Our study addressed the cytotoxic effects of different ...



Aqueous Cold Water Extracted of Shiitake Culinary-Medicinal Mushroom, Lentinus edodes (Agaricomycetes) Cross Flow Ultra Filtration Fractions Exhibit Apoptosis in Tumor CellsMiriam Sari,1* Katharina Toepler,2 Anna Nickisch-Hartfiel,3 Nicole Teusch,2 & Reinhard Hambitzer11Competence Center for Applied Mycology and Environmental Studies, Niederrhein University of Applied Sciences, Rheydter Str. 277, 41065 M?nchengladbach, Germany; 2Bio-Pharmaceutical Chemistry & Molecular Pharmacology, Technische Hochschule Koeln, Kaiser-Wilhelm-Allee, Chempark, 51368 Leverkusen, Germany; 3Department of Chemistry, Niederrhein University of Applied Sciences, Krefeld, Germany;*Address all correspondence to: Miriam Sari, 1Competence Center for Applied Mycology and Environmental Studies, Department of Food Science and Nutrition, Niederrhein University of Applied Science, Rheydter Str. 277, 41065 Moenchengladbach Germany; Miriam.sari@hs-niederrhein.deSHORT TITLE: Tumor Cell Cytotoxicity by Extract from Lentinus edodesABSTRACT: Lentinus edodes (shiitake) ranks among of the most well-known medicinal mushrooms worldwide. Immune modulating effects of extracts from Shiitake have been widely demonstrated in animals and humans. Apart from providing highly purified compounds like lentinan, the crude mushroom extracts also show antitumor activities. Especially the direct cytotoxicity of crude preparations is still to investigate. Our study’s focus lies on the molecular weight cut off distribution of beta-glucans and proteins in a cold water based extract from L. edodes. Cross flow ultrafiltration was applied to obtain six fractions of different molecular size. Beta-glucan content as well as protein content were quantified. We were able to show that only small amounts of beta-glucans were extracted and the protein content is decreasing with fraction size. The cytotoxic potential of the cold water preparation was demonstrated through analyzing growth inhibition of non-small cell lung cancer as well as triple negative breast cancer cells. KEY WORDS: beta-glucans, extract, ultrafiltration, tumor cell growth, breast cancer, non-small cell lung cancer, MDA-MB-231, A549, cytotoxicity, Lentinus edodes, cold water extraction, medicinal mushrooms ABBREVIATIONS: dm, dry mass; GOPOD, glucose oxidase/peroxidase; kDa, Kilodalton; KOH, potassium hydroxide; LEM, Lentinula edodes mycelia; MWCO, molecular weight cut off; UF, ultra filtration; U/mL, units per milliliterI. INTRODUCTIONCancer has become one of the main public health issues and represents the second cause of death worldwide. Notably, most cancer patients finally die of metastasis and recurrence, mostly due to the side effects and resistance of currently available chemotherapy. Therefore, there is a high unmet need to identify novel strategies for cancer prevention as well as to develop drugs providing high efficiency, with low side effects for cancer treatment.Shiitake, Lentinus edodes (Berk.) Singer (= Lentinula edodes; Marasmiaceae, Agaricomycetes) is also known as black forest mushroom or oak mushroom and is a popular culinary and medicinal species.1 L. edodes naturally occurs in various southeastern countries like China, Japan, Korea, Vietnam, Thailand, Burma, North Borneo, the Philippins, Taiwan and Papua New Guinea but is cultivated anywhere in the world.2,3 However, L. edodes has a long history in culinary and medicinal usage in Asia. For years the mushroom has become the focus of international medicinal research and consequently is one of the most studied mushrooms of our time, ranking third among the world’s most cultivated mushrooms.4,5L. edodes is a very promising natural resource for the prevention of clinically diagnosed tumors as well as the development for drugs.6 Its two most important preparations are LEM and lentinan. LEM is a water-soluble extract from the powdered mycelia extract containing protein-bound polysaccharides, nucleic acids and different physiological compounds.7,8 Lentinan is a highly purified, high molecular weight polysaccharide (?-1-3,1-6-glucan).4,9,10 Lentinan was first isolated by Chihara et al.6,11-13 and reported to provide antitumor properties against Sarcoma-180 also in addition to various synergic and autochthonous tumors after i.v. injection or oral administration. Notably, various studies suggest that Lentinan presumably works by modulating the immune system rather than attacking cancer cells directly.4,7,14-16 Furthermore, recent data demonstrate improvement of host immune function of cancer patients undergoing adjuvant chemotherapy.17 However, it is still a matter of debate, which molecular ingredients or combination of ingredients modulates the immune responses in particular. In contrast to the described immune modulating effects of polysaccharides, several studies have demonstrated that mushroom extracts contain other biomolecules (non-polysaccharides) displayed direct cytotoxicity to tumor cells in animals and humans.18 Molecules from mushroom extracts like phenolic compounds, sterols, alkaloids, lactones, terpenes or ceramides are also potential anticancer compounds that have to be studied further.19,20 Current debate entails the question if the whole extract from mushrooms may be more beneficial than only isolated molecules, as in purified substances like lentinan or other beta-glucans or their combinations with proteins.10,14,19 Beta-glucans linked to proteins seem to demonstrate stronger anticancer activities then free glucans.18 In this context, especially the anti-tumor activity of crude mushroom extracts could be shown in several in vitro studies. They have been reported to be capable of inducing apoptosis via caspase activation in hepatocellular carcinoma, skin epidormoid carcinoma, cervix epithelial adenocarcinoma as well as by inhibition of tumor specific telomerase activity in breast cancer cells.20-23 Different extract preparations, especially from L. edodes expressed anti-tumor activity: Hot water extracts from mycelia and fruiting bodies have been demonstrated to inhibit the growth of MCF-7 breast cancer cells.24,25 Furthermore, a hot water/ethanol extract from L. edodes mycelia induced apoptosis on HepG2 cells.26 An aqueous L. edodes extract increased apoptosis in U937 monocytic cells.27 In addition, Finimundy et al.28 demonstrated anti-tumor activity of water extracted fruiting bodies at low extraction temperatures in HepG2 and HeLa cells. Jiang and Silva29 confirmed effects of a mycelia mushroom blend on cell viability of the highly metastatic triple-negative breast cancer cell line MDA-MB-231 cells.Despite these promising initial results, the pharmacological mechanism of action of crude extracts derived from L. edodes still remains elusive. Thus, the main objective of our study was to develop a novel and economical extraction method with cold water (at 4°C) to develop fast and effective steps for purification process by ultra filtration and to establish a standardized method. Thus far, most extract preparations from mushrooms have been performed using hot water or other solvents like methanol/ethanol or other chemicals.22,29-32 Apart from that, most experiments use the entire extract while we chose the separation of the cold water extract into six fractions according to their molecular size, in order to obtain further accurate information about the effectiveness of single fractions. Since there is no standard method for the determination of the quantitative content of beta-glucans in mushroom extracts, the phenol-sulfuric-acid method (anthron reaction method) is regularly used for polysaccharide determination to date.33 However, since this method does not distinguish between shape or linking of o-glycosidic-bonds and displays only crude information about actual polysaccharide content, we used an enzyme based test kit by Megazyme Ltd. (Wicklow, Ireland) to determine the beta-glucan content. The method was previously described and is expected to deliver reliable results for beta-glucan content determination in mushrooms and mushroom preparations.34-38 Our study addressed the cytotoxic effects of different cold-water L. edodes fractions in a human breast cancer cell line MDA-MB-231 as well as in a non-small cell lung cancer (NSCLC) cell line A549 to further explore the putative mechanism of action of different extracts with respect to their molecular sizes. As previous studies showed beta-glucans with higher MWCO seem to be more efficacious than those with lower ones. The focus on cell growth inhibition tests was particularly important.20,39,40 As breast cancer is the most common solid tumor entity in females worldwide and lung carcinoma, especially non-small cell lung cancer, represents the most common tumor entity worldwide in males combined with a lack of efficient treatment strategies 41 our study specifically focusses on the effect of L. edodes fractions on the mentioned tumor entities based on the high unmet medical need.II. MATERIAL AND METHODSMushroom and Extraction 250 g lyophilisated L. edodes (fresh mushroom fruiting bodies) were harvested from substrate blocks from CNC exotic mushrooms, Hedel, Netherlands), The dried mushroom cuts (3-4 cm cuts) were extracted under stirring for 30 minutes with 5 L cold distilled water (4°C). The liquid extract was collected and the mushroom cuts were extracted twice again with 2.5 L of fresh distilled water. Thus, a complete volume of 10 L cold distilled water was used for the extraction. The yield of 7.261 L (residued water was absorbed by mushroom cut) liquid extract was pre-filtered (8-12 ?m filter paper, No. 424, VWR, Darmstadt, Germany).Cross Flow Ultra FiltrationThe prefiltered extract was ultrafiltrated with a Sartoflow? Slice 200 Benchtop crossflow system (Sartorius Stedim Biotech, G?ttingen, Germany). Five different membranes (Sartocon? slice 200 cassettes, Sartorius Stedim Biotech, Germany) with a MWCO of 300, 100, 30, 10 and 5 kDa were used. The prefiltered L. edodes extract was first filtrated with the largest filter slice (300 kDa). The obtained permeate passing through the 300 kDa MWCO was subjected to the membrane with the MWCO of 100 kDa, 30 kDa, 10 kDa and 5 kDa subsequently. The resulting retentates with >300 kDa, 100-300 kDa, 30-100 kDa, 10-30 kDa, 5-10 kDa and <5 kDa cut off were lyophilized and stored at -26°C (Fig. 1).Detection of Beta-Glucan ContentThe 1,3-1,6-beta-glucans were determined in quadruplicate using an assay kit (Megazyme Ltd., Wicklow, Ireland)41 according to the manufacturer’s instructions. For determination of total glucan content, dried and milled mushroom samples were mixed with 2 mL of sulphuric acid (12 M). After leaving the tubes in an ice-water bath for 2 h, 10 mL of distilled water was added and samples were incubated in a boiling water bath for 2 h. After a neutralization step with 10 M KOH, the samples were adjusted to 100 mL with a sodium acetate buffer (pH 5.0). An aliquot of each probe was centrifuged at 1.500 g for 10 min and was mixed with exo-1,3-beta-glucanase (20 U/mL) and beta-glucosidase (4 U/mL) and incubated in a water bath at 40°C for 60 min. 3 mL of glucose-peroxidase-reagent (GOPOD) was added and again incubated at 40°C for 20 min. For the determination of alpha-glucan content, dried extract samples were stirred with 2 mL of KOH (2 M) in an ice water bath for 20 min. After adding 8 mL of sodium acetate buffer (pH 3.8) and 0.2 mL of amyloglucosidase (1630 U/mL) and invertase (500 U/mL), the samples were incubated in a water bath at 40°C for 30 min. Aliquots were mixed with 0.1 mL of sodium acetate buffer (pH 5.0) and 3 mL of GOPOD. They were incubated again at 40°C for 20 min. The beta-glucan content of a yeast standard and an internal mushroom powder control were determined. All samples were measured photometrically at 510 nm (LKB Biochrom, Cambridge, England) against a reagent blank. In both steps, the total glucan/alpha-glucan content as well as the D-glucose in oligosaccharides, sucrose and free D-glucose content are measured. The beta-glucan content is determined by subtracting the alpha-glucan content from the total glucan content. Standard errors of approximately < 5% are achieved routinely.Determination of Protein ConcentrationProtein concentration in each fraction was determined according to the Bradford method43 using a protein assay standard kit (Bovine Gamma Globulin for calibration) and quick start dye reagent from Bio-Rad (Hercules USA). 2 mL of Quick Start Bradford Dye reagent was mixed with 40 ?L of sample material, incubated for 15 min and measured at 595 nm against a blank. Samples were determined in triplicates. Cell Viability AssayCell CulturesThe human tumour cell line A549 (lung adenocarcinoma) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), the cell line MDA-MB-231 (breast adenocarcinoma) was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). A549 cells were cultivated in DMEM medium (Gibco, ThermoFischer, Darmstadt, Germany) supplemented with 10% heat inactivated fetal calf serum, 100 U/mL penicillin and 100 ?g/mL streptomycin. MDA-MB-231 cells were grown in Leibovitz?s L15 (Biochrom, Berlin, Germany) supplemented with 15% heat inactivated fetal calf serum, 2 mM L-Glutamine 100 U/mL penicillin and 100 ?g/mL streptomycin. All cell lines were maintained at 37°C, 5% CO2, subconfluent cells were passaged two times per week.Cell Viability AssayFor determination of the toxicity of the extracts, the cell viability was measured by quantitation of ATP with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. Cells were seeded at the desired concentration in white 384-well plates. After 24 h, 2 ?L of the test compounds were added, each concentration was tested in quadruples. After the desired incubation time of 72 h, the CellTiter-Glo reagent was added and the luminescence was measured with the Infinite M200 microplate reader (Tecan Group AG, Maennedorf, Switzerland). The cell seeding, addition of compounds and reagents were conducted with the CyBi-Well 96-channel simultaneous pipettor (Analytik-Jena AG, Jena, Germany). Data AnalysisData were obtained from three or more independent experiments performed in quadruples and represent mean ± SEM. Statistical data analysis was performed with equal sample values of a minimum of nine biological values using one-way ANOVA followed by Dunnett’s post hoc test in case groups were compared with a control. P < 0.05 was used to announce statistically significant difference. Curve fitting and statistical analysis was performed in GraphPad Prism v. 6.0 (GraphPad Software, San Diego, CA, USA) using nonlinear regression.Immunofluorescence Apoptosis StainingFor the apoptosis staining cells were seeded in 96-well plates (Ibidi, Martinsried, Germany). After 24 hrs the test compounds were added in cell culture medium. After incubating for 24 hrs cells were stained with Annexin V-FITC Apoptosis staining Detection Kit (Abcam, Cambridge, England) according to the manufacturer`s instruction and fixed with 4% formaldehyde. Every washing step was performed with Annexin V-FITC Binding Buffer. For staining cell nuclei, cells were incubated for 10 minutes at room temperature with 3 ?M 4′,6-Diamidin-2-phenylindol (DAPI, Sigma, Darmstadt, Germany). Cells were analyzed by fluorescence microscopy (AxioVert A1, Carl Zeiss, Jena, Germany). III. RESULTS AND DISCUSSIONExtraction and Ultra FiltrationIn this study a cold water based extract of L. edodes was produced at 4°C and divided into fractions with ultra filtration membranes. Six different fractions of different MWCO could be obtained. A total dry weight (extract weight) of 55.681 g could be obtained with the cold extraction method. The weight of the six fractions differ strongly. The fraction Sk1.6 (< 5 kDa) with very small molecules own about 70% of the weight of the total yield (39.639 g). The five fractions with bigger molecule size weighted between 4.108 g (Sk1.1 > 300 kDa) and 5.253 g (Sk1.5, 5-10 kDa). Thus, with the extraction process under cold temperatures (4°C) mostly small molecules are extracted (Fig. 2, table 1). Cross flow ultra filtration is a non-thermic method for simple fractionating and concentrating of aqueous extracts from mushrooms. The fractionation method due to different MWCOs was described previously and the application could be interesting for separation and further experiments, as well as the production of extracts with defined molecular sized fractions.44-46 Possible disadvantages could be the loss of filtrated material during the process. The prefiltration step (8-12 ?m filter) is necessary to avoid membrane-blocking. Our previous work demonstrated, that the separation of an aqueous extract from Grifola frondosa showed quiet definite fractions with specific molecular size.32Beta Glucan ContentTable 1 gives an overview about the dry weight of all fractions, the relative and total amounts of beta-glucans and protein content from cold extracted L. edodes. The total weight of beta-glucans in all fractions adds up to only 1.187 g which equals to about 2% beta-glucan content from dried extract mass (55.681 g). Thus very small amounts of beta-glucan were extracted with this method. The fractions of pure beta-glucans entirely extracted from the 250 g of mushrooms range between 0.008 g (Fraction Sk1.2, 100-300 kDa) and 0.165 g (Fraction Sk1.4, 10-30 kDa). Thus, most parts of the obtained extract contain small sized particles under 5 kDa. This fraction Sk1.6 (< 5 kDa, dry weight: 39.639 g) represents the largest part of the extract (about 70%) and contains 0.880 g of beta glucans in total. It also includes smaller molecules like sugars, salts, smaller peptides and oligo-saccharides. Sugars like D-glucose or sucrose or oligo-saccharides will be detected with the described method and due to subtracting alpha-glucan content from the total glucan content they will be eliminated in the calculation step. Table 2 shows the relative amounts of total-, alpha- and beta-glucans obtained from the six fractions. Fraction Sk1.4 with a molecular size of 10-30 kDa has the highest amounts of total-glucans (6.837 ± 0.253 g/100 g dm) and beta-glucans (4.880 ± 0.139 g/100 g dm). Fraction Sk1.2 (100-300 kDa) has the lowest amount of total-glucans (1.779 ± 0.446 g/100 g dm), alpha-glucans (1.185 ± 0.191 g/100 g dm) and beta-glucans (0.595 ± 0.591 g/100 g dm). The high alpha-glucan content of fraction Sk1.5 (5-10 kDa) seems unusually high for Shiitake mushroom extracts (3.518 ± 0.164 g/100 g dm) and represents 70% of the total glucan content in this fraction.Protein ContentThe protein content, determined through Bradford method, shows a difference of protein concentration in the six fractions (Figure 3, Table 1). As demonstrated, the largest fraction Sk1.1 of > 300 kDa has the highest relative content of proteins (25.060 ± 0.012 g/100 g) and the smallest fraction Sk1.6 with < 5 kDa presents the lowest content (0.015 ± 0.013 g/100 g). Thus the protein content declines with the molecular weight of the six different fractions. The total amount of protein (absolute) detected in all six fractions was 1.547 g. Cell Viability AssessmentCytotoxicity induction of the six fractions was evaluated in the non-small lung cancer cell line A549 at the concentrations 1 mg/mL, 0.33 mg/mL and 0.11 mg/mL, respectively (Fig. 4). Notably, the fractions Sk1.6, Sk1.5 and Sk1.4 did not display any statistically significant cytotoxicity at the selected concentration range. In contrast, for Sk1.2 a 55% reduction of cell viability compared to the untreated control cells was noted at a concentration of 1 mg/mL. Furthermore, Sk1.1 and Sk1.3 proved significant cytotoxicity at all concentrations tested: Sk1.1 reduced cell viability of the selected lung cancer cell line by 38% at 0.11 mg/mL and by 100% at 0.33 mg/mL and 1 mg/mL, respectively. For Sk1.3 a cell viability reduction of 55% was noted at 0.11 mg/mL. At a concentration of 0.33 mg/ml Sk1.3 induced cytotoxicity in 82% of the tumor cells and at 1 mg/mL 100% of the cells underwent cytotoxicity.To further evaluate the impact of the three most efficacious fractions on a second tumor entity, the fractions Sk1.1, Sk1.2 and Sk1.3 were subjected for cytotoxicity, assessed in the triple-negative breast cancer cell line MDA-MB-231 at the concentrations 1 mg/mL, 0.33 mg/mL and 0.11 mg/mL, respectively (Fig. 5). In line with the data obtained in non-small cell lung cancer cells, Sk1.1 reduced cell viability of the selected breast cancer cell line by 20% at 0.11 mg/mL, by 95% at 0.33 mg/mL and by 100% at 1 mg/mL, respectively. For Sk1.2 a 93% reduction of cell viability compared to the untreated control cells was noted at a concentration of 1 mg/mL. At a concentration of 0.33 mg/mL Sk1.2 reduced cell viability by 20%. Furthermore, Sk1.3 proved significant cytotoxicity at all concentrations tested: For Sk1.3 a cell viability reduction of 55% was noted at 0.11 mg/mL. At a concentration of 0.33 mg/mL Sk1.3 induced cytotoxicity in 92% of the breast cancer cells and at 1 mg/mL 100% of the cells underwent cytotoxicity. Test results of the fractions Sk1.6, Sk1.5 and Sk1.4 are available in the supplements.In previous studies, Finimundy et al.28 demonstrated that aqueous L. edodes extracts prepared with different temperatures exhibit different cytotoxic activities in Hep-2 and Hela cells. Interestingly, in accordance with our results extracts prepared at lower temperatures (4°C and 22°C) demonstrated stronger cytotoxic effects on tumor cells than extracts prepared at higher temperature (50°C). Fontana et al.46 could show similar results with cold water extracts from Pleurotus.In summary, the three fractions with higher molecular sizes exhibit significant toxicological effects on the selected cancer cell lines. Noteworthy, previous studies could confirm effective antitumor effects of high molecular beta-glucan fractions from mushroom preparations compared to smaller fractions.39,47-49 It is noteworthy, that cold water extract fractions might include active enzymes or other active substances, that could possibly express bioactive activities. E. Immunofluorescence Apoptosis StainingSeveral studies could prove the apoptotic effects of different mushroom preparations on cancer cells.10,18 Jiang and Sliva showed that a mycelia blend extract of different mushrooms exhibits apoptotic effects through the inhibition of cell proliferation and cell cycle arrest at the G2/M phase in highly invasive MDA-MB-231 breast cancer cells.28 Chien et al.21 could show apoptotic effects on hepatocellular carcinoma cells which were treated with ethanolic extracts from Taiwanofungus salmoneus mycelia. Atay et al.50 could demonstrate apoptotic effects of a Ganoderma lucidum ether extract on MCF-7 breast cancer cells. Furthermore, Sia et al.27 could show apoptosis in U937 monocytic cells by an aqueous L. edodes extract.Thus, to further explore the underlying mode-of-action, non-small cell lung cancer cells were analyzed in this study for apoptosis induction. Annexin staining revealed significant exposure upon treatment with extract Sk1.1 (1 mg/mL) compared to negative control indicating apoptosis induction. The apoptosis inducer staurosporine served as a positive control (Fig. 6). Since the three effective fractions in our study include glucans as well as proteins, apoptotic protein structures should also be considered. Recent, several studies showed that also certain proteins from L. edodes (strain C91-3) like Latcripin-3 and Latcripin-13 show apoptotic effects, but the mechanisms remain unknown. These proteins have molecular weights of about 45-47 kDa, which correspond with the size of Sk1.3, which should be further investigated in the future.51,52IV. CONCLUSIONSUnfortunately, in most cases crude extracts from mushrooms like L. edodes have not been further analyzed with respect to their quantitative content of beta-glucans or proteins. Our results give evidence that certain fractions from an aqueous cold water extract from L. edodes have high potential as anticancer compounds. In contrast to previous experimental set-ups where hot water or other chemicals are used to extract putative antitumor substances, our study shows that also low temperature extraction methods with water can lead to active fractions with significant effects against various tumor cell lines. Our extraction method leads to high yield of small molecular weight particles under 5 kDa (Sk1.6) rather than bigger molecules (5-300 kDa) and only small amounts of beta-glucans and proteins can be obtained with this method in all six fractions. Thus, rather small amounts of around 7.4 g total weight of highly active molecules (dry weight of fractions Sk1.1-Sk1.3) can be extracted from 250 g L. edodes mushroom with cold water extraction. Nevertheless, these fractions show high bioactivity effects on two cancer cell lines and should be investigated further. The results from this study clearly confirm the promising potential of a fractionated cold water extract from L. edodes as an innovative approach for cancer prevention. Cross flow ultra filtration and the enzymatic method for separation and quantitative determination of beta-glucans were proven to be reliable analytical methods.ACKNOWLEDGEMENTSThis study was supported by the Ministry for Innovation, Science and Research (MIWF) of the North Rhine-Westphalia state within the program FH-STRUKTUR 2016. (AZ: 322-8.03.04.02-FH-STRUKTUR 2016/01).REFERENCES1. Wasser SP. Shiitake (Lentinus edodes). In: Encyclopedia of Dietary Supplements. New York (USA): Marcel Dekkers. 2005.2. Kaneno R, Belik AV, Frantz FG, Lorenzo JS, Fontanari LM, Castoldi L, Dias BR, Barbisan AL, da Eira AF. Antitumor and immunomodulatory effects of culinary-medicinal Shiitake mushroom Lentinus edodes (Berk.) Singer: analysis of NK activity, lymphoproliferative response, and antibody production. 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Ann XH, Lun YZ, Zhang W, Liu B, Li XY, Zhong MT, Wang XL, Cao, Ning AH, Huang M. Expression and characterization of protein Latcripin-3, an antioxidant and antitumor molecule from Lentinula edodes C91-3. APJCP. 2014;15:5055-61.FIGURE LEGENDSFigure 1. Description of applied methodical steps of a cold water extraction and ultra filtration for obtaining six different fractions from a dried and lyophilizated Lentinus edodes extract. 250 g dried L. edodes cuts were extracted with 10 L deionized water at 4°C. The obtained liquid extract was prefiltered and ultra filtration process with five different membrane cut offs was implemented. Figure. 2. Dry weights in gram of six ultra filtration fraction obtained from 250 g dried Lentinus edodes mushrooms. Figure. 3. Relative amounts (g/100 g) of beta-glucan content and protein content present in the six different Lentinus edodes fractions obtained from ultra filtration process. All values are presented as mean of quadruples or triplicates with ± SD.Figure. 4. The cell viability of A549 after 72 hrs incubation with mushroom extract fractions was measured using CellTiter-Glo assay reagent (Promega). The data represents mean value ± SEM of three independent experiments performed in four biological replicates. They were normalized against the untreated cell control. Staurosporine was used as a positive control (50 ?M). Curve fitting and data analysis using ANOVA were perfomed in GraphPad Prism (version 6.05, GraphPad Software, San Diego, CA, USA). Statistical analysis were performed using one-way ANOVA relative to the respective value of the untreated cell control (ns, not statistically significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0,0001).Figure. 5.The cell viability of MDA-MB-231 after 72 hrs incubation with mushroom extract fractions was measured using CellTiter-Glo assay reagent (Promega). The data represents mean value ± SEM of three independent experiments performed in four biological replicates. They were normalized against the untreated cell control. Staurosporine was used as a positive control (50 ?M). Curve fitting and data analysis using ANOVA were performed in GraphPad Prism (version 6.05, GraphPad Software, San Diego, CA, USA). Statistical analysis were performed using one-way ANOVA relative to the respective value of the untreated cell control (ns, not statistically significant, **** p < 0.0001).Figure. 6. Annexin V-FITC apoptosis staining. A549 cells were seeded in a 96-well plate (IBIDI). After 24 hrs incubation with mushroom extract or control compounds, cells were stained with DAPI and Annexin V-FITC.Table 1. Absolute Extract Dry Weight in Gram, Beta-Glucan Content in g/100 g (mean of quadruples) and Protein Content in g/100 g (mean of triplicate) of Six Lentinus edodes-Fractions Obtained from Ultra Filtration ProcessName of fractionMWCO of fractionDry weight of fraction (g)Beta-glucan-content (g/100 g, mean of fourfold ± SD)Total beta-glucan-content in gProtein content(g/100 g, mean of triplicate ± SD)Total protein content in g Sk1.1>300 kDa4.1080.684 ± 0.2550.02825.060 ± 0.0121.029Sk1.2100-300 kDa1.3020.595 ± 0.5910.00814.580 ± 0.0100.190Sk1.330-100 kDa1.9991.208 ± 0.3820.02412.590 ± 0.0380.252Sk1.410-30 kDa3.3804.880 ± 0.1390.1651.047 ± 0.0880.035Sk1.55-10 kDa5.2531.569 ± 1.8390.0820.495 ± 0.0090.026Sk1.6<5 kDa39.6392.221 ± 0.1930.8800.037 ± 0.0130.015Total weight55.6811.1871.547Table 2. Relative Content (g/100 g) of Total-Glucans, Alpha-Glucans and Beta-Glucans in All Six Obtained Ultra Filtration-Fractions from Lentinus edodes, Determined with a 1,3-1,6-Beta-Glucan Assay kit (Megazyme, Wicklow Ireland).Name of fractionMWCO of fractionTotal-glucan content (g/100 g, mean of fourfold ± SD)Alpha-glucan content (g/100 g, mean of fourfold ± SD)Beta-glucan content (g/100 g, mean of fourfold ± SD) Sk1.1>300 kDa3.001 ± 0.1502.317 ± 0.1270.684 ± 0.255Sk1.2100-300 kDa1.779 ± 0.4461.185 ± 0.1910.595 ± 0.591Sk1.330-100 kDa3.477 ± 0.4542.270 ± 0.1561.208 ± 0.382Sk1.410- 30 kDa6.837 ± 0.2531.957 ± 1.7314.880 ± 0.139Sk1.55-10 kDa5.087 ± 0.2143.518 ± 0.1641.569 ± 1.839Sk1.6<5 kDa4.996 ± 0.4182.775 ± 0.4512.221 ± 0.193FIG. 1.FIG. 2.FIG. 3.FIG. 4.FIG. 5.FIG. 6. ................
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