported so far. However, a variety of natural alkaloids containing echinopsine moiety showed herbicidal, insecticidal, bactericidal, anti-tumor, antifungal and antifeedant activities, etc.  showed good bactericidal, herbicidal or insecticidal activities, such as benquinox34, saijunmao35, metaflumizone36 and diflufenzopyr37. Based on the high biological activities of echinopsine and acylhydrazone structure, in order to find echinopsine derivatives with higher anti-TMV activities and summarize their structure–activity relationship, a series of echinopsine derivatives containing acylhydrazone moieties were designed, synthesized and characterized in this work . The melting points were determined on an X-4 binocular microscope melting point apparatus without correction.39 and the methods can also be found in the “The anti-TMV, insecticidal and fungicidal activities of the synthesized compounds were tested using our previously reported methodsRibavirin , chlorothalonil , carbendazim  and other reagents were purchased from commercial sources and used as received. All anhydrous solvents were dried and purified according to standard techniques. The synthetic routes were given in Fig. 40.Echinopsine was prepared according to literatureA , Cs2CO3  and acetonitrile (300 mL). The reaction suspension was stirred for half an hour at room temperature and methyl iodide  was added. The mixture was refluxed for 6 h. Water (200 mL) was added and the reaction mixture was extracted with ethyl acetate for three times. The organic phases were combined, washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue was subjected to column chromatography eluted with dichloromethane / methanol  to give compound B as a white solid ; mp 189–190 °C. 1H NMR  δ 8.53 , 8.50 , 7.71 , 7.48–7.42 , 3.93 , 3.89 ; 13C NMR  δ 174.5, 166.7, 150.1, 139.9, 132.9, 129.1, 128.0, 125.5, 115.7, 110.7, 52.3, 41.5; HRMS (ESI) calcd. for C12H12NO3 [M+H]+ 218.0812, found 218.0811.To a round bottomed flask (500 mL) were added compound B  and hydrazine hydrate  were dissolved in methanol (300 mL). The mixture was refluxed for 8 h. The mixture was concentrated under reduced pressure until a large amount of solid precipitated. The mixture was filtered, washed with a small amount of methanol to give compound C as a white solid ; mp 273–275 °C. 1H NMR  δ 10.67 , 8.86 , 8.34 , 7.88–7.81 , 7.55 , 4.58 , 4.02 ; 13C NMR  δ 175.1, 163.9, 148.3, 139.8, 133.0, 126.8, 126.0, 125.1, 117.5, 110.2, 41.2; HRMS (ESI) calcd. for C11H12N3O2 [M+H]+ 218.0924, found 218.0920.Compound C (3 mmol), one benzaldehyde from D1–D27 (3 mmol) and p-methylbenzene sulfonic acid (0.6 mmol). The reaction suspension was refluxed for 8 h. The reaction suspension was cooled to room temperature and partial methanol was evaporated under reduced pressure until a large amount of precipitation precipitated. The precipitate was filtered and washed several times with cool methanol to afford compounds 1–27. Data for compounds 1–27 can be found in the “To a round bottomed flask (100 mL) were added methanol (50 mL), compound B was carried out according to literature41 , 3 , 7  were obviouly higher than that of commercialized anti-plant virus agent ribavirin .The results of anti-TMV activities in vivo  of echinopsine and compounds 1–14), the electronic effect of the substituents on phenyl has an effect on the anti-TMV activities. The introduction of electron-withdrawing and electron-donating substituents led to the decrease of anti-TMV activities. For example, the structure–activity relationship shows the following: non-substituent (1) > p-hydroxyl (4) > p-phenoxy (8) > p-methylthio (9) > p-methoxy (5), non-substituent (1) > p-bromo substituent (13) > p-methylsulfonyl (10) > p-fluorosubstituent (11) > p-chloro substituent (12). However, there is no obvious linear relationship between anti-TMV activity and electron-donating and electron-withdrawing ability. For example, the structure–activity relationship shows the following: p-bromo substituent (13) > p-trifluoromethoxy substituent (14) > p-fluoro substituent (11) > p-chloro substituent (12), while the activity of compound 13 at 500 mg/L  is equivalent to that of ribavirin. The size of substituents also has an effect on the activities. For example, the activities of derivatives with a p-tert-butyl (3) and p-phenyl substituent (7) are higher than that with no substituents (1). Mono substitution or multi substitution on the benzene ring affected anti-TMV activity to a certain extent, for instance, compared with compounds 5 , the disubstituted compound 6  exhibited higher activity.For derivatives containing substituted phenyl (15–26 containing heterocyclic ring reduced obviously compared with that of compounds containing benzene ring (1). Compound 22, showed the highest activities at 500 mg/L , which was equivalent to that of ribavirin. However, the activity was greatly reduced when the benzene ring was changed to an anthracene ring, that is, the activities of compound 27  was lower than that of compound 1 .The anti-TMV activities of compounds 3 showed the highest activities at 500 mg/L , which is significantly higher than that of ribavirin. Thus, this compound (3) can be selected as an anti-TMV candidate drug for further study.Compound 1–27 and echinopsine against Lepidoptera pests, such as diamondback moth (Plutella xylostella), cotton bollworm (Helicoverpa armigera), corn borer , oriental armyworm (Mythimna separata) and fall armyworm (Spodoptera frugiperda (J. E. Smith)) are listed in Tables The insecticidal activities of the target compounds P. xylostella. For derivatives containing substituted phenyl (1–14) and anthranyl (27), compounds 7, 14 and 27 exhibited 100 ± 0% mortality at 600 mg/L. In particular, compounds 7 and 27 still showed 67 ± 6% and 53 ± 6% mortality even at 0.1 mg/L. Compounds 15, 21, 23, 25 and 26 containing heterocyclic ring also showed 100 ± 0% mortality at 600 mg/L, which was better than echinopsine (90 ± 0% at 600 mg/L)  against M. separata than that of echinopsine (70 ± 0% at 200 mg/L). Especially, compounds 9 and 24 showed 20 ± 0% and 30 ± 0% mortality at 50 mg/L. In addition, the compounds 5, 21, 24, and 25 showed much higher activities (100 ± 0% at 200 mg/L) against S. frugiperda than that of echinopsine (50 ± 0% at 600 mg/L). Especially, compounds 24 still showed 17 ± 6% mortality at 50 mg/L  and its derivatives 1, 3, 7 showed higher anti-TMV activities than those of ribavirin, which can be used as lead structures for the development of anti-TMV drugs. Some compounds exhibited moderate to good insecticidal activity to Supplementary Information.