Essential phospholipids (EPL) have hepatoprotective effects across many liver diseases/conditions. The impact of EPL on hepatocyte function in vitro was investigated.Effects of noncytotoxic concentrations of EPL (0.1 and 0.25 mg/ml), and its constituents, polyenylphosphatidylcholine (PPC) and phosphatidylinositol (PI) (both at 0.1 and 1 mg/ml), on membrane fluidity, apoptosis and extracellular transport versus controls were investigated in human hepatocyte cell lines . Significantly increased membrane fluidity occurred with all 3 phospholipids (PLs) in HepG2 cultures, and with PI (1 mg/ml) in steatotic HepaRG cells. Significantly decreased tamoxifen-induced apoptosis was observed in HepG2 cells with EPL, PPC and PI. Breast cancer resistance protein (BCRP) activity was significantly increased by EPL and PI in HepG2 cells. Multidrug resistance-associated protein 2 (MRP-2) activity was unaffected by any PL in HepG2 cells, and significantly increased by EPL, PI and PPC (1 mg/ml) in HepaRG cells, and by PI (1 mg/ml) in steatotic HepaRG cells. Bile salt export protein (BSEP) activity in HepG2 cells and steatotic HepaRG cells was significantly increased by EPL (0.25 mg/ml), and PPC (both concentrations), but not by PI. The PLs had no effects on HepaRG cell BSEP activity. P-glycoprotein (P-GP) activity was significantly increased by all compounds in HepG2 cells. PI (1 mg/ml) significantly increased P-GP activity in HepaRG and steatotic HepaRG cells.EPL, PPC, and PI increased hepatocyte membrane fluidity, decreased apoptosis and increased hepatocellular export, all of which may improve liver function. These in-vitro investigations provide valuable insights into the mechanism of action of EPL.The online version contains supplementary material available at 10.1186/s12944-022-01698-8. Chronic liver diseases are a major world public health problem. One worldwide estimate shows that 844 million people have chronic liver disease, with a mortality rate of 2 million deaths per year . NonalcoOf the treatments studied for use in NAFLD, EPL has the most evidence of an hepatoprotective effect –14, and Some clinical evidence exists to support the adjunctive use of EPL in liver diseases. For example, meta-analyses of clinical trials with EPL in patients with NAFLD have demonstrated significant reductions in circulating liver enzymes and lipid profiles, together with significant increases in the rate of overall improvement in steatosis and the likelihood of having no disease, and reduced likelihood of having moderate disease when used in combination with antidiabetic therapy . In a raEssentiale®, an EPL preparation, has been in use since 1957 , and > 3.5 million units were prescribed between 2001–2013 . The lipid profile of Essentiale®  consists of phosphatidylcholine (61.94 [2.23]), lysophosphatidylcholine (16.18 [1.33]), phosphatidylethanolamine (PE) (4.85 [0.48]), PI (0.47 [0.22]), phosphatidylglycerol (0.31 [0.04]), phosphatidic acid (0.40 [0.06]), diacylglycerol (1.31 [0.38]) and triacylglycerol (13.50 [2.40]) . PreclinSeveral of the potential mechanisms of EPL in preclinical studies such as disruption of membrane fluidity , apoptosPrimary human hepatocytes are the gold standard for in-vitro evaluation of cellular mechanisms, but are not readily available . Thus, iDimethyl sulfoxide (DMSO), insulin, 1,6-diphenyl-1,3,5-hexatriene (DPH), stearic acid, oleic acid, tamoxifen, valspodar (PSC833), calcein-am, sulforhodamine 101, Ko143, MK571, and mitoxantrone were purchased from Sigma-Aldrich . Hydrocortisone hemisuccinate was obtained from Santa Cruz Biotechnology . Penicillin/streptomycin, fetal bovine serum (FBS) were purchased from Biochrom . 7-β-NBD-taurocholate was custom-synthesized as described by Schneider et al.  PPC and To obtain sufficiently high concentrations of EPL, PPC and PI in aqueous cell culture media, these PLs were incorporated into liposomes. Liposomes were prepared by dual centrifugation  as described previously . The mas5 cells/cm2. This cell line was routinely cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin.The HepG2 cell line was purchased from Sigma-Aldrich . For all experiments, HepG2 cells were seeded at a density of 1.4 × 105 cells/cm2 and incubated in Williams’ medium E supplemented with 5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 5 µg/ml insulin, 50 µM hydrocortisone hemisuccinate, and 1% DMSO.Fully differentiated HepaRG cells, obtained from Lonza , were cultured as previously described by Le Guillou et al. . BrieflyTo induce steatosis, HepaRG cells were treated for a 2 week-period with a mixture of stearic acid (150 µM) and oleic acid (150 µM). Stearic acid and oleic acid were dissolved in DMSO; the final concentration of DMSO was always maintained at 1% in the cultures.2 and 95% humidity. Cell culture medium was renewed every 2 or 3 days.All cell lines were cultured at 37 °C in an atmosphere of 5% COTo enable selection of 2 noncytotoxic concentrations of each PL preparation for evaluation of their impact on hepatocyte apoptosis and transport function, cytotoxicity of all compounds was evaluated in HepG2 and HepaRG cell lines using PrestoBlue® cell viability reagent . Living cells reduce the resazurin in the solution to resorufin, which is red in colour and highly fluorescent. The colour change is detected using fluorescence measurements and correlates with cell viability .The cell lines, HepG2 and HepaRG, were seeded and cultured as described above. The PLs of interest were added to these cultures via liposome preparations (see above) and the cultures were incubated for 48 h. The concentrations of PL preparations evaluated for cytotoxicity in HepG2 cells were: EPL: 0, 0.0075, 0.0375, 0.075, 0.375, 0.75, 3.75, 7.5 and 37.5 mg/ml; PPC: 0, 0.009, 0.045, 0.09, 0.45, 0.9, 4.5, 8.9 and 44.5 mg/ml; and PI: 0, 0.0073, 0.036, 0.073, 0.36, 0.73, 3.6, 7.3 and 36.3 mg/ml. For the HepaRG cell line, all PL preparations were evaluated at 0.01, 0.02, 0.1, 0.2, 1, 2, 10 and 20 mg/ml. Following incubation, a PrestoBlue cell viability assay was performed according to the manufacturer’s instructions. For this assay, the cell viability reagent was diluted in the culture medium. After incubation for 30 min at 37 °C in a cell culture incubator the fluorescence intensities  was detected using a Tecan Fluoroscan infinite F200 pro plate reader .As a PL bilayer membrane, the plasma cell membrane is anisotropic, in which lipid resistance to motion is different in different directions; rapid motions include lateral diffusion and rotational diffusion, whereas transverse diffusion is very slow . MoleculThis technique was used by Dudeja et al.  to deterCaspase-3 and caspase-7 are involved in apoptosis . The mecThe cell lines, HepG2, HepaRG and steatotic HepaRG, were seeded and cultured as described above. The PLs were added  to these cultures via liposome preparations (see above) to detect if they had an effect on apoptosis induction in these cells. The cultures were incubated with PLs for 48 h. To induce apoptosis, the cell lines in culture were treated with tamoxifen, in the absence and presence of PL preparations. Preliminary tests showed that HepaRG cells needed slightly higher tamoxifen concentrations to show a caspase-3/7 fluorescence signal compared with HepG2 cells (data not shown). Thus, HepG2 cells were incubated for 3 h with either 42 µM or 55 µM tamoxifen, and HepaRG and steatotic HepaRG were incubated for 4 h with 45 µM or 60 µM tamoxifen.6 cells/ml in a fluorescence-activated cell sorting (FACS) buffer  and a concentration of 500 nM caspase-3/-7 green detection reagent. The samples were then incubated for 30 min at 37 °C in an atmosphere of 5% CO2. During the final 5 min of this incubation, SYTOX™ AADvanced™ solution was added at a final concentration of 1 µM. Cells were then stored on ice until analysis by flow cytometry on a FACS Fortessa LSRII (Becton Dickinson) using a 488 nm excitation and collecting fluorescence emission with a 530/30 optical filter (FITC channel) for caspase-3/-7 green detection reagent and a 695/40 optical filter (PerCP-Cy5.5 channel) for SYTOX™ AADvanced™. Cells were pre-gated for size and exclusion of doublets by forward scatter and side scatter  to these cultures via liposome preparations (see above) to detect if they had an effect on hepatocyte transport function in these cells. The cultures were incubated with PLs for 48 h. For the detection of transporter activity, the cells were treated with a model fluorogenic substrate specific to each of the transporters evaluated , 45–47.For BCRP, MRP-2 and P-GP assays, the PL pre-treated cells were incubated with the appropriate model substrate at 37 °C for a specified time in the dark; BCRP: 20 µM mitoxantrone, 3 h; MRP-2: 1 µM sulforhodamine 101, 60 min on a temperature-controlled shaker; P-GP: 1 µM calcein-am, 30 min on a temperature-controlled shaker. These incubation times and substrate/inhibitor concentrations were selected following preliminary studies, conducted to determine which combinations resulted in the highest difference in fluorescence signal. Inhibitors of these transport proteins were included as positive controls. PL pre-treated cells were incubated with appropriate inhibitor; i.e., 20 µM KO143 for BCRP inhibition for 1 h before mitoxantrone addition 20 µM (HepG2 cells) or 50 µM (HepaRG and steatotic HepaRG cells) MK571 for MRP-2 inhibition for 30 min before sulforhodamine 101 addition; and 1 µM PSC833 for P-GP inhibition for 15 min before calcein-am addition. The cells were then washed twice with ice-cold PBS to remove the fluorescent molecules on the outside of the cells. The washed cells were lysed by adding pre-warmed 1% (v/v) Triton X-100 and incubating for 30 min at 37 °C. Fluorescence was measured using Tecan Fluoroscan Infinite 200 PRO plate reader . The excitation/emission wavelengths used were: BCRP 615 nm/670 nm; MRP-2 540 nm/ 590 nm; P-GP 485 nm/535 nm.Evaluation of BSEP activity was conducted as previously described . HepG2, All authors had access to the study data, reviewed drafts of the manuscript and provided approval of the final manuscript. The study was conducted in line with International Council for Harmonisation Good Laboratory Practice.No formal sample size was determined. All data were analysed using SAS® software version 9.4 or higher (SAS institute Inc. Cary NC USA). Descriptive statistics were performed  for each parameter. Data were displayed graphically.Each experiment was conducted between 1 and 4 times, within each experiment 2 to 6 replicates per treatment were performed. The mean values for the replicates in each experiment were calculated, then the mean values across the experiments were calculated from the means of the replicates. For each cell line evaluated and for each parameter assessed, comparisons were made between each treatment group  versus untreated controls (non-PL treated cells). No statistical comparisons were made between EPL, PPC and PI or between cell lines.Cytotoxicity was not statistically analysed. For anisotropy, BCRP, MRP-2, BSEP and P-GP activity, comparisons were made using analysis of variance (ANOVA) including treatment group as a fixed factor and a Dunnett adjustment for pairwise comparisons. For caspase-3/-7 results, comparisons were made using ANOVA including treatment group, tamoxifen levels, and treatment group*tamoxifen level interaction as fixed factors and a Tukey adjustment for pairwise comparison. A threshold of 5% was used to denote statistical significance for all pairwise comparisons.Addition of EPL to HepG2 cell line cultures at concentrations ≥ 0.375 mg/ml resulted in a marked and concentration-dependent reduction in cell viability Table . Cell viMean fluorescence of resorufin in the culture medium ranged from 101.0%–123.0% (EPL), 95.9%–129.1% (PPC), and 98.8%–123.1% of control values (untreated HepaRG cells) following incubation for 48 h with EPL, PPC, or PI, respectively. Thus, none of the evaluated concentrations (0.01–20 mg/ml) of EPL, PPC and PI showed cytotoxicity in the HepaRG cell line in culture  and –0.058  with 0.1 and 0.25 mg/ml EPL, respectively. PPC also significantly reduced anisotropy measurements, at both concentrations, in HepG2 cells; LS mean differences (95% CI) versus untreated cells were –0.030  and –0.048 . Membrane fluidity (decreased anisotropy) was also significantly increased in HepG2 cells by incubation with PI; LS mean differences (95% CI) versus untreated cells in anisotropy values were –0.041  and –0.068 . At 0.1 mg/ml, the largest decrease in anisotropy from untreated cells was seen with PI (68% decrease) and the lowest decrease was with PPC (50% decrease), and EPL resulted in a 63% decrease.Figure P < 0.01)  , P < 0.001 with 0.25 mg/ml EPL. PPC significantly reduced tamoxifen-induced apoptosis (55 µM) only at the highest concentration evaluated i.e., 1 mg/ml: LS mean difference (95% CI) –39.3 (–64.0 to –14.7), P < 0.001. PI addition to HepG2 cells significantly reduced tamoxifen-induced apoptosis (55 µM) at both concentrations evaluated; 0.1 mg/ml LS mean difference (95% CI) –26.5 (–51.1 to –1.8), P < 0.05; 1 mg/ml: –49.1 (–73.8 to –24.5), P < 0.001.Figure P < 0.01), but not by the lower PI concentration of 0.1 mg/ml. In the presence of tamoxifen at 55 µM, EPL reduced the percentage of dead cells at both concentrations evaluated, which was significant for 0.1 mg/ml EPL  but not for 0.25 mg/ml EPL . PPC significantly reduced tamoxifen-induced Sytox-positive cells (55 µM) at both PPC concentrations, i.e., 0.1 mg/ml , and 1 mg/ml . For tamoxifen at 55 µM, PI significantly reduced apoptosis at both 0.1 mg/ml , and 1 mg/ml .To confirm the effects of the PLs on tamoxifen-induced apoptosis, an evaluation was also conducted of Sytox-positive cells Fig. , as thesSupplementary Fig. SP < 0.05). EPL addition to HepaRG cells had no significant effects on tamoxifen-induced (45 µM) cell death. PPC addition to HepaRG cells at 0.1 mg/ml significantly increased the percentage of dead cells in the presence of 45 µM tamoxifen ; although PPC at 1 mg/ml had no effect. With PI, the percentage of dead cells in the presence of 45 µM tamoxifen , whereas 1 mg/ml PI had no effect on this endpoint. With tamoxifen at 60 µM in HepaRG cells, neither EPL nor PI at the concentrations evaluated had any effect on the percentage of dead cells. PPC addition to HepaRG cells at 0.1 mg/ml significantly decreased the percentage of dead cells induced by 60 µM tamoxifen ; whereas 1 mg/ml PPC had no effect on this endpoint.The effects of tamoxifen, EPL, PPC, and PI on the percentage of dead HepaRG cells are shown in Supplementary Fig. STamoxifen addition to steatotic HepaRG cells slightly increased apoptosis at both concentrations evaluated  in the presence of KO143, indicating BCRP inhibition. In HepG2 cell cultures, compared with untreated controls, EPL statistically significantly decreased mitoxantrone accumulation at both 0.1 mg/ml  and 0.25 mg/ml   and 1 mg/ml  versus untreated controls, whereas PPC addition to HepG2 cells had no significant effect on the BCRP activity at either concentration tested  intracellular concentration of mitoxantrone was 123.4  and 117.4  in HepaRG and steatotic HepaRG cells, respectively, in the presence of KO143, indicating BCRP inhibition. Addition of EPL, PPC, or PI had no significant effects on BCRP activity in vitro in either HepaRG cells  intracellular concentrations of mitoxantrone were 111.4  in the presence of MK571, indicating MRP-2 inhibition. There was no significant effect of EPL, PPC, or PI addition to HepG2 cell cultures on sulforhodamine 101 accumulation at the concentrations evaluated versus untreated controls  intracellular concentration of sulforhodamine 101 was 168.9  of untreated HepaRG controls, in the presence of MK571, indicating MRP-2 inhibition. In HepaRG cells, intracellular accumulation of sulforhodamine 101 was significantly reduced by EPL addition at both 0.1 mg/ml  and 0.25 mg/ml  versus untreated cells  versus untreated cells, whereas the 0.1 mg/ml concentration had no significant effect. PI addition to HepaRG cells also significantly increased MRP-2 activity in vitro at 0.1 mg/ml  and 1 mg/ml  versus untreated cells  intracellular concentration of sulforhodamine 101 was 141.0%  of untreated steatotic HepaRG controls. In steatotic HepaRG cells, EPL or PPC addition did not significantly impact MRP-2 activity compared with untreated controls   intracellular concentration of sulforhodamine 101 was 152.9% -taurocholate in the canaliculi of cultured cells was used to assess the impact of PL addition on the bile salt export; thus, increased concentrations of this substrate in the canaliculi demonstrate increased BSEP activity.P < 0.001)   . Increased P-GP activity is demonstrated by decreased intracellular concentrations of this substrate. As a positive control, the P-GP inhibitor PSC833 was added to each cell line in the absence of PL.n = 4 experiments) in the presence of PSC833, indicating P-GP inhibition. All 3 PL preparations added to HepG2 cells in vitro significantly increased P-GP-mediated transport at all concentrations evaluated compared with control . Following PPC addition, LS mean differences (95% CI) were: 0.1 mg/ml –20.1 (–30.2 to –10.0), P < 0.001; 1.0 mg/ml –47.2 (–57.3 to –37.1). For PI addition, LS mean differences (95% CI) were: 0.1 mg/ml –32.4 (–42.5 to –22.3), P < 0.001; 1.0 mg/ml –43.3 (–53.4 to –33.2).As a percentage of untreated HepG2 cells, the mean (SD) intracellular concentration of calcein-am was 140.17  of untreated HepaRG controls, in the presence of PSC833, indicating P-GP inhibition. Addition of either EPL or PPC at the concentrations evaluated had no significant effects on P-GP transport activity in HepaRG cells versus controls  compared with controls  intracellular concentration of calcein-am was 207.92%  of untreated steatotic HepaRG controls. In steatotic HepaRG cells, EPL and PPC at the concentrations evaluated had no significant effects on P-GP transport activity versus controls  compared with controls  intracellular concentration of calcein-am was 262.1%  across different human hepatocyte cell lines.More than 90% of orally administered EPL is absorbed within 24 h in animals and humans , 48, 49.Using anisotropy as an exploratory approach to evaluate membrane fluidity, EPL, PPC, and PI produced a marked increase in membrane fluidity in HepG2 cells versus untreated controls. No differences in membrane fluidity were seen with any PL preparation in HepaRG cells. Only PI at the highest concentration tested increased membrane fluidity in steatotic HepaRG cells. The increase in membrane fluidity appeared to be greater with PI versus EPL and PPC, although the data were variable, indicating that PI has a strong membrane fluidizing effect. Several preclinical studies with EPL reported restoration and strengthening of membrane structure and increased membrane fluidity . PLs areHistopathology  and biocAnother critical aspect of the liver is hepatocyte membrane transport function. Bile salts, cholesterol and PC are transported across the apical canalicular membrane of hepatocyte by ATP-binding cassette transporters. All of the transporters evaluated in the present investigations are members of the ATP-binding cassette transporter family . TranspoClear differences in the results between the different cell lines used in these investigations were seen. One reason for such differences might be that differentiated HepaRG cells exhibit different levels of expression of drug-metabolizing enzymes and drug transporters compared with the HepG2 cell line . FurtherNAFLD and its progression is very complex and involves numerous biochemical pathways, and only 3 potential targets have been explored in the current in-vitro studies. Two other pathways disrupted in NAFLD, which are also of potential interest as targets for therapy, are inflammation  and lipiThese investigations have several strengths and weaknesses. The major strength is that several human hepatocyte cell lines were used, which are directly applicable for extrapolation to human studies. Moreover, such in-vitro techniques are ideally suited for mechanistic work of compounds of interest. In contrast, all cell lines used were immortal cancer cell lines, which are unlikely to fully represent normal liver function. Although maximum, noncytotoxic concentrations of each PL investigated were evaluated, how these concentrations in vitro relate to in-vivo concentrations, particularly at the level of the hepatocyte are unknown.In summary, in-vitro investigations in HepG2, HepaRG, steatotic HepaRG to evaluate the effects of EPL, PPC and PI provided valuable insights into the mechanism of action of EPL, which has hepatoprotective effects in patients with NAFLD and other liver conditions. The in-vitro results demonstrated increased membrane fluidity, decreased apoptosis, and increased function of hepatocellular extracellular transporters involved in bile secretion, all of which may potentially improve liver function. This first investigation of EPL  in human hepatocyte cell lines is an important contribution to understanding the mechanism of action of EPL. Further in-vitro work on the effects of EPL on pro-inflammatory cytokines and lipid-metabolizing enzymes will also provide further insights on the hepatoprotective properties of these preparations.Additional file 1:Supplementary Table S1. Cytotoxicity of EPL, PPC and PI in HepaRG cell line. Supplementary Table S2. Statistical analyses of the effects of EPL, PPC and PI on anisotropy in the HepG2, HepaRG and steatotic HepaRG cell lines. Supplementary Table S3. Statistical analyses of the effects of EPL, PPC and PI on apoptosis in the HepG2 cell line. Supplementary Table S4. Statistical analyses of the effects of EPL, PPC and PI on apoptosis in the HepaRG cell line. Supplementary Table S5. Statistical analyses of the effects of EPL, PPC and PI on apoptosis in the steatotic HepaRG cell line. Supplementary Table S6. Statistical analyses of the effects of EPL, PPC and PI on hepatocellular transport protein activity in the HepG2 cell line. Supplementary Table S7. Statistical analyses of the effects of EPL, PPC and PI on hepatocellular transport protein activity in the HepaRG cell line. Supplementary Table S8. Statistical analyses of the effects of EPL, PPC and PI on hepatocellular transport protein activity in the steatotic HepaRG cell line. Supplementary Fig. S1. Gating strategy to evaluate caspase-3/-7 activity in apoptotic cells by flow cytometry. Supplementary Fig. S2. Effect of EPL, PPC and PI on anisotropy in the HepaRG and steatotic HepaRG cell lines. Supplementary Fig. S3. Effect of EPL, PPC and PI on apoptosis in the HepaRG cell line. Supplementary Fig. S4. Effect of EPL, PPC and PI on apoptosis in the steatotic HepaRG cell line. Supplementary Fig. S5. Effect of EPL, PPC and PI on hepatocellular transport protein activity in the HepaRG cell line. Supplementary Fig S6. Effect of EPL, PPC and PI on hepatocellular transport protein activity in the steatotic HepaRG cell line.


Their structures were determined on the basis of extensive spectroscopic methods, including NMR and HRESIMS, and by comparison with the literature. Compounds 1–7 were evaluated for their in vitro α-glucosidase inhibition and cytotoxicity against KB, Hep G2, and MCF7 cell lines. Among them, compound 1 exhibited the best activity against α-glucosidase and was superior to the positive control with an IC50 value of 2.59 μM. On the other hand, compound 1 showed moderate cytotoxicity toward KB, Hep G2, and MCF7 cell lines with the IC50 values of 23.5, 19.8, and 23.7 μM, respectively. These findings provided new evidence that the stem bark of M. fruticosum is a source of bioactive flavonoid derivatives that are highly valuable for medicinal development.Three new flavonoid derivatives, melodorones A–C ( Melodorumfruticosum Lour (Annonaceae) is a shrub with fragrant yellow flowers distributed in South East Asia, more specifically indigenous to Laos, Cambodia, Thailand, and Vietnam. This plant has been used as a mild cardiac stimulant, a tonic, and as a hematinic to resolve dizziness − . This was further supported by the 1H and 13C NMR spectral data, which displayed one oxymethine, one methoxy, one olefinic methine, two methylene, two methyl, six aromatic methine, and eight quaternary carbons (1H and 13C NMR) patterns of 2 were very similar to those of 1 except for the presence of a single bond between C-2 and C-3 in the heterocyclic ring C. This flavanone moiety was proven by the 1H NMR spectrum, which showed an ABX spin system at δH 5.62 , 3.31 , and 2.82 , corresponding to H-2, H-3ax, and H-3eq, respectively 25D −73.0) with the known compound (−)-butin ([α]26D −39.9) [2 was identified as a new flavanone and was named melodorone B.Compound  carbons . The speectively . The 13C3 was obtained as a colorless gum with [α]25D −26.8 . The molecular formula C20H20O4 was determined by its HREIMS, which displayed a molecular ion peak at m/z 369.1363 [M+HCOO]− . The 13C NMR spectrum (C 198.7 (C-4), 78.4 (C-2), and 42.8 (C-3), which are frequently observed in the flavanone series − ; 1H NMR  and 13C NMR  see Melodorone A (2). Colorless gum. [α]25D −73.0 ; UV (CH3OH) λmax (log ε) 275 (4.38), 295 (4.07), 356 (3.86) nm; IR (ATR) νmax 3393, 2977, 1635, 1574, 1497, 1162, 1109 cm−1. HRESIMS m/z 399.1448 [M+HCOO]− ; 1H NMR  and 13C NMR  see Melodorone B (3). Colorless gum. [α]25D −26.8 ; UV (CH3OH) λmax (log ε) 275 (4.38), 295 (4.07), 356 (3.86) nm; IR (ATR) νmax 3392, 2977, 1634, 1450, 1217, 1122 cm−1. HRESIMS m/z 369.1363 [M+HCOO]− ; 1H NMR  and 13C NMR  see Melodorone C (1–7 and acarbose were prepared by dissolving in DMSO (400 mg/mL). Sodium phosphate buffer  was used to dissolve the α-glucosidase (0.4 U/mL) and substrate (2.5 mM pNPG). The substrate (40 μL) was added to the reaction mixture after the inhibitor (50 μL) was preincubated with α-glucosidase in 96-well plates at 37 °C for 10 min. A mixture without enzyme, sample compound, and acarbose served as blank, while the control contained only DMSO, enzyme, and substrate. The enzymatic reaction was carried out at 37 °C for 30 min and stopped by adding 0.2 M Na2CO3 (130 μL). Absorbance at 410 nm to measure enzyme activity was recorded on a BIOTEK reader. The assays were conducted in triplicates, with acarbose serving as a positive control. The IC50 values were calculated graphically using inhibition curves.The α-glucosidase inhibition of all isolates was carried out according to a method adapted from a previous report . Serial 1–7 and ellipticine against the growth of human epidermoid carcinoma (KB), human hepatocellular carcinoma (Hep G2), and human breast adenocarcinoma (MCF-7) cell lines was performed according to a previous procedure [2 environment. The tested compounds were added at concentrations ranging from 0.5 to 128 μg/mL by dissolving in DMSO (20 mg/mL) and incubated for a further 72 h in the identical condition. After the treatment, each well was filled with an MTT solution  of phosphate buffer, which was left to stand for 4 h until intracellular purple formazan crystals appeared. The MTT was removed and a 100 μL DMSO solution was added. The blank contained only a medium without any cells and MTT, as well as the solubilizing solution. The solution’s optical density (OD) at 540 nm was recorded on a BIOTEK reader. All experiments were carried out in triplicate for three independent experiments. IC50 values were computed graphically using inhibition curves.The cytotoxic evaluation of rocedure . Elliptirocedure ,34,35. TM. fruticosum afforded the isolation of three unprecedented flavonoid derivatives (1–3), including one new flavone (1) and two new flavanones (2 and 3), along with four known compounds (4–7). All isolated were obtained from M. fruticosum for the first time. Compounds 1–3 and 5 exhibited highly potent inhibition against α-glucosidase and were superior to the positive agent. Furthermore, compounds 1–5 selectively showed in vitro cytotoxicity against KB, Hep G2, and MCF7 cell lines. The results of this study reveal that M. fruticosum stem bark is a highly useful source of bioactive flavonoid derivatives that should be explored further in the medical field.The chemical investigation of the stem bark of


However, low electronic conductivity and sluggish reaction kinetics have limited its ability for lithium ion storage. To improve this, the phase engineering approach is used to fabricate orthorhombic/monoclinic MoO3 (α/h-MoO3) homojunctions. The α/h-MoO3 is found to have excessive hetero-phase interface. This not only creates more active sites in the MoO3 for Li+ storage, it regulates local coordination environment and electronic structure, thus inducing a built-in electric field for boosting electron/ion transport. In using α/h-MoO3, higher capacity (1094 mAh g−1 at 0.1 A g−1) and rate performance (406 mAh g−1 at 5.0 A g−1) are obtained than when using only the single phase h-MoO3 or α-MoO3. This work provides an option to use α/h-MoO3 hetero-phase homojunction in LIBs.With high theoretical specific capacity, the low-cost MoO Fortunately, phase engineering is an effective and powerful strategy to modulate the physicochemical properties and functionalities by tailoring transformation of different phases in nanomaterials [2 homojunctions, and found that these heterojunctions would redistribute the interfacial charges and accelerated the ion diffusion, resulting in better performance of the sodium/potassium storage than those using cubic or orthorhombic CoSe2 [3 usually exists in two structural phases, namely orthorhombic (α-MoO3), and monoclinic (h-MoO3). It is highly feasible to make and use hetero-phase homojunctions for MoO3 as electrode materials for electrochemical performance.Owing to the high energy density, flexibility in design and long cyclic life, lithium-ion batteries (LIBs) are playing essential roles in our modern society, from domestic electronic products to electric vehicles (EVs) and aerospace carriers ,2. Nonet of LIBs . It is h of LIBs ,5. Among h g−1)) ,7,8,9. Hformance ,11. In tctrolyte ,13,14, ictrolyte ,16,17, actrolyte ,19. To s of MoO3 ,21,22. Taterials ,26,27,28ic CoSe2 . It is e3 (α/h-MoO3) homojunctions were fabricated via partial phase transformation with a facile heat treatment. Compared with heterojunctions, the homojunction with interfaces exhibit stronger electron coupling due to their similar physicochemical properties and lattice matching. At the unique homojunction interfaces, the unbalanced charge distribution would also create an internal electric field, which facilitates the interfacial charge transport, accelerates ion diffusion, increases the reactive sites and improves the electrochemical surface reaction kinetics. The work demonstrates that α/h-MoO3 electrodes could delivered an extraordinarily high capacity of 1094 mAh g−1 at 100 mA g−1 with outstanding cyclic stability and a capacity of 462 mAh g−1 remaining after 300 cycles of operation at 1.0 A g−1. This work confirms that, via constructing the hetero-phase homojunction in MoO3, Li+, storage kinetics can be largely enhanced.In this work, orthorhombic/monoclinic MoO3), orthorhombic/monoclinic MoO3 (α/h-MoO3) and orthorhombic MoO3 (α-MoO3) samples were prepared as follows. For synthesis of a typical sample, 2.0 g of Ammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O) was dispersed into 10 mL deionized water before 1 mL HNO3 was added into the solution under stirring for 30 min. The mixture was transferred to a 50 mL autoclave and heated at 180 °C for 12 h. The product was collected and washed with deionized water and ethanol before being dried in an oven at 60 °C overnight. This product was named Sample A. Subsequently, Sample A was heat-treated in a tube furnace at 400 °C for 30 min to produce Sample B. Thereafter, Sample B was further treated at 450 °C for 30 min to obtain Sample C.The monoclinic . The morphology and microstructure of the samples were examined by the scanning electron microscope . The Raman spectra were performed by using a DXR Raman system spectrometer  with a 532 nm laser.3, super conductive carbon black  and polyvinylidene fluorid (PVDF) binder in N-methyl-2-pyrrolidone (NMP) with a weight ratio of 8:1:1. After stirring for 1 h, the homogeneous slurry was coated onto a copper foil. The mass loading of the active material was estimated to be about 1.0 mg cm−2. The CR2032 coin-type cells using the fabricated product were assembled in an argon-filled glovebox (oxygen/moisture concentrations < 0.01 ppm). Celgard 2400 porous polypropylene membrane was used as the separator. The electrolyte is 1 M LiPF6 in carbonate (EC)/dimethyl carbonate (DMC) with the volume ratio of 1:1.Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were conducted on a Gamry (Interface 1000 E Potentiostat) electrochemical workstation. The galvanostatic discharge and charge profiles were measured by the NEWARE-CT-4008 battery tester. The working electrodes were prepared by mixing the MoO3 with different phases. As shown in 3 (Sample A), α/h-MoO3 (Sample B), and α-MoO3 (Sample C). The characteristic peaks at 9.6°, 19.4°, 25.7°, 29.3°, 35.4°, 45.5°, and 69° can be assigned to (100), (200), (210), (300), (310), (410), and (524) of hexagonal MoO3 (PDF#21-0569) [3  [3 under air atmosphere partially transfers to α/h-MoO3 at 400 °C. The subsequent heat treatment of α/h-MoO3 leads to the formation of thermodynamically stable α-MoO3 pillar at 450 °C as confirmed in the bottom pattern in −1, 240 cm−1, 681 cm−1, 881 cm−1, and 969 cm−1 are attributed to h-MoO3 [3 shows the characteristic bands at 148 cm−1, 235 cm−1, 281 cm−1, 328 cm−1, 658 cm−1, 813 cm−1, and 990 cm−1 [3 indicating that the hexagonal and orthorhombic crystal phases coexist, which is consistent with the XRD results  and Raman scattering (RS) spectroscopy were used to investigate the phase structures and purity of MoO21-0569) ,30. No o3 top of a, indica35-0609) . It is e0), and 5 of hexag990 cm−1 . Notably results a. The ch3 samples were investigated by SEM. The homogeneous hexagonal crystals were clearly observed and shown in 3, the surface of α/h-MoO3 and α-MoO3 appears rough, which is attributed to the evolution of gases during heat treatment.The morphological characteristics of the MoO+ storage behavior of MoO3 electrodes were systematically evaluated in a coin cell configuration in a potential window of 0.01 V to 3.0 V. 3, h-MoO3 and α-MoO3. Their initial three cyclic voltammetry (CV) curves at a scan rate of 0.1 mV s−1 are shown in + insertion into MoO3 (MoO3 + xLi+ + xe−xMoO3); the irreversible peak at 0.7 V is due to the decomposition of electrolytes and the formation of the SEI layer, which disappears in the following cycles and thus causes part of the observed irreversible capacity during the first cycle [xMoO3 + (6 − x) Li+ + (6 − x) e−2O) [The List cycle . On the  + Li2O) . 2O to LixMoO3 during delithiation process, respectively [3 is formed [3 electrode during the repeated Li+ insertion and extraction. Compared to the CV profiles of h-MoO3  at high current density 5.0 A g−1. 3, h-MoO3 and α-MoO3 electrodes at various current rates. It is obvious that the charge/discharge profiles of the α/h-MoO3 electrode displays much lower polarization at varied current densities [+ transport [3 electrode. Subsequently, the long-term cycling stabilities of different electrodes was examined at 1.0 A g−1. It is obvious that the α/h-MoO3 electrode delivered a reversible capacity of 462 mAh g−1 over 300 cycles and satisfying capacity retention of 74%, which is higher than those of h-MoO3 (339 mAh g−1) and α-MoO3 (409 mAh g−1). The above results evidentially demonstrate that the homojunctions effectively alleviate the pulverization and minimize the volume change to maintain structural integrity during the Li+ insertion/extraction process [In the anodic scans, the observed two oxidation peaks around 1.2 V, 1.7 V, and 2.6 V were attributed to the reversible reaction from Mo and Liectively . The secs formed . Meanwhif h-MoO3 b, there f α-MoO3 c displayd α-MoO3 e,f. The V cycles . In succensities , illustrransport ,34, resu process .3 electrodes, the CV curves at various scan rates of 0.1–5.0 mV s−1 , a semicircle at high frequency associated with the charge transfer resistance (Rct), and at low frequency line related to Li+ diffusion (Zω) [3 electrode shows smaller diameter and larger slope, suggesting lower charge-transfer resistance and faster Li+ diffusion in the electrode, guaranteeing a much better rate performance of the α/h-MoO3 electrode. Meanwhile, the Warburg factor (σ) was obtained from the potting of Z′ vs. ω−1/2  . It is as. ω−1/2 i. It is 3 hetero-phase homojunction was prepared by a partial crystal phase transition. The product exhibited higher rate capability and better cyclic stability. The homojunctions not only showed the enhanced electronic conductivity and Li+ diffusion, but also provided more intrinsic active sites and accommodate the volume expansion during discharge/charge. All these simultaneously enhanced the charge storage capability for higher capacity and superior rate performance. Benefiting from these merits, the α/h-MoO3 electrode delivered superior electrochemical performance. Specifically, an excellent rate ability (742 mAh g−1 at 1.0 A g−1) was recorded in this work. Considering the prominent electrochemical performance of α/h-MoO3 homojunctions, in situ construction crystal phase homojunctions strategy on polymorph materials provides an alternative solution to the energy storage and related fields.In summary, the α/h-MoO


Further, Cor inhibited the NLRP3 inflammasome activation and pyroptosis in RAW264.7 and MLE-12 cells induced by LPS/nigericin and that in IEC-6 cells induced by nigericin, indicating an amelioration of Cor in II/R-induced intestinal and lung injury via inhibiting NLRP3 inflammasome activation and pyroptosis. Thus, Cor might be a potential therapeutic agent for II/R-induced inflammation and tissue injury.Intestinal ischemia reperfusion (II/R) is a clinical emergency that frequently occurs in a variety of clinical conditions. Severe intestinal injury results in the release of cytotoxic substances and inflammatory mediators which can activate local inflammatory response and bacterial translocation. This triggers multi-organ failure, including lung injury, which is a common complication of II/R injury and contributes to the high mortality rate. Corilagin (Cor) is a natural ellagitannin found in a variety of plants. It has many biological and pharmacological properties, including antioxidant, anti-inflammatory and anti-apoptosis activities. However, no studies have evaluated the effects and molecular mechanisms of Cor in alleviating II/R-induced intestinal and lung damage. In this study, Cor was found to significantly alleviate II/R-induced pathological damage, inflammatory response, oxidative stress, NLRP3 inflammasome activation, and pyroptosis in intestinal and lung tissues both  Ischemia-reperfusion (I/R) injury occurs when the blood supply is temporarily interrupted and leads to organ damage or even organ failure. Intestinal I/R (II/R) injury is a life-threatening surgical emergency that usually occurs during clinical localized interventions or as a severe complication of some systematic diseases, such as severe infection, traumatic shock, and cardiopulmonary disease . The patIncreasing evidence has shown that NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome plays crucial roles in the development of various diseases by initiating inflammatory responses . When unCorilagin Cor, , a naturin vivo and in vitro experiments, respectively. Anti-NLRP3 (19771-1-AP), anti-Caspase1 P20 (22915-1-AP), anti-ASC (10500-1-AP), anti-Bax (50599-2-Ig), anti-Bcl-2 (12789-1-AP), anti-Caspase-3 (19677-1-AP) and anti-β-actin (20536-1-AP) were obtained from Proteintech Group . Anti-GSDMD (ab219800) was brought from Abcam . Cell counting kit-8 , Lipopolysaccharides  and Nigericin (28643-80-3) were bought from MedChemExpress . Reagent kits for detecting superoxide dismutase , glutathione , malondialdehyde , lactate dehydrogenase  and myeloperoxidase  were obtained from Nanjing Jiancheng Institute of Biotechnology . ELISA kits for measurement of tumor necrosis factor alpha  and interleukin-1β  were supplied by Beyotime Biotechnology . Dulbecco’s minimum essential medium , nonessential amino acids, Fetal bovine serum  and glutamine were purchased from Gibco . Protein extraction kits and bicinchoninic acid protein assay kits  were obtained from TransGen Biotech . Rat small intestinal crypt epithelial cell line (IEC6), mouse macrophage cell line (RAW264.7) and mouse lung epithelial cell line (MLE-12) were purchased from American Type Culture Collection (ATCC). FITC (GB22403), DAPI (G1012), Hoechst 33342/PI (CA1120), triton-100X (G5060) and bovine serum albumin  were provided by Serviobio .Corilagin  was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. , and prepared at appropriate concentrations in normal saline and serum/glucose-free DMEM medium for Male C57BL/6J mice  were provided by Experimental Animal Center of Dalian Medical University (Certificate of Conformity: No. SYXK (Liao) 2018-0001). The experimental protocol was approved by the Animal Care and Ethics Committee of Dalian Medical University  and performed according to the Principles for the Care and Use of Laboratory Animals in Research . All mice were housed for 14 days under optimal conditions of 25°C, 50% humidity, 12/12 day-night cycle and free access to food and water prior to the experiment.The mouse II/R model was established as previously described . Mice wen = 6). 1) Sham group: normal saline was administered via intragastric gavage for 3 days before sham surgery; 2) II/R group: animals were subjected II/R model after being given a normal saline via intragastric gavage for three consecutive days; 3) II/R + Cor (20 mg/kg) group: animals were pretreated with Cor  once daily for 3 days before the II/R surgery; 4) II/R + Cor (40 mg/kg) group: animals were pretreated with Cor  once daily for 3 days before the II/R surgery; and 5) II/R + Cor (80 mg/kg) group: animals were pretreated with Cor  once daily for 3 days before the II/R surgery. After II/R reperfusion, executed the mice, and collected intestinal and lung tissues. Parts of the isolated intestinal and lung tissues were fixed in 4% paraformaldehyde solution for 24 h, and then used for histological analysis. The remaining parts of intestinal and lung tissues were flushed rapidly with ice-cold normal saline and immediately stored in liquid nitrogen for biochemical analysis and Western blot analysis later as previously described  to mimic II/R injury in vitro, cells were incubated in serum/glucose-free DMEM in a 5% CO2, 1% O2 and 94% N2 hypoxic chamber at 37°C to simulate ischemia by deprivation of oxygen and glucose. After incubation for 3 and 1 h, cells were cultured in normal DMEM supplemented with 10% FBS under normoxic conditions at 37°C for 12 and 1 h respectively for MLE-12 and IEC-6 cells to simulate reperfusion. RAW264.7 cells were treated with LPS (100 ng/ml) for 3 h and followed with Nigericin (10 μM) for another 4 h to active NLRP3 as previously described . The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were closed with 5% skim milk and incubated with anti-NLRP3, anti-Caspase1 P20, anti-ASC, anti-GSDMD, anti-Bax, anti-Bcl-2, anti-cleaved (cl)-Caspase 3, GAPDH and anti-β-actin overnight at 4°C at 1:1000 ratio. Then, the bolts were incubated with horseradish peroxidase-conjugated secondary antibodies  for 2 h at 37°C. The expressions of proteins were normalized to β-actin or GAPDH. Using ImageJ to count the grayscale values of protein bands. Calculate the ratio of the destination bands to β-actin or GAPDH.in vitro experiments, and data analyses were conducted according to a single-blind study design. One-way ANOVA analysis of variance with Tukey’s multiple comparisons test as post-test were used to determine statistical significance of results. All experiments were repeated at least three times and p < 0.05 was considered as statistically significant.The animal experiments, p < 0.01, Hematoxylin-eosin (H&E) staining analysis showed that the jejunal mucosal epithelium in the sham group was intact with clearly visible goblet cells (yellow arrow). Compared with the sham mice, II/R induced significant intestinal morphological changes with massive inflammatory cell infiltration (black arrow) accompanied by denuded villi (red circle), submucosal edema (blue circle) and digestion as well as disintegration of lamina propria, hemorrhage, and ulceration. The II/R-induced intestinal morphologic alterations were significantly attenuated by pre-treatment with Cor  in a dose-dependent manner with Chiu’s score markedly restored from 5.43 ± 0.14 to 2.57 ± 0.14 (p < 0.01), but also reversed the increased MDA content from 2.47 ± 0.24 to 1.62 ± 0.11 μmol/gprot (p < 0.05), as compared with II/R mice  pre-treatment not only reversed the decreased SOD activity from 3.69 ± 0.03 to 4.93 ± 0.04 U/gprot (I/R mice .p < 0.01 vs. sham group). Moreover, the lung water content ratio was significantly increased to 24.18% ± 0.83 in II/R mice compared with 15.19% ± 056 in sham group (p < 0.01). Cor pre-treatment  significantly alleviated the lung histopathological damage and decreased the tissue score and lung water content ratio to 0.29 ± 0.01 and 16.55% ± 1.24 (p < 0.01 vs. II/R group), respectively  and from 7.85 ± 0.34 to 5.47 ± 0.24 μmol/gprot (p < 0.01), respectively, and the MDA content was markedly increased from 0.91 ± 0.03 to 1.20 ± 0.04 μmol/gprot (p < 0.01) in II/R group compared with the sham mice. Cor pre-treatment  not only reversed the decreased SOD activity and GSH content to 4.27 ± 0.10 U/gprot (p < 0.01) and 6.96 ± 0.37 μmol/gprot (p < 0.05), respectively, but also reversed the increased MDA content to 0.95 ± 0.02 (p < 0.01), as compared with II/R group (p < 0.01), from 510.67 ± 28.47 to 347.17 ± 11.04 pg/ml  and from 13.47 ± 0.50 to 10.32 ± 0.23 U/g tissue , respectively . Further, the LDH activities in both intestinal and lung tissues were markedly increased from 112.42 ± 16.62 to 316.05 ± 8.11 U/L (p < 0.01) and from 497.83 ± 33.10 to 674.85 ± 18.64 U/L (p < 0.01) after II/R injury, respectively. Cor pre-treatment  inhibited II/R-induced LDH activity in a dose-dependent manner  generated from the cleavage of pyroptosis execution protein GSDMD. As shown in t manner . These rin vitro. Cells were incubated in serum/glucose-free DMEM under a hypoxic condition to simulate ischemia by deprivation of oxygen and glucose. The CCK-8 assay showed that Cor, at a concentration of 80 μM or lower, did not exert observable influence on cell viability under routine conditions  pre-treatment reduced the percentage of PI-positive cells . Cor alstivation . The resIn OGD/R-injury IEC-6 cells, the increased percentage of PI-positive cells and up-regulated expression levels of NLRP3, mature caspase-1 and GSDMD-NT induced by OGD/R injury were significantly reversed by Cor pre-treatment (1 μM). However, this reversing effect of Cor was interrupted by Nigericin, an activator of NLRP3 . These rp < 0.01) and from 100% ± 3.00 to 139% ± 3.42 (p < 0.01), respectively (p < 0.01)  + Nigericin (10 μM) to induce the specific activation of NLPR3 inflammasome pathway. For RAW264.7 cells, incubation with LPS and Nigericin did not cause significant changes in cell viability, but significantly increased the LDH activity from 239.1 ± 4.2 to 371.2 ± 6.2 U/L . And theectively . Meanwhi < 0.01) . The res < 0.01) . Moreove < 0.01) , indicatSimilarly, the prevention effects of Cor on NLRP3 inflammasome pathway and pyroptosis were also observed in LPS and Nigericin incubated MLE-12 cells , which fII/R is a clinical emergency that commonly occurs in several clinical conditions. In addition to causing local intestinal damage, II/R is often followed by distant organ injury, especially lung injury, which is associated with high morbidity and mortality. The development of innovative therapeutic strategies to mitigate II/R-induced intestinal and lung damage is of great significance.Phyllanthus niruri L., P. emblica L. and P. urinaria L. Cor is reported to possess excellent pharmacological activities, including antioxidant, anti-inflammatory, hepatoprotective, and anti-tumor activities  during reperfusion, which are converted to hydrogen peroxide (H2O2) and cleaved to hydroxyl radicals (HO−)  triggers nucleic acid fragmentation and lipid peroxidation, leading to various cellular death and changes in microvascular and mucosal permeability  depletion and inhibition of the ATPase-dependent ion pump, which further degrades ATP to hypoxanthine. Simultaneously, available oxygen generates oxidative hypoxanthine and superoxide anion radicals (Ols (HO−) . The ROSeability . In the eability . In addivia GSDMD-NT, eventually resulting in the release of inflammatory factors and LDH (Oxidative stress leads to an unfolded protein response that can cause detachment of thioredoxin from thioredoxin-interacting protein, which interacts with NLRP3 to promote activation of NLRP3 inflammasome . NLRP3 i and LDH . Pyropto and LDH . In the  and LDH . MoreoveASC aggregates into dimeric molecules, known as ASC specks. ASC specks are dynamic structures that can amplify the NLRP3 response to weak stimuli by facilitating the formation of NLRP3 inflammasome, which, in turn, activate caspase-1 . CaspaseThe active ingredients from traditional ethnic herbal medicines often have multiple targets and involve multiple pathways . PreviouIn summary, Cor alleviated II/R injury and distal lung injury. The therapeutic effects may be attributed to the prevention of NLRP3-associated pyroptosis. This study increases the drug selectivity for II/R treatment and provides a scientific reference for the standardized application of Cor in clinical practice. In the future, we will explore and illustrate the multiple targets of Cor, which will enable full exploitation of Cor for clinical applications.