The remarkable catalytic performance is attributed to the mild affiliation of MXene to NPs, which not only stabilizes NPs to maintain a good dispersion but also leaves sufficient surface active sites to facilitate the catalytic reaction.Bimetallic cost-effective CoNi nanoparticles (NPs) are conveniently supported on titanium carbides (MXene) by a simple one-step wet-chemical method. The synthesized CoNi/MXene catalysts are characterized by XPS, TEM, STEM-HAADF and ICP-AES. The as-prepared CoNi NPs with a size of 2.8 nm are well dispersed on the MXene surface. It is found that among the CoNi bimetallic system, Co 0.7Ni0.3/MXene shows the best performance toward catalyzing AB decomposition with TOF of 87.6 mol2H molcat−1 min−1 at 50 °C.Bimetallic cost-effective CoNi nanoparticles are supported on MXene by a simple one-step wet-chemical method. The Co Although, noble metal-based catalysts, such as Pt, Rh, and Ru,13 provided the highest activities to dehydrogenation of AB, the high cost and scarcity of these catalysts hinders their industrial application. Alternatively, cost-effective metals have been recently developed. Especially, it is found that bimetallic systems usually show higher catalytic kinetics than their monometallic counterparts, due to the synergistic geometric and electronic effects of bimetallic systems, such as Cu–Ni, Cu–Co, Co–Ni.14–16 However, the aggregation of these catalysts always resulted in the tremendous loss of their active sites. To address this problem, different carriers such as graphene, metal–organic frameworks and carbon nanotubes were used to disperse metal nanoparticles (NPs).17–20 The results show that these carriers can effectively restrain the agglomeration of metal NPs and improve their catalytic activities. Recently, graphene-like transition metal carbide 2) has been widely investigated as a promising carrier for new nanocatalyst.21–26 The abundant functional groups on MXene surface, such as Ti–OH and Ti–F bonds, not only stabilize the nanoparticles during reduction but also improve the hydrophilicity of the resultant catalysts.27,28 However, there is rare report on catalytic properties of Ti3C2X2 supported NPs. In this work, for the first time, we synthesize a composite material of CoNi bimetallic nanoparticles supported on MXene via a simple one-step wet-chemical method. Unexpectedly, the resultant CoNi/MXene nanocatalysts with 100% of H2 selectivity show extremely high catalytic activity and excellent durability toward AB decomposition.In order to solve the global problems caused by heavy use of fossil fuels in the world, it is highly desirable to develop clean and sustainable energy sources and in turn decrease the consumption of traditional fossil fuels.−1) and 200 μL nickel chloride (0.03 mmol mL−1) were added into the MXene solution. The resulted mixture was stirred for 20 min with a rotating shaker (220 rpm). A 0.5 mL aqueous solution of sodium borohydride (NaBH4) (1.3 mol L−1) was quickly added into the resulted mixture under vigorous stirring for 3 h at 0 °C using an ice bath to maintain the temperature. When the situ synthesis reaction was completed, 0.5 mL of aqueous solution containing 1 mmol AB was injected into the mixture using a syringe, a gas burette filled with water was connected to the reaction flask to measure the volume of released gas. For comparison, CoNi/vulcanxc-72 carbon (CoNi/XC-72), CoNi/graphene oxide (CoNi/GO) and CoNi/Al2O3 reduced by NaBH4 were synthesized respectively by the similar method.The synthesis route of the CoNi/MXene nanocatalyst is shown in Transmission electron microscopy (TEM) images and high-resolution STEM measurements were obtained using Tecnai G2 F30 S-Twin instrument with a field emission gun operating at 200 kV. X-ray photoelectron spectroscopy (XPS) measurement was performed with ESCALAB 250Xi spectrophotometer. The metal contents of the catalyst were analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES) on Leeman PROFILE SPEC. Mass spectrometry (MS) analysis of the generated gas was performed using an OmniStar GSD320 mass spectrometer, wherein Ar was chosen as the carrying gas. PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O, respectively. Additionally, the presence of F and O indicate the introduction of functional groups such as OH and F on Ti3C2X2 surface  analysis of a representative nanoparticle of Co0.7Ni0.3 suggests that the as-synthesized specimen has a clearly identified lattice fringe space of 0.2039 nm, which is less than that of the (111) plane of pure face-centered cubic (fcc) Co (0.2046 nm), and larger than that of the (111) plane of pure face-centered cubic (fcc) Ni (0.2033 nm). These results confirmed the as-synthesized Co0.7Ni0.3 NPs with an alloy structure. In addition, an energy dispersive X-ray (EDX) spectrum proves the presence of Co and Ni elements with an atomic ratio of 2.35 : 1  and high resolution TEM (HRTEM) are used. As seen in t 2.8 nm  inset. T1 Fig. S4, which i1 Fig. S4. The ele1 Fig. S4 further xNix1−/MXene (0 ≤ x ≤ 1) NPs have been applied as catalysts for the AB decomposition at 50 °C with a constant molar ratio of catalyst/AB = 0.02  increases from 0.1 to 0.7, the reaction time decreases from 6.6 min to 1.7 min and the corresponding TOF value increases from 22.7 mol2H molcat−1 min−1 to 87.6 mol2H molcat−1 min−1, respectively. However, when the molar ratio of Co reaches 1.0, namely, no addition of Ni, the reaction is completed in 3.75 min. Obviously, the catalytic activity of the CoxNix1− alloy is better than those of pure Ni and Co. The reason for the enhanced performance of CoxNix1− alloy may be mainly resulted from the synergetic effect between Co and Ni, which can efficiently tune surface electronic states of bimetallic nanoparticles, particularly related to local strain and effective atomic coordination number at the surface, leading an apparent improvement for AB decomposition.The as-synthesized CoB = 0.02 . A fast on of AB . Mass sps Fig. S5. Notably0.7Ni0.3 NPs are loaded on different carriers such as GO (Co0.7Ni0.3/GO), XC-72 (Co0.7Ni0.3/XC-72), and Al2O3 (Co0.7Ni0.3/Al2O3) and their catalytic activities toward AB decomposition at 50 °C with a constant molar ratio of catalyst/AB = 0.02 are shown in 0.7Ni0.3/GO, Co0.7Ni0.3/XC-72 and Co0.7Ni0.3/Al2O3 are completed in 5.2, 7.2 and 12.6 min, respectively, and the corresponding TOF values are 28.8, 20.6 and 11.9 mol2H molcat−1 min−1 in the same reaction condition. Compared to Co0.7Ni0.3/GO, Co0.7Ni0.3/XC-72 and Co0.7Ni0.3/Al2O3, the prepared Co0.7Ni0.3/MXene show higher catalytic kinetics. The superior catalytic performance is mainly attributed that strong interface interaction between metal and carrier, especially for the defect-rich or oxygen group-deficient surfaces on MXene. Such interface interaction is considered to favour the formation of a tunable electronic state of metal NPs, which enhances the AB decomposition.Furthermore, the carrier materials also play an important role on determining the catalytic performance for AB decomposition. In order to evaluate the effect of the carrier materials on the catalytic performances toward the AB decomposition, CoEa) of the AB decomposition catalyzed by Co0.7Ni0.3/MXene catalysts, the reactions at different temperatures (30–60 °C) were carried out. Fig. S6a2versus reaction time for AB decomposition in the presence of Co0.7Ni0.3 catalyst at different temperatures. The Arrhenius plot of ln TOF vs. 1/T for the catalyst is plotted in Fig. S6b,Ea is calculated to be 36.9 kJ mol−1.In order to obtain the activation energy  of AB to the catalyst after the reaction completion for the last run. It is evident from 0.7Ni0.3/MXene possess a moderated durability in AB decomposition. It is due to that the functional OH and F groups on Ti3C2X2 surface, as an anchor, can stabilize the CoNi NPs, avoid the aggregation of CoNi NPs during the reaction process, which is confirmed by TEM images. As clearly seen from the TEM images (Fig. S7in situ synthesized NPs are magnetic and thus can be separated from the reaction solution by an external magnet (For the practical application of catalysts, the durability/stability of catalysts is the key point. Therefore, the durability of the Cos Fig. S7, the CoNl magnet , inside,0.7Ni0.3/MXene have been proven to the most reactive nanocatalyst in this family reward to AB decomposition with 100% of H2 selectivity and excellent catalytic performance of 87.6 mol2H molcat−1 min−1 at 50 °C. Additionally, the synergetic effect between CoNi NPs and MXene also improve the kinetic toward AB decomposition. This simple synthetic method can be easily extended to facile preparation other MXene supported metal NPs.In summary, cost-effective bimetallic CoNi NPs are well dispersed on MXene surfaces by a simple one-step wet-chemical method. Wherein the MXene plays an important role in stabilizing the CoNi NPs, leading a fine dispersion and good stability of the synthesized nanocatalyst in this heterogeneous system. By optimizing the fraction of Co component in Co–Ni system, the synthesized CoThere are no conflicts to declare.RA-008-C7RA10568A-s001


Plasmodium falciparum resistance to artemisinin and partner drugs were detected in the Greater Mekong Subregion. Recently, the independent evolution of artemisinin resistance has also been reported in Africa and South America. This alarming scenario calls for the urgent development of new antimalarials with novel modes of action. We investigated the interference with protein aggregation, which is potentially toxic for the cell and occurs abundantly in all Plasmodium stages, as a hitherto unexplored drug target in the pathogen.By 2016, signs of emergence of P. falciparum proteome’s propensity to aggregation by delivering endogenous aggregative peptides to in vitro cultures of this parasite did not significantly affect their growth. In contrast, protein aggregation inhibitors clearly reduced the pathogen’s viability. One such compound, the bis(styrylpyridinium) salt YAT2150, exhibited potent antiplasmodial activity with an in vitro IC50 of 90 nM for chloroquine- and artemisinin-resistant lines, arresting asexual blood parasites at the trophozoite stage, as well as interfering with the development of both sexual and hepatic forms of Plasmodium. At its IC50, this compound is a powerful inhibitor of the aggregation of the model amyloid β peptide fragment 1-40, and it reduces the amount of aggregated proteins in P. falciparum cultures, suggesting that the underlying antimalarial mechanism consists in a generalized impairment of proteostasis in the pathogen. YAT2150 has an easy, rapid, and inexpensive synthesis, and because it fluoresces when it accumulates in its main localization in the Plasmodium cytosol, it is a theranostic agent.Attempts to exacerbate the Plasmodium significantly reduces the parasite’s viability in vitro. Since YAT2150 belongs to a novel structural class of antiplasmodials with a mode of action that potentially targets multiple gene products, rapid evolution of resistance to this drug is unlikely to occur, making it a promising compound for the post-artemisinin era.Inhibiting protein aggregation in The online version contains supplementary material available at 10.1186/s12915-022-01374-4. Plasmodium. This situation calls for immediate efforts to discover new antimalarials of easy and cost-affordable production, having several molecular targets in the pathogen and acting through new antiparasitic mechanisms not shared by currently used drugs.The available arsenal of antimalarial drugs is insufficient to progress towards eradication of the disease, a scenario that is worsened by the rampant evolution of resistance by Plasmodium falciparum, is exceptionally rich in proteins containing long glutamine/asparagine (Q/N) repeats , 1.89 , 2.40 , 2.52 , 4.50 , 7.95 , 8.85 , 8.96 ; 13C NMR  δ: 16.3 , 19.6 , 25.4 (2 CH2), 28.3 (2 CH2), 28.7 (2 CH2) , 30.5 , 59.7 , 127.9 , 137.6 , 141.5 , 142.8 , 157.6 ; HRMS-ESI + m/z calculated for [C24H38N2]2+/2: 177.1512, found 177.1513.A mixture of 1,10-dibromodecane  and 3,4-dimethylpyridine  was heated at 120 °C for 3 h. Then, isopropanol (5 ml) was added, and the reaction mixture was stirred under reflux for 1 h. The mixture was allowed to cool down to RT, the resulting brown residue was washed with ice-cold Etbis dibromide  and 4-(diethylamino)benzaldehyde  in n-butanol (5 ml) was treated with six drops of piperidine and the reaction mixture was stirred under reflux for 4 h, and then concentrated under reduced pressure. The resulting black oily residue was purified by automatic flash column chromatography (CH2Cl2 / 7 N ammonia solution in MeOH 9:1), to provide 1,1'-bis{4-[(E)-4-(diethylamino)styryl]-3-methylpyridin-1-ium} dibromide  as a red oil that solidified on standing; mp: 173–174 °C; IR (ATR) ν: 3399, 2975, 2927, 2853, 1641, 1574, 1520, 1479, 1404, 1351, 1311, 1260, 1219, 1186, 1128, 1076, 1011, 958, 807, 572 cm−1; 1H NMR  δ: 1.13 , 1.21–1.31 , 1.87 , 2.48 , 3.43 , 4.37 , 6.74 , 7.08 , 7.64 , 7.88 , 8.26 , 8.66 , 8.73 ; 13C NMR  δ: 12.5 , 16.5 , 25.5 (2 CH2), 28.4 (2 CH2), 28.7 (2 CH2) , 30.5 , 43.9 , 58.9 , 111.3 , 113.4 , 119.8 , 122.1 , 130.8 , 132.9 , 140.5 , 142.4 , 143.3 , 149.5 , 152.4 ; HRMS-ESI + m/z calculated for [C46H64N4]2+/2: 336.2560, found 336.2550.A solution of 1,1'-For its use in the assays reported below, the final product, YAT2150, was dissolved in DMSO to obtain a 9 mM stock solution.Peptide stocks prepared in DMSO were diluted in phosphate buffered saline (PBS) at a final concentration of 150 μM. After vigorous vortexing, peptides were incubated at 37 °C and 1400 rpm in a ThermoMixer®  for 48 h. After that time, peptides were further diluted in triplicates to 25 μM in PBS, ThT was added at the same final concentration in PBS, and fluorescence emission was collected from 470 to 600 nm using an excitation wavelength of 450 nm . A blank measurement of each sample was done before adding ThT.Fluorescein-labeled peptides were diluted in PBS at 150 µM and incubated for 24 h at 37 °C and 1400 rpm. After this time, peptides were further diluted in triplicates in PBS to a final concentration of 15 µM, to which the protein aggregation detection reagent ProteoStat®  was added at 1:1000 final dilution and transferred to a 96-well black plate . ProteoStat® fluorescence was quantified  using respective excitation and emission wavelengths of 550 and 600 nm. The fluorescence of a ProteoStat®-only control was also measured and subtracted from the sample values.P. falciparum proteins were conducted in the same way.For the in vitro analysis of Aβ40 aggregation, 1 mg of Aβ40  was dissolved in 500 µl of 1,1,1,3,3,3-hexafluoro-2-propanol  under vigorous stirring for 1 h and sonicated for 30 min in an ultrasound bath. Afterwards, the solution was stirred for 1 h and maintained at 4 °C for 30 min. Aliquots were prepared, HFIP was evaporated under a nitrogen stream for a few seconds and the dry peptide was stored at − 20 °C. Prior to use, these Aβ40 aliquots were dissolved in DMSO and sonicated for 10 min to ensure minimal aggregation. To assess the effect of YAT2150 on the formation of amyloid fibrils, Aβ40 DMSO solutions were diluted to 25 µM in PBS containing different concentrations of YAT2150 and incubated for 24 h at 37 °C and 1400 rpm. Alternatively, to test the effect of YAT2150 on already formed amyloid fibrils, Aβ40 DMSO solutions were diluted to 25 µM in PBS and incubated as above in order to allow fibril formation. Then, YAT2150 was added at different concentrations and the mixture was incubated in the same conditions for another 24 h. The final samples always contained less than 5% DMSO to avoid interference of this solvent on Aβ40 amyloid fibril formation. Finally, ThT treatment was performed as described above. The analyses of aggregation inhibition and disaggregation performed with aggregative peptides present in P. falciparum parasites of the 3D7  and W2  strains , and Cam 3.II (chloroquine and sulfadoxine/pyrimethamine resistance), Cam 3.II + K13 R561H, Cam 3.II + K13 R539T, 3D7 + K13 R561H, and 3D7 + K13 M579I strains . After the incubation period, 3 µl of culture from each well were mixed with 197 µl of PBS containing 0.1 µM Syto 11 (Thermo Fisher Scientific), to obtain a final concentration of ca. 1–10 × 106 cells/ml. Parasitemia was assessed by flow cytometry using a LSRFortessa flow cytometer  set up with the 4 lasers, 20 parameters standard configuration. The single-cell population was selected on a forward-side scattergram. Syto 11 fluorescence signal was detected by exciting samples at 488 nm and collecting the emission with a 530/30-nm bandpass filter. Growth inhibition was calculated taking as reference values both the growth rate of the untreated culture and the growth rate of the culture treated with chloroquine. Growth inhibition data was transformed through sigmoidal fitting and used to determine the compound’s concentration required for the reduction of P. falciparum viability by 50% (IC50).s et al.  and kinds et al. . A posit50 values for each individual compound in the mixtures were calculated and plotted in an isobologram . Fractional inhibitory concentration (FIC) values were calculated by dividing the IC50 of one of the compounds in the mixture by the IC50 of the same compound in the 1:0 or 0:1 ratio mixtures.To assess the synergistic effect of YAT2150 and artemisinin, ICP. falciparum cultures were synchronized at ring or trophozoite stages by repeated treatment with 5% sorbitol or 70% Percoll  ethanesulfonic acid (HEPES). The ghost RBC pellet was taken up in an equal volume of RPMIc: RPMI containing 5 mg/ml Albumax II  and 2 mM L-glutamine, and finally stored at 4 °C until further use.Ghost RBCs loaded with various peptides were generated as previously described . BrieflyP. falciparum was performed by establishing a new culture using late-stage parasites purified in 70% Percoll as described elsewhere -1,3-benzene disulfonate labeling reagent (WST-1) was added to each well, and the plate was incubated in the same conditions for 2 h. After thoroughly mixing by pipetting up and down, the absorbance of the samples was measured at 440 nm using a Benchmark Plus microplate reader . WST-1 in the absence of cells was used as blank, and samples were prepared in triplicate for each experiment. Percentages of viability were obtained using non-treated cells as control of survival. The compound’s concentration required for the reduction of cell viability by 50% was defined as CC50. The in vitro selectivity index was defined as CC50/IC50.Human umbilical vein endothelial cells  were plated at 5000 cells/well in 96-well plates and grown in Medium 199 with Earle’s salts supplemented with 10% FBS and 1% penicillin/streptomycin for 24 h at 37 °C in 5% COParc Científic de Barcelona (PCB). BALB/c female and male mice  were maintained with unlimited access to food and water under standard environmental conditions (20–24 °C and 12/12 h light/dark cycle). Three 100-µl doses of a drug solution prepared to administer 0.0959, 0.3069, and 0.9822 mg YAT2150/kg were tested in a total number of 6 mice/drug dose. First, the lowest dose was intravenously injected to one female and one male mouse. An oxygen stream of 4% isoflurane was used to anesthetize the mice, which were then maintained during the whole injection procedure (less than 3 min) with 2.5% isoflurane. After the administration, mice were observed and different parameters related to their behavior  and physical conditions  were followed. If after 48 h no deleterious effects were observed, the following dose was administered to two other male and female mice. All mice were observed for 14 days after treatment in order to detect long-term side effects.In vivo assays were done at the animal facility of the 10 of the protein concentration values obtained from PaxDb [Protein abundance and aggregation propensity were calculated and plotted as elsewhere described . Brieflyom PaxDb , which wCi…Cn) is each value of protein concentration from the dataset, Cmin is the minimum value of protein concentration from the dataset, and Cmax is the maximum value of protein concentration from the dataset.where (Aggregation tendency (A) was obtained using the TANGO algorithm, which estimates the cross-beta aggregation propensity in peptides and denatured proteins . For theAi…An) is each value of protein aggregation from the dataset according to the “AGG” parameter of TANGO, Amin is the minimum value of protein aggregation from the dataset, and Amax is the maximum value of protein aggregation from the dataset.where . The normal distribution of the obtained data was assessed by various normality tests , and a two-sided test of variance was performed. Afterwards, samples were analyzed by two-sided Student’s Additional file 1:Figures S1-S12 and Tables S1-S9.Figure S1. Analysis of the aggregation of six peptides present in P. falciparum proteins. Figure S2. Flow cytometry analysis of the colocalization with RBCs and pRBCs of fluorescein-labeled LMWP-conjugated peptides. Figure S3. Flow cytometry analysis of the colocalization with RBCs and pRBCs of fluorescein-labeled TAT-conjugated peptides. Figure S4. Flow cytometry analysis of the colocalization with RBCs and pRBCs of fluorescein-labeled TP2-conjugated peptides. Figure S5. Flow cytometry analysis of the parasitemia, 72 h post-infection, in P. falciparum cultures grown in regular and ghost RBCs. Figure S6. Aggregative proteins found in late and early P. falciparum blood stages represented according to their abundance and aggregation propensity normalized relative to the whole proteome. Figure S7. In vitro analysis of the aggregation of KDLLF, KVVNI and derived peptides. Figure S8. Confocal fluorescence microscopy analysis of the presence of the fluorescein-labeled peptides KDLLF and KVVNI, and of their LMWP elongations, in ghost pRBCs. Figure S9. Effect of YAT2150 on pre-aggregated Aβ40. Figure S10. ThT analysis of the effect of YAT2150 on the in vitro aggregation of aggregative peptides present in P. falciparum proteins. Figure S11. Determination of protein aggregation in live P. falciparum cultures. Figure S12. Hemozoin formation assay. Table S1. Aggregative peptides selected from the pool of 369 proteins resisting dissolution in 0.1% SDS identified in Biosca et al., 2020. Table S2. Growth inhibition assay in regular P. falciparum cultures of fluorescein-labeled aggregative peptides conjugated to CPPs. Table S3. Growth inhibition assay in ghost RBC-enriched P. falciparum cultures treated with 10 µM non-modified aggregative peptides. Table S4. Cytotoxicity assay in HUVEC cultures of the aggregative peptides which at 10 µM reduced by > 20% P. falciparum growth in ghost pRBC cultures. Table S5. Early stage P. falciparum proteins resisting dissolution in 0.1% SDS. Table S6. Aggregative peptides identified in P. falciparum E3 ubiquitin-protein ligase (C0H4K6). Table S7. P. falciparum proteins in which the peptides KDLLF and KVVNI are present. Table S8. Growth inhibition assay in ghost RBC-enriched P. falciparum cultures treated with 10 µM KDLLF and KVVNI peptides. Table S9. In vitro toxicity in HUVEC cultures of amyloid pan-inhibitors.Additional file 2. Individual data values.Additional file 3. Original, uncropped blots from Fig. 9.


Conus species, we decoded a peptide sequence from Conus lividus and named it Lv32.1 (LvXXXIIA). The folding conditions of linear Lv32.1 on buffer, oxidizing agent, concentration of GSH/GSSG and reaction time were optimized for a maximum yield of (34.94 ± 0.96)%, providing an efficient solution for the synthesis of Lv32.1. Its disulfide connectivity was identified to be 1–3, 2–6, 4–5, which was first reported for the conotoxins with cysteine framework XXXII and different from the common connectivities established for conotoxins with six cysteines. The analgesic effect of Lv32.1 was determined by a hot plate test in mice. An evident increase in the pain threshold with time illustrated that Lv32.1 exhibited analgesic potency. The effects on Nav1.8 channel and α9α10 nAChR were detected, but weak inhibition was observed. In this work, we highlight the efficient synthesis, novel disulfide linkage and analgesic potential of Lv32.1, which laid a positive foundation for further development of conotoxin Lv32.1 as an analgesic candidate.In our work of screening analgesic peptides from the conotoxin libraries of diverse  Chronic pain is a suffering, costly and long-term problem that significantly affects society and individuals ,2. The Iv1.7 and Nav1.8), voltage-gated potassium channels (Kv7), neurotensin receptor, angiotensin II receptor and α9α10 nicotinic acetylcholine receptor (nAChR) have been proved to be associated with pain and show analgesic effects with nonopioid mechanisms of action 4+) . The seq4+) . The fra4+ ion) (2O2 (3%) and K3Fe(CN)6 (2 mg·mL−1), respectively. The linear Lv32.1 was folded only in the presence of GSH/GSSG. HPLC graphs of folding products of Lv32.1 in 0.1 M NH4HCO3 buffer for different reaction times were shown in 4HCO3 buffer were found to be generally higher than in other buffers, even though they had different concentrations of GSH/GSSG. The folding rate reached the maximum (34.94% ± 0.96%) in the condition of 0.1 M NH4HCO3 buffer with 1 mM GSH/0.5 mM GSSG at 25 °C for 1 h, in which the folded Lv32.1 was synthesized for subsequent study.The molecular weight of folded Lv32.1 was determined as 3563.20 by m/z 891.80 ([M+4H]4+ ion) . The mas4+ ion) , which my23/y22 (m/z 2749.2176/2589.1869) was 160.0307, which was equal to the mass of ethyl-labeled cysteine, since IAM (iodoacetamide) was applied as an alkylated agent. It meant the cysteine at position 10 was ethylated. The observation of ion y5 (m/z 604.3235) and fragment ion RAVC(IAM) (ay m/z 459.2496) suggested that thiols in 31-Cys was also IAM-labeled. All the mass differences of y30/y29 (m/z 3507.5346/3404.5254), y22/y21 (m/z 2589.1869/2486.1778) or b11/b10 (m/z 1196.4282/1093.4190), y14/y13 (m/z 1663.7025/1560.6934) and y8/y7 (m/z 935.4186/832.4094) were 103.0092, 103.0091 (or 103.0092), 103.0091 and 103.0092, respectively, indicating that the thiols of 3-, 11-, 19- and 25-cysteines were in their reduced form. Herein, a disulfide linkage existed between Cys-II (10-Cys) and Cys-VI (31-Cys). As for the four-cysteine-alkylated product of Lv32.1 ((IAM)C(IAM)I (ay m/z 656.3007) and QHC(IAM)C(IAM)I (by m/z 699.2701) combined with ions b11 (m/z 1310.4711) and a11/10a (m/z 1282.4762/1122.4455) indicated that 10- and 11-Cys were ethylated. Cysteine at positions 3 and 31 turned out to be IAM-labeled due to the presence of ions y2 (m/z 278.1169) and b4 (m/z 392.1234) and fragment ion SC(IAM)G (ay m/z 277.0965). The detection of ions y8/y7 (m/z 935.4186/832.4049) or b25/b24 (m/z 2964.2394/2861.2303), and the fragment ion YC(IAM)Y (ay m/z 402.1482) revealed that 25-Cys and 19-Cys were the two cysteines unlabeled by IAM, revealing that 19-Cys (Cys-IV) and 25-Cys (Cys-V) were connected to form a disulfide bond, while the remaining four alkylated Cys  belonged to other groups. Considering Cys-II (10-Cys) and Cys-VI (31-Cys) had been confirmed to be linked by the two-cysteine-alkylated product (The six cysteines (Cys) in conotoxin Lv32.1 were freely connected to form three disulfide bridges, the connectivity of which remained unknown, since the one-step folding strategy. Herein, conotoxin Lv32.1 was stepwise reduced and subsequently alkylated to generate the two-cysteine-alkylated product , respectf Lv32.1 , the fra product , then thi.c.v.) administration of Lv32.1 (10 nmol per mouse). The behavioral manifestation in the hot plate test showed an evident increase in the pain threshold with time. p < 0.01). This result stated that Lv32.1 exhibited a potent effect, which was a topic worthy of in-depth studying.To further explore whether conotoxin Lv32.1 has an analgesic effect, we used a hot plate assay to detect the latent value of mice at different time points after the intracerebroventricular  and the semi-activated state (TP2). In v1.8 channel by inhibiting (21.48 ± 1.22)% and (56.13 ± 0.05)% of the currents in the complete resting state (TP1) and the semi-activated state (TP2), respectively, at a concentration of 10 μmol/L. As to the effect on α9α10 nAChR, only about 10% of the ACh-evoked currents were inhibited by Lv32.1 with a final concentration of 1 μmol/L  of this sequence has been reported by Mahardika’s group since 2019  was purchased from Beijing Enokai Technology Co., Ltd.  and used as the positive control. For current recording, the cell-planted coverslip was placed into the recording bath of an inverted microscope  and the recording electrode was contacted to the cell. After sealing the cell, the membrane voltage was clamped at −120 mV for 30 ms (microseconds) as the resting state (TP1 state). The holding potential depolarized to 0 mV for 50 ms and was recovered to −75 mV (semi-inactivation voltage) for 8 s to reach the half-inactivated state (TP2 state). Solutions of Lv32.1, positive control and negative control (DMSO) separately passed through the recording bath by gravity perfusion for 5 min, repeated thrice, at both TP1 state and TP2 state. The corresponding currents were recorded by an EPC-10 amplifier  and stored by PatchMaster  software.The blocking effect of Lv32.1 on the Nareported , by wholreported . In shor2, 2.0 mmol/L KCl, 1.0 mmol/L MgCl2, 5.0 mmol/L HEPES, pH 7.4), ND96 buffer containing antibiotic  and OR-2 buffer . Briefly, oocytes were obtained by dissecting from pre-anesthetized mature female Xenopus laevis frogs. After enzymolysis by 20 mg collagenase  incubated in 40 mL OR-2 buffer at 25 °C for 40 min, separated oocytes were gained for the subsequent expression of α9α10 nAchR. Linearized plasmids of the rat α9 and α10 subunits  were transcribed in vitro using the Mmessage MESSAGE mMACHINETM SP6 Transcription Kit . Then, α9 and α10 cRNAs were purified by a MEGAclearTM Transcription Clean-Up Kit . The cRNAs of α9 and α10 were mixed at a ratio of 1:1 and injected into oocytes (59.6 nL cRNA for each subunit) and cultured at 17 °C for 2–3 days. The oocytes were then fixed in an oocyte chamber and perfused with BSA-containing ND96 at a rate of 2 mL/min. The oocyte was given 100 μM ACh for 2 s and ND96 for 58 s. The membrane potential of the oocyte was clamped at −70 mV by two-electrode voltage patch clamp and the elicited current was recorded as control current. Conotoxin GeXIVA  was used as the positive control in our previous study [The α9α10 nAchR inhibitory activity of Lv32.1 was conducted as previously reported by two-electrode voltage patch clamp system  . Solutious study . When thv1.8 Channel were analyzed by PatchMaster  and IGOR Pro . The reaction ratio was defined as Data for α9α10 nAChR were collected by Clampfit 10.2 , and data for NaIn this work, we highlight the efficient synthesis, novel disulfide linkage and analgesic potential of Lv32.1, which laid a positive foundation for further development of conotoxin Lv32.1 as an analgesic candidate.


Spinach seedlings were transplanted into Styrofoam planting boxes filled with the substrate. Each planting box was irrigated daily by drip with a complete nutrient solution. Plants grown with MSW had a higher content of calcium. Shoot Mn increased in the biochar-containing mixes. The shoot dry weight of the plants grown in the different blends was higher than those grown in coir. Fresh yield was higher in mixes with MSW and perlite (3 kg/m2) or pine bark (2.87 kg/m2). Total phenols and DPPH antioxidant activity were not affected by the substrates. However, shoot ascorbate (AsA) content was higher or equal to those plants grown in coir. MSW and biochar are alternatives to reduce the use of coir and peat.The use of municipal solid waste compost (MSW) and biochar, two renewable resources with a low carbon footprint as components of substrates, may be an alternative to reducing peat and coir usage. The aim of this study was to assess the suitability of selectively collected MSW and biochar as components of the coir-based substrate to spinach grown. An experiment was carried out to evaluate five substrates, coir and four coir-based blends  with 12% ( Nowadays, in soilless culture in the substrate, the main goal is to reduce or replace peat as the constituent of substrates owing to high negative ecological and environmental impacts.There is increasing pressure on stakeholders (both growing media manufacturers and horticulturists) to significantly reduce their reliance on peat . For thew/w) to a soilless mixture of coconut fiber and tuff (volcanic ash) .Leaf total chlorophyll and Chl a were related to leaf Ca according to polynomial regressions: . Leaf chlorophyll and carotenoid content also increased with leaf Ca2+ excess and deficiency in Mentha Pulegium L. .Leaf Cc average values ranged from 39.69 to 54.76 mg 100 gorted in  (17–32 m FW) and  .Shoot dry weight and fresh yield were affected by the substrate . Plants S·m−1) (S m−1), plants grown in those mixes had a greater shoot dry and fresh yield than those grown in the other substrates  , higher bstrates . In the bstrates .2) were also high. This can be owing to the constant high-water potential around the plant roots and/or the cultivation period that did not subject plants to high transpiration or temperature. Under these conditions, greenhouse plants can grow with higher electrical conductivities (3 to 6 dS m−1) than in the current study , which was higher in plants grown in mixes with biochar  + 627.01, R2 = 0.8325 p < 0.01). In lettuce, the FRAP content response to different levels of calcium nitrate was also not linear [Leaf FRAP antioxidant activity was higher in plants grown in coir (235.8 mg/Trolox/100 g FW) and coir + biochar + pine bark (211.9 Trolox mg/100 g FW) than in those grown in the other mixes . Leaf FRt linear , likely 2O2 via ascorbate peroxidase (APx) [The ascorbate-glutathione (AsA-GSH) pathway comprises of AsA (ascorbate), GSH (glutathione), and four enzymes that play a vital role in detoxifying ROS . AsA levse (APx) . Plants grown in coir + biochar + perlite, coir + MSW + perlite, and coir + biochar + pine bark had higher leaf AsA than those grown in coir and coir + MSW + pine bark . This suThe average AsA levels in leaves ranged from 9.06 to 16 mg/100 g FW. These values were similar to those published in  (11 to 4Plants grown in coir had higher levels of APx (ascorbate peroxidase) and GSH than those grown in mixtures.2O2 in the cytosol and chloroplast [Ascorbate peroxidase, which predominantly scavenges Horoplast , increasoroplast . Ascorbaoroplast , beans [oroplast , and citoroplast . In the As reported in , there ap < 0.01).APx activity generally increases along with other antioxidants, such as GSH in plant tissues, and enhances plant stress tolerance ,57,58. ALeaf glutathione peroxidase (GPx) was higher in plants grown in coir + biochar + pine bark than in those grown in the other substrates.p < 0.01). This was also reported in [Leaf glutathione reductase (GR) activity was higher in plants grown in mixes with biochar than in those grown in the other substrates. However, perlite addition to the mix with biochar led to a higher increase in leaf GR. The increase in the GR activity was positively correlated with shoot Mn uptake , emitter position in relation to plants, and irrigation scheduling, adjusted to each mix on the Ca and Mn content in the leaves.Despite the differences, this study revealed that using municipal compost and biochar, two renewable wastes produced locally, allows obtaining high yields and high-quality spinach while using less coir and peat.v/v) of selectively collected municipal organic compost or biochar and 10% (v/v) perlite or pine bark was equal to or higher than those obtained in coir. Blends with municipal compost, perlite, or pine bark increased spinach fresh yield by 28% and 13%, respectively, when compared to coir. The mixes did not affect total phenols content or DPPH antioxidant activity. Phytochemical accumulation was significantly influenced by leaf calcium content. However, as there are few studies on the effect of leaf calcium on antioxidant response, more research is needed.Municipal compost and biochar are viable alternatives to reducing the use of coir and peat, contributing to increasing the sustainability of soilless culture in substrate. Spinach fresh yield in all blends with 12% , University of Évora, Portugal. The greenhouse was covered with polycarbonate and had no supplemental lighting or heating. Diurnal changes in air temperature inside the greenhouse at the plant canopy level ranged from 8 to 27 °C. Solar radiation ranged from 34 to 248 W·mS m−1, granulometry = 0–10 mm, total porosity = 95%, air  = 25, and CEC (meq/100 g) = 60–120.The experiment included five substrates: coir (control) and four mixtures. The mixtures were coir-based (Projar S.A. Spain) and mixed with other components. The coir (100% coir pith) had a pH of 5.5–6.0, electrical conductivity (EC) > 1.5 dTwo renewable resources produced in Portugal were used as mix components: municipal solid waste compost (MSW)  and Acacia wood biochar . The raw materials used in the “Nutrimais” manufacturing process include horticultural products, food scraps carefully selected from restaurants, canteens, and similar establishments, forest exploitation residues , and green residues .® Extra  were used. According to the manufacturer, pine bark is made up of 100% maritime pine bark, selected, screened, and calibrated. After sieving, it was subjected to heat treatment to eliminate the pine wood nematode (Bursaphelenchus xylophilus).To improve and adjust the characteristics of the mixes, pine bark , also a renewable product produced in Portugal, and Perlite PERLIGANThe MSW was produced from organic material selectively collected and the maximum heavy metal concentration was below the maximal levels permitted in different European countries .2O5 (1.2%), K2O (1.81%), CaO (7.06%), MgO (0.2%), and 0.3, 28.7, 15, 51.0, 0.1, 7.6, 150.0, and 32.3 mg/kg of Cd, Pb, Cr, Cu, Hg, Ni, Zn, and B, respectively. The EC and pH  were 5.4 dS.m−1 and 9.0, respectively.MSW compost in the powdery form had (expressed as a percentage of compost dry weight): organic matter (53.2%), humidity (23.7%), humic acids (13%), C/N ratio (12.4), C (29.15%), N (2.3%), PS m−1, and a granulometry of 1 to 20 mm. The pine bark had a particle size of between 8 and 15 mm, and the pH of CaCl2 was 4.5. Pine bark had (expressed as a percentage of compost dry weight): organic matter (99.1%), C/N ratio (278), C (55.6%), N (0.20%), P2O5 (0.04%), K2O (0.11%), and Mg (0.05%). The perlite was pH-neutral, chemically inert, salt-free, and had a grain size of 2 to 6 mm. Acacia wood biochar from pyrolysis had a pH of 8 to 10, an EC of 0.25 dv/v) in the mixtures is presented in The percentage of the different components . The aqueous extract of each mix was obtained by combining one part (by volume) of substrate with five parts (by volume) of distilled water. The sample was mixed and left to stand for 30 min to equilibrate. The mixture was poured into a clean funnel lined with a filter to avoid getting substrate in the solution. The pH and EC were measured using a pH meter  and a conductivity meter , respectively.Physical properties of mass wetness, moisture content, total porosity, and bulk density were determined following the methodology described in .Spinacia oleracea L. cv. Tragopan) seedlings were produced in soil blocks, with four seedlings per block. Soil blocks were obtained from a commercial nursery. The seedlings were grown in a growing medium (90% black peat and 10% brown peat) and fertilized with 0.5 g of 8-17-27 per liter of the substrate. They were transplanted into Styrofoam plant boxes, on 23 February 2021, at 22 days after emergence. The boxes (100 × 25 × 10 cm) were filled with 14 L of each mixture at the height of approximately 7 cm. The blocks were spaced at 12.5 cm in two rows per box and 10 cm between rows with a plant density of 256 plants m−2.Spinach  measured using a soil moisture probe (SM105T delta devices England), and the volume of water was drained. The nutrient solution was applied 3 to 7 times per day and averaged 10% to 35% of drainage (leaching fraction) for each application. The leaching fraction was controlled through a relay level connected to an electric valve that stopped watering when the level of leached water was within 10% to 25% of the applied water. The nutrient solution, except in the first irrigation, to moisten the growing medium was applied in each irrigation from transplanting to the day before harvesting.−1 NO3-N, 2.71 mmol L−1 NH4-N, 0.68 mmol L−1 P, 3.88 mmol L−1 K, 2.89 mmol L−1 Ca, 1.49 mmol L−1 Mg, 0.80 mmol L−1 S, 46 µmol L−1 B, 7.86 µmol L−1 Cu chelated by EDTA, 8.95 µmol L−1 Fe chelated by EDTA, 18.3 µmol L−1 Mn chelated by EDTA, 1 µmol L−1 Mo, 2 µmol L−1 Zn chelated by EDTA, 2.1 mmol L−1 Cl, and 0.7 mmol L−1 Na. The EC value of the nutrient solution ranged over time was: 1.2 ± 0.2 dS m−1 , 1.4 ± 0.2 dS m−1 (from 7 to 20 DAT), and 1.8 ± 0.2 dS m−1 (from 21 to 30 DAT). The EC values were obtained by changing the injection rate of the nutrient solution. Initially, the solution contained 8.34 mmol L3/NH4 ratio in the nutrient solution were adjusted to 4.26 mmol·L−1 NO3-N, 4.11 mmol·L−1 NH4-N, 0.67 mmol·L-1 P, 2.84 mmol·L−1 K, 2.13 mmol·L−1 Ca, 0.88 mmol·L−1 Mg, 0.47 mmol·L−1 S, 46 µmol L−1 B, 7.86 µmol L−1 Cu chelated by EDTA, 8.95 µmol L−1 Fe chelated by EDTA, 18.3 µmol L−1 Mn chelated by EDTA, 1 µmol L−1 Mo, 2 µmol L−1 Zn chelated by EDTA, 2.1 mmol L−1 Cl, and 0.7 mmol L−1 Na. The EC of the nutrient solution was 1.9 ± 0.2 dS m−1.At 31 DAT, to reduce the nitrate concentration in the leaves, the nutrient concentration and the NO3- of the drainage water from each box were measured weekly using a potentiometer (pH Micro 2000 Crison), a conductivity meter , and an ion-specific electrode , respectively, following the procedures outlined in [The pH, EC, and the concentration of NOlined in . The plants were harvested at 34 DAT (28 March 2021). The shoots of the plants were cut off at 1 cm above the substrate surface and rinsed with distilled water. Five sample plants (shoots) from each box were washed, oven-dried at 70 °C for 2–3 days, weighed, ground so that they would pass through a 40-mesh sieve, then analyzed for N, P, K, Ca, Mg, Na, B, Cu, Mn, and Zn. Total N was analyzed by using a combustion analyzer . The K and Na were analyzed by flame photometry . The P and B were analyzed using a UV/Vis spectrometer . The remaining nutrients were analyzed using an atomic absorption spectrometer .v/v), MW90 extract) for 1 min and then centrifuged at 4 °C at 6440× g for 5 min [Samples of 1000 g of spinach leaf-blade from each box were macerated in a mortar and homogenized in 8 mL of methanol/water solution  and b (Chl b), and total carotenoids (Cc) were determined in MW90 extract, as previously described in .v/v), MW80 extract) for 1 min and then centrifuged at 4 °C at 6440× g for 5 min. The extracts were stored in aliquots at −20 °C for later use.Samples of 1000 g of spinach leaf-blade were macerated in a mortar and homogenized in 8 mL of methanol/water solution , ascorbate (AsA), as well as the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging antioxidant power (DPPH) and ferric-reducing antioxidant power (FRAP) of MW80 extract were determined using the methodology described in [2 and homogenized in 5 mL of 0.12 mM phosphate buffer, pH 7.2. The supernatant obtained after centrifugation of this extract for 15 min at 15,000× g at 4 °C was collected and stored in aliquots at −20 °C (PB extract) [Samples of 1000 g of spinach leaf-blade were macerated in liquid Nextract) .Glutathione (GSH), protein content, glutathione reductase (GR), and peroxidase (POx) enzyme activities were determined in PB extract in accordance with the methodology described in . −1 cm−1 for NADPH. GPx activity was expressed in terms of nmol min−1/mg protein.Glutathione peroxidase (GPx) enzyme activity was determined in PB extract in accordance with  in a rea2O2 to H2O. The reaction was started by mixing 1 mM of ascorbate, 10 mM of H2O2, 0.1 mM of EDTA, and a suitable volume of leaf-blade PB extract in 50 mM of phosphate buffer, pH 7.0. The ascorbate consumption was followed by reading the absorbance at 290 nm, for 180 s at 37 °C. APx enzyme activity was calculated based on the slope of the reaction curves, using an extinction coefficient value of 2.8 mM−1.cm−1 for ascorbate.Ascorbate peroxidase (APx) activity was determined in PB extract in accordance with the Janda method , which iData were analyzed by analysis of variance using SPSS Statistics 25 software . Means were separated at the 5% level using Duncan’s new multiple range test. Bivariate correlation analysis between parameters was realized using Pearson’s bilateral correlation coefficient.


After single administration, blood samples were collected before administration and at different time points after administration in different groups of rats. In the study of long-term treatment effects, blood samples were collected from the animals in each group on days 1, 15, and 30 and on day 15 after withdrawal. The results showed that after a single administration, the dose change had no significant effect on the T1/2 and Tmax of agarotetrol, isoliquiritigenin, and piperine (p > 0.05). There was a certain correlation between the increase in AUC0-t and the Cmax of agarotetrol, isoliquiritigenin, piperine, and the increase in dosage, with a dose range of 0.225–0.900 g/kg. There were no significant differences in Cmax and AUC0-t of ferulic acid at different doses (p > 0.05). Meanwhile, there was no significant sex-based difference in the pharmacokinetic parameters of these four components in rats. After long-term administration, the distribution agarotetrol in various tissues of rats was kidney > liver > heart > brain; the tissue distribution in low- and medium-dose groups of isoliquiritigenin was liver > kidney > heart > brain, and in the high-dose group, kidney > liver > heart > brain. The tissue distribution of piperine in each dose group was liver > kidney > heart > brain, and that of ferulic acid in each dose group was kidney > liver > heart > brain. Through the establishment of the previously developed methodology, the pharmacokinetic properties of RYZB were analyzed after a single administration and long-term administration. Our findings confirmed this approach for the exploration and establishment of a pharmacokinetic evaluation of Tibetan medicine, to support its guiding role in clinical application, but also to accelerate research into Tibetan medicine theory and medicine and to provide a solid foundation for the translation of Tibetan medicine throughout the world.Tibetan medicine is one of the oldest traditional medicine systems in the world. Taking the Ruyi Zhenbao tablet (RYZB) as an example, which is a widely used classic oral Tibetan medicine, this article discusses the pharmacokinetics of single administration and long-term treatment and analyzed its metabolic properties and tissue distribution  Tibetan medicine, originating from Tibet, China, is one of the oldest traditional medicine systems in the world. In its natural environment with high altitude, thin air, and lack of oxygen, through long-term rich production and living practice, Tibetan medicine has unique properties and advantages over other traditional medicines, generating a complete medical theoretical system with good clinical efficacy , and thuMother-of-Pearl, Aquilariae Lignum Resinatum, Travertine, Micae Aureus Lapis, Carthami Flos, Brachyura, Caryophylli Flos, and Fructus Terminaliae Billericae. It has effects of clearing away heat, resuscitating, relaxing muscles and tendons, activating collaterals, and drying yellow water, and has mainly been used to treat diseases such as white chanel disorder , numbness of the limbs, paralysis, facial distortion, unconsciousness, arthralgia, gout, limb rigidity, and joint disadvantage (Ruyi Zhenbao tablet (RYZB) is a widely used classic oral Tibetan medicine, which was recorded in the book Tibetan Medicine Notes written by the famous doctor Gong Zhi Yuan Dan Jia Cuo. RYZB consists of 30 types of medicinal constituents, such as dvantage .In the previous pre-experiment, we did a lot of work to explore the pharmaceutical ingredients in RYZB that need to be detected. Different from chemical medicine, RYZB contains a lot of medicinal materials and there are many components in different medicinal materials. At present, pharmacokinetic research on TCM was very little, especially in Tibetan medicine. The current research studies on RYZB mainly focused on pharmacology and quality standards , but the17H18O6) present in Aquilariae Lignum Resinatum, piperine (C17H19NO3) in Piper longum L., isoliquiritigenin (C15H12O4) in Dalbergia odorifera T. Chen, and ferulic acid in licorice cream were selected.Thus, in this study, based on the composition of medicinal materials in RYZB and the prior studies on blood components, the main active components in the medicinal materials from RYZB, which were successfully detected in plasma and were more suitable for the construction of analytical methods; meanwhile, whose standard products could be purchased, were used for the pharmacokinetic study . A totalin vivo were analyzed  was purchased from Merck, United States, batch no. l1108107033 and formic acid amine was from Sinopharm Chemical Reagents Co., Ltd., China, batch no. 30011661.RYZB was purchased from Gansu Cheezheng Tibetan Medicine Co., Ltd., China, batch no. 2004007 . Agarotetrol was obtained from Chengdu Push Biotechnology Co., Ltd., China, batch no. PU0295-0025, purity 99.7%. Piperine was obtained from Dalian meilunbioThe liquid chromatography system, Waters ACQUITY UPLC H-CLASS ultra-high performance liquid chromatography, was obtained from Waters Company, United States. High-resolution mass detection was performed on a Q-Exactive-Orbitrap mass spectrometer, Thermo Fischer Scientific, United States; mass spectrometry system, 6500+ triple quadrupole tandem mass spectrometer, SCIEX Company, United States. The analytical balance was obtained from the METTLER TOLEDO XPE205 millionth analytical balance, Mettler Toledo Instrument Co., Ltd., United States and the centrifuge, EPPENDORF 5427R small desktop freezing high-speed centrifuge was from EPPENDORF Company, Germany. The vortex mixer, IKA VORTEX2, was from IKA Company, Germany.−1. A total of 24 SD rats  were randomly divided into four groups: control group, low-dose group , middle-dose group , and high-dose group , at a ratio of 1:1 males to females. Blood samples were collected from the eye socket before and at 0.25, 0.5, 0.75, 1, 2, 4, 8, 24, and 48 h after administration and heparin anticoagulation was used. Samples were centrifuged at 4°C and 10,000 rpm for 2 min, divided into aliquots, and frozen at −80°C.Clinical dosage of RYZB: each tablet weighs 0.5 g, 4–5 tablets once, twice a day . The cli−1), middle-dose group , and low-dose group , with 16 rats in each group, divided into eight males and eight female rats, of which eight rats were treated at 1 month after administration  were randomly divided into four groups: control group, high-dose group , the animals were anesthetized and blood was drawn from the abdominal aorta . Organs −1) containing internal standard was added ; mobile phase B (MPB): methanol; column temperature: 4°C; flow rate: 0.300 ml min−1; temperature of the automatic sampler: 4°C; injection volume: 5.00 μl; the chromatographic gradients are described in Chromatographic column: waters ACQUITY UPLC BEH C18, 2.1 mm × 100 mm I.D, 1.7 μm ; precolumn: ACQUITY UPLC BEH C18 VanGuard Pre-column, 130 Å, 1.7 µm, 2.1 mm × 5 mm; mobile phase A (MPA): 5 mmol LThe following mass spectrometry conditions were used: ion source: electrospray ionization (ESI); ionization mode (positive/negative); mode: multi-reaction monitoring (MRM); ion spray voltage: 5,500–4,500 V; turbo ion spray temperature: 450°C; curtain gas type: nitrogen setting, 35.0 psi; nebulizing gas; gas 1: nitrogen setting, 50.0 psi; auxiliary gas, gas 2 nitrogen setting, 50.0 psi; acquisition time: 10.0 min . The masThe tissue samples were accurately weighed and placed in a homogenizer, and pure water (0.02% formic acid) was added according to the mass-volume ratio of 1:2 to fully homogenize the samples. A 100-μl sample of uniform tissue homogenate was placed in a 2.0 ml Eppendorf tube , 900 μl of methanol (containing 0.1% formic acid) was added and the tube was vortexed for 5min, followed by ultrasonic mixing for 1 min. After centrifuging at 4°C for 5 min , 900 μl of the supernatant was transferred to a new test tube and dried in a 48°C water bath. A 100 μl equal proportion mobile phase was added to the precipitate before loading.−1; column temperature: 40°C; sampling volume: 5.00 μl; the chromatographic gradients are shown in The following liquid chromatography system and conditions were used: Waters ACQUITY UPLC H-CLASS ultra-high performance liquid chromatography; column: Waters Acquity UPLC HSS T3 column ; MPA: 0.02% formic acid water; MPB: acetonitrile: methanol = 1:1 (containing 0.02% formic acid); flow rate: 0.300 ml min2 (Top 5); polarity: positive and negative ion scanning; resolution: 70000 FWHM (Full MS); 17500 FWHM (dd-MS2); scan range: 150–800 m·z−1; spray voltage: +3.8 kV/−2.8 kV; AGC target: 3e6 (Full MS); 1e5 (dd-MS2); microscans: 1; maximum inject time: 50.0 ms (Full MS); 50.0 ms (dd-MS2); isolation window: 2.00 m z−1; dynamic exclusion: 10.0 s; sheath gas flow rate: 35.0 arbs; auxiliary gas flow rate: 10.0 arbs; sweep gas flow rate: 0 arbs; capillary temperature: 325°C; heater temperature: 350°C; stepped normalized collision energy: 20.0, 40.0, and 60.0 eV (The following mass spectrometry (MS) conditions were used: MS/MS system: Q Exactive Orbitrap Mass Spectrometer; ion source: heated electrospray ion source; scan mode: full MS/dd-MS 60.0 eV .The chromatogram collection and peak integration of analyte and internal standard were processed by MultiQuant 3.0.3 analysis software (AB Sciex). After optimizing the integration parameters, the target peak was automatically integrated and it was not allowed to integrate the peak separately or manually.2), and the regression equation (Y = a+bX) was the standard curve. The concentration of the analyte in the rat plasma was calculated according to the standard curve of the analysis batch, and the concentration unit was ng ml−1. The Phoenix WinNonlin non-compartment model module (version 8.1.0.3530) was used to calculate the pharmacokinetic parameters.Calculation of the analyte concentration was determined by obtaining the chromatographic peak areas of the analyte and the internal standard using software Analyst 1.6.3. The ratio of the analyte concentration (x) to peak area (y) in plasma was linearly regressed using the weighted least squares method (W = 1/xmax and AUC0-t) obeyed a normal distribution after logarithmic conversion. The LEVENE test was used to test the homogeneity of variance for the converted parameters. When the variance was uniform (p > 0.05), Student’s t-test was used to evaluate sex-based differences and single-factor variance analysis was used for dose difference. When the variance was uneven (p ≤ 0.05), the Mann–Whitney U-test (M–W method) was used for sex-based differences, while the Kruskal–Wallis H rank sum test was used to evaluate dose difference.The statistical analysis assumed that the main pharmacokinetic parameters , and t1/2 and Tmax were not affected by the dose change (p > 0.05) .After oral administration of RYZB in the low, middle, and high groups in rats, the main pharmacokinetic parameters AUCp > 0.05) , and t1/2 and Tmax were not affected by dose change (p > 0.05). Cmax, AUC0-t, and t1/2 of piperine changed significantly (p < 0.05), but did not have a significant effect on the Tmax (p > 0.05). There was no significant effect on Cmax, AUC0-t, t1/2, or the Tmax of ferulic acid (p > 0.05); details are given in On D1 after administration, Cmax and AUC0-t of agarotetrol and isoliquiritigenin changed significantly (p < 0.05), although t1/2 and Tmax were not affected by dose changes (p > 0.05); Cmax, AUC0-t, and Tmax of piperine changed significantly (p < 0.05), but had no significant effect on t1/2 (p > 0.05). The dosage change had no significant effects on Cmax, AUC0-t, Tmax, or t1/2 of ferulic acid (p > 0.05), details are given in On D15 after administration, Cmax and AUC0-t of agarotetrol, isoliquiritigenin, and piperine changed significantly (p < 0.05), but had no significant effect on t1/2 or Tmax (p > 0.05). The dosage change had no significant effect on Cmax, AUC0-t, Tmax, or the t1/2 of ferulic acid (p > 0.05), details are given in On D30 after administration, Cp > 0.05). On D30 of administration, sex-based differences were significant, with differences in Cmax and AUC0-t (p < 0.05), while t1/2 and Tmax were not affected by sex (p > 0.05); see Agaroterol: on D1 and D15 of administration, sex did not have an effect on the main pharmacokinetic parameters of agaroterol (p > 0.05). After D15 of administration, sex had a significant effect on Cmax (p < 0.05), but had no effect on other pharmacokinetic parameters (p > 0.05). On the D30 of administration, sex had a significant effect on AUC0-t and Tmax (p < 0.05), but had no effect on Cmax and t1/2 (p > 0.05); see Isoliquiritigenin: the pharmacokinetic parameters of isoliquiritigenin were not affected by sex on D1 of administration (p > 0.05). After D30 of administration, sex significantly influenced the pharmacokinetic parameters Cmax, AUC0-t, and Tmax (p < 0.05), but not t1/2 (p > 0.05); see Piperine: on D1 and D15 of administration, the results showed that sex had no effect on the pharmacokinetic parameters of piperine (max (p < 0.05), but had no influence on other pharmacokinetic parameters (p > 0.05). On D30 of administration, the results showed that sex had significant effects on AUC0-t and Cmax (p < 0.05), but had no significant effects on t1/2 or Tmax (p > 0.05); see Ferulic acid: on D1 and D15 of administration, sex had significant influence on CAfter 15 days of withdrawal, agarotetrol and piperine were not detected for any of the three doses; isoliquiritigenin and ferulic acid could be detected in the low dose group, while ferulic acid could be detected in the middle and high doses; see 0-t for the low, middle, and high dose groups of agarotetrol in plasma were 2.39, 0.77, and 1.10, respectively, and the Cmax ratio was 2.28, 0.65, and 1.14, respectively. The average ratios of AUC0-t and Cmax for isoliquiritigenin were 1.60, 0.63, and 1.17, respectively, and 1.48, 0.75, and 1.30 respectively. The average ratios of AUC0-t and Cmax for piperine were 2.94, 0.657, and 0.735, respectively, and 2.29, 0.38, and 0.76, respectively. The average ratios of AUC0-t and Cmax for ferulic acid were 0.27, 0.08, and 0.76, and 0.53, 0.31, and 0.74, respectively; see The average ratios of AUC−1 of RYZB, agarotetrol, ferulic acid, piperine, and isoliquiritigenin were detected in the brain, heart, liver, and kidney tissues and their concentrations were higher in the kidney and liver, but lower in the heart and brain tissues. Agarotetrol was distributed mainly to the kidney, liver, and heart, but less to the brain. Isoliquiritigenin was distributed more to the kidney and liver, but less to the brain and heart. Piperine was distributed more in the liver and distributed more to the brain than the other three substances, and was less distributed to the heart and kidney. Ferulic acid was mainly distributed to the kidney, followed by the liver, but was less distributed to the brain and heart; see After rats were given 1.50, 3.00, and 6.00 g kgAfter RYZB was administered to rats, tissue samples were analyzed and processed using a combination of full MS/dd-MS2 and the database mzCloud and ChemSpider. Fourteen components were identified in brain samples, which were 7-hydroxy-3′,4′-dimethoxyisofavone, 2′-methoxyformononetin, vestitone, sativanone, piperine, piperlongumine, pipernonaline, tryptophan, liquiritin, isocostic acid, glycocholic acid, lecithos, oleic acid, and cholan-24-oic acid. A total of 18 components were detected in the heart: agarotetrol, linolenic acid, 7-hydroxy-3′,4′-dimethoxyisoflavone, 2′-methoxyformononetin, daidzin, vestitone, sativanone, piperine, piperlongumine, tryptophan, liquiritin, isocostic acid, linoleic acid, glycocholic acid, glycochenodeoxycholic acid, corchoionoside C, oleic acid, and cholan-24-oic acid. There were 24 components detected in the liver: agarotetrol, linolenic acid, 7-hydroxy-3′,4′- dimethoxyisoflavone, 2-methoxyformononetin, daidzein, isoliquiritigenin, vestitone, sativanone, piperine, piperlongumine, pipernonaline, apigenin, tryptophan, liquiritin, ferulic acid, isocostic acid, linoleic acid, normuscone, glycocholic acid, glycochenodeoxycholic acid, lecithos, corchoionoside C, oleic acid, and cholan-24-oic acid. A total of 24 components were detected in the kidney: agarotetrol, linolenic acid, 7-hydroxy-3′,4′-dimethoxyisoflavone, 2′-methoxyformononetin, daidzein, isoliquiritigenin, vestitone, sativanone, piperine, piperlongumine, pipernonaline, apigenin, tryptophan, liquiritin, ferulic acid, isocostic acid, dehydrocostus lactone, linoleic acid, glycocholic acid, glycochenodeoxycholic acid, lecithos, corchoionoside C, oleic acid, and cholan-24-oic acid.Hydroxysafflor yellow A was the quality control index of the Ruyi Zhenbao tablet, but in the early blood components, hydroxysafflor yellow A was not detected, which was closely related to its low bioavailability. According to literature reports, hydroxysafflor yellow A was poorly absorbed by rats, and its absolute bioavailability is 1. 2%, which has a bile efflux effect . So the max of agarotetrol, isoliquiritigenin, piperine, and ferulic acid in plasma was achieved mainly between 0.25 and 4 h. The Cmax of the high dose was 89.5 ± 37.9 ng ml−1, 6.42 ± 2.02 ng ml−1, 36.8 ± 19.6 ng ml−1, and 21.1 ± 6.22 ng ml−1, respectively. AUC0-t was 768 ± 217 h ng ml−1, 22.8 ± 6.16 h ng ml−1, 110 ± 38.8 h ng ml−1, and 413 ± 76.9 h ng ml−1, respectively. The plasma t1/2 was 6.84 ± 2.40 h, 8.65 ± 5.42 h, 5.34 ± 3.44 h, and 82.6 ± 42.7 h respectively. MRT0-t was 6.69 ± 1.82 h, 5.63 ± 3.52 h, 4.33 ± 1.96 h, and 28.0 ± 3.74 h, respectively.After a single administration, the T1/2 and Tmax of agarotetrol, isoliquiritigenin, and piperine (p > 0.05). The aforementioned results showed that in the dose range of 0.225–0.900 g kg−1, the increase in the AUC0-t and Cmax values of agarotetrol, isoliquiritigenin, and piperine was related to the dose increase. There were no significant differences in Cmax and AUC0-t of ferulic acid at different doses (p > 0.05), mainly because ferulic acid was not only an effective component of TCM but also a functional phenolic acid component in plant cell walls, which was commonly found in grains and nuts .The T-test and the rank sum test were used to test sex-based differences in the main pharmacokinetic parameters in each group. The results showed that the pharmacokinetic parameters of agarotetrol, isoliquiritigenin, piperine, and ferulic acid in rats did not show significant sex-based differences (−1. Piperine was not detected in the brain, heart, and kidney, but the concentration in liver tissue was less than 6.95 ng ml−1. Ferulic acid was not detected in the brain but was present in the heart, liver, and kidney tissues.After long-term administration, the distribution order of agarotetrol in various tissues of rats was kidney > liver > heart > brain; the distribution order of tissues in the low- and middle-dose groups of isoliquiritigenin was liver > kidney > heart > brain, and that in the high dose group was kidney > liver > heart > brain. The tissue distribution of piperine in each dose group was liver > kidney > heart > brain, and that of ferulic acid in each dose group was kidney > liver > heart > brain. Isoliquiritigenin was not detected in all tissues during the recovery period, and agarotetrol was not detected in the brain, heart, or liver tissues and its concentration in kidney tissues was lower than 2.50 ng mlUnlike chemical medicines, TCM often contains many different kinds of medicinal materials, and each medicinal material can also have different ingredients. How to choose ingredients to represent the pharmacokinetics of TCM is a problem faced by the development of TCM. How to guide the clinical usage of TCM is another important problem faced by TCM. According to the current research methods and means, it is of great significance to study the pharmacology and kinetics of TCM, especially of Tibetan medicine. In future research, we should adopt a more scientific and reasonable evaluation method to evaluate the pharmacokinetics of TCM according to its characteristics, to provide a more valuable reference for clinics.In summary, by adopting and verifying a previously described methodology, the pharmacokinetic characteristics of the classic Tibetan medicine RYZB tablet after single administration and long-term administration were analyzed, which will not only play a guiding role in supporting the clinical application of RYZB, but will also accelerate research into Tibetan medicine theory and its application, thus, providing a solid foundation for the acceptance of Tibetan medicine throughout the world.


We found important basal sex differences in DM, with female rats showing better performances at baseline. Choice processes were impaired exclusively in males after BD history, associated with a decrease in impulse control in both sexes, while memory and anxiety-like behavior were not affected. Our neurobiological results demonstrate that BD did not affect basal dopamine signaling in the NAc core, regardless of the sex, but reveal changes in the sensitivity to the inhibitory effects of quinpirole in females. DM impairments were neither associated with changes in basal dopamine signaling nor pre-synaptic D2 activity. Overall, our findings show that BD affects both DM processes and dopamine transmission in the core of the NAc in a sex-related manner, further suggesting that these effects may play a role in the vicious cycle leading to BD perpetuation and the early onset of AUD. Our results may inform novel strategies for therapeutic and prevention interventions.Binge drinking (BD) is a harmful behavior for health and is a predictive factor for the development of alcohol addiction. Weak decision-making (DM) capacities could play a role in the vulnerability to BD which in turn would lead to DM impairments, thus perpetuating BD. Longitudinal preclinical studies are however lacking and necessary to understand this complex relationship. Both DM and BD are influenced by sex and involve dopamine release in the core of the nucleus accumbens, a central mechanism regulated by dopamine D2/3 autoreceptors. In this context, we used an operant self-administration procedure of BD in male and female rats, and longitudinally assessed DM capacity, memory and anxiety-like behavior. To better understand the mechanisms potentially involved in the relationship between DM and BD,  Binge drinking (BD) definition has yet to find an international consensus but it is characterized by an intense and episodic alcohol consumption, with recurring alternations between intense intoxication episodes and abstinence periods . BD is pMany factors have been associated with BD such as decision-making (DM) and impulsivity, two hallmarks of alcohol dependence. First, DM refers to a fundamental cognitive process allowing us to select a particular option among several alternatives, in order to select advantageous choices over disadvantageous one in everyday life . A receBoth DM and BD behaviors are influenced by sex. For instance, findings supported the subdivision of binge drinkers according to gender and personality dimensions . Sex difin vivo , due to the involvement of D2/3 receptors in DM. We decided to focus on the core part of the NAc due to its involvement in both DM  and the 2PO4, MgCl2, CaCl2, NaHCO3, glucose, ascorbic acid and quinpirole were purchased from Sigma-Aldrich .Ethanol (96%) was purchased from WWR  and diluted into tap water at a 20% concentration (v/v). NaCl, KCl, NaHad libitum and no enrichment. The light phase started at 7:00 a.m. for 12 h. The experiments started 1 month after their arrival. Prior to all experiments, 20 of the animals were randomly assigned to a group of voluntary alcohol administration , and the remaining 12 rats  were used as a control group for the FSCV experiments, without any exposure or behavioral training. Experiments were carried out in accordance with the guidelines of the E.C. regulations for animal use in research (CEE no. 86/609) and our local ethics committee .32 Long Evans rats  were purchased from Janvier Laboratories  at the age of 8 weeks. The animals were single housed in individually ventilated cage (IVC) with food and water Anxiety-like behavior was performed using the light dark box (LDB). The test was carried out before and after voluntary BD exposure see  timelineLearning abilities were assessed using the Novel Object Recognition test (NOR). The test was carried out before and after voluntary BD exposure see  timelineBehavioral testing took place in four identical operant chambers  in a calm room. Each chamber was standing in a dark, ventilated and sound-proof conditioning box. The chambers were divided into two equal compartments by a central plexiglass partition (0.5 × 29.5 × 30 cm), parallel to the wall and opened in its center (7 × 7 cm). They were also equipped on one side of the box with four nose-poke holes on a curved wall (dimly illuminated within with a white LED), equidistant from the food magazine at the opposite side. The nose-poke holes were equipped with an infrared detector connected to an external dispenser for the delivery of food pellets . The apparatus and data collection were controlled using the POLY software . The animals were moderately food restricted during the procedure .p = 0.9, 5 s timeout p = 0.1; P2: 2 pellets p = 0.8, 10 s timeout p = 0.2; P3: 3 pellets p = 0.5, 30 s timeout p = 0.5; P4: 4 pellets p = 0.4, 6 0 s timeout p = 0.5; see DM was assessed using a rat gambling protocol inspired from the IGT in humans and developed by Test phase consisted in daily 30 min free-choice RGT sessions. The trial design and their contingencies were the same, except that they started with the stimulus lights being turned on in all of the active holes. P1 and P2 options are considered as the advantageous choices, while P3 and P4 are considered as disadvantageous choices. If a rat chooses only one option, then the greatest number of pellets possible would be with P2 , then P1 (295), P3 (135) and P4  . The aniBD behavior was generated after the first RGT test see  timeline2PO4 1.1 mM, MgCl2 1.4 mM, CaCl2 0.5 mM, NaHCO3 18 mM, Glucose 11 mM, ascorbic acid 0.4 mM, pH 7.2–7.4) and glued into a vibratome . Coronal slices (250 µm thick) of the NAc were selected and stored in a 31°C aCSF  reservoir gassed with carbogen  for at least 1 hour. After rest, the slices were transferred to a recording chamber and perfused with aCSF .Rats were anesthetized with isoflurane  before being decapitated, and their brain were extracted and immersed in ice-cold artificial cerebrospinal fluid (aCSF) . Those electrodes were calibrated prior to the recordings using a 1 µM dopamine solution in aCSF. A triangular waveform potential ramping from −0.4 V to +1.3 V was applied to the microelectrodes at 10 Hz during the recordings. A bipolar stimulating electrode  was placed on the afferent fibers coming from the VTA, around 100 µm near the working carbon-fiber microelectrode. Phasic DA release was evoked every 5 minutes using a monophasic stimulation . The electric signal was amplified, filtered and transmitted to the Tarheel CV software . Using color plots, changes in current were plotted as a function of applied potential over time, and the oxidation current converted to DA concentration. A baseline of three consecutive stable signals was obtained for each slice. For the pharmacology, quinpirole 100 nM was applied for at least 30 min and until achieving three consecutive stable signals.After performing the behavioral experiments (48 h to 1 week), rats were euthanized and coronal slices containing the NAc were collected to measure electrically evoked dopamine transmission in the core of the NAc. Rats from the control group  were euthanized and their coronal slices collected 1 week after their arrival (57 weeks). The sensitivity to the D2/D3 receptor agonist quinpirole (100 nM) was tested.DA] is the instant extracellular concentration of DA released, f is the frequency of stimulation, [DA]p is dopamine concentration released per pulse, and Vmax and Km are respectively the velocity and affinity constants of the dopamine transporter (DAT). Km was fixed at a constant value of 200 nM (p is the reflect of the presynaptic mechanisms regulating release as those of the auto-receptors (D2/D3 activity) , with tf 200 nM . [DA]p ictivity) . Peak dopost hoc test). For single comparison, we used a Student’s t-test (2-tailed) for the data following a normal distribution and an equality of variances, and a Mann-Whitney rank sum test otherwise. For proportions, we used a χ2 test. For the correlation analysis, we used a Pearson correlation test. All data were presented as Means ± standard error means (SEM). The significance threshold was fixed at p < 0.05.Data were analyzed using SigmaPlot 11.0  with an analysis of variance test  followed by a Tukey multiple comparison test, when a significant effect was observed for the data following a normal distribution and an equality of variances. When the data did not follow these criteria, a non-parametric analysis was performed  = 20.306, p < 0.001; sex F = 2.899, p = 0.127; interaction F = 1.180, p = 0.264). Thereafter, we evaluated their baseline choice behavior in the RGT on the last 3 sessions. Male rats favored the most advantageous option (P2) and female rats favored both advantageous options (P1 and P2)  = 0.346, p = 0.573; option F = 0.1436, p = 0.257; interaction F = 5.740, p = 0.004). Female rats chose the P1 option significantly more than male rats . Distribution of poor (<33% good choices), neutral (≥33–≤66%) and good (>66% good choices) decision makers was different depending on sex (χ2(2) = 6.14, p < 0.05). Regarding the other RGT parameters, no sex differences were observed for advantageous choices, number of trials and premature responses, but there were more omitted trials in females  = 26.761, p < 0.001; option F = 0.390, p = 0.761; interaction F = 2.694, p = 0.069; Tukey p < 0.05) but not in females  = 3.006, p = 0.117; option F = 2.363, p = 0.093; interaction F = 1.314, p = 0.290). There were no effects on advantageous choices  = 1.902, p = 0.186; treatment F = 2.170, p = 0.159; interaction F = 0.380, p = 0.546) and only females displayed a small increase in their number of trials  = 0.725, p = 0.406; treatment F = 6.484, p = 0.021; interaction F = 0.643, p = 0.434; Tukey p < 0.05). Females made significantly more omissions than male rats before BD onset and they reduced their number of omissions after a voluntary BD history  = 9.378, p = 0.007; treatment F = 3.722, p = 0.071; interaction F = 12.657, p = 0.002; Tukey p < 0.001 vs. males, p < 0.01 vs. after EtOH). Both males and females made significantly more premature responses after voluntary BD history and this effect was more pronounced in females  = 0.824, p = 0.377; treatment F = 30.164, p < 0.001; interaction F = 5.697, p = 0.029; Tukey p < 0.05 males vs. females after EtOH, p < 0.05 in males before EtOH vs. after EtOH, p < 0.001 in females before EtOH vs. after EtOH). An additional analysis including the sex factor for choice behavior in each option of the RGT is provided in the supplementary material , ethanol preference during the last session of IA2BC , active lever presses during the last 5 stable FR3 15 min operant self-administration sessions , and ethanol intake during the last 5 stable FR3 15 min operant self-administration sessions . Pearson correlation tests however revealed a positive correlation in female rats between premature responses and active lever presses (p = 0.144)/ethanol intake (p = 0.0345) during the last 5 stable FR3 15 min operant self-administration sessions, but not in male rats . Outside of the ones we are showing in this manuscript, we found no correlation between the other parameters from the RGT, the BD procedure and the FSCV  and after (Tukey p < 0.001) BD  = 211.05, p < 0.001; treatment F = 0.01, p = 1.000; interaction F = 0.20, p = 0.655). The female rats explored the novel object significantly more than the familiar object, both before (Tukey p < 0.001) and after (Tukey p = 0.002) BD  = 38.12, p < 0.001; treatment F = 0.01, p = 1.000; interaction F = 0.35, p = 0.569).In the NOR test, the rats were presented with 2 objects and tested with retention in presence of a new object again 24 h later . We evalp = 0.021) and after (Tukey p = 0.02) BD  = 8.38, p = 0.010; treatment F = 3.38, p = 0.084, interaction F = 0.01, p = 0.988).In the DLB test , the ratWe evaluated the phasic dopamine signaling induced by electrical stimulation in the core of the NAc using FSCV on brain slices, in both male and female rats, after behavioral experiments. Thus, both DM capacities and dopaminergic signaling were assessed in the same animals. We tested a 100 nM dose of quinpirole on the slices to unravel potential changes in the sensitivity of the D2/3 receptors  and binger rats (Tukey p = 0.036)  = 24.715, p < 0.001; group F = 0.00594, p = 0.442; interaction F = 1.007, p = 0.399) [DA]p in control (Tukey p < 0.001) and binger rats (Tukey p = 0.002)  = 33.906, p < 0.001; group F = 0.116, p = 0.747; interaction F = 1.328, p = 0.308), and Vmax in control (Tukey p = 0.002) and binger rats (Tukey p = 0.009)  = 12.283 p = 0.002; group F = 3.787, p = 0.109; interaction F = 1.156, p = 0.354).In males, quinpirole decreased [DA]max in control (Tukey p = 0.01) but not control rats  = 8.231, p = 0.008; group F = 0.605, p = 0.472; interaction F = 1.780, p = 0.218), [DA]p in binger (Tukey p = 0.011) but not control rats  = 10.346 p = 0.004; group F = 1.828, p = 0.234; interaction F = 1.387 p = 0.294), and had no effect on Vmax  = 1.394 p = 0.292; group F = 0.949, p = 0.375; interaction F = 0.0214, p = 0.979).In females, quinpirole decreased [DA]max in binger  no poor decision makers in females; ii) males significantly preferred the optimal P2 option and iii) females preferred both P1 and P2 options. The P1 option, preferred by females, can be seen as a “risk-averse” phenotype, as it is associated with the lowest probability of punishment, as previously suggested . This obVoluntary BD impaired choice behavior specifically in males, with a decrease of the optimal P2 option choice (50% decrease), although without effects on the overall advantageous choices made. BD history in males made their choice behavior become “random” (around 25% choice for each option), as they could not effectively select the advantageous options in the task anymore. Our results are in line with those showing impaired DM in conflictual and risky situations after both forced  and voluOne of the most striking result of our study is that BD history had no effect on DM capacities in female rats. Many human studies have shown that women are more sensitive to the effects of alcohol than men, but sex differences regarding BD remain poorly investigated . Some sNo study has yet analyzed whether DM abilities can predict the vulnerability to consume ethanol in individuals who have never used ethanol before. The use of a longitudinal design allowed us to assess the vulnerability to consume ethanol in the BD procedure, but the analysis revealed no correlation between baseline DM abilities (advantageous choices: P1 + P2) and ethanol consumption in the IA2BC and operant self-administration procedures . InteresOur results on dopamine transmission demonstrate that BD does not affect basal dopamine signaling in the NAc core, regardless of sex, but reveal sex-dependent changes in the sensitivity to the inhibitory effects of quinpirole. Baseline dopamine signaling (release and uptake) was similar in both sexes in control groups. Our results are in line with those of a recent meta-analysis on 39 microdialysis studies showing that there are no sex-dependent differences in basal dopamine levels within the NAc . It is nin vivo FSCV at adulthood  sensitive to the inhibitory effect of quinpirole. It seems that autoreceptors are less sensitive to quinpirole in females with no BD history. A previous study using FSCV in female rhesus macaques showed that 1 year of daily ethanol self-administration induced greater dopamine uptake rates and sensitivity to D2 autoreceptors in the core of the NAc, thus driving a hypodopaminergic state  compared to classic passive exposures . So far, clinical results on the sex differences in the BD field of research looking at brain and cognitive deficits are still inconclusive and need more investigation. Thus, the overall difference in BD exposure raises the important question about the BD history that is also often matter of debate in human studies because it is difficult to have homogeneous population of binge drinkers with the same BD history.


C. arabica coffee cherries from Panama by dry process. The lowest concentrations of fat corresponded to Cascara 4.24 g·kg−1 and Silverskin 23.70 g·kg−1, respectively. The major fatty acids detected in all samples were palmitic, stearic, oleic, and linoleic acids, the latter two being essential fatty acids. LDA showed that 89.01% of the variability between beans and by-products was explained by lignoceric, myristic, behenic, tricosanoic, arachidic, and heneicosanoic acids. Silverskin appeared to be a good source of lignoceric, myristic, and behenic acids and had a higher concentration of dietary fiber (314.95 g·kg−1) than Cascara (160.03 g·kg−1). Coffee by-products (Silverskin and Cascara) are low-fat products enriched in dietary fiber. Their incorporation, after adjustment, into the global diet may contribute to nutrition security, the sustainability of the coffee sector, and human health.Nowadays, there is an increased interest in coffee derivatives ) due to their particular chemical composition. This study aimed to compare the content of dry matter, total fat, fatty acids, and fiber  of coffee by-products (Cascara and Silverskin) and coffee beans (green and roasted under different conditions). Coffee beans and their by-products were obtained from 100%  With constantly increasing consumption, coffee beans are one of the most traded commodities globally . AccordiThe post-harvest processing aims to obtain green coffee beans. The first step of processing starts with the pulp and hull removal using either a wet or dry method . The dryThe chemical composition of the green beans primarily depends on genetic variation, post-harvest processing techniques, bean maturation, agricultural practices, and environmental gradients. The roasting process determines coffee sensory characteristics ,9,10. DuThe coffee industry produces by-products rich in bioactive compounds such as polyphenols, alkaloids, chlorogenic acids, antioxidants, carbohydrates, proteins, and dietary fiber. These by-products can be a problem for the environment, roasters, growers, and producers, given that they have no further use . ApproxiBased on Silverskin is detached from green beans and represents the only by-products produced during the roasting process. One hundred and twenty kilograms of green coffee generate approx. 2.5 kg of Silverskin . SilversSeveral studies describe coffee by-products’ health benefits, mainly antioxidant, antimicrobial, anti-inflammatory, and anti-aging effects, while very few data have been provided on the nutritional value of them. Moreover, to the best of our knowledge, there are no previous papers available comparing the nutritional composition of unroasted and roasted coffee beans and the by-products generated during post-harvest processes such as depulping (Cascara) and roasting (Silverskin). Previous studies on the content of food contaminants, (ochratoxin A) and acrylamide ,19,20, sThe aim of the present research is to provide novel information on the nutritional composition of coffee beans and the by-products generated during coffee post-harvest processing. We applicated a statistical comparison of the fatty acid profile in green coffee beans, their roasted forms , and coffee by-products (Cascara and Silverskin). A comparative study on their chemical composition was performed with the end to stimulate their inclusion as part of a daily healthy diet for humans. At the same time, the paper aims to contribute to the sustainability of the coffee sector and nutrition security. The intake of the chemicals selected for analysis includes nutrients (fats and dietary fiber) that should be controlled to avoid the risk of non-communicable chronic diseases considered pandemics of the 21st century.Coffea arabica cherries from Central America, Panama, Hartmann micro-lot, Santa Clara, by dry method  of post-harvest processing. Silverskin was obtained as mixture from light, medium, and dark roasting processes. The samples were obtained from the local company dedicated to importing and distributing coffee, Caffé Oro Ltd. . All measurements were performed in triplicate and results are shown in g·kg−1 of dry matter.For this research, Cascara and green bean samples were obtained from 2 de-gassing valve obtained from Caffé Oro Ltd. Samples were analyzed the next day. To ensure that the right roasting level was reached, roasted samples were measured using RoastRite, Coffee Roast Analyzer . A detailed description of samples is shown in Green coffee was roasted using traditional barrel roaster technology with a gas heating system using Toper TKSMX 10 machine . Roasting programs were set as follows: Light roast (Cinnamon)—temperature 180 °C for 3 min, then 207 °C for 2 min and 50 s. Medium roast (Full City++)—the initial temperature was 180 °C for 3 min, then after 4 min the first crack occurred at 205 °C and ended at 215 °C. The second crack started at 225–230 °C (Full City +). Five seconds later, the beans reached 232 °C for 15 s. Dark roast (French)—the initial temperature was 180 °C for 3 min, then after 4 min the first crack occurred at 205 °C and ended at 215 °C. The second crack started at 225–230 °C. Then, we kept the beans at 235 °C for 45 s; samples of Silverskin were obtained from the roasting process. Silverskin was collected after each roasting process, and coffee beans were placed in a metal container to cool to room temperature, then moved to odor-free plastic bags with a COGiven the hard structure of green beans, samples were frozen at −20 °C for 30 min before the analysis to ensure easier homogenization. Samples were homogenized using Grindomix GM 200  for 60 s  and 120 s (green beans) at 10,000 rpm to achieve the required grinding thickness. The homogenized samples were then sieved through a sieve with a mesh diameter of 1 mm.−1.The dry matter was determined by moisture analyzer KERN DAB 100-3  and expressed in g·kg−1 of dry matter.Total fat content was quantified by Soxhlet extraction with petroleum ether following the procedure described by the ISO 12966-2:2017: preparation of methyl esters of fatty acids, animal and vegetable fats, and oils . Gas chrChemical reagents4 H2O , Na2SO4 , anhydrous N-hexane for chromatography , 37 component standard FAME mix 10 mg.mL−1 in methylene chloride containing C4-C24 FAME (2–4% relative concentration), manufacturer Supelco, catalog number 47885-U chromatography .Petroleum ether 40–65 °C , filter medium Hyflo Super Cel (diatomaceous earth) , methanol for chromatography , KOH , HCl 33–36% , NaHSOApparatus−1;1. Tecator Soxtec System HT 1043 Extraction unit  apparatus at a flow rate of 10 cycles/h or 10 mL·min2. Vacuum oven,  temperature adjustable (100 ± 3 °C).2SO4, and used for GC-FID analysis. Prior to loading into the column, the FAME solution was diluted in a vial, usually at a ratio of 50 μL FAME solution/950 μL n-hexane.The fat used must be liquid, dry, and clean. Approximately 200 mg of fat was weighed into a ground glass tube. A total of 5 mL of n-hexane was added to the fat by pipette. The fat was dissolved by stirring. In total, 1 mL of 2 M KOH in methanol was added with a micropipette. The tube contents were shaken vigorously and placed in a water bath set at 60 °C for 1 min and 30 s. Then, 2 mL of 1 M HCl was added, and the tube contents were gently mixed. After equilibration and separation of the layers, the upper layer containing FAME was carefully removed by pipette, filtered through anhydrous Na−1); temperature program: 50 °C for 1 min, 25 °C min−1 till the system reached 175 °C, then 2 °C min−1 to 230 °C; detector temperature: 280 °C; gas detector: hydrogen: 35 mL·min−1, air: 350 mL·min−1, nitrogen; make-up gas: 30 mL·min−1.GC-FID was performed using Agilent 6890 GC with FID (flame ionization detector), and analytical column: 60 m × 250 μm × 0.15 μm DB-23 (Agilent 122-2361) was used. Experimental conditions were set as follows: injector temperature: 250 °C; injected volume: 1 μL; dividing ratio: 1/10; carrier gas: helium; head pressure: 238.96 kPa  and linear discriminant analysis (LDA) were used to describe any possible significant differences between the analyzed samples. This statistical analysis was performed using Microsoft Office Excel 365 for iOS and XLStat, Addinsoft (2022). XLStat is a statistical and data analysis solution .LDA is a statistical method based on reduction of a classification. This common technique provides class separability by drawing a decision region between the different classes. This advanced method focuses on pattern recognition and machine learning to find a linear combination of characteristics that separates observations into classes, which is used for dimensionality reduction and better classification  ,25.−1. Our results indicated a relationship between roasting degree and DM values. Under dark roasting conditions, the highest values of DM were obtained, being on average 983.10 g·kg−1. Medium and light values were 981.20 and 959.85 g·kg−1, respectively. Our previous research showed that medium roasted coffee could have from 967.7 to 998.2 g·kg−1 of DM [−1 for light, 982.8 ± 10.4 g·kg−1 for medium, and 987.4 ± 9.1 g·kg−1 dark roasting conditions. Cascara DM value was of 890.5 g·kg−1, similar to 911.3 ± 9.66 g·kg−1 reported by Ameca et al. (2018) (911.3 ± 9.66 g·kg−1) [−1. Significant differences were observed between each analyzed group (p < 0.0001).Green beans reached an average DM of 932.20 g·kg−1 of DM . Results−1 of DM , which w g·kg−1) . The Sil−1) > GC (99.42 g·kg−1) > MRC (73.22 g·kg−1) > LRC (68.64 g·kg−1) > SS (23.70 g·kg−1) > CC (4.24 g·kg−1). The results indicated that fat content increased in relation to the roasting process. Endeshaw and Belay (2020) [The fat content of the samples in decreasing order was as follows: DRC (135.84 g·kgy (2020)  showed ay (2020)  associat−1 of fat. Similar results were obtained in our research. Results and significant differences based on ANOVA Duncan and REGWQ tests are shown in Regarding coffee by-products, our results are in accordance with the findings presented by Iriondo-DeHond et al. (2020) , who repThirteen acids of the 40 analyzed were detected in the coffee and/or its by-products . The res−1) > GC (43.67 g·kg−1) > MRC (32.54 g·kg−1) > LRC (30.19 g·kg−1) > SS (5.88 g·kg−1) > CC (1.32 g·kg−1) and palmitic acid DRC (47.61 g·kg−1) > GC (34.93 g·kg−1) > MRC (25.39 g·kg−1) > LRC (24.40 g·kg−1) > SS (6.03 g·kg−1) > CC (1.34 g·kg−1). Dong et al. (2015) [Linoleic acid was the most abundant fatty acid in the samples under study, which in decreasing order are as follows: (60.56 g·kg. (2015) . Our res−1 of oleic and 6.96 g·kg−1 of stearic acid, respectively. After light and medium roasting processes, we observed a significant decrease of both fatty acids. However, under dark roasting conditions their concentration significantly increased compared to the green beans. Coffee by-products contained significantly lower values (Cascara 0.24 g·kg−1 and Silverskin 1.12 g·kg−1) than those found in green and roasted coffee beans. Tsegay et al., 2020 [−1 of palmitic acid, 8.92 g·kg−1 of oleic acid, and 51.6 g·kg−1 of linoleic acid in green coffee beans. They reported a relationship between the concentration of fatty acids and geographical origin, namely the altitude of the growing area. Our results are in accordance with this research . According to our results, 98.11%, thus the majority, of variability can be described by two factors . Polyunsaturated fatty acids (PUFA) are defined as hydrocarbon chains containing two or more double bonds. The characterization either as an n-3 PUFA or n-6 PUFA refers to the position of the first double bond relative to the methyl end of the fatty acid. In nature, double bonds are usually in the cis form . Monounsp < 0.001) in coffee beans compared to their by-products.−1. The content of PUFA in roasted beans increased during the roasting process (LRC < MRC < DRC). The lowest concentration of PUFA was observed in coffee by-products . Omega-3 and omega-6 fatty acids are essential PUFA (polyunsaturated fatty acids). These acids must be a part of our diet, given that they cannot be synthesized in the human body. The balance between dietary n-6 and n-3 fatty acids is crucial regarding human health [PUFA content in green coffee was 45.05 g·kgn health ,42. PUFAn health ,42,43.−1 of monounsaturated fatty acids (MUFA). The roasting process significantly increased their content. The highest concentration of MUFA reached beans roasted on dark level (11.60 g·kg−1). As expected, Silverskin (1.21 g·kg−1) and Cascara (0.24 g·kg−1) showed low levels of them. Schwingshackl and Hoffmann (2012) [Green coffee beans contained 7.56 g·kgn (2012)  stated t−1) > GC (46.09 g·kg−1) > MRC (33.16 g·kg−1) > LRC (31.46 g·kg−1) > SS (16.01 g·kg−1) > CC (1.80 g·kg−1).The values of SFA copied the following decreasing order: DRC (61.06 g·kg−1. The range of the Σn6/Σn3 ratio in the roasted samples ranged from 30.17 g·kg−1 (LRC) to 31.48 g·kg−1 (MRC). Silverskin contained higher values (12.29 g·kg−1) of Σn6/Σn3 ratio compared to the Cascara sample. The values of the n3/n6 ratio in green, light, medium, and dark were identical (0.03 g·kg−1). The observed difference of the ratio n3/n6 was in coffee by-products .The Σn6/Σn3 ratio in green coffee reached 31.57 g·kgThe PUFA/SFA ratio is known to be a crucial parameter regarding human health and nutrition, given that specific saturated and polyunsaturated fatty acids might have different effects in metabolism. Foods with a low PUFA/SFA rate are considered unhealthy and may induce hypercholesterolemia. When this ratio is analyzed separately, there are some restrictions, considering all SFA can increase blood cholesterol, thus ignoring the hypocholesterolemic effect of MUFA . Foods wp < 0.0001). Two factors are needed to explain the majority (95.24%) of the variability between green, roasted coffee beans, and their by-products (Silverskin and Cascara). Factor F1 explains 82.28% of the variability and correlates with PUFA, MUFA, SFA, and ratio Σn6/Σn3. Factor F2 is responsible for 12.96% variability and correlates with Σn3/Σn6  having the highest values, 284.59 g·kg−1, which is higher by 25% than the dark roasting level (DRC 213.45 g·kg−1). Similarly, Endeshaw and Belay (2020) [−1.y (2020)  reportedy (2020)  showed t−1 of fiber. Iriondo-DeHond et al. (2020) [−1); however, Cascara had only 88.34 g·kg−1 of fiber.Costa et al., 2018 , determi. (2020)  reportedHoltzapple 2003  stated tp < 0.0001) in both parameters. Nevertheless, the dark roast caused a significant decrease (p < 0.0001). The medium roasting level reached the highest value of ADF and NDF, 41.82 g·kg−1 and 60.45 g·kg−1, respectively.A similar pattern as in the fiber was observed within ADF and NDF, which means that the roasting process caused a significant increase  [C. arabica from Mexico. They observed that samples contained 52.14% of ADF and 55.19% of NDF. However, their samples of Cascara were obtained using wet post-harvest processing. Klingel et al. (2020) [−1), insoluble dietary fiber (313.2 ± 15.1 g·kg−1), and soluble dietary fiber (161.2 ± 11.5 g·kg−1) in Cascara. Guglielmetii et al. (2019) [−1).The by-products reached relatively high values of ADF and NDF too. When comparing the content of ADF, we observed that Silverskin contained 38.64 g·kg. 2018)  evaluate  evaluat. (2020)  also ide. (2019)  state thThe roasting degree differently affected the composition of the green beans. Dark roasted coffee showed the highest values of dry matter, fat, and the major fatty acids among the beans under study. Our results confirmed that coffee beans contain mainly linoleic, palmitic, oleic, and stearic acids. In conclusion, the analysis of the selected chemical parameters allowed for differentiation between green and roasted beans submitted to different degrees of roasting and coffee by-products. The factors obtained by LDA proved that 89.08% of the variability between coffee beans and their by-products correlates with lignoceric, myristic, behenic, tricosanoic, arachidic, and heneicosanoic acids. Light and medium roasting caused a significant decrease in the identified fatty acids. Only behenic, cis-11-eicosenoic, and lignoceric acids showed stability in their content with light and medium roasting temperatures.Silverskin appeared to be a good source of lignoceric, myristic, and behenic acids and had a higher concentration of dietary fiber than Cascara. Silverskin showed the highest values of the dietary fiber among the studied samples and the second lowest in fat after Cascara. In general, the data confirmed that coffee beans (green and roasted) and their by-products are healthy foods attending to their composition in nutrients, including those studied in the present research. The results suggest that, given the fatty acids content and dietary fiber, coffee by-products might be used for the production of enriched and innovative foods.