Rubus idaeus L.) have not been investigated systematically. In the present study, the phenolic profiles of leaves (LE), fruit pulp (FPE), and seed extracts (SE) in raspberry were analyzed by HR-HPLC-ESI-qTOF-MS/MS method, and their antioxidant activities and digestive enzymes inhibitory abilities were also investigated. The molecular docking analysis was used to delineate their inhibition mechanisms toward type II diabetes related digestive enzymes. Regardless of LE, FPE, or SE, 50% methanol was the best solvent for extracting high contents of phenolic compounds, followed by 50% ethanol and 100% methanol. The LE of raspberry displayed the highest total phenolic content (TPC) and total flavonoid content (TFC). A total of nineteen phenolic compounds were identified. The quantitative results showed that gallic acid, ellagic acid, and procyanidin C3 were the major constituents in the three extracts. The various parts extracts of raspberry all exhibited the strong antioxidant activities, especially for LE. Moreover, the powerful inhibitory effects of the three extracts against digestive enzymes (α-glucosidase and α-amylase) were observed. The major phenolic compounds of the three extracts also showed good inhibitory activities of digestive enzyme in a dose-dependent manner. The underlying inhibitory mechanisms of the main phenolic compounds against digestive enzymes were clarified by molecular docking analysis. The present study demonstrated that the various parts of raspberry had strong antioxidant activities and inhibitory effects on digestive enzymes, and can potentially prevent oxidative damage or diabetes-related problems.The anti-oxidative phenolic compounds in plant extracts possess multiple pharmacological functions. However, the phenolic characterization and in vitro bio-activities in various parts of raspberry ( Nowadays, incidences of metabolic syndrome-related diseases have rapidly increased all over the world, such as oxidative damage, diabetes, obesity, hypertension, and cardiovascular disease . Food nuRubus idaeus L.) belongs to the Rosaceae family, which is widely cultivated in Asia, Europe, and North America . α-glucosidase was performed as the previous reported method with modifications + and its fragments ions at m/z 291.0503 [C15H14O6+H]+, which can be tentatively identified as three isomers of procyanidin dimers. Among them, compound 7 can be identified as procyanidin B2 according to MS spectrum data and the retention time of the standard. Compounds 6 and 8 showed the parent ions [M+H]+ at 867 and its MS/MS fragments ions at 579.1502 [C30H26O12+H]+ and 291.0155 [C15H14O6+H]+, respectively, which can be likely identified as two isomers of procyanidin trimer. By comparing the MS fragments information and the retention time of the standard, compound 8 can be identified as procyanidin C3. Ellagic acid pentoside was easily identified by the parent ion [M+H]+ at m/z 435 and its ion fragment at m/z 303.0142 [C14H6O8+H]+. Compound 10 was identified as rutin by the parent ion [M+H]+ at m/z 611 and its ions fragments m/z at 303.0563 [M-glc+H]+ and 163.1221 [M-C15H10O7]+. Compound 11 was kaempferol-galactoside-glucoside by its ions [M+H]+ at m/z 611 and two fragments ions at m/z 449.1338 [M−glc+H]+ and 287.0716 [C15H10O6+H]+. Compound 13 with [C14H6O8+H]+ ion at m/z 303 was easily identified as ellagic acid. Compounds 14 and 15 with [M+H]+ ions at m/z 465 giving MS/MS fragments ions m/z at 303 [C15H10O7+H]+ and 161 [M-C15H10O7+H]+ were very likely to be two isomers of quercetin glucosides. By comparing the retention time of two standard compounds, they were identified as quercetin-3-O-galactoside and quercetin-3-O-glucoside, respectively. Compound 16 with [M+H]+ ion m/z at 435 giving fragment ion at 303.0500 [C15H10O7+H]+ was identified as avicularin. Kaempferol-7-O-glucuronide (Compound 17) was easily identified by the [M+H]+ ion m/z at 463 and its MS/MS fragment ion at 287.0546 [C15H10O6+H]+. Quercetin-7-O-glucuronide (Compound 18) was identified by the ion [M+H]+ at m/z 479 and its fragment ion at 303.0506 [C15H10O7+H]+. Compound 19 was easily identified as kaempferol-3-O-glucuronide by the [M+H]+ ion at m/z 463 and its ion fragment at [C15H10O7+H]+.The phenolic compositions in different samples were identified by comparing their retention times and MS spectrum data with the authentic standards and reported data . Table 1O-glucuronide, quercetin-7-O-glucuronide and kaempferol-3-O-glucuronide) except for rutin and avicularin were only found in LE and SPE of raspberry. Meanwhile, it can be found that there also existed high contents of some invididual phenolic compounds in SE, such as gallic acid (127.15 ± 3.21 μg/g DW), procyanidin C3 (29.12 ± 0.11 μg/g DW) and ellagic acid (48.32 ± 0.23 μg/g DW), which showed that SE may be used as a good food ingredient enriched in phenolic compounds. Qin et al. (2018) have confirmed that high levels of gallic acid and ellagic acid existing in the form of bound phenolics were found in raspberry fruit and seed extracts, which was consistent with the results of our study [The quantification results of fifteen phenolic compounds are shown in ur study .+, and OH− free radical scavenging activities and ferric reducing antioxidant activity (FRAP) were used to perform the antioxidant activity assays. In order to fully reflect the antioxidant capacity of the samples extracts, four well-known chemical test methods including DPPH, ABTS50 values of the samples/controls. Notably, the lower the IC50 values indicated, the stronger the antioxidant activity. For the DPPH assay, the IC50 value of LE (5.60 ± 0.31 μg/mL) was lower than that of Vc (16.80 ± 1.31 μg/mL) and Trolox (39.90 ± 0.67 μg/mL), about one-third of that of FPE (18.71 ± 1.35 μg/mL), and one-sixth of that of SE (32.33 ± 1.42 μg/mL). LE also exhibited the strongest ABTS+ free radical scavenging activity. The IC50 value of LE (3.70 ± 0.17 μg/mL) was lower than that of Trolox (41.50 ± 1.97 μg/mL), FPE (29.43 ± 1.83 μg/mL), and SE (33.15 ± 2.19 μg/mL). Meanwhile, three various parts’ extracts in raspberry  also exhibited the strong OH− free radical scavenging activity, and their corresponding IC50 values were 3.01 ± 0.13 μg/mL, 6.40 ± 0.27 μg/mL, and 8.61 ± 0.52 μg/mL, respectively. The IC50 value of LE was also lower than that of Vc (19.83 ± 0.37 μg/mL) and Trolox (33.91 ± 1.82 μg/mL). The insets of It was found that LE exhibited the strongest antioxidant activity, followed by FPE, and SE performed the weakest antioxidant activity. All of the samples’ extracts showed the antioxidant activities in a concentration-dependent manner A–D. The r = −0.807, p < 0.01; ABTS vs. TPC, r = −0.875, p < 0.01; OH− vs. TPC, r = −0.792, p < 0.01), suggesting that the phenolic compounds significantly contributed to the antioxidant activities of three samples extracts. Qin et al. (2018) reported that the antioxidant activities of raspberry fruits and seeds extracts were strongly correlated with the released phenolic contents during in vitro digestion [50 values of 2.24 μg/mL and 4.80 μg/mL, respectively [The correlation co-efficient analysis results between the phenolic contents at different concentrations and antioxidant activities of the three extracts clearly verified that there was a good correlation between these two parameters  was evidently stronger than that of FPE (IC50 = 265.41 ± 20.7 μg/mL) and SE (IC50 = 218.5 ± 17.53 μg/mL) (p < 0.01). However, the α-glucosidase inhibition potency of acarbose (IC50 = 267.47 ± 19.72 μg/mL) was lower than that of LE (p < 0.05). FPE also possessed good inhibition activity against α-glucosidase, but there is no statistically significant difference with acarbose (p > 0.05). 50 value for α-glucosidase inhibition activity of PC was 93.37 ± 5.79 μg/mL, which was significantly lower than that of GA (IC50 = 590.34 ± 15.71 μg/mL) and EA (IC50 = 976.32 ± 41.72 μg/mL) (p < 0.01). For α-amylase inhibition activity assay, LE have the lowest IC50 values (IC50 = 118.42 ± 2.79 μg/mL), followed by FPE (IC50 = 388.27 ± 2.47 μg/mL) and SE (IC50 = 891.12 ± 25.71 μg/mL). Moreover, the IC50 value of acarbose (IC50 = 442.23 ± 19.74 μg/mL) was higher than that of LE (p < 0.05), which indicates that LE may be used as a potential good anti-diabetic resource. Among the three main phenolic compounds, PC showed the strongest α-amylase inhibition activity (IC50 = 92.31 ± 3.51 μg/mL), followed by EA (IC50 = 516.73 ± 25.29 μg/mL), and GA (IC50 = 397.37 ± 12.37 μg/mL) (p < 0.01). α-glucosidase inhibition potency vs. TPC, r = 0.781, p < 0.05; α-amylase inhibition potency vs. TPC, r = 0.854, p < 0.01). Many researches have confirmed that phenolic-rich extracts from leaf-tea, edible fruits, and natural products possess good inhibitory ability on digestive enzymes [α-glucosidase inhibitory capacity [α-glucosidase inhibitory capacity, but ellagic acid possessed the weakest inhibitory ability of α-glucosidase, which was consistent with the results of our study [The correlation analysis results between the TPC and two digestive enzymes inhibition potency clearly verified that there was a good positive correlation between these two parameters  with five amino acid residues . The distances of H-bonds ranged from 1.896 Å to 2.600 Å. EA formed six H-bonds (the shortest distance was 1.700 Å and the longest distance was 2.433 Å) with five amino acid residues . PC formed thirteen H-bonds within 4 Å (the distances ranged from 1.776 Å to 2.665 Å) with eleven amino acid residues . However, it was found that acarbose formed sixteen H-bonds within 4 Å (distances ranged from 1.776 Å to 2.732 Å) with ten amino acid residues . Some amino acid residues  of α-glucosidase at least interacted with the above three investigated molecules, suggesting that these amino acid residues may play important roles in exerting the catalytic reaction of α-glucosidase.Molecular docking analysis was further done to analyze the digestive enzymes inhibitory mechanisms of the main phenolic compounds including GA, EA, and PC. The results clearly revealed that the structures of phenolic compounds significantly affect their inhibitory effects on α-amylase with the investigated phenolic molecules. The C-Scores of three main phenolic molecules and acarbose were all ≥ 4. α-amylase and gallic acid. The five amino acid residues with the active site were Asp 197, Arg 195, Glu 233, His 299, and His 305, respectively. The average distance of six H-bonds was 2.115 Å. Ellagic acid formed H-bonds with four amino acid residues with the active site, namely Asp 197, Asp 300, Arg 195, and His 299. The distances of H-bonds ranged from 1.937 Å to 2.735 Å. However, procyanidin C3 formed nine H-bonds with the active sites of six amino acid residues . The shortest distance was 1.864 Å and the longest distance was 2.724 Å. It was found that acarbose formed eleven H-bonds (the distances ranged from 1.776 Å to 2.732 Å) with seven amino acid residues, namely, Asp 300, Gln 63, Glu 240, Gly 306, Tyr 151, Lys 200, and His 305.α-glucosidase or α-amylase, the higher numbers of H-bonds and amino acid residues with active site were formed in the complex of digestive enzymes and acarbose. As a result, acarbose showed very good inhibitory effect of α-glucosidase and α-amylase. The numbers of H-bonds and amino acid binding sites formed by the two phenolic molecules (GA and EA) docked with α-glucosidase were equal, but there existed significant differences in inhibitory capacities of α-glucosidase. It may be due to that different interaction sites (amino acid residues) existed in between these molecules and α-glucosidase. Both of GA and EA all interacted with the amino acid residues His 351, Asp 215, Asp 352, and Arg 213 of α-glucosidase. EA also interacted with the amino acid residue Glu 411 of α-glucosidase. Some researchers have confirmed that some active sites (Glu 411) of α-glucosidase may inhibit the catalytic activity of this enzyme [α-glucosidase inhibitory effect. At least three investigated molecules formed H-bonds with the amino acid residues of Asp 69, Asp 215, and Asp 352, which may exert its α-glucosidase inhibitory effect. Hua et al. (2018) have also reported that Asp 69, Asp 215, and Arg 442 were the important residues involved in H-bond formation during the binding with α-glucosidase [α-glucosidase played important roles in exert its α-glucosidase inhibitory effect [α-amylase was: Acarbose (7) = PC (7) > GA (5) > EA (4). The order of H-bonds number was: Acarbose (11) > PC (9) > GA (6) > EA (5). Consequently, the order of the docking T-Score was: Acarbose > PC > GA > EA, which was consistent with the results of α-amylase inhibition activity. Moreover, at least three investigated molecules interacted with the amino acid residues Asp 197 and His 305 of α-amylase, indicating that these two amino acid residues may play critical roles in the catalytic reaction of α-amylase. Many reports have confirmed that the amino acid residues Asp 197 and His 305 played critical roles in the catalytic reaction of α-amylase [It can be found that the numbers and distances of the H-bonds play important roles in exerting the catalytic reaction of the complex of digestive enzymes and ligands, and thereby cause the differences in inhibitory activity of digestive enzymes. Regardless of acarbose docked with s enzyme . Consequcosidase . Zhang ey effect . Similar-amylase ,41. The -amylase . The resThe solvents have significant impacts on the extraction of total phenolics and total flavonoids in various parts of raspberry. Fifty percent methanol was the best solvent for extracting high contents of phenolic and flavonoid compounds. The LE in raspberry displayed the highest TPC and TFC. A total of 19 phenolics compounds were identified. Gallic acid, ellagic acid, and procyanidin C3 were the main phenolic compositions existing in various parts of raspberry. Higher levels of phenolic compounds in raspberry showed the stronger anti-oxidant activities and inhibitory potency of digestive enzymes. The major phenolic compounds that were found in various parts of raspberry all showed good digestive enzyme inhibitory activities, especially for PC. Molecular docking analysis revealed the underlying inhibition mechanisms of these three main phenolic compounds against digestive enzymes, and the theoretical analysis data explained the experimental results very well.


Piper aduncum seedlings were inoculated by spores of the arbuscular mycorrhizal fungi (AMF) Rhizophagus clarus and Claroideoglomus etunicatum. P. aduncum seedlings were inoculated by spores of R. clarus and C. etunicatum and then, development parameters, root colonization, lipoxygenase (LOX) activity, and essential oil (OE) chemical composition were monitored at 30, 60 and 90 days’ post-inoculation (dpi). The inoculation had influenced the plant height and root length at 30 and 90 dpi and microscopic analysis of roots showed the presence of hyphae, arbuscules and vesicles in the inoculated plants. Phenylpropanoids and sesquiterpene hydrocarbons were the main compounds in the EO. In the leaves, the concentration of phenylpropanoids showed a decrease, mainly at 60 dpi, with increased sesquiterpene hydrocarbon production. The main compounds were dillapiole, myristicin, and germacrene D; the dillapiole concentration decreased in all treatments. LOX activity had an increase in the leaves and roots at 90 dpi. These results suggest that alterations in the secondary metabolites of P. aduncum can be induced by its mechanisms of resistance during AMF interaction.The aim of this study was to evaluate the changes in the production of secondary metabolites  Piperaceae have wide distributions in tropical and subtropical regions, and are known as a pantropical family with approximately 2700 species mainly of the genus Piper  has demonstrated several biological properties, such as antifungal , and kept in a greenhouse under 70% shading. The commercial nutrient solution (Biofert Root) was applied to promote root development and reapplied after 15 days, and the cuttings were moistened daily. After 21 days, the roots had developed, and seedlings were transplanted into polypropylene bags of approximately 9 cm in diameter, on a commercial substrate containing a mixture of limestone, castor oil, bone meal, and expanded vermiculite type B.Rhizophagus clarus and Claroideoglomus etunicatum) were obtained from rhizosphere soil samples from the southeast Pará State, Amazon region (Brazil). They were multiplied in a greenhouse in sterile sand, using Brachiaria brizantha as trap culture  and AMF (inoculated by AMFs). Roots and leaves were collected at 30, 60 and 90 days post inoculation (dpi) to monitor the mycorrhizal colonization, plant development, secondary metabolites, and LOX activity. All analyzes were performed in biological triplicates.® RM 2245, Nussloch, Germany), the sections were stained with safranine and astra blue . Photomicrographs were obtained using in Cannon digital camera (model A65015), coupled to a Zeiss microscope (model 426126.)For the visualization of mycorrhizal colonization, usual techniques in plant anatomy were employed , number of leaves, plant basal stem (mm), number of nodes, root length (cm), and the fresh mass of leaves and roots (g) for each plant per replicate. The fresh leaf biomass production was based in the total weight per plant and the fresh root biomass in the total weight of the roots per plant.P. aduncum were obtained by simultaneous distillation–extraction process using a Likens-Nickerson apparatus for 2 h and n-pentane (3 mL) as solvent. After extraction, an aliquot (1.0 μL) of the organic phase was analyzed by gas chromatography. Qualitative analysis was carried out on a GC–MS (Shimadzu QP2010 plus instrument) under the following conditions: Rtx-5MS silica capillary column (30 m × 0.25 mm × 0.25 mm film thickness); programmed temperature, 60–240 °C (3 °C/min); injector temperature, 200 °C; carrier gas, helium, adjusted to a linear velocity of 1.2 mL/min; injection type, splitless; split flow was adjusted to yield a 20:1 ratio; septum sweep was a constant 10 mL/min; EIMS, electron energy, 70 eV; temperature of the ion source and connection parts, 200 °C. The retention index was calculated for all the volatile constituents using a homologous series of n-alkanes   of The substrate was prepared using 78 μL of linoleic acid  and 90 μL Tween 20 (Sigma-Aldrich), mixed with 10 mL of boiling water and a few drops of sodium hydroxide (0.5 N). The final volume was adjusted to 25 mL, resulting in a sodium linoleate solution (10 mM), which was stored at − 20 °C. The LOX activity determination was carried out with 5 μL of crude leaf extract and 50 μL of sodium linoleate (10 mM), mixed with 1950 μL of sodium phosphate buffer (50 mM) at pH 6.5. The absorbance at 234 nm for the reaction was monitored for 60 s, using a UV–Visible spectrophotometer -β-ocimene (0.12%), terpinen-4-ol (0.11%), α-copaene (0.25%) and n-tetradecanol (1.54%). At 60 dpi, there was an increase in the contents of β-caryophyllene (2.16–5.58%), germacrene D (3.19–5.49%), myristicin (2.71-4.70%), and a greater decrease of dillapiole (83.57–44.73%). Several monoterpenes and sesquiterpenes were produced only by inoculated plants, such as (Z)-β-ocimene (1.10%), (E)-β-ocimene (2.84%), and β-selinene (1.33%). At 90 dpi, only inoculated plants produced the monoterpene allo-ocimene (1.64%), also displaying a decrease in the concentrations of dillapiole (48.66–39.36%), myristicin (3.26–2.41%), and apiol (2.54–1.83%).At 30 dpi, quantitative and qualitative changes were observed in the leaves. Quantitatively, a decrease in the dillapiole content (93.74–86.11%), and an increase of β-caryophyllene 0.27–2.62%) and germacrene D production (1.28-2.78%) was observed  was identified only in the roots of inoculated plants ,4-diene (1.34%). The phenylpropanoids myristicin (19.25–18.47%) and dillapiole (54.27–51.96%) showed a decrease as well as the sesquiterpene hydrocarbons δ-elemene (0.40–0.29%) and α-copaene (1.91–1.63%). The minor compounds produced in the roots showed a behavior different than leaves, with a decrease of monoterpenes (Z)-β-ocimene (0.86–0.68%) and (E)-β-ocimene (0.77–0.57%) in inoculated plants  and a slight increase in the production of myristicin 18.38–19.93%) and elemicin (2.22–3.13%) was observed  occurred, presumably due to the recognition of signaling molecules of the plant by AMF, after the exchange of biochemical signals between fungus and host  and rosemary  inoculated with G. clarum spores, which showed an increase of 45.49 and 25.93%, respectively -β-ocimene, (Z)-β-ocimene and allo-ocimene -β-ocimene and (Z)-β-ocimene showed a gradual increase during inoculation by AMF in the leaves and a decrease in the roots. These compounds are emitted by plants to response to herbivore attack and changes in abiotic factors  (Mora-Romero et al. P. divaricatum seedlings infected by Fusarium solani f. sp. piperis (Meireles et al. LOX is involved in the production of volatile compounds in leaves and roots such as alcohols, ethers, and aldehydes, which are considered both signaling and defense compounds (Liavonchanka and Feussner P. aduncum presents many biological activities reported in the literature, which are attributed to the phenylpropanoid dillapiole (Bernard et al.


V1.8-expressing neurons. Importantly, transcriptomic analysis shows that these neuronal populations are nonoverlapping. Using in vivo GCaMP imaging in live mice we show that the vast majority of acute cold-sensing neurons activated at ≥1 °C do not express NaV1.8, and that the loss of NaV1.8 does not affect acute cold-sensing behavior in mice. Instead, we show that cold-responding neurons are enriched with Trpm8 as well as numerous potassium channels, including Kcnk9. By contrast, NaV1.8-positive neurons signal prolonged extreme cold. These observations highlight the complexity of cold sensing in DRG neurons, and the role of NaV1.8-negative neurons in cold sensing down to 1 °C.The cellular correlate for cold sensing has been ascribed to either Trpm8-expressing or Na V1.8 and NaV1.9, as well as the TRP channel Trpm8, have been shown to contribute to cold sensation in mice. Surprisingly, transcriptional profiling shows that NaV1.8/NaV1.9 and Trpm8 are expressed in nonoverlapping neuronal populations. Here we have used in vivo GCaMP3 imaging to identify cold-sensing populations of sensory neurons in live mice. We find that ∼80% of neurons responsive to cold down to 1 °C do not express NaV1.8, and that the genetic deletion of NaV1.8 does not affect the relative number, distribution, or maximal response of cold-sensitive neurons. Furthermore, the deletion of NaV1.8 had no observable effect on transient cold-induced (≥5 °C) behaviors in mice, as measured by the cold-plantar, cold-plate (5 and 10 °C), or acetone tests. In contrast, nocifensive-like behavior to extreme cold-plate stimulation (−5 °C) was completely absent in mice lacking NaV1.8. Fluorescence-activated cell sorting (FACS) and subsequent microarray analysis of sensory neurons activated at 4 °C identified an enriched repertoire of ion channels, which include the Trp channel Trpm8 and potassium channel Kcnk9, that are potentially required for cold sensing above freezing temperatures in mouse DRG neurons. These data demonstrate the complexity of cold-sensing mechanisms in mouse sensory neurons, revealing a principal role for NaV1.8-negative neurons in sensing both innocuous and acute noxious cooling down to 1 °C, while NaV1.8-positive neurons are likely responsible for the transduction of prolonged extreme cold temperatures, where tissue damage causes pan-nociceptor activation.The ability to detect environmental cold serves as an important survival tool. The sodium channels Na V1.8 and NaV1.9, have all been associated with robust cold-insensitivity phenotypes. The genetic deletion of Trpm8 significantly attenuates cold sensitivity in mice, and ablation of the Trpm8-expressing population of neurons almost completely abolishes cold sensitivity down to 0 °C  were used to investigate the relative distribution of cold-sensitive neurons within mouse DRG that innervate the sciatic nerve (lumbar regions L3–L5)  mice, as Cre replaces both copies of the Scn10a gene, while keeping the promoter region intact; therefore, these mice retain no residual NaV1.8 function  did not express NaV1.8 as defined by reporter fluorescence  of neurons negative for NaV1.8 expression  and 79.28% (941/1187) of neurons being observed in DRG from NaV1.8Cre/+ and NaV1.8Cre/Cre null mice, respectively .To identify cold-sensitive neurons in vivo, we used a previously developed in vivo imaging technique to study the responses of individual DRG neurons in situ . Mice cos L3–L5) A. Importehaviors , 10. In function . The appfunction B. On averescence C and D. pression E. Furtheull mice F and G. −1, every 5 s, from an initial baseline of 32 °C. The application of this protocol identified several populations of cold-sensitive neurons, exhibiting discrete activation thresholds ranging from 25 to 5 °C  to study the responses of threshold-specific cold-sensitive neurons. To investigate the activation thresholds of specific cold-sensitive neurons, we applied a cooling protocol that stepped down by ∼5 °C, at a rate of 8 °C.s to 5 °C A. Intere to 5 °C B. As obsull mice C and D.SI Appendix, Fig. S2 A and B). To further investigate the response profiles of individual cold-sensitive neurons, we applied a stepped cooling protocol, immediately followed by a drop cooling protocol to the plantar surface of the mouse, and measured the resulting change in GCaMP3 fluorescence in WT mice . In keeping with our previous results, we were able to identify similar activation thresholds of cold-sensitive neurons, with all cold-sensitive neurons being activated at 1 °C . Furthermore, the majority of cold-sensitive neurons were small, ranging in size between 100 and 450 μm2, and exhibited the same threshold of activation whether stimulated by a stepped or drop cooling protocol .Next, we investigated how populations of threshold-specific cold-sensitive neurons responded to the intensity of the cold stimulus applied. We applied a series of transient cooling steps, ranging from 1 to 25 °C, to the plantar surface of the mouse, with each lasting 5 s, from a baseline of 32 °C. Using this protocol, we were able to identify discrete populations of cold-sensitive neurons that exhibited specific thresholds of activation, ranging from 5 to 25 °C A. We fouV1.8-null, as well as NaV1.8-diphtheria toxin (DTA) mice, where the NaV1.8-expressing population of sensory neurons has been ablated through the action of diphtheria toxin  caused a decrease in the latency of paw withdrawal , the average temperature at time of paw withdrawal was 19.61 (±1.13) °C for WT and 19.66 (±1.34) °C for NaV1.8-null mice. Furthermore, the deletion of NaV1.8 caused no change in the cold-plate response at either 10 or 5 °C, or the acetone response, compared with WT; however, the ablation of NaV1.8-expressing neurons led to a significantly increased response (V1.8Cre/+ mice displayed no difference in the cold-plate response (10 or 5 °C) compared with NaV1.8Cre/Cre null mice . Acute nocifensive responses to mechanical or heat stimulation were also performed on WT and NaV1.8-null mice. Although there was no difference in the latency of paw withdrawal to a noxious heat stimulus, NaV1.8-null mice did exhibit an increase in mechanical withdraw latency of the tail, compared with WT, consistent with previous observations .To understand how the neuronal imaging results relate to behavioral responses, we performed cold-plantar, cold-plate, and acetone tests on WT and Naia toxin . Importathdrawal A. Correlresponse B and C. rvations   s; however, none of the NaV1.8Cre/Cre null mice exhibited any jumping behavior for the duration of the assessment period (V1.8-expressing (+ve) and nonexpressing (−ve) DRG neurons from NaV1.8Cre/+ and NaV1.8Cre/Cre null mice, using in vivo imaging. Extreme prolonged cold stimulation (1 °C for 5 min) caused the activation of cold-sensitive neurons within all populations of DRG neurons imaged, which typically subsided within 75 s from the initiation of the cold stimulus  at 4 °C. By separating GCaMP3-only neurons from tomato-positive neurons, we were able to isolate a purified population of cold-sensitive, NaV1.8-negative neurons (Dataset S1). Interestingly, the putative cold-sensing candidates Trpm8 and Kcnk9 showed increased expression in the GCaMP3-only population, whereas the gene encoding NaV1.8, Scn10a, as well as other ion channel genes, including Kcna1, Kcna2, Scn11a, and Trpm3 showed greater expression in tomato-positive neurons. Enriched ion channel genes specific to the GCaMP3-only or tomato-only populations are summarized in i and ii. Importantly, far fewer GCaMP3-only neurons were obtained when the FACS was performed at 37 °C (51 neurons), compared with 4 °C , thus supporting the use of 4 °C FACS to isolate putative cold-sensing DRG neurons.Due to our in vivo imaging and behavioral data, we wanted to investigate the identity of cold-sensitive neurons that reside outside of the Na neurons A and B.  neurons C. Gene eV1.8 has been identified as a major contributor to pain in cold conditions, despite showing limited overlap with Trpm8  behaviors in mice, as measured by the cold-plantar, cold-plate, or acetone tests. In contrast, nocifensive behaviors to prolonged extreme temperatures appeared to be dependent on NaV1.8, giving rise to the hypothesis that NaV1.8 is necessary for detecting cold-induced damage.Over a decade ago, the identification and characterization of Trpm8 provided a substantial mechanistic link to our understanding of how sensory neurons sense a cooling environment . Since tth Trpm8 . Importath Trpm8 , 13, 15.V1.8 in neuronal conduction at low temperatures, it is surprising that the majority of acute cold-sensitive DRG neurons activated at temperatures ≥1 °C reside outside of the NaV1.8 population. The functional loss of NaV1.8 has been shown to prevent action potential initiation in cultured neurons at 10 °C, which is supported by a significant reduction in nocifensive behavior measured at 0 °C , or acetone tests. In contrast, we observe that nocifensive behaviors in response to extreme cold, as measured by exposure to a −5 °C cold plate for up to 5 min, is completely absent in NaV1.8-null mice compared with WT mice, suggesting that while NaV1.8 is not required for acute cold sensing at temperatures ≥1 °C, it is required for prolonged extreme-cold stimulation. In support of this, using in vivo imaging, we show that prolonged extreme-cold stimulation causes the delayed activation of a subset of NaV1.8-expressing neurons, in a NaV1.8-dependent manner. This observation is consistent with previous data showing that the number of cold-activated nociceptors increases in line with the intensity of the stimulus applied, with up to 100% of nociceptors being activated at temperatures <0 °C  will principally affect NaV1.8-expressing neurons.Given the essential role of Na at 0 °C , 11. Impchannels . This ises <0 °C , 17. As 8 to 85% , 18, 19,V1.8-null mice, DTA-mediated ablation of NaV1.8 neurons significantly increased the cold sensitivity of affected mice at temperatures ≥5 °C. Although the explanation behind this observation is currently unclear, it was previously reported that the ablation of Trpm8 neurons distorts normal thermal responsive behavior to preferred temperatures in mice, potentially caused by reduced aversive input from the Trpm8 neuronal population , or high-threshold cold-responsive neurons  . AlthougV1.8 gene, Scn10a. In addition, we also observed a significant enrichment in Kcna1 and Kcna2 that encode the Shaker-like voltage-gated potassium channels KV1.1 and KV1.2, respectively. These channels are responsible for generating the potassium brake current (IKD), which acts to limit the excitability of sensory neurons through the generation of a voltage-dependent hyperpolarizing current . To ablate the neuronal population of DRG neurons expressing NaV1.8, NaV1.8-Cre mice were crossed with Cre-dependent DTA mice  were anesthetized using 120 mg/kg ketamine  and 1.2 mg/kg medetomidine (Orion Pharma). The depth of anesthesia was assessed by pedal reflexes, breathing rate, and whisker movement. Throughout the experiment the body temperature of the animal was maintained at 37 °C using a heated mat (VetTech). A dorsal laminectomy was performed at spinal level L3–L5 and the DRG was exposed for imaging as previously described  was constantly perfused over the exposed DRG during the procedure to maintain tissue integrity. All in vivo imaging experiments were performed using a Leica SP8 confocal microscope  (Leica). Bidirectional scan (800 Hz) confocal images were taken at a frame rate of 1.54 frames per s−1 and at a resolution of 512 × 512 pixels. The pinhole was kept at 1 a.u. Laser lines of 488 and 552 nm were used to excite GCaMP3 and tdTomato, respectively. The collection of the resulting emission for both GCaMP3 and tdTomato was system optimized to maximize yield and minimize cross-talk . Laser power was <5% for both 488- and 552-nm laser lines. The stimulation was applied to the left hind paw . Cold stimulation was performed by transiently immersing the hind paw with cold water (0 °C) or acetone, or by a Peltier-driven thermal stimulator .NaF/F0 and were analyzed as previously described  and analyzed using ImageJ (NIH). All images were stabilized for XY movement using the TurboReg plug-in , with alescribed . Where aV1.8Cre/+ and NaV1.8Cre/Cre null mice were terminally anesthetized with pentobarbitone sodium . Both mouse strains also expressed the Cre-dependent tdTomato reporter gene. Whole-body fixation was then achieved through the transcardial perfusion of heparinized saline [0.1 M PBS (1× PBS) with 0.01 mL/L−1 heparin] followed by paraformaldehyde (4% in 1× PBS). Once fixed, whole DRG were removed from lumbar regions L3–L5 and immediately placed into a sucrose solution overnight (30% wt/vol in 1× PBS). Sections of DRG were then mounted  and sections were taken (10 μm) using a cryostat (Bright). Sections were immediately placed in washing buffer (1× PBS with 0.1% Triton 100) for 5 min, followed by 60 min in blocking buffer (1× PBS with 10% goat serum). After the blocking step, a primary antibody against NeuN  was added to each section and incubated overnight at 4 °C. After 16 h, the incubated sections were washed (1× PBS with 0.1% Triton 100) and a secondary antibody was added  and the sections were incubated at room temperature (∼23 °C) for 1 h in the dark. The sections were then washed (1× PBS with 0.1% Triton 100) and mounted  for imaging. All images were taken using a Leica SP8 confocal microscope using laser lines 488 nm (NeuN:Alexa Fluor 488) and 552 nm (tdTomato).Na2 blunt conical probe  at 4 °C, or 37 °C for control. Neuronal populations were gated based on their fluorescence. A nonfluorescent sample was used before each sorting experiment to ensure the exclusion of autofluorescent neurons. Sorted populations were immediately placed into lysis buffer and were used for RNA extraction. Cell lysis and RNA extraction was performed using the PureLink RNA Micro kit (Invitrogen). Extracted RNA was then used for microarray analysis .DRG neurons were extracted, acutely dissociated, and resuspended in extracellular solution containing (in mM): 140 Na, 4 KCl, 1.8 CaClSupplementary FileSupplementary File


Pectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020.4)2SO4, 4.33; K2HPO4, 1.36; MgSO4.5H2O, 0.05; KCl, 0.05; and FeSO4.5H2O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h.The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL  was produced in a medium composed of (g/L): pectin, 32.22; (NHStatistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions. Pectic substances are a class of complex glycosidic polysaccharide compounds with a high molecular weight. They constitute the main components of the middle lamella and primary plant cell wall. Pectinase EC 3.2.1.15) is an enzyme that hydrolyzes these biopolymers; it breaks down pectin in plant materials. This enzyme attacks and depolymerizes pectin through hydrolysis and esterification reactions. Pectinase splits polygalacturonic acid (pectate polymer) into monoglacturonic acid by opening the glycosidic linkages and breaking ester bonds between carboxyl and methyl groups .15 is an, 5. It iBacillus spp., yeasts (Yarawia lipolytica and Saccharomyces cerevisiae), and many filamentous fungi, such as Aspergillus niger, A. oryzae, A. awamori, A. sojae, Trichoderma viridiae, T. virens, Penicillium griseoroseum, and Phanerochaete chrysosporium, are potential pectinase producers [A. niger has attracted the most attention as a pectinase bioreactory because of its long history in fermentation industries and its status as a Generally Regarded As Safe (GRAS) organism, according to the United States Food and Drug Administration [A large number of bacterial strains, mainly roducers , 11–17. stration . DespiteC. sinensis) and lemon (C. limon) [C. reticulate) are popular citrus fruits.Fermentation medium accounts for a large portion of the production cost. Alternative substrates for pectinase production include cheap carbon sources including wheat bran, soybean meal, sugar cane molasses, and agro-industrial wastes, especially from citrus fruits such as sweet orange (. limon) , 11. In A. niger NRC1ami [n experiments to investigate a maximum of 4n-1 factors at two levels [In this work, mandarin orange peel was used as a substrate for pectinase production at a semi-industrial scale, using a high yield-producing fungal strain,  NRC1ami . First, o levels . The Boxo levels –23. Thiso levels . The optThe pectin present in mandarin peel was extracted according to the method described by Huong and Luyen . BrieflyAspergillus niger NRC1ami was obtained from the National Research Center culture collection . This strain was isolated from citrus fruit and exhibits high extracellular pectinase production [A. niger spores were collected in saline solution.oduction . Once th4)2SO4, 3.33; K2HPO4, 1.0; MgSO4.5H2O, 0.05; KCl, 0.05; and FeSO4.5H2O, 0.10. The pH was adjusted to 5.5 before sterilization.The initial pectinase production medium was composed of (g/L): pectin, 30.0;  at 150 rpm and 30 °C. The propagated vegetative cells were used as inoculum for either shake-flask or bioreactor cultivations at a ratio of 5%.Shake flask cultivation was carried out in a 250-mL Erlenmeyer flasks with a 50 mL working volume, which were inoculated with 1 × 10i(impeller diameter) = 85 mm; dt(tank diameter) = 214 mm, di/dt = 0.397). Sterilization was carried out at 121 °C for 30 min. The agitation was adjusted to 200 rpm throughout the cultivation, and aeration was performed using sterile air at a rate of 1 v/v/min. Foam was suppressed during cultivation using Struktul anti-foaming agent . The pH and dissolved oxygen were determined during the cultivation using pH and DO polarographic electrodes . The pH-controlled culture was adjusted to 5.5 by connecting the pH controller to an acid/base feeding peristaltic pump containing 5 M HCl and 5 M NaOH solutions.A stainless steel, double-jacketed, 16-L stirred tank bioreactor was used  with a working volume of 6 L. The stirrer was equipped with two, 6-bladed Rushton turbine impellers (d4)2SO4, K2HPO4 and MgSO4.5H2O; thus, these medium components were selected as variables. The low level (− 1) and high levels (+ 1) of individual nutrients are given in Table The Plackett-Burman design was applied to determine the medium components affecting pectinase production. The four key medium components were pectin, (NH level −  and high4)2SO4 and K2HPO4. These three factors were further studied for the optimal range in the Box-Behnken design using Minitab 16 software . The low, center, and high levels are shown in Table The factors affecting the response of the Plackett-Burmann design were pectin, 2SO4, K2HPO4; g/L), β0 is the intercept term, and βi, βii, and βij are the linear, squared, and interaction coefficients, respectively [where Y is the predicted pectinase activity (U/mL), ectively . The preDuring shake flask cultivation, samples were withdrawn intermittently for analysis. For the bioreactor cultivation, aliquots (20 mL) of the culture were taken through a sampling system. Samples were filtered using dry, pre-weighed Whatman filter paper. The supernatant was extracted to determine pectinase activity. The filtered biomass was washed twice using distilled water and dried in an oven at 100 °C until it reached a constant weight .w/v) citrus pectin as the substrate; 0.3 mL of enzyme was added to 0.7 mL of substrate and mixed for 15 min at 40 °C. The liberated galacturonic acid concentration was determined using the method described in Esawy et al. [Pectinase determination was carried out using 1.0% 2SO4, 3.3; K2HPO4, 0.50; MgSO4.5H2O, 0.50. Moreover, the coefficients of determination (R2) for both cell biomass and pectinase activity were 86.94 and 85.31%, respectively, indicating that this design had a good model fitting.Fractional factorial design was applied to find out the most significant medium components affecting pectinase production by 4)2SO4 and K2HPO4, were the most significant factors affecting pectinase production. This can be seen based on the obtained model F-value and the lower p-value (p- < 0.05). Accordingly, magnesium sulphate was excluded from the second step of optimization, since its effect on pectinase production was insignificant (p- = 0.23).However, based on the analysis of variance (ANOVA) for pectinase activity Table , Fig. 1,4)2SO4 and K2HPO4 in the culture medium according to the three levels  studied and shown in Table The BB experimental design was further applied to optimize the concentrations of pectin, 2SO4 and K2HPO4) and the response of pectinase production. Figure 4)2SO4 concentration (1.0–6.0 g/L) and pectin concentration (10–50 g/L) while keeping K2HPO4 concentration constant at 1.25 g/L. Pectinase production increased with increasing pectin concentration to reach its maximal production at 32.22 g/L of pectin, while (NH4)2SO4 was less effective on pectinase production. Figure 2HPO4 concentration (0.5–2.0 g/L) and pectin concentration (10–50 g/L), while keeping (NH4)2SO4 concentration constant at 3.5 g/L. Results showed that highest pectinase production will be obtained at the most optimum pectin concentration (32.22 g/L), while 1.36 g/L K2HPO4 was suitable for producing maximal pectinase production. On the other hand, the combined surface plot for (NH4)2SO4 and K2HPO4 2SO4, respectively. Furthermore, from the model results compared to our experimental runs, it can be seen that the maximal obtained experimental pectinase production (90 U/mL) was in good agreement with the model predicted production (92.48 U/mL) with a desirability of 0.915. It can be generally seen, that the evaluated components are significant for pectinase production. Carbon and nitrogen sources are basic components of fermentation medium, where they are used for cellular growth and metabolic machinery, which is finally reflected on the production of pectinase.Response surface plots Fig.  represenPO4 Fig. , where pYP/X) results showed that the maximal yield obtained in optimized medium  was almost 3-folds of the maximal yield obtained in un-optimized medium .This part of the work was designed to compare the kinetics of cell growth and pectinase production on both un-optimized and statistically optimized media in shake flasks Fig. . Cells gv/v/min, under uncontrolled pH. This cultivation was compared with cultivation conducted at controlled pH conditions at 5.5 by the continuous addition of H2SO4/NaOH using a computerized pH control system  were comparable in both cultivations . Additionally, both produced maximal pectinase concentrations were higher by about 30.4 (uncontrolled pH) and 43.6% (controlled pH) than the maximal pectinase produced in optimized shake flask cultivation (76.35 U/mL).The optimized production conditions were transferred from shake-flask level to bioreactor level to gain insights about process performance in semi-industrial scale bioreactor. Cultivations were run in a 16-L stirred tank bioreactor at an aeration rate of 1 YP/X) obtained in the fed-batch cultivation recorded 75,762 U/g cells, which was almost 63.7% higher than the highest production yield obtained in pH-controlled batch cultivation .Based on the previous data obtained from batch cultivations, fed-batch cultivation was conducted under controlled pH conditions in 16-L stirred tank bioreactor Fig. . The culFinally, Table 4)2SO4 and K2HPO4, were the most significant factors affecting pectinase production. Furthermore, determination coefficients (R2) for cell biomass and pectinase activity were 86.94 and 85.31%, respectively, representing a good model fitting. These results are in good agreement with those previously reported for statistical medium optimization for pectinase production using either fungal or bacterial microorganisms [4 is not significantly affecting pectinase production during their statistical optimization experiments. Although Mg+ 2 acts generally as an inducer or co-factor for enzyme production, however, few reports have suggested that magnesium sulphate can inhibit glucose 6-phosphate dehydrogenase, G6PD [Pectinase enzymes constitute a major sector in the enzyme food market , 5, 7. Arganisms , 28. Autse, G6PD , 30. G6Pse, G6PD .4)2SO4 and K2HPO4. We found that highest pectinase activity was obtained using the middle levels of all tested three components  was the best level for the production, and that pectinase levels decreased significantly when the higher levels were used (+ 1). The model for pectinase activity was used to calculate the coefficient of determination (R2), which can be defined as the ratio of the expected deviation to the overall deviation. Our results showed that R2 has a value of 93.69% for pectinase response, which indicates a good fitting of the model and that the model can explain 93.69% of the deviation from the expected values, and that only 6.31% of the deviation cannot be explained by the proposed model. Moreover, as the value of R2 comes closer to 1.0, this means that the model correlates well [p-value reflects the relationship between the variables/factors and the response variable, i.e. p-value lower than 0.05 indicates that the applied model is significant.Secondly, Box-Behnken design was performed in order to further optimize the concentrations of the obtained most significant components; pectin, , which are in good agreement with our results . Statistlus spp. . They weA. niger is an aerobic microorganism and thus needs a continuous supply of oxygen during cultivation for growth and efficient metabolite production [oduction . Therefooduction , 35. Moroduction , 36. Furoduction . Additiooduction .A. niger and they were able to overcome such problems by increasing the agitation from 300 to 600 rpm and the aeration rate from 0.5 to 1.2 v/v/min once they reached their maximal growth rate [A. niger [On the other hand, both bioreactor cultivations showed lower rates of growth and pectinase production than in the case of shake flask cultivations. This decrease can be attributed to the nature of growing fungal hyphae, which under bioreactor conditions tend to have condensed morphology and thus affecting the viscosity of the fermentation broth. Consequently, the cultivation will suffer from decreased oxygen transfer between the condensed hyphae and the viscous broth leading to decreased growth and production rates , 39. Morwth rate . AccordiA. niger , 41, 42.A. niger [A. niger. They found that feeding sucrose to pectin containing medium greatly improved pectinase production due to its inductive effect. Moreover, Phutela et al. used wheat bran medium for the production of pectinase by A. fumigatus [Batch cultivation is generally terminated earlier due to exhaustion of important nutrient components from the cultivation medium, which greatly affects the overall productivity of the cultivation process. Therefore, fed-batch mode of cultivation was developed to overcome problems encountered in batch cultivations , 43. OurA. niger . They foumigatus . They foAspergillus niger NRC1ami, isolated from citrus fruit, showed high pectinase production using a statistically optimized medium. The medium variables affecting enzyme production were pectin, (NH4)2SO4 and K2HPO4, while MgSO4 was found to be insignificant. The optimum medium composition statistically optimized (g/L) was: pectin, 32.22; (NH4)2SO4, 4.33; K2HPO4, 1.36; MgSO4.5H2O, 0.05; KCl, 0.05; and FeSO4.5H2O, 0.10. Medium optimization enhanced pectinase production by about 2.8 folds (from 27.2 to 76.35 U/mL). The enzyme yield was further improved by about 4-folds upon transferring the process from shake flasks to bioreactor under controlled pH. Further improvement was achieved by fed-batch cultivation with constant carbon source feeding on a semi-industrial scale in 16-L stirred tank bioreactor. Maximal volumetric enzyme production increased by about 16.5-folds from the initial starting conditions. Finally, the high yield obtained in the semi-industrial scale bioreactor supports the scaling-up and industrialization of this process.In the present work, a new strain,


Silybum marianum) is a medicinal plant that has been used for thousands of years as a remedy for a variety of ailments. The main component of S. marianum fruit extract (silymarin) is a flavonolignan called silybin, which is not only the major silymarin element but is also the most active ingredient of this extract, which has been confirmed in various studies. This compound belongs to the flavonoid group known as flavonolignans. Silybin’s structure consists in two main units. The first is based on a taxifolin, the second a phenyllpropanoid unit, which in this case is conyferil alcohol. These two units are linked together into one structure by an oxeran ring. Since the 1970s, silybin has been regarded in official medicine as a substance with hepatoprotective properties. There is a large body of research that demonstrates silybin’s many other healthy properties, but there are still a lack of papers focused on its molecular structure, chemistry, metabolism, and novel form of administration. Therefore, the aim of this paper is a literature review presenting and systematizing our knowledge of the silybin molecule, with particular emphasis on its structure, chemistry, bioavailability, and metabolism.Milk thistle ( Silybum marianum L. Gaernt.), sometimes called wild artichoke, is a medicinal plant that has been used for thousands of years as a remedy for a variety of ailments. -2,3-dihydro-3,5,7-trihydroxy-4H-1-benzopyran-4-one. Silybin B has a configuration of 2R, 3R, 10S, 11S and IUPAC name -2--2,3-dihydro-3,5,7-trihydroxy-4H-1-benzopyran-4-one , while silybin B has an c 0.28, acetone) [2O) forms yellowish flat crystals with a melting point of 162–163 °C, while silybin B generates yellow grain crystals (MeOH–H2O) with a melting point of 158–160 °C [The silybin diastereoisomers also have different optical rotation parameters: silybin A has an acetone) . The dif.07° c 0., acetoneSilybum marianum, flavonolignans are formed by combination of flavonoid and lignan structures. This occurs through oxidative coupling processes between flavonoids and a phenylpropanoid. The first hypothesis of this process was described by Freudenberg [In udenberg  and confudenberg ,35. Silyudenberg . The firudenberg ,37.After oral administration, silybin undergoes extensive enterohepatic circulation. The elimination half-life of silybin is approximately 6 h . It has O-demethylatedsilybin (major), and both mono- and dihydroxy-silybin (minor) metabolites. The presence of these metabolites was also confirmed by Gunaratna and Zhang [Using nuclear magnetic resonance (NMR), Jancova et al.  demonstrnd Zhang  using lind Zhang .Silybin monoglucuronides and diglucuronides, as well as silybin monosulfates and silybin diglucuronides sulfate, are all formed during phase II of silybin’s biotransformation . ExperimThe silybin glucuronides formed in phase II are then transported by biliary flow to the intestinal tract, where bacterial enzymes cleave sugar moieties and release silybin aglycones, which can be absorbed again, thus promoting enterohepatic circulation ,51.1/2 of about 6–8 h). However, as mentioned previously, the absorption efficiency is rather low [Due to its highly hydrophobic and non-ionizable chemical structure, silybin displays poor water solubility of less than 50 μg/mL, and this has a great influence on its bioavailability . Howeverther low ,53,54. Sther low .Silybin bioavailability in the gastrointestinal tract is dependent on various factors, such as the concentration of the preparation and the presence of additional substances with a solubilizing character  .1/2 parameters were 2.5 and 3.8 h [® capsule, and silymarin tablets—on 24 healthy volunteers was made by Kim et al. [Silybin concentrations in blood after oral administration of conventional preparations based only on silymarin extract are considered to be low. One of the first studies of silybin bioavailability was made by Lorenz et al. . In theind 3.8 h . A compam et al. . Each suSilybin bioavailability can be significantly improved by adding solubilizing substances to the standard silymarin pharmaceutical product . Other pAnother formulation that was created to increase silybin bioavailability after oral administration is a compilation of silybin, phosphatidylcholine, and vitamin E, which considerably enhanced silybin’s solubility . A studyN-methylglucamine, after oral administration the relative bioavailability of the mixed micelles was 252.0%. The Cmax values were 107.0 and 94.7 ng/mL, respectively, but the AUC values were 13,372.4 and 505.8 ng/mL × h. Tmax was similar for both preparations—1 h. A creation of silymarin-loaded liposomes containing bile salt (SM-Lip-SEDS) produced a 4.8-fold enhancement in the oral bioavailability of silybin in rats. Cmax and AUC values (after treatment of 20 mg/kg dose) for SM-Lip-SEDS were 1.296 ± 0.137 µg/mL and 18.406 ± 1.481 µg/mL × h, respectively, while for silymarin powder these values were 0.640 ± 0.132 µg/mL and 3.824 ± 0.355 µg/mL × h [The next proposition for enhancing silybin’s oral bioavailability is the formation of bile salts mixed micelles. Many authors ,71,72,73g/mL × h .o/w) microemulsion under gentle agitation following dilution by aqueous phases, with a particle size of less than 100 nm [® capsule and Legalon®, respectively. The relative bioavailability of the SMEDDS to the Legalon® was 227%, although in the case of the Tmax parameter, no significant difference was observed (p > 0.05) for the two formulations. Additionally, in Yu et al.’s experiment, the silymarin was successfully solubilized 327-fold by SMEDDS (130.8 mg/mL), which was closer to that in polysorbate 20 [®. After its oral administration in rats (at dose 140 mg/kg), the bioavailability of the drug from the SMEDDS was a few times higher than the reference capsule: Cmax values were 24.79 ± 4.69 μg/mL and 3.47 ± 0.20 μg/mL, respectively, while AUC (0–6 h) were 81.88 ± 12.86 µg/mL × h and 22.75 ± 3.19 µg/mL × h, respectively. Additionally, Tmax for SMEDDS was 0.5 h, whereas for Legalon® was 1.1 h [The next most promising way of enhancing silybin’s bioavailability is the Self-Micro Emulsifying Drug Delivery System (SMEDDS). This is a drug delivery system that uses a microemulsion achieved by chemical rather than mechanical means. Drugs coated in this way are well known for their potential as alternative strategies for delivery of hydrophobic drugs . The forn 100 nm ,78,79,80n 100 nm . Addition 100 nm . The sturbate 20 . Wu et arbate 20  examinedas 1.1 h .One of the most effective methods of intensifying silybin bioavailability is creation of specific nanoemulsions and nanosuspensions. Silybin nanoemulsion can consist of sefsol-218 as an oil, Tween 80 as a surfactant and ethanol as a co-surfactant (having a nano-droplet size and low viscosity). Parveen’s results indicated that the stability of silymarin can be enhanced in nanoemulsion formulation using Tween 80 as a surfactant . After o1/2 (h) 7.830 ± 3.204 vs. 2.938 ± 0.694 [Another very interesting method of improving the dissolution and bioavailability of silymarin is use of solution-enhanced dispersion by supercritical fluids (SEDS). A study performed on rats orally administered 20 mg/kg silymarin as SEDS or silymarin powders showed that SEDS has much better bioavailability and pharmacokinetic parameters than silymarin powder: Cmax (μg/mL) 1.093 ± 0.249 vs. 0.57 ± 0.143, AUC (µg/mL × h) 5.017 ± 0.35 vs. 2.054 ± 0.074. Additionally SEDS, also increased t ± 0.694 .In one of the most promising studies performed in the last few years, surface-attached silymarin-loaded solid dispersion with an improved dissolution profile and enhanced oral bioavailability was formulated using silymarin, polyvinylpyrrolidone (PVP), and Tween 80. The drug solubility of the optimized solid dispersion prepared with silymarin/PVP/Tween 80 at a weight ratio of 5/2.5/2.5 increased by almost 650 times compared to standard silymarin powder. After oral administration in rats , this formulation caused an increase in Cmax and AUC values (44.85 ± 11.42 μg/mL and 366.49 ± 93.62 µg/mL × h vs. 16.74 ± 1.63 μg/mL and 157.04 ± 36.29 µg/mL × h) .In summary, silybin is a very interesting chemical compound, the use of which as a dietary supplement is increasing all around the world, and this explains the recent studies aimed at increasing its oral bioavailability.


The compound with the highest activity was used for an in vivo assay with Leishmania amazonensis.A series of eight substituted bis-2-hydroxy-1,4-naphthoquinone derivatives was synthesized through lawsone condensation with various aromatic and aliphatic aldehydes under mild acidic conditions. The title compounds were evaluated for antileishmanial activity  Yield: 83% yellow solid. mp: 229–232 °C dec. (229 °C)  (3b). Yield: 54%, orange solid. mp: 205–208 °C dec. (213 °C)  (3c). Yield: 34%, orange solid. mp 210–211 °C (169–170 °C)  (3d). Yield: 89%, yellow solid. mp 215–217 °C (202–204 °C)  (3e). Yield: 93%, yellow solid. mp 222–223 °C (220–222 °C)  (3f). Yield: 98%, yellow solid. mp 175–176 °C (175–177 °C)  (3g). Yield: 92%, yellow solid. mp 143–146 °C. (177–179 °C)  (3h). Yield: 83%, yellow solid. mp 202–204 °C (193–195 °C) [max/cm−1: 3414, 3348, 1666, 1625, 1593, 1508, 1458, 1365, 1342, 1276, 1230, 1161, 1041, 833, 725; 1H-NMR  δ 6.00 , 7.00 , 7.28 , 7.77 , 7.91 , 7.97 ; 13C-NMR  δ 37.7, 114.5, 123.5, 126.0, 126.5, 130.3, 132.6, 133.5, 135.1, 137.4, 156.5, 159.8, 162.2, 181.6, 184.0. Anal. Calcd. C27H15FO6: C, 71.37; H, 3.33. Found: C, 71.16; H, 3.44.–195 °C) . IR (KBrL. braziliensis promastigotes (MHOM/BR/87/BA788) were obtained from Dra. Valéria de Matos Borges at the Gonçalo Moniz Research Center, Fiocruz_BA. L. amazonensis promastigotes (MHOM/BR/77/LTB0016) were obtained from Dr. Eduardo Caio Torres dos Santos at the Oswaldo Cruz Institute-Fiocruz_RJ. The parasites were maintained in vitro in Schneider’s medium supplemented with 10% FBS and 2% human urine at 27 °C in BOD incubator.2.The adherent-phenotype macrophage line was cultured in Dulbecco’s Modified Eagle’s medium  supplemented with 10% FBS at 37 °C, 95% humidity and 5% CO5 cells per well in a complete culture medium with 10% FBS at 37 °C. After 1 h, the wells were washed with HBSS to remove non-adherent cells, leaving approximately 1 × 105 adherent macrophages. The cells were cultured in DMEM complete medium supplemented with 10% FBS. The compounds and pentamidine were added at serial concentrations (0.1–100 µM). The cells were also cultured in media free from compounds, a vehicle  or media with DMSO 0.1% (vehicle control). The positive control (dead cells) was obtained through cellular lyses with 1% of Triton X-100 in DMEM complete medium. After 48 h, the cytotoxicity was evaluated using the MTT assay [post-hoc tests.To evaluate the cytotoxicity activity against the J774 cell line, the host cells were plated in 96-well vessels at 2 × 10TT assay . The datin vitro in Schneider’s medium supplemented with 10% FBS and 2% human urine. Novel bis-lawsone analog stock solutions and pentamidine  were prepared in DMSO immediately before use. The cytotoxicities of the bis-lawsone analogs and pentamidine against the promastigotes were determined. Stationary phase L. braziliensis and L. amazonensis promastigotes were plated in 96-well vessels  at 1 × 105 cells per well in Schneider’s medium supplemented with 10% FBS and 2% human urine. Each compound solution was added at increasing concentrations (0.1–100 mg to the extract and its phases or 0.1–100 µΜ to the isolates and pentamidine). The cells were also cultured in a medium free of compounds, a vehicle  or with DMSO 0.1% (vehicle control). After 48 h, the extracellular load for L. braziliensis and L. amazonensis promastigotes was estimated by counting the promastigotes in Schneider’s medium using a CELM automatic cell counter  [post-hoc tests.The parasites were maintained  Brazil) . The dat5 stationary promastigotes (5 days of culture in Schneider’s medium) of L. amazonensis (MHOM/BR/77/LTB0016) were subcutaneously inoculated into the right ear dermis of 6-week-old female BALB/c mice weighing ca. 20 g. and were later intraperitoneally treated with 3a or glucantime at 30 μmol/kg × 28 days. The lesion size was measured using a paquimeter [post-hoc tests.This study was approved (protocol no. 2013.02) by the Ethics Committee for Animal Experimentation of the Federal University of Alagoas (Brazil). All animals received humane care in compliance with the “Principles of laboratory animal care” formulated by the National Society for Medical Research and the “Guide for the care and use of laboratory animals” prepared by the National Academy of Sciences . Next, 1 × 10quimeter . The parquimeter . Complexin silico screening using the program OSIRIS [Bis-lawsone analogs were submitted to m OSIRIS  to analym OSIRIS  of the bin vitro cytotoxic activities of the derivatives synthesized were evaluated against L. braziliensis, where they showed a maximum effect greater than 60%. The bis-lawsone analogs 3a, 3b, 3e and 3h present efficacies and potencies similar to the reference drug pentamidine without cytotoxicity to the host cells. Finally, 3a presented the activity 30 μmol/kg × 28 days (i.p.); the L. amazonensis lesion size on the infected ear of BALB/c mice decreased, but the number of parasites in the infected ear and draining lymph nodes did not decrease. In summary, these findings show that 3a, 3e and 3h are antileishmanial drug candidates and suggest a useful starting point for rationally designing new agents against leishmaniasis. Clearly, further studies are necessary for exciting advances in the medicinal use of drug candidates in this class of secondary products.The present study reports the synthesis and leishmanicidal evaluation of a series of substituted bis-2-hydroxy-1,4-naphthoquinones prepared from lawsone. The


Clostridium perfringens is a common opportunistic pathogen endangering livestock and poultry breeds. Here, using enhanced green fluorescent protein as screening marker, a recombinant lactobacillus tetravalent vaccine constitutively expressing α, ϵ, β1, and β2 toxoids of C. perfringens was developed, and its immunogenicity in mice was investigated via oral administration. This probiotic vaccine could effectively induce antigen-specific secretory IgA (sIgA)-based mucosal and IgG-based humoral immune responses, and significantly high levels (p< 0.05) of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and IFN-γ were produced in immunized mice. Moreover, lymphoproliferation and percentage of CD4+ and CD8+ T cells significantly increased in mice of the probiotic vaccine group. Challenge experiments were performed in mice with C. perfringens toxinotypes A, C, and D crude toxins to evaluate protection efficiency of the probiotic vaccine, using a commercial inactivated C. perfringens vaccine made by C. perfringens toxinotypes A, C, and D as vaccine control. We observed 80% protection rate in the probiotic vaccine group, which was higher than commercial vaccine group, whereas all mice in control groups died and obvious histopathological changes were observed in liver, spleen, kidney, and intestines of mice. Significantly, we compared the immunogenicity and protection efficiency of lactobacillus constitutive expression system and lactobacillus inducible expression system, and results showed that lactobacillus constitutive expression system has obvious advantages. Our study clearly demonstrated that the probiotics vaccine could effectively induce mucosal, humoral, and cellular immunity, and provide effective protection against C. perfringens toxins, suggesting a promising strategy for the development of oral vaccine against C. perfringens. Clostridium perfringens (C. perfringens), a Gram-positive, spore-forming anaerobic bacillus that is widely distributed in natural environments and can usually be found in the gastrointestinal tract of livestock and poultry as an opportunistic pathogen, is associated with human food-borne diseases as well as animal diseases [C. perfringens toxin typing scheme, C. perfringens is classified into five toxin types  based on the major toxins α, β, ϵ, and ι produced by each toxinotype [C. perfringens strains should be classified into seven toxinotypes, A to G [C. perfringens infections that are responsible for gas gangrene in sheep [C. perfringens but is the only toxin produced by toxinotype A [C. perfringens β2-toxin produced by newly characterized β2-toxigenic C. perfringens shows a low homology to β1-toxin, and is involved in enterocolitis [C. perfringens can cause various diseases in different animals, including gas gangrene, acute enterotoxemia, and enteritis syndrome. Therefore, the development of an effective vaccine against C. perfringens toxins is of great importance.diseases ,2, and exinotype , while a, A to G . The α-tin sheep , and humin sheep , which cnotype A ,8. The βnotype A ,10. The ocolitis ,12. The ocolitis . C. perfC. perfringens infections in livestock and poultry, antibiotics can result in many negative effects on the environment as well as human health [C. perfringens is an opportunistic pathogen in the gastrointestinal tract of animals and its enterotoxins are absorbed via intestinal mucosa. Therefore, effective oral vaccines that can induce a more efficacious sIgA-based protective mucosal immunity are urgently needed for the prevention of C. perfringens diseases. An ideal oral vaccine would not only effectively deliver antigenic matter to the immune system to induce protective mucosal and systemic immune responses, but also the delivery vehicle itself should be safe and harmless.Currently, although in-feed antibiotic, such as virginiamycin and tylosin, is the most effective approach for the control of n health . Parenten health . HoweverLactobacillus is a Gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria, which is the most common probiotic found in food and the intestinal tract of humans and animals [ animals . Studies animals  and prev animals , anti-in animals , modulat animals  and sIgA animals , regulat animals ,23. More animals ,25. Ther animals –32.C. perfringens α, ϵ, β1, and β2 toxoids as antigens, a genetically engineered Lactobacillus casei 393 (L. casei 393) strain pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 with non-antibiotic resistance constitutively expressing toxoids of C. perfringens α, ϵ, β1, and β2 toxins were successfully constructed with a lactobacillus constitutive expression plasmid pPG-T7g10-PPT constructed in our lab, and its immunogenicity and immune protective efficacy were evaluated in BALB/c mice via oral vaccination, and subjected to comparison with the lactobacillus inducible expression system pPG-2-α-β2-ϵ-β1/L. casei 393 with chloromycetin resistance constructed previously by our lab [In the present study, using enhanced green fluorescent protein (EGFP) as a screening marker for recombinant lactobacillus and  our lab .C. perfringens toxinotype A (C57-1), toxinotype C (CACC-61), and toxinotype D (CCVC-81) were purchased from the China Institute of Veterinary Drug Control . L. casei ATCC393 was grown anaerobically in de Man, Rogosa and Sharpe (MRS) broth  at 37°C without shaking. The plasmid pMD19-T-EGFP containing the gene encoding EGFP (GenBank accession no. U57607) and the plasmid pMD18-T-α-β2-ϵ-β1 containing the fusion genes encoding toxoids of C. perfringens α (H68G), β2 (C234G), ϵ (H106P), and β1  was constructed by our laboratory [Bacillus subtilis for surface-displaying heterologous protein on the cell membrane, and an rrnBT1T2 terminator was constructed by our laboratory [L. casei 393 with Chloromycetin resistance was constructed previously by our lab [boratory ,33. The boratory ,31. A xyC. perfringens α, β2, ϵ, and β1 toxoids were subcloned into the constitutive expression vector pPG-T7g10-PPT, giving rise to pPG-α-β2-ϵ-β1. Next, the gene encoding the EGFP was inserted upstream of the fusion gene α-β2-ϵ-β1 and linked with α-β2-ϵ-β1 by a linker, generating the plasmid pPG-E-α-β2-ϵ-β1. Subsequently, the chloramphenicol-resistant gene in pPG-E-α-β2-ϵ-β1 was deleted by enzymatic digestion with NcoI and StuI followed by ligation of the blunt-ended DNA, giving rise to recombinant pPG-Δ-E-α-β2-ϵ-β1 with EGFP marker. Finally, the recombinant pPG-Δ-E-α-β2-ϵ-β1 was electroporated into L. casei 393 competent cells, followed by screening of the positive recombinants by flow cytometry. Briefly, the strain pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 was grown in MRS medium at 37°C for 16 h without shaking, followed by centrifuging and washing with sterile PBS, the bacteria were subjected to screening by flow cytometry, and the bacterial strain with green fluorescent signal was separated, and cultured in 96-well cell plate containing MRS agar medium at 37°C. Overall, the EGFP-marked recombinant lactobacillus strain pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 was successfully constructed.Recombinant expression plasmids were constructed as shown in L. casei 393 was grown in normal MRS medium at 37°C for 16 h without shaking, and then the bacterial culture was centrifuged at 10,000 × g for 5 min followed by washing with sterile PBS for three times, resuspended in PBS, and then the expression of EGFP was analyzed by ultra-high resolution microscopy and flow cytometry. For identification of constitutive expression of C. perfringens α, β2, ϵ, and β1 toxoids, the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 was cultured overnight in basic MRS medium supplemented with raffinose, sucrose, lactose, trehalose, glucose, and fructose as carbon sources, respectively. Then, the bacterial cultures were centrifuged at 10,000 × g for 5 min, and the cell pellets were washed twice with sterile 25 mmol/L Tris-Cl (pH8.0) and lysed with 2× SDS buffer solution followed by boiling for 10 min. After centrifugation at 12,000 × g for 10 min, the supernatant was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were then electrotransferred onto a PVDF membrane, followed by immunoblots developed with mouse anti-α/β2/ϵ/β1 toxoid monoclonal antibody (diluted at 1:500) as the primary antibody prepared by our lab followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  diluted 1:2000 as the secondary antibody. The immunolabelled bands were visualized using the chemiluminescent substrate reagent  according to the manufacturer’s instruction.For identification of EGFP expression, the recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 was cultured to an OD600 of 1.0 and then centrifuged at 5000 × g for 10 min followed by washing twice with sterile PBS. The bacteria pellets were then resuspended to a concentration of 1010 CFU/mL. Next, the recombinant lactobacilli were labeled with 5ʹ (and 6ʹ)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) at 37°C for 25 min, and then washed twice with sterile PBS to remove excess CFDA-SE. Finally, the recombinant lactobacilli were resuspended in sterile PBS, and the labeling rate was detected by flow cytometry. A total of 24 mice were orally administrated with approximately 109 CFU/mL of the CFDA-SE-labeled recombinant lactobacilli per mice, and groups of three mice each were sacrificed on days 1, 3, 5, 7, 9, 11, 13, and 15, and then the duodenum, jejunum, ileum, and colon were extracted and cut longitudinally, and visible residual food particles or fecal material was removed from individual sections prior to the detection of adhering CFDA-SE-labeled lactobacilli. After dislodging microbes from the mucosal surfaces of each intestinal section with PBS and being fixed with 0.75% formaldehyde (v/v) as previously described [The recombinant pPG-Δ-E-α-β2-ϵ-β1/escribed , flow cyL. casei 393 was adjusted to a concentration of 1 × 1010 CFU/mL. BALB/c mice were randomly divided into five groups. Mice in the probiotic vaccine group (n = 64) were orally immunized with 100 μL of recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393. Mice in the control group (n = 64) were immunized with an equivalent dose of pPG-T7g10-PPT/L. casei 393. Mice in the mock control group (n = 64) received 100 μL of PBS. Mice in the recombinant lactobacillus control group (n = 64) were immunized with an equivalent dose of pPG-2-α-β2-ϵ-β1/L. casei 393. Mice in the vaccine control group (n = 40) were injected intramuscularly with commercial inactivated C. perfringens vaccines produced for sheep that were made by C. perfringens toxinotypes A, C, and D  according to the manufacturer’s instruction. Oral immunization protocol was performed on three consecutive days , and a booster immunization was given at days 14, 15, and 16, and a second booster was given at days 28, 29 and 30 [Prior to oral immunization, recombinant pPG-Δ-E-α-β2-ϵ-β1/9 and 30 .C. perfringens α, β2, ϵ, and β1 toxoid proteins that were expressed by E. coli and purified in our lab. Next, the plates were washed thrice with PBS containing 1% Tween-20, and then saturated with PBS containing 5% skimmed milk at 37°C for 2 h. The sera, intestinal mucus, and fecal extraction were used as the primary antibody, and horseradish peroxidase-conjugated goat anti-mouse IgA or IgG  was used as a secondary antibody, and followed by color development using tetramethylbenzidine (TMB)  as the substrate, and then absorbance was measured at 450 nm. Moreover, the levels of cytokines IL-4, IL-12, IL-10, IL-17, IL-2, and IFN-γ in the sera samples were detected by ELISA Kits according to the manufacturer’s instructions .Sera, intestinal mucus, and fecal samples were prepared on days 0, 7, 14, 21, 28, 35, and 42 after immunization and used for detecting specific IgA or IgG antibodies by ELISA. Briefly, polystyrene microtiter plates were, respectively, coated overnight at 4°C with 10 μg of L. casei 393 administered on days 28, 29, and 30, spleen lymphocytes were collected from the mice in the vaccine and control groups, and were adjusted to 2 × 106/mL. Then, the lymphocytes were stained with FITC anti-mouse CD4, APC anti-mouse CD3, and PE anti-mouse CD8 fluorescence-labeled antibody  at room temperature for 30 min, respectively. After washing thrice with PBS, flow cytometry was performed using a FACS Caliber  to determine the T cell subsets.Seven days after a second booster immunization with pPG-Δ-E-α-β2-ϵ-β1/6 cells/mL) in eight duplicates were incubated in a 96-well plate containing RPMI 1640 medium plus 10% fetal bovine serum at 37°C in a 5% CO2 incubator for 8–12 h. The cells were then restimulated with 0.01 mg/mL, 0.1 mg/mL, 1.0 mg/mL, and 10 mg/mL of purified recombinant α-β2-ϵ-β1 protein for 60 h, respectively (culture medium used as blank control). Next, 10 μL of thiazolyl blue tetrazolium bromide (MTT) at 5 mg/mL was added into each well and continually incubated at 37°C for 4 h. Lymphocyte proliferation was assessed by the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay according to the manufacturer’s instructions , followed by absorbance measured at 600 nm. The stimulation index was calculated as follows: SI = OD600 (sample)/OD600 (blank control).On day 42 after the primary immunization, the immunized mice were euthanized and spleen cells were obtained for a lymphocyte proliferation test. In brief, 100 μL of spleen cell suspensions . The mice in one subgroup were intramuscularly injected with 1× LD100 (60 μg toxin/g body weight) of the C. perfringens toxinotype A, C, and D crude toxin mixture. The mice in another subgroup were orally challenged with 200 μL of the C. perfringens toxinotype A, C, and D mixture (1 × 1010 CFU/mL). The daily survival rate of the mice in each group was recorded post-challenge, and the histopathological changes in the liver, kidney, heart, colon, spleen, and brain of the mice immunized with recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393, commercially available inactivated C. perfringens vaccine, pPG-T7g10-PPT/L. casei 393, and PBS were observed, respectively.To evaluate the protective efficacy of recombinant pPG-Δ-E-α-β2-ϵ-β1/p values: p < 0.05 (*) and p < 0.01 (**).The data were presented as mean ± standard deviation (SD). Statistical significance was determined using SPSS software (IBM) for ANOVA, and expressed as the following L. casei 393 cultured in normal MRS medium was identified by ultra-high resolution microscopy and flow cytometric analysis, and the results showed that significant green fluorescence was observed on the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 , butasei 393 : B). Floasei 393 : A). In asei 393 , which cL. casei 393 was labeled with CFDA-SE  and its colonization ability in the intestinal tracts was measured via oral administration of CFDA-SE-labeled lactobacilli to mice. Flow cytometric analysis showed that the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 could survive and adhere to the duodenum, jejunum, ileum, and colon of mice  of anti-α/β2/ϵ/β1 toxoid protein-specific systemic IgG  observed in the mice of pPG-T7g10-PPT/L. casei 393 group and PBS group before and after immunization. By contrast, the IgG and IgA antibody levels in the mice induced by the constitutive recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 were significantly higher (p< 0.01) than that induced by the xylose-inducible expression recombinant pPG-2-α-β2-ϵ-β1/L. casei 393 after the second booster. Furthermore, the levels of cytokines IL-4, IL-12, IL-10, IL-17, IL-2, and IFN-γ in the sera samples collected on days 35 after the primary immunization were determined by ELISA kits, and the results showed that significant levels of cytokines were detected in the sera of the mice orally immunized with pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 , and in and 60% ), respecC. perfringens, the liver, intestine, kidney, spleen, heart, and brain of mice in each group were isolated for histopathological observation. As shown in L. casei 393; slight congestion and hemorrhage changes were found in liver and kidney of the mice in the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 group and the commercial C. perfringens vaccine group. The most obvious histopathological changes in the intestine of the mice in the commercial vaccine group, pPG-T7g10-PPT/L. casei 393 group, and PBS group was observed, including severe disruption of the intestinal structural integrity, intestinal epithelial necrosis, and shortening of the villi. Moreover, hyperemia, hemorrhages, tubular epithelial cell degeneration/necrosis, and inflammatory cell infiltration were obvious in the histological sections of the kidney and liver of the mice in the pPG-T7g10-PPT/L. casei 393 and PBS groups.After being challenged with C. perfringens is a classical opportunistic pathogen in the gastrointestinal tract of livestock and poultry, causing a wide range of diseases such as enterotoxemia, dysentery, and hemorrhagic enteritis [C. perfringens are its exotoxins, which are mainly absorbed through the intestinal tract to cause diseases. Although there are many very successful commercial vaccines (toxoids and bacterins) that have been used for decades for a range of C. perfringens diseases of animals, there are a few clostridial diseases of animals for which convention vaccines have not been very effective. Thus, the development of vaccines that can effectively induce sIgA-based protective intestinal mucosal immunity against the toxins is of great importance for the control of C. perfringens diseases.nteritis ,35. The C. perfringens, which could induce effective systemic immune responses together with mucosal immunity through the mucosal immune system. However, vaccine antigens can easily be destroyed by gastric juice before reaching the intestine tract, generally resulting in a poor immunization effect. Therefore, an ideal antigen delivery carrier is crucial for developing oral vaccines. The delivery of vaccine antigens via viable vectors not only can stimulate the intestinal immune response, but can also produce specific immune responses against specific antigens that allow the body to achieve full protection. Compared to attenuated Salmonella as mucosal antigen delivery vehicles [L. casei in the food industry has been demonstrated to be non-pathogenic, and has been designated as “generally recognized as safe”. Currently, L. casei is recognized as a promising candidate used for delivering therapeutic proteins or antigen to the mucosal surface as a live carrier, which could effectively induce both mucosal and systemic immunity [Mucosal vaccination by oral administration is a better vaccine formulation against the toxins of vehicles –38, lactvehicles ,23,30,39L. casei as an antigen delivery vector, the EGFP-marked recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 expressing α, β2, ϵ, and β1 toxoids proteins of C. perfringens was successfully constructed with a constitutive expression plasmid pPG-T7g10-PPT. Currently, inducible expression systems such as xylose [L. casei to express α, β2, ϵ, and β1 toxoids proteins of C. perfringens, and our results showed that the fusion protein α-β2-ϵ-β1 could be constitutively expressed by the recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 cultured in MRS broth using raffinose, sucrose, lactose, trehalose, glucose and fructose as carbon sources, respectively, and can be recognized by anti-α/β2/ϵ/β1 toxoid monoclonal antibody, indicating that the recombinant L. casei constructed in this study can use different carbon sources to grow and express the protein of interest.In the present study, using s xylose ,33 and ns xylose  are wides xylose ,31, was L. casei 393, and the results showed that the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 could survive and adhere to the duodenum, jejunum, ileum, and colon of mice. Although the colonization rate of the recombinant lactobacilli was gradually decreased due to the metabolism of mice and the effects of the digestive tract environments, the CFDA-SE-labeled recombinant lactobacilli in the intestinal tract still remained detectable levels as analyzed by flow cytometry on day 15 after the oral administration.As a live mucosal vaccine, the adherence and colonization ability in the intestinal tract is important and desirable. Before oral immunization, we investigated the colonization potentiality of the recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 via oral immunization. Our data demonstrated that the recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 constructed in this study displayed a better immunogenicity, and significant levels (p< 0.01) of anti-α/β2/ϵ/β1 toxoid sIgA-based mucosal immune responses and anti-α/β2/ϵ/β1 toxoid IgG-based systemic immune responses were effectively elicited in mice orally immunized with the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 after the second booster, compared to the pPG-T7g10-PPT/L. casei 393 or PBS groups. Expectedly, significant levels (p< 0.01) of IgG and IgA antibodies could be elicited by the constitutive recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 compared with the xylose-inducible expression recombinant pPG-2-α-β2-ϵ-β1/L. casei 393 after the second booster.An effective mucosal vaccine should effectively induce intestinal mucosal and systemic immune responses. Of which, sIgA is the predominant antibody at the mucosal surface, which plays a key role in host protection against pathogenic infections. Thus, we evaluated the immunogenicity in mice of the recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 can also induce cellular immune responses, and higher levels of Th1-type  and Th2-type  cytokines were elicited in mice orally immunized with the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 than those in the control groups. CD4+ and CD8+ T cells are important effector cells for cell-mediated immunity. In this study, the spleen lymphocytes of mice in each group were isolated after immunization, and the percentage of CD4+ and CD8+ T cells was determined by flow cytometry. The results showed that the percentages of CD4+ and CD8+ T cells in splenocytes of mice from the recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 group were significantly increased, indicating that the immunization with recombinant lactobacillus promoted the proliferation of CD4+ and CD8+ T lymphocytes. These results demonstrated that oral vaccination with the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 can induce potent mucosal, cellular, and humoral immune responses. Moreover, using mouse as animal model which is one of the target species of C. perfringens infection, the immunoprotection of the recombinant lactobacillus pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 against C. perfringens toxins and C. perfringens were tested, respectively. In parallel, a commercially available inactivated C. perfringens vaccine and a xylose-inducible expression recombinant pPG-2-α-β2-ϵ-β1/L. casei 393 were used as positive immune control. The protection rates for mice oral immunized with the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393, pPG-2-α-β2-ϵ-β1/L. casei 393, and commercially available vaccine against crude C. perfringens toxins were 80%, 70%, and 60%, and against C. perfringens were 80%, 70%, and 50%, respectively, while all the mice in the control groups died, suggesting that the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 is a potential candidate vaccine against the diseases caused by C. perfringens, which indicates that constitutive expression lactobacillus system could provide more effective immune protection that inducible expression lactobacillus system. Moreover, the immune protection effect of the probiotic vaccine for other susceptible animals such as rabbits, cattle, and sheep/goat is underway to explore.Moreover, there was a positive correlation between lymphocyte proliferation rate and cell immunity; thus, we were able to detect the proliferation of spleen lymphocytes in immunized mice. Our results showed that the recombinant pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 was successfully developed by a flow cytometry-based screen. Prior to immunization with the probiotic vaccine, we had evaluated the segregational stability and structural stability of the recombinant strain pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 during serial subcultures until 50 generations, in the absence of selection pressure, and results showed that the recombinant strain had good stability properties (data not shown). However, before releasing the probiotic vaccine as commercial use in practice, there are more evaluations that need us to perform.Moreover, antibiotic resistance genes such as erythromycin, kanamycin, and chloramphenicol are often used as markers to screen for genetically engineered LAB ,33,41,42C. perfringens α, ϵ, β1, and β2 toxoids were developed in this study, and its immunogenicity as an oral probiotic vaccine was investigated. Our results showed that the pPG-Δ-E-α-β2-ϵ-β1/L. casei 393 could effectively induce mucosal, cellular, and humoral immune responses via oral administration and provide more effective protection against C. perfringens toxins and C. perfringens infections than inducible expression lactobacillus system, suggesting a promising candidate for the development of oral vaccine against C. perfringens. Actually, we will hardly have a problem with all these toxinotypes in the field. Therefore, it would be of great value to construct and test the separate toxinotypes.In conclusion, an EGFP-marked genetically engineered lactobacillus strain constitutively expressing