Đề tài Khoa học effect of selenium enrichment on antioxidant activities and chemical composition of lentinula edodes (berk.) pegl. mycelial extracts

da ex ol Wars Keywords: Lentinula edodes Antioxidant activities Selenium Phenolics Free radicals Len ds extracts from mycelium of L. edodes (Berk.). To elucidate the cause of enhanced antioxidant activity of n in C (Turło et al., 2008a). Lentinan, a highly purified polysaccharide frac- tion extracted from L. edodes fruit bodies or mycelium, is an ap- proved drug used in cancer treatment as well as in AIDS research broth harvested from submerged liquid culture. Commercial prep- arations are available as tablets, capsules or elixirs, and sold in most Orient countries as well as the USA, New Zealand, Australia and Europe. Selenium is one of the trace elements of fundamental impor- tance to human health, and part of the antioxidant enzymes that protect cells against the effects of free radicals (Roy et al., 1994; Tapiero et al., 2003; Dodig and Cepelak, 2004). Another important health effect of selenium relates to proper function of the immune system. Selenium appears to be a key nutrient in cancer prevention and the inhibition of HIV progression to AIDS (Schamberger and Abbreviations: DS, dietary supplements; LEM, Lentinula edodes mycelium extract; LAP, alcohol-insoluble fractions prepared from L. edodes culture medium; DETBA, 1,3-dietyl-thiobarbituric acid; DPPH, 1,1-diphenyl-2-picrylhydrazyl; RP- HPLC, reversed phase high performance chromatography; DAN, 2,3-diaminonaph- thalene; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; MBRT, methylene blue reduction test; GAEs, gallic acid equivalents. * Corresponding author. Tel.: +48 22 5720649; fax: +48 22 5720631. Food and Chemical Toxicology 48 (2010) 1085–1091 Contents lists availab Food and Chemi journal homepage: www.elsevE-mail address: jadwiga.turlo@wum.edu.pl (J. Turło).Japan as Shiitake, is one of themost valuablemedicinalmushrooms, renowned for thousand of years both as a food and medicine (Bre- ene, 1990). This medical mushroom contains proteins composed of 18 different amino acids, including all the essential amino acids, in a ratio similar to the ideal for humans (Vetter, 1995). Shiitake mush- rooms contain significant quantities of vitamin D (the highest vita- min D levels of any plant), C, B1, B2, PP and B12 (Mattila et al., 2000). While the vitamin B12 level in Shiitake fruit bodies is low (Mattila, 2001), cultivation in cobalt-enriched media induces Shiitake myce- lium to synthesize a ten thousand fold higher amount of cobalamin edodesmycelium extract-LEM, and alcohol-insoluble fractionswere prepared from the culture medium-LAP) contain strong antitumor activity (Sugano et al., 1982; Suzuki et al., 1990; Iizuka, 1997), and alcohol extracts from Lentinula edodes fruit bodies contain strong antioxidant, reducing and free radical scavenging activity (Cheung and Cheung, 2005). L. edodes can be consumed whole or as concentrated extracts, or dietary supplements (DS) (Wasser et al., 2000). Several types of DS are derived from L. edodes: dried and pulverized fruit bodies, hot water and alcohol extracts, biomass or extracts of mycelium, or1. Introduction Lentinula edodes (Berk.) Pegl., know0278-6915/$ - see front matter  2010 Elsevier Ltd. A doi:10.1016/j.fct.2010.01.030extracts, a preliminary selenium speciation by specific oxido-reduction reaction was performed. Se- enrichment enhanced antioxidant activity, reducing power and free radical scavenging effect of mycelial extracts by almost 100–400%. Increase of activity was particularly high for diluted extracts (concentra- tions 0.1–0.5 mg/ml). The chemical composition of extracts from both Se-enriched and non-enriched mycelium was compared by determination of polyphenols, proteins, carbohydrates and lipids. Results showed that Se-enrichment enhanced antioxidant activities of mycelial extracts, likely by high amounts of organic Se-compounds (–II oxidation state) and elemental red selenium, and by increased polyphenols content. Our results suggest that Se-enrichment is a good method for enhancement of important activi- ties of human dietary supplements, including Shiitake preparations.  2010 Elsevier Ltd. All rights reserved. hina as Xiang-gu and in (Ajinomoto Co., Information Publication 1984; Chang, 1996; Was- ser and Weiss, 1999; Gunde-Cimerman, 1999). Hot water and alco- hol extracts from L. edodes mycelium and culture media (LentinulaAccepted 29 January 2010 anticancer activity. L. edodes mycelium enriched with organic forms of selenium like selenized yeast pos- sess putative, higher cancer preventive properties. The objective of this study was to test the effect of enrichment in selenium on antioxidant, reducing and free radical scavenging activity of water and alcoholEffect of selenium enrichment on antioxi of Lentinula edodes (Berk.) Pegl. mycelial Jadwiga Turło *, Bo _zenna Gutkowska, Franciszek Her Department of Drug Technology, Medical University of Warsaw, Banacha 1 St., 02-097 a r t i c l e i n f o Article history: Received 10 August 2009 a b s t r a c t Preparations derived from ments containing compounll rights reserved.nt activities and chemical composition tracts d aw, Poland tinula edodes (Berk.) Pegl. mycelium are worldwide used as dietary supple- active as immune system enhancers, demonstrating chemopreventive and le at ScienceDirect cal Toxicology ier .com/locate / foodchemtox man, 2000; El-Bayoumy, 2001). The mechanism by which selenium lial selenium and preliminary Se speciation by specific oxido- ing Inc., gradient system (SCL-10AVP controller, two LC-10AT vp pumps, CTO-10AC 2.5. Determination of antioxidant, free radical scavenging and reducing power 2.5.1. Reducing power spectrophotometer. A higher absorbance indicated a higher reducing power. of ethyl acetate was added, the mixture was mixed and centrifuged at 2000 rpm and A1 the absorbance in the presence of sample. 2.6.1. Preparation of the samples for the total Se determination l Tovp oven) with fluorescence detector (LaChrom L-7480 Merck-Hitachi Darmstadt, Germany). A C18 column 250  4 mm (Phenomenex, Luna 2) with an appropriate guard column was used. For fluorescence determinations, a LaChrom L-7480 (Merck-Hitachi Darmstadt, Germany) was used and for UV–Vis absorbance deter- minations, we used a Shimadzu UV–Vis spectrometer. Mycelial cultures were cultivated in a 5 l Infors AG-CH 4303 fermenter. 2.2. Standards and reagents The linoleic acid standard was ordered from Fluka. The 2,3-diaminonaphthalene (DAN) for selenium derivatization, 1,3-dietyl-thiobarbituric acid (DETBA) gallic acid standard, the sodium selenite standard, 2-tert-butyl-4-methoxyphenol (BHA), 2,6- di-tert-butyl-4-methylphenol (BHT), Bradford reagent, Folin–Ciocalteau reagent and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were all purchased from Sigma. HCl, HNO3 and HClO4 GC, acetonitrile, methanol and water HPLC were purchased from Merck. 2.3. Microorganism and cultivation media The Lentinula edodes strain used in this study was ATCC 48085. The seed culture was grown in 500 ml flasks containing 150 ml of basal medium (glucose 5% (w/v), yeast extract 1%, casein hydrolysate 1%, KH2PO4 0.1%) at 26 C on a rotary shaker at 110 rev/min for 7 days. Mycelia were grown in submerged culture in a 5 l fermenter. Fermentation medium was inoculated with seed culture to 10% (v/v) and cultivated at 26 C. The culture media contained glucose 5%, yeast extract 1%, soybean extract 1.5%, KH2PO4 0.1% (w/v), and was either not fortified or enriched with selenium at a con- centration of 10 lg/ml by the addition of sodium selenite. Fermentation was per- formed under the following conditions: aeration rate, 2 vvm; agitation speed, 200 rev/min; initial pH 6.5; and working volume, 3 l. 2.4. Sample preparationreduction reactions was carried out (Dauchy et al., 1994; Johansson et al., 1995; Goldberg et al., 2005). The chemical composition of ex- tracts from both Se-enriched and non-enriched mycelium was compared by determination of polyphenols, proteins, carbohy- drates and lipids. 2. Materials and methods 2.1. Apparatus HPLC analysis of selenium was carried out using a Shimadzu USA Manufactur-of L. edodes could be a source for a new immunomodulating drug, chemopreventive preparation or food supplement. Therefore, here we evaluated the effect of selenium enrichment on antioxidant properties, reducing power and free radical scavenging activity of water and alcohol extracts from selenated and non-selenated mycelium of L. edodes. To analyze the effect of Se-enrichment on the antioxidant activ- ity of mycelial extracts, RP-HPLC determination of the total myce-exerts anticancer and immunomodulating activity differs from that of L. edodes extracts and polysaccharide fractions, but a similar pharmacological effect suggests a possible synergism of these two agents. We hypothesize, therefore, that high concentrations of selenium in mycelial biomass would enhance the anticancer and inhibition of HIV progression activity of mushroom mycelial extracts, as well as the antioxidant and free radical scavenging activity. Thus, Se-enriched mycelial preparations could be an alter- native to Se–yeast chemopreventive agents. Our previous works show that mycelial cultures of Lentinula edodes very effectively accumulate selenium from cultivation medium in a bioavailable form (Turlo et al., 2007). We expect that Se-enriched myceliumFrost, 1969; Yu et al., 1991; Spallholz, 1994; Hehr et al., 1997; Ray- 1086 J. Turło et al. / Food and ChemicaMycelia were harvested by filtration and freeze-dried. Methanol and water ex- tracts were prepared from freeze-dried mycelium in a Soxhlet extractor. The dried extracts were redissolved in methanol or water to the appropriate concentration.A sample of mycelia (0.06 g) dried at 100 C was placed in a Kjeldahl flask and digested with 2.5 ml of 65% HNO3 and 1 ml of 65% HClO4. Samples were heated for 2 h at 80–120 C, 1 h at 120–150 C, and 40 min at 150–180 C. When the samples had cooled, 1 ml of 4 M HCl solution was added (to reduce Se(VI) to Se(IV)), and samples were heated for 15 min at 150 C. A blank digest was performed in the same way.2.6. Selenium determination and speciation in mycelial extracts The content of total selenium in tested extracts was determined by the RP-HPLC method, after precolumn derivatization with diaminonaphtalene (DAN). For each extract, speciation of selenium was carried out by specific oxido-reduction reac- tions (Fig. 1).for 15 min. Ethyl acetate was separated and its absorbance was measured in a spec- trofluorometer with fluorescence excitation at 515 nm and emission at 555 nm. The antioxidant activity was expressed as the percentage of lipid peroxidation, with a control containing no sample set as 100%. A higher percentage indicated a lower antioxidant activity. The conjugated diene method according to Lingnert et al. (1979): Hundred microlitre of each extract in deionized water or methanol (0.1–30 mg/ ml) was mixed with 2 ml of 10 mmol/l linoleic acid emulsion in 0.2 mol/1 sodium phosphate buffer pH 6.6 in test tubes, and the tubes were placed in the dark at 37 C to accelerate oxidation. Mixtures were incubated for 15 h, and 6 ml of 600 ml/l methanol in deionized water was then added. The absorbance of the mix- ture was measured at 234 nm against a blank. The antioxidant activity was calcu- lated as follows: antioxidant activity (%) = [(A0  A1)/A0]  100, where A0 was the absorbance of the control reaction and A1 the absorbance in the presence of sample. A value of 100% indicated the strongest inhibitory ability. 2.5.3. Scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals Scavenging effects on free DPPH radicals of water and methanol extracts of sel- enated and non-selenated L. edodes mycelium was determined using the method by Shimada et al. (1992). Various concentrations of extracts from mushrooms (4 ml) were mixed with l ml of methanol solution containing l,l -diphenyl-2-pic- rylhydrazyl (DPPH) radicals, resulting in a final concentration of the DPPH of 0.2 mM. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at 517 nm. The capability to scavenge DPPH radicals was calculated using the following equation: DPPH scavenging effect (%) = [(A0  A1)/A0]  100, where A0 was the absorbance of the control reaction2.5.2. Antioxidant activity The antioxidant activity was determined by two different methods. The DETBA (1,3-diethyl-2-thiobarbituric acid) method, according to Furata et al. (1997): 50 ll of linoleic acid emulsion (2 mg/ml in 95% ethanol) were added to 50 ll of various concentrations of mycelial extract (0.1–1 mg/ml). The mixture was incubated at 80 C for 60 min, and cooled in an ice bath. The following were then sequentially added: 200 ll of 20 mM butylated hydroxytoluene (BHT, Sigma), 200 ul of 8% sodium dodecyl sulphate, 400 ul of deionized water, and 3.2 ml of 12.5 mM DETBA in sodium phosphate buffer (pH 3.0). The mixture was mixed, placed in an oven at 95 C for 15 min, and then cooled in an ice bath. After 4 mlThe reducing power was determined according to the method of Oyaizu (1986): 2.5 ml of various concentrations of methanol or water extracts (0.5–30 mg/l) were mixed with 2.5 ml of 200 mM sodium phosphate buffer pH 6.6 and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50 C for 20 min. After 10% trichloroacetic acid (2.5 ml) was added, the mixture was centrifuged at 650 rpm for 10 min. The upper layer (5 ml) was mixed with 5 ml of deionized water and l ml of 0.l% ferric chloride. The absorbance was measured at 700 nm in a ShimadzuMethanol and water extracts of selenated and non-selenated mycelia were examined for antioxidant activity, reducing power and scavenging effect on DPPH free radicals. Tests were performed using dilutions ranging from 0.1 to 30 mg/ml. Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used as controls.xicology 48 (2010) 1085–10912.6.2. Preparation of the samples for Se(IV) direct determination A total of 0.05 g of sample was diluted in 10 ml of methanol or water (Suprapur, Merck) and used for direct Se(IV) RP-HPLC determination. was calculated as the difference between the obtained result and the Se(IV) amount al ex al Todetermined by the direct determination (without sample digestion) described below. 2.6.4. Se derivatization with 2,3-diaminonaphtalene (3,5-benzopiazselenol formation) A 0.1% solution of 2,3-diaminonaphtalene (DAN) in 0.1 M HCl was prepared by dissolving 100 mg of the reagent in 100 ml of 0.1 M HCl. Prepared DAN reagent was stored at 4 C in the dark. The digested samples were dissolved in 8 ml of2.6.3. Preparation of the samples for Se(VI) determination A sample of mycelia (0.06 g) was placed in a Kjeldahl flask and digested with 1 ml of 4 M HCl solution (to reduce Se(VI) to Se(IV)), and samples were heated for 15 min at 150 C. A blank digest was performed in the same way. Se(VI) content Fig. 1. Selenium speciation in myceliJ. Turło et al. / Food and ChemicSuprapur water and the pH was adjusted to 1.8–2 by the addition of HCl or 7 M NH3H2O solution. One milliliter of DAN reagent was added to each sample, and the mixtures were heated for 45 min at 50 C. After cooling, 3 ml of cyclohex- ane was added, and the test tubes were vigorously shaken for one minute to ex- tract the fluorescent piazselenol. Samples were stored in dark prior to determination. 2.6.5. RP-HPLC determination of Se The procedure used was a modified HPLC method with fluorescence detection (Handelman et al., 1989; Hawkes and Kutnik, 1996; Pedro et al., 2004). The cyclohexane extracts obtained as described above were injected into the column. Elution was isocratic, and the eluent was acetonitrile. Temperature was 25 C, injection volume 20 ll and flow rate 1.4 ml/min. The chosen excitation wavelength was 378 nm and emission wavelength 557 nm. Piazselenol retention time was 3.1 min. All determinations were performed three times. Three standard additions (four replications of each) and peak area measurements were used for quantification. The calibration curve of 3,5-benzopiazselenol was linear in the range of determined concentrations (Turło et al., 2009). 2.6.6. Inorganic selenium determination (elemental selenium and selenites) Mycelial extracts were tested for inorganic selenium using the Methylene Blue Reduction Test (MBRT) according to the method by Nagodawithana. (Feigl and West, 1947; Nagodawithana and Gutmanis, 1985). The sample (0.5 g powder) was placed in a vial, and 5 ml of ‘‘reducing solution” containing a 20% solution of 1-thioglycerol in 0.2 M phosphate buffer pH 5.5 was added. The vial was closed and shaken for 10 s. After 3 min (at t = 0), two drops of solution containing 2% (w/ v) methylene blue in Suprapur water were added, and the vial was closed and sha- ken for 10 s. The time at which decolorization was essentially complete, which indi- cates the presence of inorganic selenium, was recorded; this was referred to as the MBRT time. A standard curve was constructed for non-enriched mycelium supple- mented with sodium selenite, as relationship between MBRT time, and inorganic Se concentration.2.7. Determination of composition of selenated and not selenated mycelial extracts To evaluate differences in chemical composition, we determined the content of polyphenols, proteins, carbohydrates and lipids in Se-enriched and non-enriched mycelial extracts. 2.7.1. Determination of total polyphenol content The concentrations of polyphenols in the Se-enriched and non-enriched meth- anol and water extracts, expressed as gallic acid equivalents (GAEs), were measured according to the method of Singleton and Rossi (1965). One milliliter of sample (water or alcohol extracts at a concentration of 0.5 mg/ml) was mixed with l ml of Folin–Ciocalteu reagent. After 3 min, l ml of saturated Na2CO3 was added and dis- tracts by oxido-reduction reactions. xicology 48 (2010) 1085–1091 1087tilled water was added to a total volume of 10 ml. The reaction was kept in the dark for 90 min, after which the absorbance at 725 nm was read. A calibration curve was constructed with different concentrations of gallic acid (0.01–0.04 mg/ml) as standard. 2.7.2. Determination of total protein content (Bradford method) A total of 100 ll of sample (water or alcohol extracts at a concentration of 20 mg/ml) was mixed with 5 ml of Bradford reagent. After 5 min, the absorbance at 595 nm was read. A calibration curve was constructed with different concentra- tions of albumin (0.25–2.0 mg/ml) as the standard. 2.7.3. Determination of carbohydrate content by the phenol–sulphuric acid method (Dubois method) One milliliter of sample (water or alcohol extracts at a concentration of 0.3 mg/ ml) was mixed with l ml of 5% phenol solution and 5 ml of concentrated H2SO4. The mixture was kept at room temperature for 10 min, and then heated at 30 C for 20 min. Absorbance at 490 nm was measured. A calibration curve was constructed with different concentrations of 1:1 w/w galactose–mannose mixture (25–100 lg/ ml) as the standard. 2.7.4. Determination of lipids based on the sulfo-phospho-vanillin reaction A total of 50 ll of sample (water or alcohol extracts at a concentration of 20 mg/ ml) was mixed with 2 ml of concentrated H2SO4. The mixture was heated in a boil- ing water bath for 20 min. When cool, the samples were mixed with 4 ml of phos- pho-vanillin reagent (vanillin in phosphoric acid) and kept for 20 min at room temperature, after which the absorbance at 525 nm was read. Cholesterol solution at a concentration of 6 mg/ml was used as a standard. 2.8. Statistical analysis All analyses were performed in triplicate. The data were expressed as mean ± standard deviation, and one-