Thalassophryne maculosa, a venomous fish commonly found in Latin America whose envenomation produces an injury characterized by delayed neutrophil migration, production of major pro-inflammatory cytokines, and necrosis at the wound site, as well as a specific systemic immune response. However, there are few studies on the protein structure and functions associated with it. Here, the toxin was identified from the crude venom by chromatography and protein purification systems. TmC4-47.2 shows high homology with the Nattectin from Thalassophryne nattereri venom, with 6 cysteines and QPD domain for binding to galactose. We confirm its hemagglutinating and microbicide abilities independent of carbohydrate binding, supporting its classification as a nattectin-like lectin. After performing the characterization of TmC4-47.2, we verified its ability to induce an increase in the rolling and adherence of leukocytes in cremaster post-capillary venules dependent on the α5β1 integrin. Finally, we could observe the inflammatory activity of TmC4-47.2 through the production of IL-6 and eotaxin in the peritoneal cavity with sustained recruitment of eosinophils and neutrophils up to 24 h. Together, our study characterized a nattectin-like protein from T. maculosa, pointing to its role as a molecule involved in the carbohydrate-independent agglutination response and modulation of eosinophilic and neutrophilic inflammation.TmC4-47.2 is a toxin with myotoxic activity found in the venom of  Lectins are a comprehensive group of proteins with carbohydrate-binding properties. Due to their binding singularity, many natural lectins, purified from several sources such as plants, algae, and fungi, have prospective applications in biotechnology, medical research, and crop protection. Additionally, many groups have identified lectins in organs and tissues  of various fish species . LectinsThalassophryne nattereri + 1533.85) and angiotensin II ([M+H+]+ 1046.54)  were used as external calibrants.To determine the toxin mass, we used 10 μL of the sample injected into an LC-MS Surveyor MSQ Plus system (Thermo Finnigan) by direct infusion in a flow of 50 μL/min of aqueous acetonitrile solution (1:1) containing 0.1% formic acid under capillary voltage conditions 3.1 kV, 75 V cone, scan lasting 1 s in the 0 beta 8 . For MAL−1 was added to 8 μL of the toxin solution and incubated 18 h at 37 °C. The samples were analyzed using a nanoflow LC/MS/MS system customized with a PepFinder Kit. Aliquots of 10 μL were initially charged onto a reversed-phase peptide trap column in a of 10 μL/min flow rate for 3 min. Then peptides were eluted and partitioned on a reversed-phase capillary column . Solution A comprised 0.1% formic acid, and Solution B acetonitrile and 0.1% formic acid. The flow rate began at 100% A at 10 μL/min for 3 min. Next, it was raised to 70 μL/min for 6.9 min at 100% A and the gradient commenced at 100% A and 0% B. The gradient was increased to 50% B in 60 min, then to 90% B in 5 min and later diminished to 0% B in 5 min and sustained at 100% A for 10 min. The complete program duration was 110 min. By the PepFinder Kit, the flow was separated in a 1:100 ratio. Hence, the factual flow rate injected into the mass spectrometer was 0.5 μL/min. The HPLC was coupled to a Finnigan LCQ Deca XP Plus ion trap mass spectrometer supplied with a nanospray ionization font. Spray voltage was established at 2.5 kV, and the equipment was managed in a data-dependent method, in which one MS scan was captured in the 300−1600 m/z range followed by MS/MS acquisition using collision-induced segregation of the 10 most intense ions from the MS scan. Dynamic peak exclusion was used to avoid the same m/z being chosen for the following 120 s. Tandem mass spectra were extracted by Xcalibur software . The subsequent MS/MS spectra were searched through a MASCOT search engine  against the NCBI NR database in the taxa Chordata with a 1.20 Da parent tolerance and 0.60 Da fragment tolerance. Iodoacetamide derivatives of cysteine and oxidation of methionine were detailed in MASCOT as fixed and variable modifications, respectively. Scaffold  was used to confirm MS/MS-based peptide and protein description. Peptide distinguishing were credited if they exceeded certain database search engine thresholds. MASCOT descriptions required ion scores higher than the associated identity scores and 20, 30, 40, and 40 for singly, doubly, triply, and quadruply charged peptides. X! Tandem identifications needed at least –Log (Expect Scores) scores of greater than 2.0. Protein identifications were considered if they had at least 2 identified peptides.The purified toxin was used for the identification of disulfide bridges, according to Yang, Liu, and Liu . The tox−1 of N-glycosidase F  or 25 mU.50 μL−1 O-glycosidase was added, and the mixture was incubated for 18 h at 37 °C. The deglycosylation profiles were evaluated by SDS-PAGE.In denaturing conditions, for protein deglycosylation, toxin (3.4 μg) were set in 20% SDS for 1 min at 95 °C. After adding 0.2 M sodium phosphate buffer, 0.08% sodium azide, 0.5 M EDTA pH 8, 10% nonidet P-40, incubation was prolonged for 2 min at 95 °C. After cooling, 1 U.μLT. maculosa venom was analyzed by 12% SDS-PAGE under non-reducing conditions. Venom from T. nattereri (VTn) was used as a control. After SDS-PAGE and transfer to nitrocellulose membrane , the toxin was detected using plasma from mice sensitized with T. nattereri venom (1:20 dilution) or from Nattectin-sensitized mice followed by Goat anti-mouse IgG HRP  as a secondary antibody (Ab).The purified protein (10 μg) of Galanthus nivalis agglutinin specific to mannose: α(1–2), α(1–3), or α(1–6) linked to mannose; SNA: Sambucus nigra agglutinin specific to Sialic acid: α(2–6) to GalNAc; MAA: Maackia amurensis agglutinin specific to Sialic acid: α(2–3) to galactose N-linked α(2–3) to galactose O-linked; PNA: Arachis hypogaea Peanut agglutinin specific to Core and terminal galactose: Gal-β(1–3)-N-acetylgalactosamine; and DSA: Datura stramonium agglutinin specific to Core galactose: Gal-β(1–4)-N-acetylglucosamine.The toxin was cut from 12% SDS-PAGE gel and eluted in PBS to identify toxin binding specificity using a competition assay performed according to Haab  using thM. luteus A270, E. coli SBS 363, and C. albicans MDM8, as described by Rossi et al. [2O containing 86 mM NaCl at pH 7.4; 217 mOsM for M. luteus and E. coli and 1.2 g potato dextrose in 100 mL of H2O at pH 5.0; 79 mOsM for C. albicans), at 37 °C under agitation. The absorbance at 595 nm was set on, and one aliquot of this solution was taken to get cells in logarithmic growth (A595 nm ~0.6) and diluted 600 times (A595 nm = 0.0001). The isolated toxin or Nattectin, both at 10 μg, was dissolved in sterile Milli-Q water to 100 μL of PB broth. Tetracycline and Gomesin were used as inhibitor controls. After 18 h of incubation at 30 °C, the inhibition of bacterial growth was determined by measuring absorbance at 595 nm.Human erythrocytes (type A) were collected in 0.15 M citrate buffer, pH 7.4, and washed 3 times by centrifugation with 0.15 M PBS, pH 7.4. To assess the hemolytic activity, aliquots of 10 µL of the selected toxin at 0.01, 0.1, 1, or 10 µg were put in 50 µL in a 3% suspension of erythrocytes in wells of U-shaped bottom plates and incubated for 3 h at room temperature. Solutions of isolated toxin at 10 µg previously incubated with D-Galactosamine  or D-Mannose  both at 10 or 30 mM for 1 h were also usedThe hemagglutinating activity was verified by reading the absorbance at 595 nm of each well in a plate reader. Erythrocytes incubated with water were used as positive control (100% hemolysis). Antimicrobial potential was monitored by a liquid growth inhibition assay against i et al. . Pre-ino−1, 30 min before TmC4-47.2 injection. Control animals received the same amount of control isotype IgG. Negative control was injected with intrascrotal sterile PBS. The study of the microvascular system was performed with an optical microscope  coupled to a photographic camera  through a 10/025 longitudinal distance objective/numerical aperture and 1.6 optovar. The surgical cremaster preparation was handled as described previously [®, Agener União, São Paulo, Brazil) and with 0.5 g Kg−1 of ketamine . The scrotum was exposed, and the cremaster muscle reached. Following the incision with cautery and spreading the muscle over a cover glass, the epididymis and testis were mobilized and pinned aside, allowing the microscopic access to the muscle microcirculation. The exposed tissue was superfused with 37 °C warmed bicarbonate-buffered salines, pH 7.4. The post-capillary venules with a diameter of 25–40 µm were chosen, and the interaction of leukocytes with the luminal surface of the venular endothelium was evaluated, counting the number of rolling leukocytes every 10 min after application of inflammatory agent for 30 min. Rolling leukocytes were defined as those moving at a velocity less than erythrocytes and demonstrated a clear rolling motion. The number of adherent cells was expressed as the number per 100 μm length of venule. The experiments were carried out under the National Council for Animal Experiment Control (CONCEA) and approved by the Butantan Institute’s Animal Use Ethics Commission (CEUAIB #275/06).Initially, Balb/c mice were injected by the intrascrotal route with 50 μL of TmC4-47.2 at 10 μg and rested for 3 h. Independent groups of mice were pre-treated with 500 µL of intraperitoneal (i.p.) injection of the purified rat anti-mouse CD29 , hamster anti-mouse CD49a , rat anti-mouse CD49b , rat anti-mouse CD49e , and rat anti-mouse CD106—VCAM-1, 14-1061-82, Thermo Fisher eBioscience) monoclonal antibodies at 10 μg mLeviously . Mice we+), eosinophils (CCR3+), and macrophages (F4/80+) were identified by flow cytometry and based on staining and morphologic characteristics using a light microscope Axio Imager A1  with an AxioCam ICc1 digital camera (Carl Zeiss).Balb/c mice were intraperitoneally injected with TmC4-47.2 at 10 μg in 500 μL, according to Lima et al. . As a nep < 0.05 using GraphPad Prism .All values were expressed as mean ± SEM. Experiments using 3 to 5 mice per group were performed independently two times. Parametric data were evaluated using analysis of variance, followed by the Bonferroni test for multiple comparisons. Non-parametric data were assessed using the Mann-Whitney test. Differences were considered statistically significant at


Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease, characterized by bile duct inflammation and destruction, leading to biliary fibrosis and cirrhosis. The purpose of this study was to investigate the utility of T1 and T2 mapping parameters, including extracellular volume fraction (ECV) for non-invasive assessment of fibrosis severity in patients with PSC.t test, ROC analysis and Pearson correlation were used for statistical analysis.In this prospective study, patients with PSC diagnosis were consecutively enrolled from January 2019 to July 2020 and underwent liver MRI. Besides morphological sequences, MR elastography (MRE), and T1 and T2 mapping were performed. ECV was calculated from T1 relaxation times. The presence of significant fibrosis (≥ F2) was defined as MRE-derived liver stiffness ≥ 3.66 kPa and used as the reference standard, against which the diagnostic performance of MRI mapping parameters was tested. Student P < 0.001) and ECV  correlated with MRE-derived liver stiffness. To diagnose significant fibrosis (≥ F2), ECV revealed a sensitivity of 84.2%  62.4–94.5%) and a specificity of 84.6% (CI 57.8–95.7%); hepatic native T1 revealed a sensitivity of 52.6% (CI 31.7–72.7%) and a specificity of 100.0% (CI 77.2–100.0%). Hepatic ECV (area under the curve (AUC) 0.858) and native T1 (AUC 0.711) had an equal or higher diagnostic performance for the assessment of significant fibrosis compared to serologic fibrosis scores (APRI (AUC 0.787), FIB-4 (AUC 0.588), AAR (0.570)).32 patients with PSC (age range 19–77 years) were analyzed. Both, hepatic native T1 (r = 0.66; Hepatic T1 and ECV can diagnose significant fibrosis in patients with PSC. Quantitative mapping has the potential to be a new non-invasive biomarker for liver fibrosis assessment and quantification in PSC patients. PSC has a strong male predominance and is often associated with other immune-mediated diseases such as inflammatory bowel disease  and autoimmune hepatitis (AIH). To date, there are several reports of therapy showing effect in PSC, but no established medical therapy with proven effect on transplant-free survival [Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease, leading to biliary fibrosis and cirrhosis. PSC is believed to be immune-mediated, however, the etiopathogenesis of the disease has still not been completely investigated and remains unclear. The main feature of PSC is a long-term, progressive inflammation followed by fibrosis of the intra- and extrahepatic bile ducts , 2. PSC survival , 4. Accosurvival , 6. As fsurvival , 8.Over the last decades, MRI techniques have undergone significant advancement from a qualitative to quantitative approach, offering the opportunity for the development of objective and reproducible imaging biomarkers that can be incorporated into clinical routine . To dateInitially extensively used in cardiac imaging for the detection and quantification of cardiac fibrosis and inflammation, quantitative T1 and T2 mapping techniques , might aThis study was approved by the institutional review board and was conducted in compliance with the ethical standards set in the 1964 Declaration of Helsinki as well as its later amendments. Written informed consent was obtained from all participants prior to MRI examination. Consecutive patients of the University Hospital of Bonn with diagnosis of PSC were prospectively enrolled from January 2019 to July 2020. Diagnosis of PSC was based on diagnostic criteria of PSC established by the EASL . Biochem3, scan duration/breath hold 15.3 s, 3 slices. The system configuration was based on an active pneumatic driver connected via plastic tube with a passive driver, which was placed at the patient's right upper quadrant. MRE involves (a) generation of shear waves in the tissue, (b) acquisition of MR images, (c) depicting the propagation of the induced shear waves, and (d) postprocessing of the share waves to generate quantitative liver stiffness maps using implemented vendor´s software . For hepatic T1 mapping, we used a heart rate independent 10-(2)-7-(2)-5-(2)-3-(2) modified Look-Locker inversion recovery (MOLLI) acquisition scheme with internal triggering [3, reconstructed voxel size 1.13 × 1.13 × 10 mm3, scan duration/breath hold 14 s. For the post-contrast T1 maps the same technique was used after 10 min of contrast agent application in the same positions as pre-contrast examinations. For contrast enhanced T1 mapping, a gadolinium-based contrast agent  was administered as a single bolus with an injection rate of 1.5 ml/s. Hepatic T2 mapping was performed using a six-echo gradient spin echo sequence (GraSE) with the following parameters [All liver MRI were performed on a clinical whole-body 1.5 T system  equipped with 32-channel abdominal coil with digital interface for signal reception. Liver MRE and T1 and T2 mapping were performed in addition to morphological sequences. For liver MRE, a 2D gradient-recalled echo with added cyclic motion encoding gradients (MEGs) sequence with the following parameters was applied: time of repetition (TR)/time of echo (TE) 50/20 ms, flip angle (FA) 20°, parallel imaging factor 2.3, active driver frequency 60 Hz, active driver power 100%, field of view (FOV) 450 × 403 × 32 mm, acquired voxel size 1.50 × 4.74 × 10 mm, reconstructed voxel size 1.17 × 1.17 × 10 mmiggering . Technicrameters : TR/TE 42) in at least three different representative regions of the liver. Based on MRE-derived liver stiffness, all study participants were divided into two groups, first, without (< fibrosis stage F2) and second, with significant fibrosis (≥ F2). To differentiate between patients with and without significant liver fibrosis a cutoff value of 3.66 kPa was chosen [An experienced board-certified radiologist  performed image analyses, blinded to the clinical data. For the assessment of T1 and T2 relaxation times, the mean relaxation time of three representative regions of interest (ROI) was calculated. ROIs were drawn centrally in the hepatic segments II, IVa and VII within liver parenchyma away from confounding factors like organ borders, vessels or bile ducts. Blood pool T1 values were derived from the abdominal aorta. ECV was calculated with ROI-based on pre- and post-contrast T1 values according to the previously published equation . Hematocs chosen .t test was used for comparison of continuous variables between two different groups. Dichotomous variables were compared using the χ2 test (with the cell count > 5) and Fisher test (with a cell count ≤ 5). Bivariate Pearson correlation coefficient or Spearman’s rank correlation coefficient were used for a correlation analyses, as appropriate. Receiver operating characteristic analysis (ROC) was used to determine the cut-points with the highest combined sensitivity and specificity, positive predictive values (PPV), negative predictive values (NPV) and accuracy using MRE-derived liver stiffness as a reference standard. DeLong method was used to compare areas under the curves (AUCs) [P < 0.05.All data were analyzed using software . Patient characteristics are presented as mean ± standard deviation, as absolute frequency or median, as appropriate. Student s (AUCs) . MRE-derP = 0.03). The mean age of the disease onset in the group of patients without significant fibrosis was 38.4 ± 7.5 years, with significant fibrosis 31.1 ± 12.5 years. At the time of MRI examination, 40.6% (13/32) patients received therapy with ursodeoxycholic acid (UDCA) alone; 31.3% (10/32) patients received a combination of 5-aminosalicylic acid (5-ASA) with UDCA due to accompanying IBD; 12.5% (4/32) patient the combination of corticosteroids (budesonide) with UDCA due to overlap with AIH. 20.0% (5/32) patients received no therapy at the time of MRI examination. Clinical characteristics are summarized in TableA total of 32 patients with diagnosis of large-duct PSC were included in this study. Based on MRE-derived stiffness values, 40.6% 13/32) patients had no (< F2) and 59.4% (19/32) had significant (≥ F2) fibrosis. 15.8% (3/19), 21.1% (4/19), and 63.1% (12/19) patients had fibrosis stages F2, F3 and F4, respectively. 18.7% (6/32) patients had additional features of AIH. There were 61.5% (8/13) patients with intrahepatic biliary changes only and 38.5% (5/13) patients with both intra- and extrahepatic bile duct changes in patients without significant fibrosis (< F2) and 73.7% (14/19) and 26.3% (5/19) in the group of patients with significant fibrosis (≥ F2), respectively. In patients without significant fibrosis (< F2), there were 15.4% (2/13) patients with and 84.6% (11/13) with no imaging features of portal hypertension . In patients with significant fibrosis (≥ F2) there were 52.6% (10/19) patients with and 47.4% (9/19) patients without imaging features of portal hypertension (3/32 patiP = 0.024). We found significant correlations between liver stiffness measurements derived by TE and MRE  as well as hepatic ECV . ECV was significant higher in patients with significant fibrosis according to TE .Based on liver stiffness measurements derived by TE 44.4% (8/18) patients had no (< F2) and 55.6% (10/18) had significant fibrosis (≥ F2). The mean value of liver stiffness measurements derived by TE in patients without (< F2) was 5.7 ± 0.8 kPa and in patients with significant fibrosis (≥ F2) 23.1 ± 20.3 kPa . Hepatic native T1 showed also high diagnostic performance with an AUC of 0.870, a sensitivity of 77.8% and a specificity of 88.9% to diagnose significant fibrosis . Hepatic T2 achieved an AUC of 0.753, a sensitivity of 55.6% and a specificity of 100.0% .P = 0.043, and 30.5 ± 4.4% vs. 26.3 ± 1.9%, P = 0.003, respectively . Also, fat fraction differed not significantly between both groups . All MRI parameters are given in Table Hepatic native T1 as well as ECV were remarkably increased in the group of patients with significant fibrosis ≥ F2) compared to the group of patients without significant fibrosis (< F2): 559.6 ± 56.3 ms vs. 522.8 ± 33.2 ms,  comparedP = 0.113). Hepatic ECV provided a sensitivity of 84.2%  62.4–94.5%), and a specificity of 84.6% (CI 57.8–95.7%). Hepatic native T1 provided a sensitivity of 52.6% (CI 31.7–72.7%) and specificity of 100.0% (CI 77.2–100.0%). Diagnostic performance of hepatic ECV was significantly higher when compared to the evaluated fibrosis scores FIB-4 and de-Ritis ratio  and equal when compared to the APRI and MELD scores . Furthermore, in contrast to ECV, diagnostic performance of hepatic native T1 was comparable with that of all evaluated serological fibrosis scores: APRI , FIB-4 , de-Ritis ratio . Hepatic T2 also performed well, however, with diagnostic performance expressed as AUC significantly lower when compared to hepatic ECV  and equal when compared to hepatic native T1 . Hepatic T2 provided a sensitivity of 57.9% (CI 36.3–76.9%) and a specificity of 92.3% (CI 66.7–98.6%). All values of diagnostic performance statistics for evaluated laboratory and mapping parameters are presented in Table Analysis of the diagnostic performance of MRI-derived mapping parameters for diagnosing significant fibrosis ≥ F2) was performed. According to the ROC analysis among all mapping parameters, hepatic ECV and native T1 demonstrated the best diagnostic performances with an AUC of 0.858 and 0.711, respectively, which were also comparable  hepatic ECV and native T1 correlated strong with MRE-derived liver stiffness and, (2) for the diagnosis of significant fibrosis (≥ F2), hepatic ECV and native T1 revealed the highest diagnostic performance in patients with PSC.P < 0.001) in patients with PSC  are needed to establish the results of this study and confirm the accuracy and usefulness of MRI mapping in patients with chronic liver disease.In our observational prospective study, MRI mapping parameters, including ECV calculation showed strong correlations with MRE-derived liver stiffness. Especially, T1 mapping techniques with estimation of ECV have a potential to be a new non-invasive biomarker for assessment and early detection of significant fibrosis in patients with PSC by providing additional information, without adding costs to examination.


P1 despite being achiral. Two classical hydrogen bonds link the molecules to form a layer structure.The title compound crystallizes in space group  16H13N3O2S, one hydrazinic nitrogen atom is essentially planar, but the other is slightly pyramidalized. The torsion angle about the hydrazinic bond is 66.44 (15)°. Both hydrazinic hydrogen atoms lie antiperiplanar to the oxygen of the adjacent carbonyl group. The molecular packing is a layer structure determined by two classical hydrogen bonds, N—H⋯O=C and N—H⋯Nthiazole. The space group is P1 with Z = 1, which is unusual for an achiral organic compound.In the molecule of the title compound, C Compound 3 was prepared by the reaction of 2-(benzo[d]thiazol-2-yl)acetohydrazide (2) with benzoyl chloride in the presence of pyridine at room temperature. The structure of 3 was initially determined on the basis of spectroscopic data and elemental analysis. In order to establish the structure of the product unambiguously, its crystal structure was determined and is presented here.Heterocycles represent a link between organic synthesis and pharmaceutical chemistry, thereby encouraging researchers to discover new hetereocyclic drug candidates. One of the most prominent heterocycles is benzothiazole, a privileged scaffold in the field of synthetic and medicinal chemistry °]. Each hydrazinic hydrogen atom lies antiperiplanar to a carbonyl oxygen atom across the respective N—C bond. The interplanar angle between the benzothiazol group and the phenyl ring is 75.65 (3)°.The structure determination confirms the formation of compound 3 Fig. 1. Bond leab plane —N torsion angle of JEBQOZ is −109.79 (19)° in contrast to −152.41 (11)° in the title structure. The average database bond lengths C2—S and C2—N for the benzo[d]thiazole ring system were calculated; for 444 hits (600 different molecules) the values were 1.750 (16) and 1.300 (29) Å, respectively, virtually unchanged from the values we obtained previously  for other structures containing the same benzothiazol-acetylhydrazide moiety gave only one hit, refcode JEBQOZ, with a P1, which may be considered as a moderately rare space group; of the over 1.1 million structures in the Cambridge database, only 9843 are in P1 .Anecdotal evidence, combined with previous experience, would suggest that it is unusual for an achiral compound to crystallize in space group P1 structures in the CSD, particularly those with Z = 1, were achiral. Unfortunately, there is at present no means of identifying, labelling and searching for chirality or chiral (‘asymmetric’) atoms using the standard ConQuest search routines, and it is clearly unfeasible to check all the P1 structures by hand. We therefore began by simply considering the small and possibly non-representative subset of 20 P1 structures (13 with Z > 1) that were determined by PGJ. Of these, 14 were pure enantiomers; for 12 of these, the absolute configuration was determined. Of the remaining six, five were not organic compounds , and the remaining structure  displayed planar chirality, but contained no ‘asymmetric’ atom. On this limited basis, we would therefore postulate that is very rare for achiral organic compounds to crystallize in P1, especially with Z = 1. An extension of this survey to all P1 structures in the CCDC is being planned.We therefore wished to see how many of the d]thiazol-2-yl)acetohydrazide 2 (0.08 mol) and pyridine (10 mL) was stirred for 15 min at room temperature. Benzoyl chloride (0.16 mol) was then added gradually to the reaction mixture, which was stirred for 15 min at 273 K. The reaction mixture was left to stand at room temperature for another 3 h, then poured onto ice water and neutralized with HCl. The precipitate thus formed was filtered off and dried to produce a white solid product 3. This was washed with ethyl acetate and recrystallized from ethanol; yield 85%, m.p. 487 K.A mixture of 2-(benzo[−1): υ 3429–3284 (NH), 2974 (CH aromatic), 1696, 1662 (2CO); 1H NMR : δ 4.23 , 7.43 , 7.49–7.53 , 7.58 , 7.91 , 7.99 , 8.09 , 10.48 , 10.55 ; 13C NMR : δ 39.4 (CH2), 122.5, 122.8, 125.5, 126.5, 127.9, 128.9, 132.4, 132.8, 136.9, 152.7, 165.0 (Ar-C), 166.0, 167.1 (2CO). Analysis: calculated for C16H13N3O2S (311.36): C 61.72; H 4.21; N 13.50%; found: C 61.70; H 4.22; N 13.55%.IR . The U(H) values were fixed at 1.2 times the equivalent Uiso value of the parent carbon atoms.Crystal data, data collection and structure refinement details are summarized in Table 3The compound contains no chiral centres and crystallizes only by chance in a chiral (Sohncke) space group.10.1107/S2056989021007672/yk2155sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989021007672/yk2155Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021007672/yk2155Isup3.cmlSupporting information file. DOI: 2099652CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Mitochondrial and intracellular reactive oxygen species (ROS) were overproduced and associated with disrupted mitochondrial membrane potential and respiratory capacity. Additionally, the mitochondrial network was fragmented with downregulated fusogenic proteins, MFN1/2 and L-/S-OPA1, and upregulated fission-related proteins, DRP1 and FIS1. Autophagy was also activated as evidenced by increased expression of autophagosome formation-related proteins, Beclin, p62, and LC3. Enhanced mitochondrial fragmentation and autophagy indicate that mitophagy was activated. ROS-induced cytotoxicity was reversed by the addition of N-acetylcysteine, confirming ROS overproduction as a contributor. Taken together, pardaxin demonstrated promising anticancer activity in OC cells, which warrants further preclinical development of this compound.Most ovarian cancer (OC) patients are diagnosed with stage III or higher disease, resulting in a poor prognosis. Currently, paclitaxel combined with carboplatin shows the best treatment outcome for OC. However, no effective drug is available for patients that do not respond to treatment; thus, new drugs for OC are needed. We evaluated the antimicrobial peptide, pardaxin, in PA-1 and SKOV3 cells. Pardaxin induced apoptosis as determined by MTT and TUNEL assays, as well as activation of caspases-9/3, Bid, t-Bid, and Bax, whereas Bcl-2 was downregulated. The IC Ovarian cancer (OC) is the fifth leading cause of cancer-related deaths in females  with ove2•−), hydroxyl radicals (•OH), hydrogen peroxide (H2O2), and singlet oxygen [Reactive oxygen species (ROS) are of particular interest since their deleterious effects in cell physiology and pathology were reported by Droge et al. . ROS inct oxygen . In cellt oxygen . To prevt oxygen ,10. Overt oxygen .Mitochondria are double-membraned organelles that vary considerably in size and structure . They arAutophagy is a cytoprotective mechanism that involves homeostatic functions such as cytoplasm, protein, and organelle turnover . FundamePardachirus marmoratus. It contains a helix–hinge–helix structure that is common in peptides able to lyse mammalian and bacterial cells [Oceans are a treasure trove for developing novel chemotherapeutics, since they account for nearly 70% of the total global area and contain a diversity of ecosystems much greater than that of terrestrial systems. Antimicrobial peptides (AMPs) represent a large family of marine compounds that exhibit an overall high efficacy against Gram-positive and Gram-negative bacteria ,23,24,25al cells , with ital cells . In thisN-acetylcysteine (NAC) and MTT, respectively, were prepared in DMSO and PBS as stock solutions which were then freshly diluted in the proper media at concentrations of interest. An In Situ Cell Death Detection Kit, Fluorescein, used in the Terminal Transferase-mediated dUTP Nick-End Labeling (TUNEL) method, was acquired from Roche Life Science . DAPI , FITC-Annexin V/PI assay kit, the chloromethyl derivative of 2,7-dichlorodihydro- fluorescein diacetate (CM-H2DCFDA), MitoSOXTM Red, and JC-1 were acquired from Molecular Probes, Inc. . A Seahorse XF Cell Mito Stress Test kit that contained enzymatic inhibitors carbonyl cyanide-4-(trifloromethoxy)phenylhydrazone (FCCP), oligomycin, and rotenone, were acquired from Agilent Technologies . The plasmid, pcDNA 3.1(+)-DsRed2-Mito-7, for the study of mitochondrial morphology was acquired from Addgene Corporation . CellROX® was acquired from Life Technologies . NAC was acquired from Santa Cruz Biotechnology , while DRAQ7TM was acquired from Abcam .PBS  was used to prepare pardaxin stock solution which was stored at −20 °C. A powder of 3--dimethylthiazol(-z-y1)-3,5-diphenyltetrazolium bromide (known as MTT) and dimethyl sulfoxide (DMSO) reagent were acquired from Sigma-Aldrich . ® CRL-1572™, Homo sapiens ovary epithelial teratocarcinoma), were cultured with the Eagle’s Minimum Essential medium. The human OC cell line, SKOV3 cells  were cultured with the RPMI 1640 medium (Life TechnologiesTM). Both media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco®), 2 mM glutamine, 50 U/mL penicillin and 50 mg/mL streptomycin (Sigma-Aldrich®). The cell lines were cultured under a humidified atmosphere of 5% CO2 mixed in 95% air at 37 °C, and they were examined under a Leica DMI 3000B phase contrast microscope  regularly. After microscopic examination, these cells were used for the following experiments.The human OC cell line, PA-1 cells  and placed overnight in a 37 °C incubator equipped with a humidified atmosphere mixing with 5% CO2 and 95% air. The OC cells were challenged with 0, 0.01, 0.1, 1, 2.5, 5, 10, and 20 μΜ of pardaxin for 24 or 48 h. Subsequently, the drug-containing medium was replaced with the MTT solution, and the OC cells were further incubated in the incubator for 4 h to allow MTT being reduced to an insoluble formazan crystal through viable cell dehydrogenases. Each concentration was performed in triplicate. The Leica DMI 3000B phase contrast microscope was used to observe the cells during the experimentation. DMSO was used to redissolve the formazan crystal. Absorbance at 570 nm was recorded using a Dynatech Laboratories ELISA reader  for calculating cell viability, and the results are showed as mean ± SD. A MTT-based assay was performed to assess cell viability as described in the literature . A densiIn Situ Cell Death Detection Kit, Fluorescein was used for TUNEL analysis as per manufacturer’s user manual. Afterwards, green fluorescent TUNEL-positive cells were detected by an immunofluorescence microscope acquired from Lecia Microsystems . In addition, the TUNEL-positive cells showing green fluorescence were spotted with the assistance of DAPI staining and by cellular morphology. The sample slides were observed using a fluorescence-detectable microscope, and green fluorescence was fixed at 520 nm. A SPOT CCD RT-slider integrating camera  was used to captured images. The cells stained green suggested apoptotic cells, while cells fluoresced blue represented DNA fragmentation.The TUNEL assay was conducted to probe DNA fragmentation using fluorescence microscopy. The terminal deoxynucleotidyl transferase (TdT) included in the assay kit can catalyze fluorescein-dUTP incorporation at free 3′-hydroxyl ends of the fragmented DNA, allowing it to be detected. In brief, the OC cells plated on sample slides with pardaxin at 0, 0.1, 1, 2.5, and 5 μΜ for 24 h were fixed with pH 7.4, 4% methanol-free formaldehyde for 5 min on ice. Following ice-cold PBS rinse, the 5 cells/mL was prepared. One hundred (100) μL of the solution (6 × 104 cells) were then transferred to a 5 mL tube. The resulting samples were subjected to FITC-Annexin V labeling as per manufacturer’s user manual. In brief, 100 μL of 1X Annexin V binding buffer from BD Biosciences  were used to resuspend the cells which were then fluorescently labeled by mixing 5 mL of FITC-Annexin V and 5 mL of 1 mg/mL PI (BD Biosciences) to each sample. After gentle mixing and incubation for 30 min at room temperature in a dark room, 1 mL of the 1X binding buffer was added to each sample. The yielded samples were analyzed using a flow cytometer  with Cell Lab Quanta™ SC analysis software. At least 104 cells/sample were analyzed.To detect mode of cell death, FITC-Annexin V/PI method was used in flow cytometric analyses, of which FITC is a fluorophore. Annexin V-positive signals are an indicator of cellular apoptosis, while PI-positive indicates necrotic or late apoptotic cells, due to disrupted integrity of cell and nuclear membranes. Viable cells are not responsive to both staining probes. Briefly, the OC cells were challenged with pardaxin at 0, 0.1, 1, 2.5, and 5 μΜ for 24 h, and they were harvested and cold PBS washed. After resuspending the cells in 1X binding buffer, a solution containing a concentration of 6 × 102O2) level using a chemical probe CM-H2DCFDA. In the presence of H2O2, cell-permeable CM-H2DCFDA is processed by intracellular esterases and oxidized into a fluorescent adduct of 2,7-dichlorodifluorescein (DCF) which is detectable using flow cytometry. Prior to use, the CM-H2DCFDA probe was protected from air. Briefly, 6 × 105 cells/well were seeded in 6-well plates with the specified media and placed for 16–18 h for cell attachment on the plate bottom in the 37 °C incubator equipped with a humidified atmosphere mixing with 5% CO2 and 95% air. After washing with PBS, the culture media were replaced with pardaxin-containing media at concentrations of 0–5 μΜ and incubated for 4 h at 37 °C. Subsequently the cells were incubated with 5 μΜ CM-H2DCFDA for additional 30 min at 37 °C. Following washing and resuspending in PBS, the Beckman Coulter cytometer was used to detect fluorescence signals with excitation/emission at 495/520 nm and analyzed with Cell Lab QuantaTM SC software. At least 104 cells/sample were analyzed.The following procedures were conducted as described in the literature ,37. Intr•−) level was analyzed by cell-permeable MitoSOXTM Red , a redox-sensitive dye believed to specifically target mitochondrial matrix, and red fluorescence is emitted upon oxidation. A density of 6 × 105 cells/well were seeded with the specified media and placed for 16–18 h for cell attachment on the plate bottom in the 37 °C incubator equipped with a humidified atmosphere mixing with 5% CO2 and 95% air. After being treated with pardaxin at 0–5 μM for 4 h at 37 °C, the cells were rinsed with PBS once. The MitoSOXTM Red at 10 mM was added to the OC cells which were incubated for 10 min at 37 °C, rinsed, and redissolved in PBS. The cytometer of Beckman Coulter and Cell Lab Quanta™ SC software was used for analysis. The absorption/emission wavelengths were set at 510/580 nm. At least 104 cells/sample were analyzed.The following procedures were conducted as described in the literature ,37. Mito® Green is a fluorogenic dye that measures oxidative stress in live cells. The cell-permeable probe emits weak fluorescence under a reduced state, whereas upon oxidation by ROS, it exhibits bright green fluorescence and subsequently binds to DNA with absorption and emission peaked at ~485 and 520 nm, respectively. Before use, the reagent was placed at −20 °C and protected from light. In brief, a density of 6 × 105 cells/plate were seeded with the specified media and placed for 16–18 h for cell attachment on the plate bottom in the 37 °C incubator equipped with a humidified atmosphere mixing with 5% CO2 and 95% air. After being treated with pardaxin at 0–5 μM for 4 h, the OC cells were rinsed with PBS once. CellROX® Green at 5 mM was added to the cells which incubated for 10 min at 37 °C, rinsed, and redissolved in PBS. The cytometer of Beckman Coulter and Cell Lab Quanta™ SC software was used for analysis. At least 104 cells/sample were analyzed.CellROX5 cells/well with specified media and placed for 16–18 h for cell attachment on the plate bottom in the 37 °C incubator equipped with a humidified atmosphere mixing with 5% CO2 and 95% air. After treatment with indicated pardaxin concentrations, the cells were added to JC-1 working solution, incubated for 20 min at 37 °C, and washed with HBSS solution. Following the removal of the media, the OC cells were detached and resuspended in 1 mL PBS. A CytoFLEX flow cytometer from Beckman Coulter  was used to record red and green JC-1 fluorescence intensity. At least 2 × 104 cells/group were analyzed using Beckman software CytExpert 2.0, and red/green fluorescence intensity ratio calculated accordingly.The following procedures were conducted as described in the literature . A fluor5 cells/well with specified media and placed for 16–18 h for cell attachment on the plate bottom in the 37 °C incubator equipped with a humidified atmosphere mixing with 5% CO2 and 95% air. They were then challenged with pardaxin (0–5 μM) for 24 h at 37 °C. Subsequently, the cells were washed with PBS prior to adding acridine orange at 1 µg/mL and incubated for 15 min at 37 °C. Following that, the OC cells were harvested, washed with PBS, and diluted into 1 mL cold PBS. The Beckman Coulter cytometer was used for conducting the AVO experimentation. The data were analyzed using the software of Cell Lab Quanta™ SC. At least 104 cells /sample were analyzed.The OC cells were seeded at a density of 4 × 10Before Western blotting, the OC cells were challenged with pardaxin  for 24 h. Both floating and attached OC cells were collected and washed with ice-cold PBS, followed by adding lysis buffer with RIPA reagent of Sigma-Aldrich  and incubating for 30 min at 4 °C prior to centrifugation at 13,500 rpm for 15 min. Utilizing 8–12 % SDS-PAGE, the resultant proteins were separated and then transferred to polyvinylidene fluoride (PVDF) membrane  which was soaked in 5% milk-containing Tris-buffered saline or Tween 20 to block the membrane for 1 h. Afterwards, it was incubated overnight with primary antibodies designed to capture the proteins of interest at 4 °C overnight . Followi5 cells/well) with specified media and placed for 16–18 h for cell attachment on the plate bottom in the 37 °C incubator equipped with a humidified atmosphere mixing with 5% CO2 and 95% air. The culture media were then replaced with pardaxin-containing medium at 0, 0.1, 1, 2.5, and 5 μΜ for 24 h of incubation. Following rinsing the OC cells with 1 mL of Seahorse medium (sodium bicarbonate-free DMEM), 675 μL of the Seahorse medium were added to each well. Four measurements of the basal OCR were recorded and averaged under basal conditions, then sequentially adding 1 μM of oligomycin, 250 nM of FCCP, and 2 μM of rotenone. Subsequently, the OC cells were washed with ice-cold PBS and lysis buffer with RIPA reagent for 30 min at 4 °C, followed by centrifugation at 13,500 rpm for 15 min. The concentrations of proteins from the resultant supernatant were determined with a DC protein assay kit . A standard curve of protein concentration was generated using bovine serum albumin. To compare results, data were expressed as OCR in pmol/min/mg protein and ECAR in mpH/min/ mg protein, and their values were calculated after normalizing with the protein concentration.A Seahorse XF24 Extracellular Flux Analyzer purchased from Seahorse Bioscience  was used to detect consumption of oxygen in a cell. The OC cells were seeded in Seahorse cell culture 24-well plates  in serum-free medium for additional 16 h, and added 4% paraformaldehyde for fixation. After the drug treatment, the nuclei were DAPI stained. After being processed with Sigma-Aldrich’s mounting media, the slides were viewed using a Leica Microsystems TCS SP5 II confocal microscope at a magnification of 2000× . A semi-quantitative analysis scale was employed to evaluate the degree of mitochondrial network formation using the procedures as described in the literature [To prepare transfection, a mixture of 0.5 mg of pcDNA 3.1(+)-DsRed2-Mito-7 and 1 mL of Lipofectamine 2000  in a total volume of 100 μL was incubated for 20 min to allow the formation of DNA-Lipofectamine complexes. Each well containing 1 × 10terature . The valt-test were expressed as means ± SE from three independent trials, where * p < 0.05 and ** p < 0.01 are considered to be statistically significant.Statistical analysis was carried out with SPSS version 13.0, Windows version. The results of Student’s 50) values for pardaxin were 4.6–3.0 μM after 24 and 48 h, respectively  or a lower range of 0–5 μM to study the effects of pardaxin. To determine the mechanism of cell death induced by pardaxin, flow cytometric studies were implemented to analyze Annexin V and PI-stained OC cells. In the Annexin V/PI chart, the lower right quadrant (Annexin Vpositive/PInegative) represents a group of early apoptotic cells, whereas the upper right quadrant (Annexin Vpositive/PIpositive) indicates a group of late apoptotic cells [To measure cell viability following pardaxin treatment, a range of concentrations  were evaluated in PA-1 and SKOV3 cells using the MTT assay. After a 24 h incubation in PA-1 cells, the percentage of cell viability at 0, 0.01, 0.1, 1, 2.5, 5, 10, and 20 μM of pardaxin was 100 ± 6.9, 101 ± 9.0, 100.9 ± 0.7, 79.9 ± 16.5, 49.6 ± 7.6, 32.1 ± 1.31, 13.3 ± 0.6, and 2.7 ± 0.4, respectively, whereas in SKOV3 cells, the percentages were 100 ± 13.2, 99.2 ± 2.9, 99.1 ± 1.8, 97 ± 4.5, 71.6 ± 9.6, 46.9 ± 7.1, 3.1 ± 1.5, and 1.4 ± 0.5, respectively. The same pardaxin concentrations were also evaluated at 48 h in both OC cells. The half-maximal inhibitory concentration  levels were significantly increased to 25.8 ± 0.5 and 60.0 ± 1.0 % at 5 μM in PA-1 and SKOV3 cells, respectively, compared with the controls , Bcl-2, and Bcl-2-associated X protein (Bax). Bcl-2 is an apoptosis suppressor, whereas the other proteins are pro-apoptotic. In PA-1 cells, the t-Bid/Bid ratio and Bax levels were significantly increased to ~7.8-fold and ~7.3-fold relative to the untreated cells, respectively, following 5 μM of pardaxin treatment. In contrast, Bcl-2 levels were markedly reduced to ~0.6-fold. Similar results were observed in SKOV3 cells. The t-Bid/Bid ratio, Bax level, and Bcl-2 level were altered to ~7.7-fold, ~5.0-fold, and ~0.7-fold, respectively, relative to untreated cells I. Taken Acridine orange (AO) is a dye that has been used for detecting autophagic events. The cytoplasm and nucleus of AO-stained cells, respectively, fluoresce as dim red bright and green colors, whereas the acidic compartments appear bright red ,41. The Mitochondrial respiration capacity can be measured through oxygen consumption rate (OCR) of the OXPHOS: basal, ATP-linked, ATP-independent, and ETC-independent respiration. Each OCR is derived by sequentially adding inhibitors against enzymatic complexes of the OXPHOS. Therefore, we sequentially added oligomycin for inhibiting ATP synthase , FCCP for uncoupling the OXPHOS, and rotenone for inhibiting complex I. Since mitochondria are affected by pardaxin, we evaluated respiration OCR parameters following treatment with pardaxin at 0, 0.1, 1, 2.5, and 5 μM for 4 h in PA-1 and SKOV3 cells. After treatment with 5 μM pardaxin in PA-1 cells, the results indicated that mitochondrial basal respiration, ATP-linked respiration, ATP-independent respiration, and ETC-independent respiration were significantly decreased to 22.1 ± 7.2, 17.2 ± 5.2, 4.9 ± 5.8, and 2.0 ± 3.4 pmoles/min/mg protein, relative to the control at 164.2 ± 21.5, 122.1 ± 15.4, 42.0 ± 7.2, and 39.6 ± 16.9 pmoles/min/mg protein, respectively. Similarly, for SKOV3 cells, the values were significantly decreased to 26.2 ± 8.6, 20.1 ± 5.8, 6.2 ± 5.8, and 20.2 ± 8.2 pmoles/min/mg protein, relative to the control at 114.5 ± 12.4, 102.9 ± 12.0, 15.5 ± 6.4, and 72.6 ± 6.9 pmoles/min/mg protein, respectively A–D. The TM. Our findings indicate that mitochondrial network was fragmented after 5 μM pardaxin were incubated for 16 h in both cell lines , acts as an antioxidant within cells to deplete ROS [TM Green, CM-H2DCFDA, MitoSOXTM Red, or AO and subjected to flow cytometry for analysis. The results indicated that NAC did not cause apoptosis, and apoptotic cells were elevated significantly following pardaxin treatment, which was significantly rescued by NAC pretreatment (TM Green, DCF, and MitoSOXTM Red signals (The reducing agent, lete ROS . We pretreatment A,B. Simi signals C,H. Simi signals I,J. TakeMost OC types are epithelial and approximately two-thirds of ovarian epithelial cancer patients are diagnosed at stage III or IV . In the Traditionally, most new cancer drugs are primarily sourced from the development of natural products, with more than 60% of the FDA-approved drugs from terrestrial animals and plants, but relatively few from marine creatures ,53. In pThe 33-amino acid pardaxin compound exhibited cytotoxic activity against PA-1 and SKOV3 cells. Since pardaxin contains cationic and amphipathic amino acids, it can readily interact with the anionic membranes of tumor cells, which enhances entry into the cell. Pardaxin demonstrated a cytotoxic mechanism of action by inducing ROS overproduction in mitochondria, resulting in mitochondrial membrane depolarization that further caused an imbalance in mitochondrial membrane potential and initiated the activation of pro-caspases 9 and 3. Mitochondria-mediated apoptosis induced by pardaxin was also buttressed by the upregulation of t-Bid and Bax, because t-Bid is situated on the mitochondrial membrane. Since the OXPHOS enzymatic complexes were simultaneously attenuated by pardaxin-induced ROS, additional ROS generation is likely to occur that further contributes to total ROS levels. Additional studies of mitochondrial morphology showed that pardaxin fragmented the mitochondrial network, which is consistent with other ROS-generating marine peptides, such as TP3 , althougAutophagy and apoptosis mostly occurs in a sequence in which autophagy precedes apoptosis  when a s2•− in part, with an estimate that 0.2–2.0% of oxygen consumed by mitochondria is converted to O2•− [2•− is quickly converted to H2O2 by dismutases including superoxide dismutases 1 and 2. Therefore, both O2•− and H2O2 yielded from this process are considered mtROS and the levels of H2O2 are positively correlated with O2•−. Because of its negatively charged and short half-life characteristics, O2•− hardly passes from the mitochondrial outer membrane to the cytosol, and is thus unable to stimulate signal transduction molecules in the cytosol. Instead, O2•− prefers having a radical–radical reaction with nitric oxide to yield an adduct of peroxynitrite within mitochondria, a harmful oxidant known to cause DNA damage, disruption of mitochondrial integrity, and detrimental modification of proteins [2O2 is neutrally charged and more stable than O2•−, enhancing its ability to cross the mitochondrial membrane to influence intracellular components and signal transduction pathways. In the present study, we found that the O2•− levels were elevated in mitochondria and intracellular H2O2 was positively correlated with mitochondrial O2•− levels, which indicates that pardaxin-induced ROS are initially generated in the mitochondria.Under normal conditions, the ETC process in the OXPHOS is not considered to be ideal. Leakage of electrons from complexes I and III results in reduction of oxygen to form O to O2•− . Subsequproteins . In contTM Red and CellROXTM Green fluorescence, and the resulting ROS may break DNA in an efficient manner. Besides, most tumor types reside in a complex microenvironment that is hypoxic, which impedes ROS generation by ionizing radiation, hence decreasing radiotherapeutics efficacy. A probable tactic to mitigate hypoxia is to downregulate OXPHOS capacity. This strategy is based on the idea that inhibiting OXPHOS decreases oxygen consumption by mitochondria, thus increasing oxygen availability for use in the cytosolic compartments and nearby tissues [DNA damage induced by ionizing radiation is the primary mechanism through which radiation therapy kills cancer cells. Thus, inducing additional ROS within cancer cells is beneficial for enhancing the radiotherapeutic effect. Pardaxin-induced ROS has been found in mitochondria and the nucleus, as evidenced by MitoSOX tissues . Our fin


As prostate-specific antigen (PSA) plays an important role in the response evaluation of this therapy, and the aim of this study was to prospectively assess the prognostic value of early PSA measurements. We found PSA changes as early as four weeks after the first administration of PSMA-RLT to be predictive of both long-term biochemical and PET imaging response, as well as overall survival. We then evaluated relevant predictive thresholds in PSA change at that time point, as the early detection of long-term (non-)response to PSMA-RLT can be of great benefit in the clinical management of terminally ill mCRPC-patients.Radioligand therapy with [177Lu]PSMA-617 (PSMA-RLT) is a promising therapeutic option for metastatic castration-resistant prostate cancer (mCPRP). This study assessed the prognostic value of early PSA measurements during PSMA-RLT. Methods: 27 patients with mCRPC scheduled for PSMA-RLT were prospectively enrolled for a serial short-interval PSA-assessment. Change in PSA (∆%PSA) during two treatment cycles was correlated with biochemical response (BR) and change in tumor volume on PET (TV) after 16 weeks (w16), as well as overall survival (OS). PCWG3 criteria and the recently recommended threshold of ∆%PSA ≤ −30% were assessed for their predictive value. Results: ∆%PSA first correlated with BR, TV and OS after 4 weeks (c1w4). At c1w4, ∆%PSA ≤ −30% was associated with the biochemical response at w16 (p = 0.003) and a longer median OS (p = 0.025), whereas the PCWG3-derived threshold of ∆%PSA ≤ −50% showed no such correlation. In contrast, ∆%PSA ≥ 25% at c1w4 was associated with biochemical progression at w16 (p = 0.003) and a shorter median OS (p < 0.001). Conclusion: PSA changes as early as four weeks after PSMA-RLT allow a significant prediction of later biochemical and PET-based imaging response, as well as OS. At this early time point, a more lenient threshold for a PSA decrease of at least 30% appears better-suited for the prediction of a positive biochemical response and longer OS. In contrast, the PCWG3-derived threshold for PSA increase (+25%) reliably anticipates biochemical progression and shorter OS.Purpose: Radioligand therapy with [ Two cycles of PSMA-RLT (at 6 GBq (162.16 mCi) per cycle) were administered six to eight weeks apart, followed by biochemical (PSA) and PSMA PET/CT-based restaging six to eight weeks later . Subsequently, following in-house standards, patients who showed no obvious signs of progression  or high-grade toxicity received further cycles of RLT, while patients with clinical and/or imaging-based progression were offered the best standard of care treatment. To assure consistency and comparability, prospective PSA assessment in this study was therefore confined to the first two treatment cycles up to restaging. For each cycle , serum PSA  was determined at our facility during the 3- to 4-day hospitalization period immediately before administration (d0), as well as 24 (d1), 48 (d2) and 72 (d3) hours later; during ambulatory visits the following week ; during follow-up examination four weeks after administration (w4) and at the restaging after two cycles (w16). See Radiosynthesis of the radioligand PSMA-617 was performed as previously described , with slThe change of PSA at each time point relative to the baseline (c1d0) was calculated using the following formula:Whole-body PSMA PET scans were acquired before therapy and at w16. Additionally, to assess the clinical treatment effects, the pain status was assessed at c1d0 and w16, utilizing the “Quality of Life Questionnaire C30” (QLQ-C30) developed by the “European Organization for Research and Treatment of Cancer” (EORTC). Pain intensity was estimated on a verbal rating scale of “none”, “a little”, “quite a bit” and “very much” .Referring to PCWG3 recommendations, PSMA-RLT was considered a cytotoxic treatment. Therefore, the primary control/relieve/eliminate endpoint was the biochemical response (BR) at w16, with a quantified change of the whole-body tumor volume at w16 as a secondary endpoint for validation. Additionally, overall survival (OS) was assessed for all enrolled patients in September 2021 (51 months after the inclusion of the first patient). Following the PCWG3 criteria , BR at wt-tests and Tukey’s HSD post-hoc analysis when appropriate. Chi-square test, utilizing Cramer-V, and Fisher’s exact test were used to assess the cross-table correlation between the qualitative PSA responses, categorized by different thresholds, after four and 16 weeks. Univariate linear regression was applied to correlate the PSA change at given time points with the change in tumor volume at w16, utilizing adjusted R2 (adj. R2) for the effect size assessment. The relation between various baseline parameters and the primary endpoints was evaluated using linear regression for changes in the PSA at w16 and Cox regression models for the OS, with only significantly univariate variables (p ≤ 0.05) included in the multivariate analysis. Kaplan–Meier analysis and Cox proportional hazard regression were applied to determine associations between changes in the PSA and OS, utilizing log-rank tests and hazard ratios (HR) to compare survival data between the biochemical response groups. Finally, an exploratory retrospective receiver operating characteristic (ROC) analysis, including Youden’s J, was used to retrospectively ascertain the early PSA thresholds, predictive of BR at w16.SPSS 24.0.0.0 was used for statistical analyses. Data were presented as the mean ± standard deviation, range or 95% confidence intervals (95% CI). Normal distribution was assessed using Shapiro–Wilk tests. Mixed analysis of variance (ANOVA) was used to analyze and compare serial PSA change during the first two cycles between patient groups categorized by BR at w16, employing Greenhouse–Geisser adjustment to correct the violations of sphericity and Levene’s test to assess the homogeneity of error variances, as well as Welch’s In 27 patients, 59 cycles of PSMA-RLT were administered with an average dose of 5.99 ± 0.12 GBq . Detailed patient characteristics, including previous therapies, are given in w16 −77 ± 13). Four patients (15%) displayed PSA progression (mean ∆%PSAw16 103 ± 62), and three patients were biochemically stable (mean ∆%PSAw16 −19 ± 22). In all patients, the biochemical response status was confirmed in the PSA follow-up. ∆%PSAw16 had no correlation to previous treatments or other relevant pretherapeutic parameters , while, concurrently, former opioid intake could be reduced to NSAID in five responders.At w16, 16 patients (59%) achieved a biochemical response according to the PCWG3 criteria (mean ∆%PSAp = 0.012) and TV50 (p = 0.014) at the baseline were predictive of OS (negative correlation). Detailed univariate and multivariate cox regression results are shown in According to a multivariate Cox regression model based on prior univariate Cox regression analyses of relevant pretherapeutic parameters, only PSA .Additionally, independent univariate Cox regression showed that the number of PSMA-RLT cycles correlated positively with the OS (HR 0.55 (95% CI 0.38–0.81), A mixed ANOVA was performed, differentiating the PSA development of the later biochemical response (according to PCWG3) over eleven time points across two cycles. Please also refer to p < 0.001, partial η2 = 0.64). ∆%PSA differed significantly between the response groups, from four weeks after the first administration of PSMA-RLT (c1w4) until the end of the observation period (p < 0.001). At c1w4, each response group was distinguishable by ∆%PSA from the other two groups (Tukey HSD p < 0.015).Due to the expected dependence between within-subjects variables (∆%PSA) and between-subjects factors (response), there was a statistically significant interaction between the time and response groups  = 17.39, c1d1–c1w1 −5 ± 18, range −49 to 69) but approximately normally distributed (p > 0.095).Earlier changes in the PSA during the first two weeks after administration showed no such significant correlation with long-term BR, as PSA values were fluctuant  = 16.39 p < 0.001, n = 23; Cramer-V = 0.844) and the response at w16 (χ2(4) = 4.54, p = 0.033, n = 23; Cramer-V = 0.44), respectively. At that time point, ∆%PSAc1w4 ≥ 25% showed a sensitivity of 75% (3/4) and specificity of 100% (19/19) for biochemical progression, while ∆%PSAc1w4 ≤ −30% offered a sensitivity of 63% (10/16) and a specificity of 86% (6/7) for the biochemical response. The respective contingency table and waterfall plot comparing the two time points (c1w4 and w16) are given in At c1w4, PSA thresholds of ∆%PSAc1w4 ≥ 25% and ∆%PSAa PSA decrease of at least 50%, b PSA decrease smaller than 50% or PSA increase smaller than 25% and c PSA increase of at least 25%; χ2(4) = 11.91, p = 0.003, Cramer-V = 0.622.Data are presented as the number of patients with percentage of patients per row in parentheses. c1w4 ≤ −50% narrowly failed to be a significant predictor of the future biochemical response (p = 0.059), as only six out of the 16 responders at w16 were correctly categorized  revealed ∆%PSAc1w4 ≤ −10% to have the highest discriminatory power for the biochemical response at w16, providing a sensitivity of 81% (13/16) and specificity of 87% (6/7) (area under curve (AUC) 0.88 (95% CI 0.68–0.98), p < 0.001, Youden’s J 0.67). Conversely, a ∆%PSAc1w4 > 10% proved optimal for the detection of biochemical progression at w16, with a sensitivity and specificity of 100%, respectively  (AUC 1.0 (95% CI 0.85–1.0), p < 0.001, Youden’s J 1.00).The ROC analysis of ∆%PSAp < 0.001, adj. R2 = 0.73). A corresponding significant correlation with ∆%TV50 for early ∆%PSA occurred for the first time at c1w4  = 8.85, p = 0.007, adj. R2 = 0.26).∆%PSA at w16 correlated strongly with ∆%TV50  = 59.69, p = 0.015).To assess the relation between PSA change during the first two cycles of PSMA-RLT and OS, continuous univariate Cox regression was used for all time points until restaging (w16). c1w4 was again the first time point at which ∆%PSA was significantly associated with OS (HR 1.007 (95% CI 1.001–1.013), c1w4 ≥ 25% to be significantly associated with shorter median OS (6.4 (95% CI 3.1–9.7) months vs. 21.8 (95% CI 14.6–28.9) months, p < 0.001; HR 7.63 (95% CI 2.28–25.64), p = 0.001; c1w4 ≤ −30% exhibited significantly longer median OS (27.8 (95% CI 17.1–38.4) months vs. 12.0 (95% CI 6.9–17.1) months, p = 0.025; HR 0.37 (95% CI 0.15–0.91), p = 0.031; Kaplan–Meier analyses at that time point revealed ∆%PSAw16 ≥ 25% (6.2 (95% CI 3.0–9.2) months vs. 23.7 (95% CI 17.2–29.4) months p < 0.001; HR 7.7 (95% CI 0.14–0.97), p < 0.001) and ∆%PSAw16 ≤ −30% (24.6 (95% CI 16.4–32.7) months vs. 11.0 (95% CI 6.8–15.2) months, p = 0.038; HR 0.36 (95% CI 0.13–0.87), p = 0.045).This differentiation of OS persisted up until w16 for both thresholds: ∆%PSAp = 0.039; HR 0.36 (95% CI 0.13–0.95), p = 0.048) and was not found at earlier time points, e.g., at c1w4 (p = 0.507).For the PCWG3-derived response criterion of ∆%PSA ≤ −50%, an association with a significantly longer median OS was only observed at w16 (25.8 (95% CI 17.8–33.9) months vs. 11.3 (95% CI 7.7–14.9) months, c1w4 ≤ −10%) exhibited significantly longer median OS (26.3 (95% CI 16.5–36.0) months) than all the other patients  months, p = 0.024; HR 0.39 (95% CI 0.16–0.91), p = 0.03). Conversely, a PSA increase of more than 10% (∆%PSAc1w4 > 10%) was significantly associated with a shorter median OS (7.4 (95% CI 3.7–11.1) months vs. 22.9 (95% CI 15.3–30.6) months, p = 0.002; HR 4.1, (95% CI 1.6–10.2), p = 0.003).Using the PSA thresholds identified by exploratory retrospective ROC analyses, patients with a PSA decrease of at least 10% at c1w4  or the secondary endpoint (∆%TV50 at w16). Thus, no prognostic information can be derived from ∆%PSA assessments during this early period.However, beginning in week four after the first treatment, continued periodic ∆%PSA assessments revealed a significant correlation between the PSA change and BR and OS, respectively. At that time point, ∆%PSA thresholds of −30% and 25% were highly predictive of both BR at w16, as defined by the PCWG3 criteria , as wellA PSA increase of at least 25% after four weeks was especially prognostic of both a shorter OS (3.5 times) and biochemical progression at w16 (specificity 100%), as PSA flare-up (PSA increase of at least 25% succeeded by PSA decrease at w16), occurring, e.g., in antihormonal treatment , was notA PSA decrease threshold of −30% after four weeks was predictive for the biochemical response and a significantly longer median OS (27.8 vs. 12.0 months). This more lenient, a priori defined threshold for PSA decrease was evaluated in accordance with data from other recent studies ,22,23. ICorresponding to that idea, we also retrospectively performed exploratory ROC analyses to ascertain PSA changes at week four, with the optimal discriminatory power for BR at week 16. In-line with our previous observations, these analyses provided even lower PSA thresholds, as PSA changes of more than ±10% were most indicative of both the future biochemical response and progression, respectively, while also being significantly associated with the corresponding differences in the OS. However, considering the high variability of the PSA levels we observed in the first weeks after the first treatment cycle, the reliability of such low thresholds has to be assessed very carefully and needs to be validated in future prospective studies.This study had some limitations. Despite the prospective nature of our serial short-interval PSA assessment, PSMA-RLT itself was still performed under clinical routine conditions. Moreover, besides the relatively small sample size, the results may be biased by the heterogeneity of previous and, especially, further therapies. For instance, treatment was primarily continued in responsive patients, demonstrated, for example, by the significant correlation between the OS and the number of administered cycles. In addition, despite the concordance of the biochemical and PET-based quantified imaging responses, an established assessment system integrating, e.g., PCWG criteria and PET is still warranted. Moreover, due to logistical reasons , the PSA at week three of each cycle could not be evaluated systematically.This study shows that PSA changes in patients with mCRPC as early as four weeks after the first administration of PSMA-RLT are predictive of both long-term biochemical and PET imaging responses, as well as overall survival. At this early stage, a PSA decrease of at least 30% was found to be more suitable to detect future responders, as well as a longer overall survival compared to the threshold of 50% proposed by the PCWG for long-term biochemical evaluation. An early increase in PSA (+25%) is a strong predictor for biochemical progression and shorter overall survival. Larger prospective studies are warranted to validate these findings and provide criteria for the early assessment of PSMA-RLT, including even more lenient thresholds, as suggested by our explorative retrospective ROC analysis.


Portulaca oleracea is a well-known species for traditional medicine and food homology in Taiwan. In traditional medicine, P. oleracea is also used to treat gastrointestinal disorders, liver inflammation, fever, severe inflammation, and headaches. We investigated antioxidant, anti-tyrosinase, and anti-α-glucosidase activities of various solvent extracts and major bioactive components from P. oleracea. Ethanol and acetone extracts showed potent DPPH, ABTS, and hydroxyl radical scavenging activities. Chloroform and n-hexane extracts displayed significant superoxide radical scavenging activity. Furthermore, ethyl acetate and acetone extracts of P. oleracea showed potent anti-tyrosinase and anti-α-glucosidase activities. Examined and compared to the various solvent extracts for their chemical compositions using HPLC analysis, we isolated seven major compounds and analyzed their antioxidant, anti-tyrosinase, and anti-α-glucosidase activities. Seven active compounds of P. oleracea, especially quercetin, rosmarinic acid, and kaempferol, exhibited obvious antioxidant, anti-tyrosinase, and anti-α-glucosidase activities. The molecular docking model and the hydrophilic interactive mode of tyrosinase and α-glucosidase revealed that active compounds might have a higher antagonistic effect than commonly inhibitors. Our result shows that the active solvent extracts and their components of P. oleracea have the potential as natural antioxidants, tyrosinase and α-glucosidase inhibitors. Our results suggest that the active solvent extracts of P. oleracea and their components have potential as natural antioxidants, tyrosinase and α-glucosidase inhibitors. Free radicals are substances produced after oxygen metabolism in the body, and are molecules or molecular fragments whose atoms contain unpaired electrons or contain other oxygen species, including molecular orbitals or simple reactive oxygen species, which are very active and can react strongly with any substance . An imbaNumerous studies have shown an inverse relationship between the dietary intake of antioxidant-rich foods and incidence of human disease . HoweverDisturbed carbohydrate metabolism contributes to harmful health problems such as obesity, oral disease and diabetes worldwide. Recent studies have shown that oxidative damage plays a major role in the occurrence of many degenerative and chronic diseases, and oxidative damage to pancreatic islet tissue can lead to diabetes . FurtherWhen it comes to enzymes related to oxidation, tyrosinase is also a common oxidase. Tyrosinase is a copper-containing enzyme mainly involved in the biosynthesis of melanin. It catalyzes melanin biosynthesis in human skin, and epidermal hyperpigmentation may lead to various skin diseases such as age spots and melasma ,11. In aPortulaca oleracea L. (PO) is an annual herb with reddish stems and alternate leaves from the Portulacaceae family. It is distributed in many parts of the world, especially in tropical and subtropical regions [P. oleracea has been used as a traditional remedy for a variety of ailments, including gastrointestinal disorders, respiratory disorders, liver inflammation, kidney and bladder ulcers, fever, insomnia, severe inflammation, headaches, etc. In our studies on the antioxidant and anti-inflammatory activities of plants in Taiwan, many species have been screened for these effects, and P. oleracea was found to be an active species. This report describes the evaluation of the antioxidant, anti-tyrosinase, and anti-α-glucosidase activities of various solvent extracts from P. oleracea and their main bioactive components. regions . P. olerp-Nitro-phenyl-α-D-glucopyranoside (p-NPG) and ferric chloride (FeCl3) were purchased from Alfa Aesar . Folin–Ciocalteu’s reagent, ethylenediaminetetraacetic acid (EDTA), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), tyrosinase, α-glucosidase, hydrogen peroxide solution, and ascorbic acid were supplied by Sigma-Aldrich . Potassium peroxodisulfate, sodium carbonate, disodium hydrogen phosphate, and potassium dihydrogen phosphate were obtained from the SHOWA Chemical Co. Ltd. . Phenazine methosulfate (PMS), 2,2-diphenyl-1-hydrazyl (DPPH), nitro blue tetrazolium (NBT), phloroglucinol, deoxyribose, and 2-thiobarbituric acid (TBA) were purchased from Tokyo Chemical Industry Co., Ltd. .Nicotinamide adenine dinucleotide (NADH), trichloroacetic acid (TCA), and butyl hydroxytoluene (BHT) were obtained from Acros Organics . P. oleracea was collected from Xichang Dist., Taipei City, Taiwan, in September 2020 and identified by Prof. J.-J. Chen. A voucher specimen was deposited in the Department of Pharmacy, National Yang Ming Chiao Tung University, Taipei, Taiwan. Samples were collected, air-dried, and ground to powder (0.5 to 2 mm). Preparation of P. oleracea extract was performed as described previously [The whole plant of eviously .P. oleracea was pulverized and extracted three times with MeOH (0.5 L each) for 3 days. The MeOH extract was concentrated under reduced pressure at 37 °C, and the residue (95 g) was partitioned between EtOAc and H2O (1:1) to provide the EtOAc-soluble fraction . Fraction A (32.3 g) was chromatographed on silica gel , eluting with n-hexane, gradually increasing the polarity with EtOAc to give 10 fractions: A1–A10. Fraction A3 (4.06 g) was further isolated by silica gel chromatography column (CC) and purified by preparative TLC  to afford kaempferol (4.40 mg), p-coumaric acid (6.22 mg), and caffeic acid (3.85 mg). Part (210 mg) of fraction A6 was further purified by preparative TLC  to afford quercetin (14.40 mg). Part (195 mg) of fraction A8 was further isolated by preparative TLC  to afford rosmarinic acid (4.52 mg). Part (180 mg) of fraction A9 was further purified by preparative TLC  to afford chlorogenic acid (5.52 mg).The dried whole plant (250 g) of ® 100 RP-18 Endcapped  purchased from Merck KGaA, Darmstadt, Germany. HPLC-PDA chromatographic fingerprints were obtained with an Agilent 1260 Infinity II HPLC instrument equipped with a 1260 Infinity II quaternary pump, a 1260 Infinity II degasser, a 1260 Infinity II vialsampler, a 1260 Infinity II column thermostat, a 1260 Infinity II diode array detector HS, and a PC with the Agilent ChemStation software. All of them were purchased from Agilent Technologies . Gradient separation using 0.2% acetic acid in water (v/v) (solvent A) and acetonitrile (solvent B) as mobile phase was as follows: 0–15 min, linear gradient from 95 to 88% A; 15–35 min, 88% A with isocratic elution; 35–55 min, 75% A with isocratic elution; 55–65 min, 60% A with isocratic elution; 65–90 min, 40% A with isocratic elution; 90–95 min, linear gradient from 40 to 0% A; 95–100 min, back to initial condition at 95% A; and 100–105 min, at 95% A. The flow rate was 1.0 mL/min, and the injection volume was 500 μL. Peaks were detected at 280 nm. Different compounds were identified by retention time. To guarantee peak purity, DAD acquisition from 200–650 nm was conducted to register UV-spectra. For the quantitative analysis of seven compounds in the extracts, aliquots of samples were dispersed in 10 mL of a methanol solution by sonication for 5 min. Following this, the samples were centrifuged for 15 min at 3500 rpm, and the supernatant extracts were filtered through 0.45 μm PTFE syringe filters . Quantification of seven components from Portulaca oleracea in each solvent extract was described as above.Reversed-phase separations were performed using a LiChrospherThe total phenolic content (TPC) of various solvent extracts was measured as described with Folin–Ciocalteu’s method with a slight modification .w/v) aluminum chloride solution and 0.1 mL of 0.1 mM potassium acetate solution. The mixture was kept at room temperature (25 °C) for 30 min. Then the maximum absorbance of the mixture was measured at 415 nm using a UV–VIS spectrophotometer. The TFC was expressed as milligram quercetin equivalent per gram of dry extract (mg QCE/g dry extract).The total flavonoid content (TFC) of different solvent extracts was determined with slight modification of the aluminum chloride colorimetric method described by Chang et al. . In brieThe DPPH radical scavenging assay was determined by the reference method .The ABTS cation radical scavenging assay was carried out using the reference method .2•−) scavenging activity was described earlier with slight changes [Superoxide anion radical (O changes . Briefly0 − A1)/A0 × 100, where A1 is the absorbance of the test sample and A0 is the absorbance of the control.Superoxide anion radical scavenging activity (%) = . After incubating at room temperature for 10 min, 20 μL of mushroom tyrosinase (1000 units/mL) was added, and the mixtures were incubated for another 30 min. Absorbance was measured at 490 nm and the percentage of anti-tyrosinase activity of sample was calculated from,0 − A1)/A0 × 100, where A1 is the absorbance of the test sample and A0 is the absorbance of the control. Arbutin was used as positive control.Inhibition (%) = (Ap-nitro-phenyl-α-D-glucopyranoside (p-NPG) (0.53 mM), and 20 μL of α-Glucosidase solution (1 units/mL) were mixed in 0.1 M potassium phosphate buffer (pH 6.8). After incubating at 37 °C for 20 min, 500 μL of sodium carbonate (0.1 M) was added in order to quench the reaction. The anti-α-glucosidase activity of the sample was determined by calculating the decrease in absorbance measured at 405 nm by the following equation,α-Glucosidase inhibitory activity was established in accordance with the described method  with sli0 − A1)/A0 × 100, where A1 is the absorbance of the test sample and A0 is the absorbance of the control. Acarbose was used as positive control.Inhibition (%) =  software. Primarily, the structure of the docked compound is energy minimized until the default derivative convergence criterion of 0.01 kcal/mol is met. The crystal structure (PDB: 3A4A or 2Y9X) is retrieved from the Protein Databank and hydrogen atoms are added to prepare the docked receptor. This protein structure is subsequently used in the CDocker program to dock the compound into the active site. Ten different docking poses are calculated and ranked by using the PLP score scoring function. The top-ranked docking solution is visually analyzed to determine the binding mode of the docked compound.U test. A probability of 0.05 or less was considered statistically significant. All the experiments were performed at least 3 times.All data are expressed as mean ± SD. Statistical analysis was carried out using the Mann–Whitney P. oleracea. n-hexane, chloroform, dichloromethane, ethyl acetate, acetone, methanol, and ethanol extracts from P. oleracea. The yields of different solvent extracts from P. oleracea ranged from 19.2 ± 1.25% (chloroform extract) to 38.2 ± 1.43% (methanol extract). We found TPC in all the different solvent extracts, with the ethanol extract having the highest amount of TPC (219.27 ± 4.13 mg/g), followed by acetone (34.61 ± 4.87 mg/g), ethyl acetate (30.91 ± 3.09 mg/g), dichloromethane (30.12 ± 2.79 mg/g), chloroform (28.99 ± 1.30 mg/g), methanol (27.80 ± 2.86 mg/g) and n-hexane (19.67 ± 3.33 mg/g).We investigated the total phenol contents (TPC), total flavonoid contents (TFC) and yields in different solvent extracts of n-hexane (36.41 ± 5.81 mg/g), and methanol (27.21 ± 0.74 mg/g). The TPC of the ethanolic extract was about 7 to 11 times higher than that of other solvent extracts, and the TFC was about 4 to 16 times higher, indicating that the suitable relative polarity of the solvent for extracting phenolics and flavonoids from P. oleracea is about 0.654.On the other hand, differences were found in TFC among different solvent extracts, of which ethanol extract contained the highest amount of TFC (437.38 ± 13.14 mg/g) followed by ethyl acetate (115.49 ± 8.85 mg/g), dichloromethane (107.75 ± 7.28 mg/g), acetone (89.65 ± 8.53 mg/g), chloroform (66.60 ± 5.81 mg/g), n-hexane, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, and methanol) from the whole plant of P. oleracea is first conducted in our study. This can provide a guide for the selection of appropriate solvents in TPC and TFC extraction applications.The comparative evaluation of the total phenolic content (TPC) and total flavonoid content (TFC) of various solvent extracts (50 = 41.33 ± 2.89 μg/mL) exhibited DPPH free-radical scavenging activity, which was comparable to BHT (IC50 = 39.88 ± 1.30 μg/mL). In addition, acetone (IC50 = 49.79 ± 3.34 μg/mL), methanol (IC50 = 55.36 ± 2.08 μg/mL) and ethyl acetate (IC50 = 64.24 ± 3.03 μg/mL) extracts also exhibited DPPH scavenging activity.DPPH is relatively stable in aqueous or ethanol solution. Antioxidants could interact with DPPH radical and transfer an electron or hydrogen atom to DPPH radical so as to neutralize free radicals . The DPP50 = 45.40 ± 6.06 μg/mL) and acetone (IC50 = 45.40 ± 6.06 μg/mL) extracts exhibited stronger ABTS radical scavenging activity, followed by ethanol (IC50 = 64.69 ± 3.97 μg/mL), methanol (IC50 = 69.85 ± 6.76 μg/mL), dichloromethane (IC50 = 79.03 ± 6.52 μg/mL), chloroform (IC50 = 97.91 ± 7.36 μg/mL) and n-hexane (IC50 = 296.79 ± 10.16 μg/mL).As shown in 50 = 6.80 ± 1.77 μg/mL) and n-hexane (IC50 = 14.36 ± 2.17 μg/mL) exhibited strong superoxide radical scavenging activities (50 > 400 μg/mL).The extracts of chloroform (ICtivities . However50 = 23.23 ± 0.28 μg/mL), followed by methanol (IC50 = 3n8.31 ± 0.54 μg/mL), ethyl acetate (IC50 = 68.60 ± 5.76 μg/mL), acetone (IC50 = 85.92 ± 5.99 μg/mL), dichloromethane (IC50 = 115.66 ± 17.68 μg/mL), chloroform (IC50 = 115.95 ± 7.01 μg/mL) and n-hexane (IC50 = 132.94 ± 12.85 μg/mL).The ethanol extract showed the strongest activity for scavenging hydroxyl radical  from the whole plant of P. oleracea is first proposed in our study. This can provide an indication for the selection of appropriate solvents in antioxidant extraction applications.Comparative evaluation of antioxidant assays  of various solvent extracts (50 = 396.20 ± 24.73 μg/mL) and acetone (IC50 = 412.22 ± 9.52 μg/mL) extracts of P. oleracea showed more potent anti-tyrosinase activity, followed by ethanol (IC50 = 497.24 ± 32.99 μg/mL), dichloromethane (IC50 = 545.29 ± 29.29 μg/mL), methanol (IC50 = 571.35 ± 25.73 μg/mL), n-hexane (IC50 = 621.43 ± 41.33 μg/mL), and chloroform (IC50 = 659.87 ± 24.89 μg/mL).Tyrosinase inhibitors are considered important for their potential applications in improving food quality and preventing pigmentation disorders and other melanin-related health problems in humans. As shown in P. oleracea had the highest anti-α-glucosidase activity (IC50 = 328.24 ± 52.67 μg/mL), followed by dichloromethane (IC50 = 416.34 ± 13.58 μg/mL), n-hexane alkane (IC50 = 418.31 ± 11.95 μg/mL), ethanol (IC50 = 440.31 ± 5.94 μg/mL), ethyl acetate (IC50 = 472.92 ± 2.45 μg/mL), chloroform (IC50 = 527.06 ± 25.64 μg/mL), and methanol (IC50 > 800 μg/mL). The acetone extract has comparable anti-α-glucosidase activity compared to the positive control acarbose (IC50 = 319.52 ± 24.46 μg/mL).Natural products with anti-α-glucosidase activity have received extensive attention for their potential use in the treatment of diabetes. As shown in n-hexane, chloroform, dichloromethane, ethyl acetate, acetone, ethanol, and methanol) from the whole plant of P. oleracea is first conducted in our study. This can provide an indication for the selection of appropriate solvents in natural tyrosinase and α-glucosidase inhibitors extraction applications.The comparative assessment of anti-tyrosinase and anti-α-glucosidase assays of various solvent extracts  of the standard solutions. The linear regression parameters of calibration curves, correlation coefficient, LOD, and LOQ were shown in R2 > 0.9993) between Y  and X (concentration of the standards) was achieved in the tested stage.The HPLC methods using reverse-phase column for the quantification of seven components isolated from P. oleracea by reverse-phase HPLC analyses. The contents of seven compounds in each solvent extract were shown in n-hexane > dichloromethane > acetone > ethyl acetate. The EtOH extract exhibited more of the seven active components than the other extracts. Among the seven active compounds in the organic solvent extract, kaempferol was the most abundant, followed by quercetin, trans-ferulic acid, chlorogenic acid, p-coumaric acid, caffeic acid, and rosmarinic acid . Furthermore, all seven compounds showed high ABTS radical scavenging activity, especially kaempferol (IC50 = 8.75 ± 0.90 μg/mL) and quercetin (IC50 = 11.46 ± 1.82 μg/mL). In the superoxide radical scavenging assay, quercetin (IC50 = 44.08 ± 3.07 μg/mL) showed the strongest activity, followed by caffeic acid (IC50 = 57.34 ± 7.44 μg/mL) and rosmarinic acid (IC50 = 61.62 ± 6.43 μg/mL). In addition, rosmarinic acid (IC50 = 6.56 ± 4.77 μg/mL), quercetin (IC50 = 9.75 ± 4.39 μg/mL) and kaempferol (IC50 = 11.46 ± 5.60 μg/mL) showed strong hydroxyl radical scavenging activity.Compounds isolated from P. oleracea exhibited good antioxidant activity against DPPH, ABTS, superoxide, and hydroxyl radical scavenging. The comparative evaluation of antioxidant assays of active compounds from the whole plant of P. oleracea is first conducted in our study.In conclusion, all seven compounds isolated from 50 = 10.12 ± 0.84 μg/mL), quercetin (IC50 = 11.20 ± 0.59 μg/mL), p-coumaric acid (IC50 = 22.20 ± 1.14 μg/mL) and chlorogenic acid (IC50 = 79.96 ± 13.00 μg/mL) have good inhibitory effect against tyrosinase, and their IC50 values are much lower than that of the positive control, arbutin (IC50 = 185 ± 10.56 μg/mL).Tyrosinase inhibition assay showed that rosmarinic acid (IC50 = 7.88 ± 2.07 μg/mL) and quercetin (IC50 = 8.11 ± 1.66 μg/mL) showed strong anti-α-glucosidase activities. In addition, caffeic acid (IC50 = 169.69 ± 11.27 μg/mL) and rosmarinic acid (IC50 = 185.36 ± 22.76 μg/mL) were more effective than the positive control, acarbose (IC50 = 331.24 ± 15.40 μg/mL).With further analysis of α-glucosidase inhibitory activity, kaempferol  of tyrosinase from mushroom complexed with tropolone, an inhibitor for tyrosinase, shows that the substrate binding site of tyrosinase is primarily formed by five α-helices as well as several loops, and it mainly contains hydrophilic residues such as six histidines, His 61, 85, 94, 259, 263 and 296, which interact with two copper ions . Once trTo understand how rosmarinic acid b might mAs the positive control, the docking model for arbutin a is alsoThe 3′,4′-dihydroxyphenylacetic acid moiety of rosmarinic acid and the phenolic group of arbutin both can locate at the tropolone binding position of tyrosinase’s active site to make the same π-π interaction with His 263 as tropolone. Furthermore, the phenolic group and the glucosyl group of arbutin make two H-bond interactions with His 85 and Asn 260. However, the 3′,4′-dihydroxyphenylacetic acid moiety of rosmarinic acid makes three H-bond interactions with Asn 260, His 263 and Val 283, so rosmarinic acid exerts more hydrophilic interaction than arbutin in the tropolone binding site. Additionally, the caffeic acid moiety of rosmarinic acid makes one H-bond interaction with Ala 323 and can exert weak interaction with the loop of the substrate binding pocket to enhance the binding affinity by its ethenyl group with His 85 and 3-hydroxyl group with Asn 81.Based on the results mentioned above, it is highly suggested that rosmarinic acid should have better antagonistic activity than arbutin.Ruminococcus obeum complexed with voglibose (PDB: 6C9X) reveals that its active site is formed by six β-sheets, two loops and one α-helice. Once voglibose enters the substrate binding pocket, it is surrounded by some hydrophilic and hydrophobic residues including Pro 75, Trp169, Asp 197, Ile 198, Ile 234, Trp 305, Asp 307, Arg 404, Trp 417, Asp 420, Phe 453 and His 478. Voglibose mainly interacts with the substrate binding pocket by the significant H-bond interactions including when (1) the 1-hydroxymethyl and 2-hydroxyl group interact with Asp 197 by acting as the H-bond donors; (2) the 4-hydroxyl group contacts with Arg 404 by serving as the H-bond acceptor; (3) the 1-hydroxypropan-2yl-amino group makes H-bond interaction with Asp 420.The 3D crystal structure of α-glucosidase complexed with acarbose showed that it mainly contains numerous structural domains including N-terminal domain, the barrel domain in which the active site is located and C-terminal domain. The α-glucosidase substrate binding site is primarily formed by numerous β-sheets and several loops or α-helices. The crystal structure of α-glucosidase from Saccharomyces cerevisiae and to try to interpret how kaempferol might exert its antagonistic effect, the docking model of kaempferol was generated by the Discovery Studio 2019  CDocker modeling program. The crystal structure of α-glucosidase from Saccharomyces cerevisiae is not available now, so the crystal structure (PDB: 3A4A) of Saccharomyces cerevisiae containing 72% sequence homology with the α-glucosidase from Saccharomyces cerevisiae is usually used to perform the docking study and also employed in this study [Ruminococcus obeum but it is deep and narrow. In the active site, its co-crystalized ligand, α-D-glucopyranose, locates deeply in the substrate binding pocket and makes three essential H-bond interactions including when (1) the 2-hydroxyl group interacts with Glu 277 and Asp 352 by acting as the H-bond donor; (2) the 3-hydroxyl group makes H-bond contact with Asp 352 by acting as the H-bond donor, and also serves as the H-bond acceptor to interact with Arg 213; (3) the 4-hydroxyl group acts as the H-bond donor to interact with His 351 and Asp 352.To further study the interaction between kaempferol b and theis study . In the As shown in As the positive control, the docking model for acarbose a is alsoSaccharomyces cerevisiae complexed with kaempferol or acarbose, further research is needed to further establish the nature of their respective interaction.Unlike acarbose, kaempferol can reside in the middle of the substrate binding pocket and occupy the whole pocket. Especially when kaempferol makes four H-bond interactions with the residues on different secondary structures in the pocket to allow it to stay long enough for exerting its antagonistic effect. For acarbose, its A ring can locate at the similar position as the A ring of kaempferol but its D ring partially protrudes out of the substrate binding pocket. The binding mode of acarbose shows that acarbose highly replies on its A ring to make contact with the residues nearby the substrate binding site. However, the B and C rings of acarbose exert only one hydrophilic interaction. More importantly, the B and C rings of acarbose are surrounded by the hydrophobic residues including Tyr 158, Phe 159 and Phe 178, so their numerous hydroxyl moieties might not be able to exhibit significant hydrophobic interaction as the A ring of kaempferol. Furthermore, the D ring of acarbose protrudes out of the pocket and exerts its interactions with the residues outside the pocket. These interactions out of the substrate binding pocket might not contribute significant binding affinity to the antagonistic effect. All the results mentioned above highly indicate that kaempferol should be able to exert more antagonistic effect than acarbose. However, to generate the crystal structure of the α-glucosidase from Agaricus bisporus tyrosinase could be detected. Furthermore, quercetin was bound with ASP 69, ARG 442, GLN 279, and GLN353 through conventional hydrogen bonds, while other interactions  were also observed with GLU 277, GLU411, TYR 72, and Val 216. These allowed quercetin and protein to form a stable complex.In addition, it was found that quercetin exhibited a strong inhibitory effect on both of the tyrosinase and α-glucosidase. As shown in A. bisporus tyrosinase and S. cerevisiae α-glucosidase. This indicated that these compounds may deserve further investigation as natural tyrosinase and α-glucosidase inhibitors.According to the above data, the docking scores of rosmarinic acid, kaempferol, and quercetin were higher than those of arbutin and acarbose, indicating their better binding capability. In this study, the active ingredients, rosmarinic acid, kaempferol and quercetin, possessed not only anti-tyrosinase and anti-α-glucosidase activity, but also the better binding potential with the active sites of P. oleracea in an effort to evaluate these different metabolites. We found that various metabolites have different degrees of biological activity due to differences in solvent polarity.Several methods have been developed and utilized to extract natural products, i.e., plants, fungi and herbs, as an alternative to modern medicines. The most traditional method applied for many years is to boil or make a decoction with water, which is a very simple and economical procedure. Today, studies based on medicinal plants have been published and many studies are ongoing as many metabolites with health benefits are found in natural products. More recently, in more advanced studies, organic solvents have been used to obtain natural product extracts including various metabolites depending on the polarity and property of the components of interest . Other f3+) complex to ferrous iron (Fe2+) complex by antioxidants present in the samples [P. oleracea showed high antioxidant activities among all solvent extracts via DPPH and hydroxyl radical scavenging assays. The differences in antioxidant capacities of the extracts may be owing to the different extents of TPC or the composition of antioxidant compounds in the extracts.DPPH and ABTS radical scavenging assays have been widely utilized to assess the antioxidant activities of natural components. Both assays are mainly related to the proton radical scavenging or hydrogen donating ability of the target compound . Superox samples . In our n-hexane, chloroform, dichloromethane, EtOAc, acetone, EtOH, and MeOH) from the whole plant of P. oleracea is first mentioned in this study. This can provide a guide for the selection of appropriate solvents in TPC, TFC, and antioxidant extraction applications. According to the antioxidant data, rosmarinic acid and quercetin displayed potent antioxidant properties. In addition, the contents of rosmarinic acid and quercetin in the ethanolic extract were the highest among all solvent extracts. This was consistent with the result that the ethanolic extract has high antioxidant activity.The comparative evaluation of the total phenolic content (TPC), total flavonoid content (TFC), and antioxidant assays  of various solvent extracts  and relatively high total flavonoid contents (TFC). The overall antioxidant capacity of the ethanolic extract of P. oleracea was superior to that of the other solvent extracts, consistent with the results of TPC and TFC assay. Furthermore, the acetone extract of P. oleracea showed the highest anti-α-glucosidase activity among all solvent extracts. EtOAc and acetone extracts of P. oleracea showed more potent anti-tyrosinase activity than other solvent extracts.Various solvent extracts of P. oleracea were quantified by HPLC and identified as chlorogenic acid (1), p-coumaric acid (2), caffeic acid (3), trans-ferulic acid (4), quercetin (5), rosmarinic acid (6), and kaempferol (7). Moreover, the comparative evaluation for the identification and quantification of seven active components of different solvent extracts  from the whole plant of P. oleracea by HPLC analysis is first conducted in our study.Seven active compounds from p-coumaric acid had a potent inhibitory effect against tyrosinase. Further molecular model docking analysis also confirmed their binding sites and binding abilities.Biological activity analysis showed that these seven compounds had potent antioxidant activity in general on the scavenging of DPPH, ABTS, superoxide and hydroxyl radicals, especially rosmarinic acid, kaempferol and quercetin. Kaempferol and quercetin showed strong anti-α-glucosidase activity. Rosmarinic acid, quercetin, and P. oleracea affects total phenolic and flavonoid contents, antioxidant activities, and bioactive component levels. Ethanol is the most suitable solvent for extracting the effective components of P. oleracea among all solvents. The ethanol extract and its active components  of P. oleracea can be used as natural antioxidant, anti-tyrosinase, and anti-α-glucosidase agents.In conclusion, this study demonstrated that extraction solvent for