Cutaneous melanoma exhibits heterogeneous metastatic patterns and prognosis. In this regard, liver metastasis, which is detected in ~ 10–20% of stage 4 patients, came to the fore of melanoma research, as it recently evolved as decisive indicator of treatment resistance to immune checkpoint inhibition.Hepatic metastases were induced by intrasplenic injection of five different murine melanoma cell lines. The efficiencies of hepatic colonization, morphologic patterns, gene expression profiles and degree of vascularization were analyzed and Sorafenib was applied as anti-angiogenic treatment.WT31 melanoma showed the highest efficiency of hepatic colonization, while intermediate efficiencies were observed for B16F10 and RET, and low efficiencies for D4M and HCmel12. RNAseq-based gene expression profiles of high and intermediate metastatic melanomas in comparison to low metastatic melanomas indicated that this efficiency predominantly associates with gene clusters involved in cell migration and angiogenesis. Indeed, heterogeneous vascularization patterns were found in the five models. Although the degree of vascularization of WT31 and B16F10 metastases differed, both showed a strong response to Sorafenib with a successful abrogation of the vascularization.Our data indicate that molecular heterogeneity of melanomas can be associated with phenotypic and prognostic features of hepatic metastasis paving the way for organ-specific anti-angiogenic therapeutic approaches.The online version contains supplementary material available at 10.1186/s12967-022-03255-4. Cutaneous melanoma (CM) is a phenotypic and molecular heterogeneous disease arising from melanocytes of the skin and preferentially spreads to the skin, lymph nodes, lung, liver and brain . In rece+ monocytes [+ T-cells from the periphery, which underwent apoptosis, and, as consequence, induced systemic immunosuppression and reduced response to ICI [Recent studies have provided insight into mechanisms of global therapy resistance mediated by hepatic metastasis. In murine models of colorectal carcinoma (CRC) and melanoma, subcapsular injection of MC38 CRC or B16F10 melanoma cell lines into the liver abolished the response of corresponding subcutaneous tumors to ICI. The authors tie this to infiltration of regulatory T-cells into the subcutaneous tumors which in turn recruit CD11be to ICI .Current clinical research focuses on sequential and combined treatment approaches of ICI and targeted therapies . MoreoveIn general, liver metastases most commonly arise from CRC, pancreatic, lung or mammary carcinoma. Regarding CRC or pancreatic cancer, the liver is the first organ in line of the vascular tree and therefore could be considered as sieve for disseminating tumor cells. In contrast, this does not apply for lung or mammary carcinoma, uveal melanoma (UM) or CM. Paget postulated that both tumor-intrinsic factors (“seeds”) as well as the microenvironment of the target organ (“soil”) need to be taken into account to understand organotropic metastasis . UM is tResearch has often focused on phenotypic characteristics of liver metastasis in these tumor entities. Morphologic characterization identified pushing, replacement and desmoplastic histological growth patterns (HGPs) of breast cancer, colorectal, UM and CM liver metastases –13. In CIn regard to tumor cell-intrinsic mechanisms regulating organotropic liver metastasis epithelial-to-mesenchymal transition controlled by miR-200c and the PKCζ/ADAR2 axis is a decisive step to hepatic metastasis of CRC . MoreoveIn CM, mutations of both NRAS and BRAF correlate with brain and liver metastasis . HoweverTumor intrinsic features associated with hepatic colonization of CM have not been characterized in detail. Therefore, this study comparatively analyzed five genetically different murine melanoma cell lines, reflecting the genetic heterogeneity of melanomas, and their molecular and phenotypic features underlying the different efficiencies of liver metastasis formation in mice.For in vivo experiments female C57Bl/6 wildtype mice were purchased by Janvier. For metastasis experiments mice were age-matched and were used at least at 10 weeks of age. All animals were hosted in single ventilated cages  in a 12 h/12 h day/night cycle under Specific-pathogen-free conditions and fed ad libitum with a standard rodent diet .luc2 was purchased from Perkin Elmer . RET1 melanoma cells were generated from metallothionein-I (MT)/RET transgenic mice [Tyr∷NrasQ61K/°; INK4a−/− mice [CA;Ptenlox/lox mice [luc2, WT31 and RET cells were maintained in RPMI 1640 media  with 10% (v/v) fetal calf serum (FCS) and 100 U/ml penicillin/streptomycin at 37 °C, 5% CO2. HCmel12 cells were cultured in RPMI with 10% (v/v) FCS, Hepes, ß-Mercaptoethanol, 1% L-glutamine, 100 U/ml penicillin/streptomycin, 1% Natrium Pyruvate and 1% NEAA. D4M cells were maintained in Advanced DMEM/F-12  with 5% (v/v) FCS, 100 U/ml penicillin/streptomycin and glutamine. For in vivo experiments always the same passage of corresponding cell lines was used. After thawing they were not passaged more than three times and maximum culture time prior to in vivo experiments was one week.All used melanoma cell lines were from murine origin. The melanoma cell line B16F10 nic mice  and kind−/− mice  and was −/− mice  and kindlox mice  and was luc2, RET, D4M or HCmel12 ranged from 0.5 × 105 to 3.0 × 105, please refer to individual Figure legends. Regarding WT31 melanoma cells, 0.1 × 105 or 0.3 × 105 were used for spleen injection. For i.v. injection, 1.25 × 106 to 2.5 × 106 WT31 cells were injected. The animals were sacrificed at indicated time points, at least at day 21. Organs were removed and analyzed for melanoma colonization.Spleen injection of melanoma cells was performed as described previously . Corresp5 WT31 melanoma cells intrasplenically. From day 1 to 18 mice received daily i.p. injections of Sorafenib  , 12.5% ethanol, and 75% sterile saline) or vehicle/solvent control . At day 19 mice were sacrificed and metastases were quantified.Mice were injected with 0.3 × 10Mice were sacrificed by cervical dislocation. Livers were fixed in 4% PFA at 4 °C for 24 h, followed by paraffin embedding according to standard protocols. As well, livers were embedded in OCT .Deparaffinization and rehydration of paraffin sections (1–5 µm) was performed according to standard protocols. Antigen retrieval was carried out with epitope retrieval solution  at either pH 6, pH 8 or pH 9. Cryosections (8 µm) were fixed with 4% PFA or re-hydrated in PBS when PFA perfused, pre-fixed livers were cut. First antibody was incubated over night at 4 °C, secondary antibody was applied for 1 h at room temperature after three washing steps with PBS. Sections were mounted with Dako fluorescent mounting medium . Staining of paraffin sections was performed as previously described . For hemPictures of routine histology stainings were acquired by Nikon Eclipse Ni-E  using a 10x/0.45 plan apochromat objective. During acquisition data were not compressed. Pictures were processed in NIS-Elements .Analysis of fluorescent-labelled sections was performed with a TCS SP5 DS laser scanning spectral confocal microscope . For excitation wavelengths were set to 488, 543 and 633 nm. To visualize Alexa Fluor 488, Cy3 and Alexa Fluor 647 conjugates the emission maxima were detected at 518, 570 and 673 nm. Three representative areas per samples were chosen and images were acquired in a sequential mode. Processing of images was performed by Leica confocal software  and ImageJ software . In detail, color thresholds were set in relation to the whole image (= fluorescent area). Endothelial marker expression was quantified using Image J. First, the metastatic area was marked manually and auto thresholding was applied. Then the lower threshold was adjusted to represent the positive signal. Afterwards, the areas positive for marker expression inside and outside the metastatic area were calculated and measured as area fraction. Last, the percentage of a certain marker expression was set in relation to the total intratumoral vessel area.Primary antibodies: Rabbit anti-cleaved Caspase 3 , rabbit anti-Ki-67 , rat anti-CD31 , goat anti-CD32b , rabbit anti-Stabilin-2 peptide 15 antibody , goat anTotal RNA from sub-confluent (50%), cultured melanoma cells was extracted with innuPREP RNA Mini Kit , then treated with TURBO DNA-free Kit . Samples were prepared and RNA concentration and quality were measured by a NanoPhotometer NP80  and a 2100 Bioanalyzer . The library preparation and the sequencing with an Illumina HiSeq 4000 sequencing system  were then performed by BGI . The raw and normalized gene expression profiling data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE185539.log2-counts per million (logCPM) using the voom-function from the limma package [https://www.genome.jp/kegg/pathway.html). The GSEA was made with R.Most of the procedure was done with R and Bioconductor using the NGS analysis package systempipeR . The qua package . Differe package . Pathway package  and the  package  in R usihttps://www.ncbi.nlm.nih.gov/tools/primer-blast/). For mRNA specificity, qPCR primers were designed to span an exon-exon junction where possible. Primers were tested with no template controls, original RNA, and melt curve analysis. Primer sequences are listed in Additional file We performed reverse transcription with Maxima Reverse Transcriptase  and Oligo(dT)18 primers  according to the manufacturer’s instructions. innuMIX qPCR SyGreen Sensitive  was used on a qTOWER 3 G touch thermal cycler  for quantitative PCR (qPCR) of cDNA. qPCR primers were designed with NCBIs PrimerBLAST  test was used. For grouped analysis, a Dunn’s multiple comparisons test was applied when data were not distributed normally. For analysis of qPCRs a one-way ANOVA was performed followed by a correction for multiple comparisons by a Tukey test. Differences between data sets with P < 0.05 were considered statistically significant.All statistical analyses and graphical displays were performed with GraphPad Prism7  and mean ± SEM is presented. For statistical analysis, an unpaired, two-tailed luc2 and RET showed a medium colonization efficacy and lower numbers of liver metastases. D4M and HCmel12 melanoma cells formed liver metastases at the highest cell concentrations injected indicating the lowest efficiency of hepatic colonization. Macroscopically, most of the tested melanoma cell lines developed black or grey round tumor nodules, except for D4M, which formed white, pinpoint-like lesions. In cell culture, cell pellets of WT31 melanoma were the most pigmented ones  and WT31 the largest (1.92 mm2 ± 0.9)   showed the largest necrotic areas as compared to RET (5.7% ± 1.6) and HCmel12 (6.1% ± 1.7)  and HCmel12 (7.6% ± 3.6) revealed the most cCasp3-positive cells at the metastatic center  indicating a smaller cell size  (HIM-melanoma) were therefore compared to the ones with low metastatic efficiency (D4M or HCmel12) (LM-melanoma). To select for commonly regulated genes and pathways, only significant genes with the same direction of regulation were considered among HIM-melanoma. First, uniformly regulated significant genes of HIM-melanoma were compared in relation to D4M identifying a gene set of 6386 regulated significant genes   indicating a mixed sinusoidal and capillarized molecular phenotype in a minor fraction of HCmel12 vessels . In both models Sorafenib led to central pseudocystic, hemorrhagic degradation of metastases with a thin residual rim of viable tumor cells in around 80–90% of metastases  and similar the induction of pseudocystic degradation was significantly higher in WT31 (73% vs. 60.3%) indicating stronger dependence on vascularization than B16F10 luc2   melanoma compared to low metastatic (LM-) melanoma showed the most marked differences in pathways involved in cell migration, angiogenesis, EMT, oxidative phosphorylation or TNF-α signaling. Cell adhesion and migration are the first steps of organ colonization in the metastatic cascade. In this regard, integrin alpha2 is associated with enhanced hepatic colonization while integrin alpha4 is associated with lymph node metastasis in B16 melanoma , 47, 48.Angiogenesis also stood out as a differentially regulated process between HIM- and LM-melanoma. Angiogenesis is well known as determinant of hepatic metastasis in general . In CRC Aside from angiogenesis itself, vessel maturity which is regulated by vessel pruning and regression is an important process involved in tumor blood supply . Detectiluc2). Although this response was even stronger in WT31, both cell lines showed a solid treatment response. Necrosis or pseudocystic degeneration is not seen when subcutaneous B16 melanoma is treated with Sorafenib [The tumor vasculature can be disrupted by anti-angiogenic agents such as bevacizumab, an anti-VEGF antibody, or Sorafenib, a multi-kinase-inhibitor , 54. Firorafenib . This inorafenib  a small orafenib , a combiorafenib .Altogether, molecular and phenotypic diversity of murine melanoma determine liver metastatic propensity involving cell migration and angiogenesis as major processes. Metastatic vascularization correlates with metastatic size. Interestingly, both highly and poorly vascularized hepatic lesions responded to Sorafenib indicating a broad efficacy for Sorafenib in this specific context. Therefore, anti-angiogenic therapy is an appealing approach to treat liver metastasis in advanced melanoma patients.Heterogeneity of hepatic metastasis of cutaneous melanoma was studied using a murine orthotopic model with five genetically different cell lines. Efficacies and phenotypic features of liver colonization of these melanoma cell lines were comprehensively compared. Migration and angiogenesis were among the differentially regulated functions identified by comparative RNA-seq analysis. Overall, molecular and phenotypic heterogeneity of hepatic melanoma metastasis revealed angiogenesis as a targetable determinant of hepatic colonization paving the way for organ-specific anti angiogenic treatment.Additional file 1:Figure S1. Morphological characterization of melanoma cell lines. A. A selection of representative pictures of hepatic metastases of WT31, B16F10 luc2, RET, D4M and HCmel12 melanoma after spleen injection. Besides, liver metastases of WT31 melanoma after i.v. injection are shown. Scale bar = 1 cm. B. Images of cell pellets of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. C. Representative pictures of lungs, brain, tibia, kidneys and spleen with metastases after intravenous injection of WT31 melanoma. Scale bar = 1 cm. Figure S2. Sirius red and PAS stainings of hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M. A. Images of Sirius red (SR) stainings of hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. Scale bar = 200 µm, n ≥ 5. B. Pictures of liver metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma stained by PAS. Scale bar = 200 µm, n ≥ 5. Figure S3. Heat map of top25 up- and downregulated genes and heat map of melanocytic differentiation genes. A. A heatmap of the 25 most up- and downregulated genes of HIM-melanoma in comparison to LM-melanoma is shown. Please also refer to Fig. 3A, B for definition of this gene set. B. A heat map of common melanocytic differentiation genes is presented. A red color code presents an upregulation of genes, whereas the green color code shows downregulation. Clustering of genes is presented on the left side. Figure S4. Heat map GOBP—cell migration. A heat map of the GOBP cell migration gene set is presented for B16F10, RET, WT31, HCmel12 and D4M melanoma cells. The heat map was split in two parts to fit the page. A red color code presents an upregulation of genes, whereas the green color code shows downregulation. Clustering of genes is presented on the left side. Figure S5. Heat map GOBP—angiogenesis. A heat map of the GOBP angiogenesis gene set is presented for B16F10, RET, WT31, HCmel12 and D4M melanoma cells. A red color code presents an upregulation of genes, whereas the green color code shows downregulation. Clustering of genes is presented on the left side. Figure S6. A. Additional pictures of immunofluorescences for CD31, CD32b and DAPI of hepatic metastasis of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma at the border to adjacent liver. Scale bars = 100 µm, n ≥ 5. B. Additional pictures of immunofluorescences for Collagen IV, CD31 and DAPI of hepatic metastasis of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma at the border to adjacent liver. Scale bars = 100 µm, n ≥ 5. Figure S7. Analysis of peri- and intratumoral Stab2 expression as well as extracellular Collagen I and Collagen III matrix deposition in hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. A. Immunofluorescence images of Stab2 and DAPI of B16F10 luc2, RET, WT31, D4M and HCmel12 melanoma liver metastases at the metastatic border. White dotted lines present the border of hepatic metastases to adjacent liver tissue. Scale bars = 100 µm, n ≥ 5. B. Immunofluorescences of Collagen I of hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. Scale bar = 100 µm, n ≥ 5. C. Immunofluorescences of Collagen III of liver metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. Scale bar = 100 µm, n ≥ 5. Figure S8. Treatment with Sorafenib leads to loss intratumoral vessels and cystic degeneration of hepatic melanoma metastases. A. WT31 melanoma cells (0.3 × 105 cells) or B16F10 luc2 melanoma cells (1.5 × 105 cells) were injected intrasplenically. From day 1 to 18 mice received daily i.p. injections of Sorafenib (60 mg/kg KG) or vehicle/solvent controls. At day 19 mice were sacrificed. Macroscopic visible metastases were quantified. A Mann-Whitney Utest was performed . Representative images are shown. Scale bars = 1 cm. Number of animals analyzed = 8 , 9 , 6 , 6 . B. The size of hepatic metastases of WT31 (left graph) or B16F10 luc2 melanoma (right panel) in mice treated with Sorafenib or vehicle was measured. Mann-Whitney U-tests were performed . Number of animals analyzed = 6 , 6 , 5 , 4 . Number of metastases analyzed = 48 , 32 , 57 , 28 . C. Comparison of the percentage of pseudocystic WT31 or B16F10 luc2 melanoma metastases after treatment with Sorafenib. MannWhitney U-tests were performed (p = 0.1970). Number of animals analyzed = 6 , 5 . Data information: *P < 0.05, **P < 0.01, *** P < 0.0001, n.s. = not significant.Additional file 2: Table S1. Statistic analysis of metastatic efficiency and number of hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M after spleen injection. Table S2. A. Percentage of animals with hepatic metastasis after spleen injections of melanoma cells. B. Number of hepatic metastases of each individual animal after spleen injections of melanoma cells. Table S3. A. Percentage of animals with hepatic metastasis after intravenous injections of WT31 melanoma cells. B. Number of hepatic metastases of each individual animal after intravenous injections of WT31 melanoma cells. Table S4. Sequences of primers used for qPCRs. Table S5. Statistic analysis of qPCRs of Kazn, St6galnac3, Glrb, Tyr, Unc5c, Thbs2, Tfap2b, Tenm3, Stambpl1 and Ly6e in WT31, B16F10 luc2, RET, D4M and HCmel12 melanoma cells.


Prunus salicina Lindl. (named as Niuxin plum) is a remarkable material for brewing fruit wine owing to its suitable sugar-acid ratio, characteristic aroma and bioactive compounds. This study intends to modify the fermentation technology, identify and quantify nutritional compositions and volatile profiles, as well as bioactive substances in Niuxin plum wine, as well as evaluate the antioxidant and hypoglycemic activities in vitro of major bioactive components from Niuxin plum wine. According to single-factor and orthogonal tests, the optimal fermentation conditions of 13.1% vol Niuxin plum wine should be Saccharomyces cerevisiae Lalvin EC1118 at 0.1% and a fermentation temperature of 20°C for 7 days. A total of 17 amino acids, 9 mineral elements, 4 vitamins, and 55 aromatic components were detected in plum wine. Polysaccharides from Niuxin plum wine (named as NPWPs) served as the major bioactive components. The NPWP with a molecular weight over 1,000 kDa (NPWP-10) demonstrated extraordinary DPPH free radical scavenging capacity and α-glucosidase inhibitory activity among all NPWPs having different molecular weight. Moreover, the structural characterization of NPWP-10 was also analyzed by high performance liquid chromatography (HPLC), fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectra studies. NPWP-10 was composed of mannose, rhamnose, arabinose, galactose and galacturonic acid with molar ratios of 2.570:1.775:1.045:1.037:1. NPWP-10 contained α-configuration as the main component and β-configuration as the auxiliary component. This study highlights NPWP-10 is an importantly biological polysaccharide from Niuxin plum wine, as well as provides a scientific basis for developing the plum wine industry.With the increase in demand of fruit wine year by year, it is necessary to develop novel fruit wine with high functional activities.  Prunus salicina Lindl.) wine.Evaluation of bioactive compounds and bioactivities in plum ( Prunus salicina Lindl., known as “Niuxin plum” in Chinese, is a crop tree species native to China, and cultivated mainly for the fresh fruit market. At present, plum also is typically processed into dried fruit, jam and jelly (E)-2-hexenal, (E)-2-octenal, (E)-2-nonenal, decanal, 2-nonanone, 2,6,6-trimethyl-2-cyclohexene-1,4-dione, 2,4-di-tert-butylphenol, p-cymene and dipentene, were identified in plum cultivars . Saccharomyces cerevisiae Lalvin71B, Lalvin EC1118, Lalvin D254, Lalvin RC212, Lalvin RC2323, Lalvin K1, Lalvin U43, Lalvin KD, and Lalvin R-HST were from Longbao Commercial and Trading Co. . Saccharomyces cerevisiae Angel BV818, Angel SY, and Angel RW were provided by Angel Yeast Co. . HPLC-grade rutin, glucose, phenol, gallic acid, 4-hydroxypiperidine, acarbose, α-glucosidase (100 UN), Folin-Ciocalteu reagent, 2-diphenyl-1-picrylhydrazyl (DPPH), and p-nitrophenyl-β-d-galactopyranoside (PNPG) were provided by Sigma-Aldrich Co. . Overall, chemicals were at an analytical grade unless specified otherwise.The Niuxin plum trees were planted in the hill districts of the Guangxi province of China and harvested in June 2017. For the tests, Niuxin plum fruits were rinsed and separated into pulp and seeds. The plum fruit pulp having high maturity (15.6°Brix) was stored at −20 °C for further research.Saccharomyces cerevisiae was added when the juice temperature was reduced to 40°C, and fermentation was carried out at 20°C for 7 days. Three factors were introduced for single-factor testing, including Saccharomyces cerevisiae strain, yeast amount, and the temperature of fermentation . The hydrolyzed plum juice was further mixed with 178.7 g of sucrose and then sterilized under the temperature conditions of 75°C for 10 min inside the water bath. entation . Based o+ ionization mode, 80 μA emission current, 70 eV electron energy, 250°C interface temperature, 200°C source temperature, and 1,000 V detector voltage. The temperature of the GC oven was first set to 40°C for 3 min, and it was then up-regulated by 5°C/min to 90°C and by 10°C/min to 230°C. The heating under the temperature condition of 230°C lasted for 7 min. Helium at a flow rate of 0.8 mL/min served as the carrier gas.The amino acids, mineral elements, and vitamins as nutrients in Niuxin plum wine were evaluated by modified methods –19. The The content of total polysaccharides was measured using the phenol-sulfuric acid approach, in which glucose served as the extrinsic criterion . ResultsNiuxin plum wine was first decompressed for distillation treatment under the temperature condition of 60°C and then condensed to a viscous fluid. The concentrated sample (100 mL) was mixed with 900 mL of absolute ethanol and stirred adequately, and preserved under the temperature condition of 4°C. The sediments were collected as Niuxin plum wine polysaccharides (NPWP). NPWP solution (250 mL) was separated by the MinimatePall ultrafiltration system  with 1,000, 500, 300, 100, 50, 10, 5, 3, and 0.65 kDa-ultrafiltration membranes. Ten kinds of NPWP solutions (50 mL) were mixed with 450 mL of absolute ethanol and preserved under the temperature condition of 4°C for 24 h. All the sediments collected were freeze-dried. The antioxidant and anti-hyperglycemic activities of 10 NPWPs were detected using the methods described below. The NPWP possessing the highest bioactivities were selected to identify the chemical structure.The monosaccharides from NPWP were hydrolyzed and released by the modified method . The mix−1. 1H NMR (600 MHz) and 13C NMR (150 MHz) spectra of NPWP were recorded using Bruker Advance 600M spectrometer , with deuterated H2O (D2O) as the solvent and tetramethylsilane (TMS) as the intrinsic criterion. Chemical shifts (δ) were expressed as ppm in contrast to TMS  of Niuxin plum wine was assessed following the improved Folin-Ciocalteu assay . TPC wasThe total alkaloid content (TAC) of Niuxin plum wine was analyzed by the modified method . TAC wassample – Ablank)/Anegative control] × 100%.DPPH radical-scavenging activity in Niuxin plum wine was assessed with an improved method . The per2 – A3)/A1] × 100%, in which A1 is blank absorbance, A2 is sample or acarbose absorbance, and A3 is the absorbance of sample short of α-glucosidase and PNPG.The α-glucosidase-inhibitory activity of Niuxin plum wine was evaluated by using PNPG as the substrate . Data coP < 0.05).Data present the mean and standard deviation of three replicates. Statistical research was carried out by variance analysis (ANOVA) and SPSS 17.0 statistical software . Duncan's test was the most effective solution for detecting the notable discrepancy of means  than others, implying that the yeast amount acts as a significant factor. As seen in P < 0.05) than others, which indicated that temperature plays a crucial role in fermentation.According to Saccharomyces cerevisiae strain (A), yeast amount (B), and fermentation temperature (C) as the significant influence factors fit for orthogonal testing. According to Saccharomyces cerevisiae strain as the foremost influence factor of plum wine SES. The ANOVA of orthogonal experiment results also confirmed that. In comparing the values of three factors, namely k1, k2, and k3, the optimal combination of A1B1C2 was determined. It meant that the optimal conditions of fermentation for plum wine should be Saccharomyces cerevisiae strain of Lalvin EC1118, yeast amount of 0.10%, and fermentation temperature of 20°C. Under these conditions, the SES of plum wine was 92.0 ± 0.5, and alcohol content of the produced wine was 13.1% vol.Based on the findings of single-factor testing, we considered P < 0.05), followed by alanine (3.77 ± 0.01 mg/100 mL), aspartic acid (3.74 ± 0.02 mg/100 mL) and proline (2.77 ± 0.02 mg/100 mL). Seven essential amino acids accounted for 33.43% of the total amino acid content, and included threonine (1.57 ± 0.01 mg/100 mL), valine (2.69 ± 0.03 mg/100 mL), methionine (2.45 ± 0.02 mg/100 mL), isoleucine (1.46 ± 0.04 mg/100 mL), leucine (2.42 ± 0.03 mg/100 mL), phenylalanine (0.98 ± 0.05 mg/100 mL) and lysine (1.13 ± 0.01 mg/100 mL). There also existed a small fraction of histidine (0.61 ± 0.03 mg/100 mL), arginine (1.11 ± 0.03 mg/100 mL), glycine (2.32 ± 0.01 mg/100 mL), serine (2.02 ± 0.03 mg/100 mL), tyrosine (1.03 ± 0.02 mg/100 mL) and cysteine (2.02 ± 0.01 mg/100 mL) in Niuxin plum wine.The total amino acid content of Niuxin plum wine was detected as 37.99 ± 0.05 mg/100 mL. Among the 17 amino acids found in plum wine, glutamic acid (5.90 ± 0.02 mg/100 mL) was the most abundant (P < 0.05) element was potassium (123.38 ± 0.01 mg/100 g), followed by calcium (20.54 ± 0.02 mg/100 g), magnesium (8.30 ± 0.01 mg/100 g), zinc (4.60 ± 0.03 mg/100 g), sodium (1.63 ± 0.03 mg/100 g), manganese (0.29 ± 0.02 mg/100 g), boron (0.06 ± 0.01 mg/100 g), barium (0.05 ± 0.01 mg/100 g) and strontium (0.03 ± 0.01 mg/100 g).In Niuxin plum wine, the inductively coupled plasma mass spectrometry with external standard method, was used for the determination of mineral elements. The most widely distributed (P < 0.05), and it was successively followed by vitamin E (6.96 ± 0.01 μg/100 mL), vitamin B9 (0.33 ± 0.01 μg/100 mL) and vitamin A (<0.1 μg/100 mL).In Niuxin plum wine, four important vitamins were determined selectively through qualitative and quantitative analysis. Among them, vitamin C (590 ± 0.01 μg/100 mL) exhibited the maximum content . Therefore, polysaccharides are considered as the major functional components in Niuxin plum wine, and were further isolated for selecting the fraction with the highest antioxidant and hypoglycemic activities, illustrating its structural characterization and analyzing the structure-activity relationship.The antioxidant and hypoglycemic activities of polyphenols, alkaloids, and polysaccharides in Niuxin plum wine were verified preliminarily. Phenolic extracts from Niuxin plum wine scavenged activity against DPPH radicals with 40.362 ± 1.171% and inhibited α-glucosidase activity with 7.726 ± 0.842%, as well as alkaloid extracts from Niuxin plum wine possessed DPPH radical scavenging activity (43.692 ± 1.915%) and inhibiting α-glucosidase capacity (6.918 ± 0.350%). However, polysaccharides extracted from Niuxin plum wine exhibited significantly higher DPPH radical scavenging activity (77.646 ± 0.916%) and α-glucosidase inhibitory capacity (50.446 ± 0.826%) than those of both phenolic and alkaloid extracts , 3–10 kDa (NPWP-2), 10–30 kDa (NPWP-3), 30–50 kDa (NPWP-4), 50–100 kDa (NPWP-5), 100–300 kDa (NPWP-6), 300–500 kDa (NPWP-7), 500–750 kDa (NPWP-8), 750–1,000 kDa (NPWP-9), and >1,000 kDa (NPWP-10). According to In the present study, the anti-hyperglycemic abilities of 10 NPWPs were detected using acarbose as control. From The HPLC chromatograms of monosaccharides hydrolyzed from NPWP-10 along with the seven standards are shown in −1, possibly as a result of the O–H bond's stretching vibration on carboxylic acids. The absorption peak at 2,932.63 cm−1 should be attributable to the C–H bond's stretching vibration. Absorption peaks at 1,725.20 cm−1 and 1,608.01 cm−1 were associated with symmetrical and asymmetrical stretching vibration of the carboxyl (–COOH) C = O bond. The absorption peak at 1,411.71 cm−1 might be caused by the stretching vibration of C–N in the amide bond. The stretching vibration of the C–O bond's caused the absorption peak at 1,259.22 cm−1. A strong absorption peak was observed at 1,047.55 cm−1, indicating that this NPWP contained a pyranose ring and had the stretching vibration of the C–O–C bond. The pyranose ring's symmetrical stretching vibration caused the absorption peak at 609.49 cm−1.The FT-IR spectrum of NPWP-10 was shown in 13C NMR spectrum of NPWP-10  21], Guangxi Scientific Research and Technological Development Projects (Grant No. Gui Ke AD19110141), and Foundation of Fundamental Research Project from Guangxi Academy of Agricultural Sciences .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mandragora autumnalis has been used as a traditional medicinal plant for the treatment of numerous ailments. In light of this, the current study was designed to isolate and identify the chemical constituents of the flavonoids fraction from M. autumnalis ripe fruit (FFM), and evaluate its DPPH scavenging, anti-lipase, cytotoxicity, antimicrobial and antidiabetic effects. An ethyl acetate extract of M. autumnalis was subjected to a sequence of silica gel column chromatography using different eluents with various polarities. The chemical structures of the isolated compounds were identified using different spectral techniques, including 1H NMR and 13C NMR. FFM’s anti-diabetic activity was assessed using a glucose transporter-4 (GLUT4) translocation assay, as well as an inhibition against α-amylase and α-glucosidase using standard biochemical assays. The FFM anti-lipase effect against porcine pancreatic lipase was also evaluated. Moreover, FFM free radical scavenging activity using the DPPH test and antimicrobial properties against eight microbial strains using the micro-dilution method were also assessed. Four flavonoid aglycones were separated from FFM and their chemical structures were identified. The structures of the isolated compounds were established as kaempferol 1, luteolin 2, myricetin 3 and (+)-taxifolin 4, based on NMR spectroscopic analyses. The cytotoxicity test results showed high cell viability (at least 90%) for up to 1 mg/mL concentration of FFM, which is considered to be safe. A dose-dependent increase in GLUT4 translocation was significantly shown (p < 0.05) when the muscle cells were treated with FFM up to 0.5 mg/mL. Moreover, FFM revealed potent α-amylase, α-glucosidase, DPPH scavenging and porcine pancreatic lipase inhibitory activities compared with the positive controls, with IC50 values of 72.44 ± 0.89, 39.81 ± 0.74, 5.37 ± 0.41 and 39.81 ± 1.23 µg/mL, respectively. In addition, FFM inhibited the growth of all of the tested bacterial and fungal strains and showed the greatest antibacterial activity against the K. pneumoniae strain with a MIC value of 0.135 µg/mL. The four flavonoid molecules that constitute the FFM have been shown to have medicinal promise. Further in vivo testing and formulation design are needed to corroborate these findings, which are integral to the pharmaceutical and food supplement industries.Since ancient times,  Humankind has been fascinated with botanicals since antiquity because of their exceptional nutritional, cosmetic and medicinal characteristics . A growiMandragora autumnalis Bertol.  is a highly valued wild medicinal herb. It is a perennial herbaceous plant in the Solanaceae family with purple or violet blooms and orange or yellow mature fruits (berries). It is widely distributed throughout the Mediterranean Basin, including Palestine. It is still used in traditional Arabian medicine, especially Palestinian herbal medicine, to treat pains, insomnia, cough, throat pain, bronchitis and various genital organs diseases + at m/z 287.0556. The 13C-NMR spectrum of 1 displayed resonance for all of the 15 carbons present in the molecule as indicated in the 13C-NMR (DEPT) , 7.40  and 6.89 , as well as the presence of four OH peaks. The HSQC and HMBC experiments supported the structure of 2 +, suggesting that it had the chemical formula C15H10O8. The 1H-NMR spectra revealed that 3 was a flavonol with six OH peaks and four aromatic proton peaks (1H- and 13C-NMR (3 was discovered to be a myricetin skeleton.Flavonoid R (DEPT) . The 1H-spectrum  of 1 werempferol ,23. Compramework  due to ture of 2 ,22. Thuson peaks . When 1H 13C-NMR  data wer4 was isolated as a pale yellow solid and its chemical formula was established as C15H12O7 based on ESI-HRMS m/z 305.0531 [M + H]+ and NMR spectral data. Compound 4 had 15 carbon atoms, including 8 quaternary carbons and 7 methine carbons, according to the DEPT and HMQC spectra  and a doublet at 4.99 (J = 11.2 Hz) corresponding to H-3 and H-2, respectively. The carbon resonances at 72.0, 83.5 and 198.3, designated C-3, C-2 and C-4 of the flavanonol C-ring, respectively, confirmed this identification. H-2 and H-3 were trans, as shown by their coupling constants of 11.2 Hz. The 1H-NMR spectrum  had long-range correlations with carbon signals at δC 128.5 and 72.0, showing carbon signals at δC 128.5 and 72.0 to be due to C-1′ and C-3, respectively  into the bloodstream. Fourteen GLUT proteins, including GLUT 1-12, myoinositol transporter (HMIT) and GLUT 14, are expressed in humans . Both glThe effect of FFM on GLUT4 translocation to the PM in the L6 skeletal muscle cell line was examined in this work. The results indicate that FFM dramatically increased the amount of GLUT4 in the PM in both the presence and the absence of insulin. These findings suggest that FFM may include anti-diabetic chemicals. Flavonoids have been shown to have anti-diabetic action via distinct mechanisms. Certain flavonoids have been shown to enhance glucose uptake by hepatocytes and myocytes . The stiMost notably, kaempferol reduced GLUT4 and AMPK expression in muscle and adipose tissue in obese mice while reversing the high-fat diet. In obese diabetic mice, kaempferol therapy improved hyperglycemia and glucose tolerance . These fα-amylase and α-glucosidase activity. The FFM displayed potent α-amylase inhibitory action with an IC50 value of 72.44 ± 0.89 µg/mL, lower than the commercial anti-diabetic medication acarbose (IC50 = 28.18 ± 1.22 µg/mL). These findings are consistent with those published earlier by Proenca et al., who used a microanalysis screening method to examine the capacity of 40 structurally similar flavonoids to inhibit α-amylase activity [α-amylase with an IC50 value of 118 ± 7, 78 ± 3 and 107 ± 6 µM. Moreover, this plant fraction displayed powerful α-glucosidase inhibitory activity with an IC50 value of 39.81 ± 0.74 µg/mL, which is comparable to that of acarbose (IC50 = 37.15 ± 0.33 µg/mL).The findings of this study demonstrated that FFM has potent activity . It was α-amylase and α-glucosidase [α-amylase and α-glucosidase inhibitory activities highlighted that various flavonoids displayed higher α-glucosidase inhibitory activity than acarbose [These findings add to prior research indicating that natural polyphenols, particularly flavonoids, can inhibit the activity of carbohydrate-hydrolyzing enzymes such as cosidase . A revieacarbose ,35. Two 50 values of 5.37 ± 0.41 µg/mL, being comparable with the standard antioxidant trolox (IC50 = 2.23 ± 1.23 µg/mL). Previous studies demonstrated that flavonoids and other polyphenols are able to scavenge the DPPH free radicals by donating their hydrogen [50 values 12, 5, and 4 μM [As a result of various endogenous systems and exposure to various physico-chemical conditions, or pathological states, our bodies produce reactive oxygen and reactive nitrogen species, known as radicals. For proper physiological function, a balance between antioxidants and free radicals is required. Oxidative stress occurs when free radicals overwhelm the body’s ability to regulate them. Free radicals alter lipids, proteins and DNA, resulting in a wide range of human diseases. Hence consumption of antioxidants’ rich diet can assist in coping with this oxidative stress ,37. Our hydrogen . This reand 4 μM .50 value of 39.81 ± 1.23 µg/mL, compared with that of orlistat (IC50 = 12.56 ± 0.35 µg/mL). Shimura et al. reported that kaempferol, luteolin and myricetin inhibited lipase activity with IC50 of 0.72, 2.03 and 2.54 µg/mL, respectively [Obesity and overweight are chronic metabolic disorders caused by an imbalance of energy expenditure and intake. They are major risk factors for cancer as well as cardiovascular, metabolic and endocrine diseases . The lipectively .Streptomyces toxytricini bacteria. This clinically approved medication is intended for the treatment of obesity and overweight by decreasing the digestion of lipids [In fact, orlistat is a potent inhibitor of pancreatic lipase enzyme produced from f lipids .K. pneumoniae strain, with a MIC value of 0.135 ± 0.01 µg/mL, being comparable with the reference drug ciprofloxacin (MIC = 0.13 ± 0.02 µg/mL) and higher than ampicillin (MIC = 1 ± 0.02 µg/mL). Furthermore, FFM extract showed higher antibacterial activity against S. aureus, E. coli and MRSA with MIC values of 1.5 ± 0.07, 2.25 ± 0.07 and 25 ± 1.01µg/mL, respectively, than the reference drug ampicillin . It exhibits antibacterial activity against P. aeruginosa with a MIC of 12.5 ± 0.98 µg, comparable to ampicillin. The FFM showed potent antifungal activity against C. albicans, compared with the commercial antifungal drug fluconazole with MIC values of 6.25 ± 0.48 and 1.56 ± 0.01 µg/mL, respectively. Finally, this plant fraction exhibited a weak anti-mold activity (E. floccosum) with a MIC value of 12.5 ± 0.88 µg /mL, compared with fluconazole, which was used as a positive control and has a MIC value of 0.78 µg /mL.The microdilution assay was utilized to assess the FFM’s antimicrobial activity. The results showed that FFM has potent antibacterial activity against the M. autumnalis fruits’ ethanolic extract displayed potential antimicrobial activity against E. coli, P. aeroginosa, MRSA and C. albicans [Obeidat demonstrated that albicans .Extracts of numerous medicinal plants encompassing flavonoids have been previously stated to possess antimicrobial activity ,46. FlavM. autumnalis fruits. More research is needed to establish these putative pharmacological characteristics of the FFM extract of M. autumnalis in order to assess its impact in animal models and build suitable pharmaceuticals or food supplement formulations from this fraction.To the best of the authors’ knowledge, this is the first study that has looked at the chemical content and biological activity of α-MEM), standard culture medium and all of the other tissue culture reagents were purchased from . Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies were obtained from . Polyclonal anti-myc (A-14), the 3--2,5-diphenyl tetrazolium bromide (MTT) reagent, methoxyamine hydrochloride, pyridine, N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) and other standard chemicals were purchased from .The rat L6 muscle cell line and stably expressing myc-tagged GLUT4 (L6-GLUT4myc cells) were obtained from . Fetal bovine serum, modified Eagle’s medium  and trolox were purchased from Sigma-Aldrich . Finally, Mueller-Hinton broth, fluconazole, Sabouraud dextrose agar and dimethyl sulphoxide (DMSO) were acquired from Himedia .All of the solvents used were HPLC-grade and acquired from . Tris-HCl, silica gel 60 GF254 thin-layer chromatography, silica gel (100–200 and 200–300 mesh) for column chromatography, porcine pancreatic amylase and lipase enzymes were acquired from . Corn starch was purchased from . Ampicillin, 3,5-dinitrosalicylic acid, ciprofloxacin and orlistat were acquired from . Acarbose, 1H- and 13C-NMR spectra were obtained using a Bruker AV-400 instrument and DMSO-d6 as a solvent. Spectrophotometer , microtiter plate reader , stir-mixer , weighing scale , multichannel micro-pipet , sterile syringe filter hydrophobic  and an incubator with CO2  were used to complete the current study; the microplate reader was from Anthos, Biochrom .The M. autumnalis were harvested in the Nablus highlands of Palestine. The plant was validated in the Herbal Products Laboratory at An-Najah National University and deposited in the same laboratory under the voucher specimen code Pharm-PCT-1509. M. autumnalis fruits were cleaned with distilled water and dried in the shade at room temperature (25 ± 2 °C). After four weeks, the total dry fruits were finely crushed and kept in a tightly sealed glass jar for future use.In May 2020, the golden mature fruits of M. autumnalis fruit powder (200 g) was macerated with methanol (0.5 L), and the suspension was agitated at 35 °C for 24 h. The resultant extract was filtered, and the residual solid was extracted again with 0.5 L of methanol under the same conditions. Methanol was removed under reduced pressure to offer dark green sticky oil. Water (50 mL) was added to the concentrated green extract before extracting it with pentane to remove lipids, waxes and nonpolar chemicals and ethyl acetate to extract the flavonoids and other polyphenols. The biological effects of the ethyl acetate fraction of the methanolic extract of M. autumnalis fruits were studied.1 (6 mg). Fraction 3 (0.58 g) was subjected to silica gel column chromatography eluted with hexane–acetone (95:5–9:10) to obtain two fractions. Fraction 3a was subjected repeatedly to silica gel column chromatography eluted with hexane–acetone (95:5) and was finally purified by a preparative thin-layer chromatography column eluted with CH2Cl2-EOAc (7:3) to give compound 2 (6 mg). Fraction 3b was subjected to silica gel column chromatography using gradient elution with hexane–acetone (99:1–90:10) followed by preparative TLC to give compound 4 (7.6 mg). Fraction 4 (0.72 g) was subjected to repeated silica gel column chromatography using gradient elution CH2Cl2-EtOAc (9:1–6:4) led to the isolation of compound 3 (4.3 mg) [The ethyl acetate extract (3 g) was subjected to silica gel column chromatography utilizing gradient dichloromethane–EtOAc (10:0–1:1) to obtain four fractions. Fraction 2 (0.687 g) was subjected to silica gel column chromatography employing gradient elution (hexane: acetone 10:0–10:1) to give compound (4.3 mg) .2 in α-MEM supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 0.1 mg/mL streptomycin.Cells from the rat L6 muscle cell line, stably expressing myc-tagged GLUT4 (L6-GLUT4myc), obtained from Kerafast , were maintained under an atmosphere of 95% air and 5% CO4 cells/well in 96-well plates with 100 µL of culture media in each well. After 24 h, FFM was administered at increasing doses (0–1 mg/mL) for 20 h. The cell media was then changed with 100 µL of new medium/well containing MTT (0.5 mg/mL) and cultured for 4 h in the cell’s incubator in the dark. The supernatant was withdrawn from each well, and 100 µL of isopropanol/HCl (1 mM HCl in 100% isopropanol) was added. Using a microtiter plate reader, the absorbance at 620 nm was measured. A microtiter plate reader was used to detect absorbance at 620 nm. To serve as controls, two wells on each plate were left unfilled. All of the experiments were conducted three times [The cellular metabolic activity was assessed as an indication of cell viability using the MTT test methodology devised by . This pree times . To illuv/v) goat serum, then reacted with polyclonal anti-myc antibody (1:200) for 1 h at 4 °C before being washed 10 times in PBS. Following that, cells were incubated for 1 h at 4 °C with horseradish peroxidase-bound goat anti-rabbit secondary antibody (1:1000) before being washed 15 times with PBS. Cells were then treated with 1 mL of phenylenediamine dihydrochloride reagent and allowed to develop in the linear range for 20–30 min in the dark at room temperature. The reaction was quenched by the addition of 1 mL of 3N HCl to each well. Supernatants were collected, and absorbance at 492 nm was measured. All of the results were subtracted from the background absorbance obtained in the absence of anti-myc antibodies.Surface myc-tagged GLUT-4 was assessed in intact, non-permeabilized cells using an anti-myc antibody followed by a secondary antibody coupled to horseradish peroxidase, as previously described . Cells wα-amylases enzyme (2 units/mL) was made by combining 12.5 mg of this enzyme powder with a few drops of 10% DMSO, and a buffer solution was added to achieve 100 mL. The starch solution was made by dissolving 1 g of corn starch in distilled water (100 mL). A combination of 200 μL of FFM stock solution, 200 μL of α-amylase stock solution and 200 μL of corn starch solution was incubated in a water bath at 30 °C for 15 min. In addition, 3,5-dinitrosalicylic acid was added to the mixture and cooked for 10 min in a water bath at 85–90 °C. After allowing the mixture to cool to room temperature, 5 mL of distilled water was added. The blank solution was created by substituting the plant portion with 200 μL of the buffer. Acarbose was employed as a positive control, and absorbance was measured at 540 nm using a Spectrophotometer (UV-Vis). The α-amylase inhibitory concentrations were estimated using the following equation:25 mg of FFM was dissolved in 10% DMSO (25 mL). The solution was diluted using 0.02 M sodium phosphate buffer (pH 6.9) to generate the following dilutions: 10, 50, 70, 100 and 50 μg/mL. In parallel, a working solution of the rcentage .α-glucosidase solution (1 unit/mL) were then mixed with 50 μL of phosphate buffer and incubated in a water bath at 37 °C for 15 min. Thereafter, 20 μL of P-NPG solution was added to the mixture and incubated for 20 min at 37 °C before terminating the reaction with 50 μL of 0.1 M Na2CO3. The blank solution was prepared by replacing the FFM solution with phosphate buffer. Acarbose was employed as a positive control, and absorbance was measured at 405 nm using a UV-Vis spectrophotometer. The α-glucosidase inhibitory activity was calculated using the following formula:α-glucosidase [The stock solution was prepared by dissolving a portion of the FFM (1 mg) in phosphate buffer (1 mL). The resulting solution was diluted with phosphate buffer to achieve different concentrations . Twenty microliters of the stock solution and cosidase .A 1 mg/mL working solution was made by dissolving 100 mg of FFM in 100 mL of methanol, which was then diluted with methanol to yield serial concentrations . Methanol (1 mL) was added to the FFM stock solution (1 mL), followed by DPPH solution (1 mL), and the combination was incubated for 30 min at room temperature under the light exclusion. The blank solution was prepared by replacing the plant fraction solution with methanol. Trolox was employed as a positive control, and absorbance was measured at 517 nm using a UV-Vis spectrophotometer and compared to the positive control. The DPPH scavenging activity was calculated using the following equation:activity .In a beaker, 100 mg of FFM was dissolved in 10% DMSO to achieve a series of concentrations . Right before use, a lipase enzyme stock solution (1 mg/mL) was produced by dissolving lipase enzyme powder (25 mg) in 10% DMSO (25 mL). The p-nitrophenyl butyrate (PNPB) stock solution was prepared by dissolving PNPB (20.9 mg) in acetonitrile (2 mL). Then, 0.2 mL of each of the FFM serial dilutions was mixed with lipase enzyme stock solution (0.1 mL) and Tris-HCl solution was added to the mixture to bring the total volume to 1 mL. After 15 min of incubation at 37 °C in a water bath, a 100 μL of PNPB solution was added and the mixture was incubated for 30 min at 37°C. The blank solution was prepared by mixing 100 μL of the lipase enzyme solution (1 mg/mL) with a Tris-HCl solution (0.9 mL). Orlistat was used as a positive control. The absorbance was measured using a spectrophotometer (UV-Vis) at 405 nm. Lipase enzyme inhibitory percentage was calculated employing the next equation :I (%) = Staphylococcus aureus (ATCC 25923), Enterococcus faecium (ATCC 700221) and Methicillin-Resistant Staphylococcus aureus  belong to Gram-positive strains. In addition to Shigella sonnie (ATCC 25931), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 13883) and Escherichia coli (ATCC 25922), which are classified as Gram-negative species.The bacterial and fungal strains used in the current investigation were obtained from the American Type Culture Collection (ATCC) and clinical isolates. Epidermatophyton floccosum (ATCC 10231) and Candida albicans (ATCC 90028) were also evaluated. The antimicrobial activity of the FFM against fungal and bacterial strains was evaluated using the microdilution technique. Mueller–Hinton broth (8.8 g) was autoclaved and kept at 4 °C after 400 mL of distilled water was added. The extract was first dissolved in sterile distilled water at a concentration of 1 mg/mL. In each well of the 96-well microdilution plate, 100 µL of the prepared broth was distributed; subsequently, serial dilution of the extract solution was performed obtaining concentrations ranging from 500 to 0.5 µg/mL. Well number 12 was left free from the plant material and was considered a control for microbial growth. A fresh bacterial colony was picked from an overnight agar culture and was prepared to match the turbidity of the 0.5 McFarland standard to provide a bacterial suspension of 1.5 × 108 colony forming units (CFU)/mL. This suspension was diluted with broth by a ratio of 1:3 to reach 5 × 107 CFU. Then, 1 µL of the bacterial suspension was added to all of the wells except well number 11, which was considered as a negative control for microbial growth. The plate was then incubated at 35 °C for 18 h.The effect of the FFM against the fungal strains including C. albicans was performed as described above, except that after matching the yeast suspension with the McFarland standard, which was diluted with NaCl (0.9%) by a ratio of 1:50, followed by 1:20, and 100 µL was placed in the wells. The plate was incubated for 48 h instead of 18 h.The microdilution method for the yeast Epidermatophyton floccosum. Sabouraud dextrose agar (1 mL) was placed in each test tube and kept at 40 °C in a water bath. Then, FFM (1 mL) was mixed with Sabouraud dextrose agar (1 mL) in the first tube and serial dilutions were performed in six tubes. Tube number 6 was kept free of any plant material and was considered as a negative control for microbial growth.The agar dilution method was carried out to investigate the antimold activity of FFM against After the Sabouraud dextrose agar was solidified, the spores of the mold culture were dissolved in distilled water containing 0.05% Tween 80 and scratched from the plate for comparison with McFarland turbidity. Twenty microliters of the obtained suspension was pipetted into all of the tubes except tube number 5, which was considered as a negative control. The tubes were incubated at 25 °C for 14 days .In the current antimicrobial experiments, ampicillin, ciprofloxacin and fluconazole, (powder purity ≥98%), which all obtained from Sigma-Aldrich (Germany), were utilized as positive controls and prepared via the above-mentioned method. Each experimental work was repeated in triplicates.t-test statistical calculations conducted with the SPSS version 21.0 software. When comparing samples, differences were considered statistically significant when p < 0.05.The conducted experiments were determined in triplicates. The results are presented as means ± standard deviation (SD). GLUT4-myc translocation results are presented as the mean ± SEM. The data were analyzed using M. autumnalis ripe fruit flavonoid fraction and evaluated its antidiabetic, DPPH scavenging, antiobesity, cytotoxic and antimicrobial effects. Four flavonoids were isolated and characterized from the flavonoids fraction, namely kaempferol 1, luteolin 2, myricetin 3 and (+)-taxifolin 4. The results indicate that FFM dramatically increased the amount of GLUT4 in the PM in both the presence and the absence of insulin. These findings suggest that FFM has anti-diabetic properties. Furthermore, FFM revealed α-amylase and α-glucosidase inhibitory characteristics and was shown to have strong DPPH scavenging and porcine pancreatic lipase inhibitory activities. In addition, FFM inhibited the growth of all of the tested bacterial and fungal strains and showed the greatest antibacterial activity against the K. pneumoniae strain. Moreover, the cytotoxicity screening showed that FFM is found to be safe up to 1 mg/mL. Future plans are required to confirm these very important outcomes by in vivo trials and to design suitable formulations to be used in the pharmaceutical and food supplement industries.The current investigation documented for the first time the chemical components of


O-glycan cores.Glycan microarrays have played important roles in detection and specificity assignment of glycan recognition by proteins. However, the size and diversity of glycan libraries in current microarray systems are small compared to estimated glycomes, and these may lead to missed detection or incomplete assignment. For microarray construction, covalent and noncovalent immobilization are the two types of methods used, but a direct comparison of results from the two platforms is required. Here we develop a chemical strategy to prepare lipid-linked probes from both naturally derived aldehyde-terminating and synthetic amino-terminating glycans that addresses the two aspects: expansion of sequence-defined glycan libraries and comparison of the two platforms. We demonstrate the specific recognition by plant and mammalian lectins, carbohydrate-binding modules and antibodies and the overall similarities from the two platforms. Our results provide new knowledge on unique glycan-binding specificities for the immune receptor Dectin-1 toward β-glucans and the interaction of rotavirus P[19] adhesive protein with mucin  Recognition of glycans by proteins is crucial to understand molecular mechanisms in health and disease. Carbohydrate microarrays, including those of polysaccharides  and sequNatural glycans cannot be arrayed directly due mainly to their highly hydrophilic nature and the incompatibility of the functional groups of carbohydrate molecules with readily available microarray slides. Various approaches were developed to convert glycans into forms suitable for printing and immobilization on different surface-modified glass slides used for arrays of nucleic acids and proteins. As carbohydrate molecules cannot be cloned, their isolation from natural glycome sources  or synthAlthough debatable, it has been estimated that there are 100,000–500,000 glycan structures in the mammalian glycome  present Given the different ways of constructing glycan microarrays and the vast diversity of carbohydrate molecules with different structural and chemical/physical properties over nucleic acids and proteins, careful comparisons across different platforms are necessary for widening the scope and future use of microarrays in diverse applications and in deriving glycan-binding specificities with confidence. In light of this, there have been studies comparing glycan-binding profiles obtained with microarrays that use different chemistries for glycan derivatization, glycan linker types, glycan probe densities and modes of presentation . HoweverThe present work has aimed to address the two aspects: the need to expand libraries of sequence-defined glycans and for comparison of two of the major microarray platforms, by developing a new lipid reagent, which is suitable for preparation of NGL probes from amino-terminating glycans. This opens the way for NGL microarrays to be sourced from both naturally derived aldehyde-terminating and synthetic amino-terminating glycans, and therefore expansion of the microarray coverage. It is now also possible to compare the two platforms using the very same amino-terminating glycans as probes for both covalent and noncovalent microarrays after their conversion into NGLs . Althougsn-glycero-3-phosphoethanolamine (DHPE) is used to conjugate aldehyde-terminating glycans by reductive amination to form NGLs -DHPE (DHPC) (A carboxyl-terminating lipid was designed for amide condensation with amino-terminating sugars. The amino group of DHPE reacted with succinic anhydride to form E (DHPC) , which cN-hydroxybenzotriazole (HOBT), leaving the free aldehyde for linking to amino-terminating glycans by reductive amination. The aldehyde-terminating lipid product N-(4-formylbenzamide)-DHPE (DHPA) was obtained only in a moderate yield (63%) due mainly to the poor solubility of DHPE in the reaction solvent (dichloromethane or chloroform).An aldehyde-terminating lipid was also designed by reaction of DHPE and hetero-bifunctional 4-carboxybenzaldehyde . The car2CH2-NH2 , was used as the model sugar.For assessing the use of the two lipid reagents in preparation of NGLs the aminoethyl glycoside of galactose, Galβ-O-CH2 via amide condensation was carried out in the presence of the activation reagents EDC and NHS , two lipids (DA2) and methylated single lipid (DA1 + Me), as revealed by HPTLC . The conjugation efficiency was thereby increased to ~50% (data not shown).HPTLC analysis showed that the yield of these products (including NGLs with single and double lipid and methylated) was greater than 80% after 6–24 h for most of the oligosaccharides used in this work . ConjugaAleuria aurantia lectin (AAL), Ulex europeus agglutinin (UEA-1) and the anti-blood group H type 1 and H type 2 antibodies; the β-galactoside specific Ricinus communis agglutinin I (RCA120); the core 1 specific peanut agglutinin (PNA); the α-GalNAc-specific proteins, human macrophage galactose-type lectin (MGL), Vicia villosa lectin (VVL) and Helix pomatia agglutinin (HPA); and the O-β-GlcNAc-specific antibody CTD110.6  with degree-of-polymerization (DP) of 12, 13 and 15  with known specificities . The micThe GalNAcα-Ser/Thr (Tn antigen) specific lectins human MGL , VVL/VVAThe α2,6-sialyl-Tn antigen has been identified as a ligand for human Siglec-15 . Here, hO-GlcNAc mAb CTD110.6, showed strong binding to GlcNAcβ-Ser/Thr , respectively , GlcNAc1-β6/β3- terminating core 4 (#47), and α2,6-sialyl-Tn-Ser/Thr . The fucose-specific lectin AAL and the anti-blood group H type 1 and type 2 antibodies showed the specific binding to the terminal fucosylated DA-NGL probes .The terminal Manα1-, Galβ1- and GlcNAcβ1,4-probes also elicited specific binding signals with the plant lectins Concanavalin A (ConA), RCAectively . AdditioL probes . AAL alsCmCBM6–2 bound with similar intensities to the linear β1,3-glucan probes with DP12 and DP15 (#53 and #54) and to the branched β1,3/1,6-glucan DP13 (#55) , GlcNAcβ1-O-Ser (#26), and GlcNAcβ1-terminating core 2 , core 3 (#41) and core 4 .The two bacterial CBMs showed different binding profiles in the DA-NGL microarrays, in accord with their specificities toward β1,3-glucans  and topo13 (#55) , whereasar DP 12 , compareAs the glycan probe repertoire in the NGL microarray system has now been increased to include synthetic amino-terminating glycans, it allowed a more comprehensive study and better understanding of glycan–protein interactions than in the previous investigations using conventional NGL arrays as exemplified by the mammalian immune receptor Dectin-1  and the Dectin-1 interaction with glucan polysaccharides is highly specific for backbone sequences of β1,3-linked glucose with a minimum chain length of DP10 , 2015. TO-glycan cores and the blood group H type 1 sequence  and H type 1 histo-blood group antigen (HBGA) precursors using different types of microarrays  and only sporadically humans. It is postulated that the P[19] genotype may represent an early evolutionary stage that started adapting to human receptors but retaining the binding specificities to the short chain mucin cores 2 and 4, and also H type 1 HBGAs. A more recent study using microarrays of O-glycans isolated from a porcine mucin by the beam search strategy  was from Acros Organics . 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), N-hydroxybenzotriazole (HOBT) and N, N-diisopropylethylamine (DIPEA) were from Aladdin . All other chemicals were analytical reagent grade. HPTLC plates were from Merck . Column chromatography was conducted by elution of a column of silica gel (200–300 mesh). 1H and 13C NMR spectra were recorded with a JEOL JNM-ECP-600 (600/150 MHz) or an Agilent DD2–500 (500/125 MHz) instrument. Chemical shifts were reported on the δ scale. CDCl3 (δ = 7.26 ppm) or tetramethylsilane (δ = 0.00 ppm) was used as an internal reference. ESI-MS of synthetic amino-terminating saccharides were carried out on a Waters Q-TOF-type mass spectrometer .DHPE was from Fluka . Sodium cyanoborohydride –C3–NH2, and methylamino-terminating sugars Glc–C3–NHMe, Gal–C3–NHMe and Lac–C2–NHMe is described in Supplementary Methods. The glycine-terminating glycans as N-glycosides were prepared by conjugation of the respective reducing sugars with glycine as described (2 and Glc13(B10)-C15-NH2 were synthesized using solid-phase chemistry by automated carbohydrate synthesizer as described , and Core 1-Ser was from Dextra . Fully protected, Core 1-Thr and Core 4-Thr were from Sussex Research  and used after deprotection.Glc15-C2-NH3/MeOH, 6:1). The mixture was then concentrated before dilution with hydrochloric acid (15 mL). The reaction product was extracted with CHCl3 (10 mL× 3). The combined organic extracts were dried with Na2SO4, and concentrated to afford compound DHPC  as a white solid. ESI-MS: calculated for C41H81NO9P [M-H]− 762.5649, found m/z 762.5682.DHPE  was dissolved in 8 mL tetrahydrofuran, followed by addition of succinic anhydride  and triethylamine . The reaction mixture was stirred for 2 hr at 50°C. The completion of the reaction was indicated by TLC analysis , EDC , HOBT  and DHPE  were sequentially added at 0°C. The reaction mixture was stirred for 30 min, followed by 24 h at room temperature. HPTLC of an aliquot of the reaction mixture revealed a major product, Rf 0.3 , visualized under 254 nm UV light or under 365 nm UV light after primulin staining. The mixture was concentrated, and the residue was purified by silica gel chromatography  to give DHPA  as a white solid. 1H NMR : δ 10.03 , 8.02–7.99 , 7.94–7.91 , 7.48 , 4.04–3.98 , 3.89–3.83 , 3.65–3.60 , 3.56–3.46 , 3.43–3.36 , 1.53–1.46 , 1.30–1.16 , 0.84 ; 13C NMR : δ 192.19, 167.47, 138.91, 138.16, 71.69, 70.57, 70.08, 64.97, 63.79, 40.84, 31.79, 29.81, 29.56, 29.52, 29.51, 29.47, 29.39, 29.22, 25.95, 25.89, 22.52 and 13.73. HR ESI-MS: calcd. For C45H81NO8P [M-H]− 794.5700, found m/z 794.5765.4-Carboxybenzaldehyde  was dissolved in 10 mL CH3CN . For example, 200 nmol DHPA  was added to 20 nmol dried saccharide in a glass microvial. The mixture was evaporated to dryness under a nitrogen stream and dissolved in 50–100 μL CHCl3/MeOH or CHCl3/EtOH (1:1). The mixture was incubated at 60°C for 6–24 h. For the aromatic amine-functionalized glycans (Hep-4-NS-PhNH2 and Hep-4-NAc-PhNH2), incubation was conducted at 80°C for 48 hr. Aliquots of the reaction mixtures were analyzed by HPTLC using aluminum-backed silica gel plate (Merck) and solvent system CHCl3/MeOH/H2O (65:35:8) with primulin and orcinol staining  were incubated with 10 equivalents of DHPA and 15 equivalents of reducing reagent NaBHstaining . Aiming 3/MeOH/H2O (25:25:8) until analysis.DA-NGLs were isolated from reaction mixtures by semipreparative TLC or silica cartridge  as described . Purifie3/MeOH/H2O (25:25:8) at ~10 pmol/μl and 0.5–1 μL was deposited on the sample target together with 1 μL of matrix of 2′,4′,6′-trihydroxyacetophenon. For MS the laser energy at an attenuation scale 80 and for CID-MS/MS a collision gas Ar (2 bar) and collision energy at 80–140, depending on specific samples, were used.MALDI-MS of the DHPA-NGLs was carried out on an AXIMA Assurance linear TOF instrument (Shimadzu) and MALDI-CID-MS/MS on an AXIMA Resonance QIT-TOF instrument (Shimadzu). NGLs were dissolved in a solvent of CHClInformation on the glycan probes, generation of the microarrays, imaging and data analysis are described in the Supplementary MIRAGE  documentFor construction and validation of the noncovalent microarrays the 60 DHPA-NGLs derived from amino-terminating sugars with single lipid tail  were useFor construction of the microarrays via covalent immobilization, 46 of the amino-terminating sugars  were priThe protein samples analyzed, their preparation, concentration and detection antibodies are detailed in 2 and 2 mM MgCl2 (3% BSA/TBS-Ca-Mg), followed by incubation for 2 h with the different lectin solutions, prepared in the binding buffer (1% w/v BSA in TBS-Ca-Mg).The plant lectins were analyzed using a single-step overlay protocol for biotin-tagged samples. In brief, the DA-NGL subarrays were blocked with 3% bovine serum albumin  in 20 mM Tris–HCl pH 8.5, 150 mM NaCl, 2 mM CaClThe antibodies were analyzed using specific biotinylated secondary antibodies for detection. In brief, after blocking with 3% BSA in 100 mM phosphate-bufferred saline pH 7.4 (3%BSA in PBS), the microarrays were probed for 2 h with the solutions of the antibodies prepared in binding buffer (1% BSA in PBS), followed by incubation for 1 h with 10 μg/ml biotinylated anti-mouse IgG in the binding buffer.The His-tagged CBMs were analyzed at a final concentration of 10 μg/mL pre-complexed with mouse monoclonal anti-poly-histidine (Ab1) and biotinylated anti-mouse IgG (Ab2) antibodies, both from Sigma, at a ratio of 1:3:3 (by weight). While blocking with 3% BSA in TBS-Ca-Mg, the CBM-antibody complexes were prepared by preincubating Ab1 with Ab2 for 15 min at ambient temperature, followed by addition of CBMs, incubation for a further 15 min, and diluted in the binding buffer (1% BSA in TBS-Ca-Mg) for microarray overlay. The binding of His-tagged murine Dectin-1 was detected following a similar procedure but without pre-complexing the protein with the detection antibodies: after overlaying with the Dectin-1 solution (30 μg/mL in 1% BSA in TBS-Ca-Mg) for 2 h, the slides were incubated for 1 h with the antibody pre-complexes at a final concentration of 10 μg/mL in the binding buffer.The human Fc-tagged MGL was analyzed as a pre-complex with the biotinylated anti-human IgG (Vector) detection antibody . While blocking the slides with 3% BSA in TBS-Ca-Mg, the MGL-antibody complex was prepared by preincubating the protein and antibody for 1 h at 4°C and diluted at a final concentration of 10 μg/mL in the binding buffer (1% BSA in TBS-Ca-Mg) for microarray overlay. For analysis of Fc-tagged Siglec-15 two conditions were used: 1) the arrayed slides were blocked with 2%BSA in PBS and the protein was analyzed at 2 μg/mL as a pre-complex with the biotinylated anti-human IgG detection antibody , which was prepared by preincubating the protein and antibody, diluted in the final required volume of blocking buffer, for 1 h at 4°C; and 2) the arrayed slides were blocked with 3%BSA in PBS and the protein was analyzed at 20 μg/mL in the binding buffer (1%BSA in PBS) followed by detection with biotinylated anti-human IgG.2. The protein was analyzed at 50 μg/ml in the blocking buffer, followed by detection with rabbit anti-GST polyclonal antibody (Santa Cruz) and biotinylated anti-rabbit IgG (Sigma), both at 1:200 in the blocking buffer.For analysis of the GST-tagged P[19] VP8* viral protein, the slides were blocked with 0.02% casein (Pierce) and 1% BSA in HBS  with 5 mM CaCl2, 2 mM MgCl2, 0.05% Tween 20).The analysis of protein binding in covalent microarrays was carried out using the same procedures and buffer systems described above for the different proteins, but with the two differences: 1) omission of the 1 h blocking step and 2) addition of 0.05% Tween 20 to the binding and washing buffers. One exception was the GST-tagged P[19] VP8* protein that was analyzed in a binding buffer without casein  was used for fluorescence readout. Imaging and data analysis were essentially as described . The parW.C. conceived the project. C.L. designed the chemical strategies and A.S.P. designed and performed microarray binding experiments and carried out data analysis. P.Z. carried out the synthesis of the novel lipid reagents and related glycan standards. C.G., Z.L. and F.T. performed microarray binding experiments and contributed to data analysis. W.C. and Y.Z. carried out analysis of glycan probes and L.M.S. performed microarray printing. M.W., P.H.S., L.M.L., V.P., J.Y. and U.W. contributed to the key glycan probes. W.C., A.S.P. and C.L. wrote the paper and P.Z., J.Y. and U.W. wrote the synthetic part. All co-authors edited and approved the manuscript.DHPA-Supporting_Information-revised_cwab037Click here for additional data file.


Scientific Reports 10.1038/s41598-020-64664-3, published online 12 May 2020Correction to The original version of this Article contained errors in the qPCR analysis of the gene expression levels and mitochondrial DNA content. The calculations were based on the incorrect assumption that the amplification efficiency was 100%. The Authors have now reanalysed the data using the actual values for amplification efficiency , and found that the p-values marked by asterisks in panel (b) of Figure The description of the statistical significance annotations in these figures has been corrected in the figure legends, where in the legend of Figure “Data are presented as mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001, n = 3.”now reads:“Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3.”In the legend of Figure “Data are presented as mean ± SD. **P < 0.01, ***P < 0.001, n = 3.”now reads:“Data are presented as mean ± SD. **P < 0.01, n = 3.”Figures Additionally, in the Results and discussion section under the subheading ‘SAS tumour spheres with stem cell-like features showed increased expressions of metabolism-associated and pluripotency genes.’,OCT4 (P = 0.03) and SOX2 (P = 0.0004), and the proteins they encode  were confirmed by qPCR  and SOX2 (P = 0.052), and the proteins they encode  were confirmed by qPCR . The raw data from the microarray analysis were deposited in Gene Expression Omnibus . The raw data for the other analyses are provided as supplementary datasets.”Finally, Supplementary Tables S6 and S7 and Supplementary Datasets S4 and S6 have been corrected to include the values for amplification efficiency.The original Supplementary Information file which includes Tables S6 and S7, and the original Supplementary Datasets S4 and S6 are provided below.The original Article has been corrected.Supplementary Information.Dataset S4.Dataset S6.