Phone: Fax: 1-203-456-6161 1-203-456-616 Begell House, Inc. Journal Production 50 North Street Danbury, CT 06810 E-Mail: [email protected]Dear Corresponding Author, Attached is the corresponding author pdf file of your article that has been published. Please note that the pdf file provided is for your own personal use and is not to be posted on any websites or distributed in any manner (electronic or print). Please follow all guidelines provided in the copyright agreement that was signed and included with your original manuscript files. Any questions or concerns pertaining to this matter should be addressed to [email protected]. Thank you for your contribution to our journal and we look forward to working with you again in the future. Sincerely, Vicky Lipowski Begell House Production Department
Transcript
Phone:Fax:
1-203-456-61611-203-456-616
Begell House, Inc.Journal Production50 North StreetDanbury, CT 06810
Attached is the corresponding author pdf file of your article that has been published.
Please note that the pdf file provided is for your own personal use and is not to be posted on anywebsites or distributed in any manner (electronic or print). Please follow all guidelines providedin the copyright agreement that was signed and included with your original manuscript files.
Any questions or concerns pertaining to this matter should be addressed [email protected].
Thank you for your contribution to our journal and we look forward to working with you againin the future.
Sincerely,
Vicky LipowskiBegell House Production Department
International Journal of Medicinal Mushrooms, 18(2): 109–121 (2016)
Protective Effect of Antioxidant Extracts from Grey Oyster Mushroom, Pleurotus pulmonarius (Agaricomycetes), Against Human Low-Density Lipoprotein Oxidation and Aortic Endothelial Cell DamageMohamad Hamdi Zainal Abidin,* Noorlidah Abdullah, & Nurhayati Zainal Abidin
Faculty of Science, Institute of Biological Sciences, Mushroom Research Centre, University of Malaya, Kuala Lumpur, Malaysia
*Address all correspondence to: Mohamad Hamdi Zainal Abidin, Faculty of Science, Institute of Biological Sciences, Mushroom Research Centre, University of Malaya, 50603 Kuala Lumpur, Malaysia; Tel: +603-7967 4371; Fax: +603-7967 4178; [email protected]
ABSTRACT: This study evaluated the in vitro antioxidant capacities of extracts from Pleurotus pulmonarius via Folin-Ciocalteu, 1,1-diphenyl-2-picrylhydrazyl free radical scavenging, metal chelating, cupric ion reducing anti-oxidant capacity, and lipid peroxidation inhibition assays. Extract compositions were determined by phenol–sulfuric acid; Coomassie Plus (Bradford) protein; Spectroquant zinc, copper, and manganese test assays; and liquid chro-matography–tandem mass spectrometry (LC/MS/MS) and gas chromatography–mass spectrometry (GC/MS). Methanol-dichloromethane extract, water fraction, hot water, aqueous extract and hexane fraction exhibited the most potent extracts in the antioxidant activities. LC/MS/MS and GC/MS showed that the extracts contained ergo-thioneine, ergosterol, flavonoid, and phenolic compounds. The selected potent extracts were evaluated for their inhibitory effect against oxidation of human low-density lipoproteins and protective effects against hydrogen peroxide–induced cytotoxic injury in human aortic endothelial cells. The crude aqueous extract was deemed most potent for the prevention of human low-density lipoprotein oxidation and endothelial membrane damage. Ergothioneine might be the compound responsible for the activities, as supported by previous reports. Thus, P. pulmonarius may be a valuable antioxidant ingredient in functional foods or nutraceuticals.
ABBREVIATIONS: CA: crude aqueous; CD: conjugated diene; CHW: crude hot water; CUPRAC: cupric ion reducing antioxidant capacity; DCM: dichloromethane; DPPH: 1,1-diphenyl-2-picrylhydrazyl; EAF: ethyl acetate fraction; ELISA: enzyme-linked immunosorbent assay; GC/MS: gas chromatography–mass spectrometry; HAEC: human aortic endothelial cell; HF: hexane fraction; LC/MS/MS: liquid chromatography–tandem mass spectrometry; LDL: low-density lipoprotein; MD: methanol + DCM; MDA, malondialdehyde; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PF30: protein fraction 30; PF60: protein fraction 60; PF90: protein fraction 90; PP: partially purified polysaccharide; TBARS: thiobarbituric acid reactive substances; WF: water fraction
I. INTRODUCTION
Grey oyster mushroom, Pleurotus pulmonarius (Fr.) Quél. (Pleurotaceae, Agaricomycetes) is a commonly consumed mushroom cultivated worldwide. Species of the genus Pleurotus, including P. pulmonarius, represented the third place of global edible mush-room production, after species Agaricus spp. and Lentinus edodes.1 More recently, production has likely increased because P. pulmonarius is easily grown at varied conditions without temperature and humidity control.2 In addition to having good flavor and texture,
it has medicinal properties.3 Oxidative stress is a sig-nificant factor in many pathological conditions, such as atherosclerosis, cancer, hypertension, and neuro-degenerative and inflammatory diseases.4 Oxidation results in excessive production of free radicals, which can cause cell damage and death.4 Therefore, synthetic antioxidants, including butylated hydroxyanisole and tert-butylhydroxyquinine, have been developed as potential antioxidant drugs. In the food industry, application of antioxidants has become increas-ingly important. Unsaturated fatty acids are targets
International Journal of Medicinal Mushrooms
M.H.Z. Abidin, Abdullah, & N.Z. Abidin110
for oxidation in food, because when oxidized, they cause food quality to deteriorate. Hence, the food industry has considered synthetic antioxidants, such as those mentioned previously, as food additives.5 Unfortunately, these synthetic antioxidants have sus-pected carcinogenic effects, leading to demands for natural antioxidants as alternatives.6
Our study aims were threefold. The first objec-tive was to investigate antioxidant activities of extracts that were prepared according to the sol-vent fractions, protein- and polysaccharide-rich fraction from P. pulmonarius. The second aim was to standardize the chemical composition of proteins, carbohydrates, and select antioxidant metal ions in the extract exhibiting the greatest antioxidant activ-ity for each bioassay tested. The third aim was to investigate the effects of potent antioxidant extract on low-density lipoprotein (LDL) oxidation and endothelial membrane damage. This study will pro-vide valuable information for the pharmaceutical, nutraceutical, food, and preservatives industries.
II. MATERIALS AND METHODS
A. Mushroom Material
P. pulmonarius fruiting bodies were purchased from Mdm. Cheng’s farm (Agro-Tech Sdn. Bhd.,Taman Pelangi,Selangor Darul Ehsan, Malaysia). We identified the species as P. pulmonarius (based on morphological characteristics and DNA sequence data of internal transcribed spacer regions). The culture was then deposited in the Mycological Laboratory of the Mushroom Research Centre, University of Malaya, with the registration number KUM61119. A voucher of the specimen was deposited in the University of Malaya Herbarium (registration num-ber KLU-M1234). Mushrooms were immediately washed with water to remove surface impurities.
1. Preparation of the Methanol-Dichloromethane Extract and Liquid-Liquid Solvent Fractions
Crude methanol-dichloromethane extract (MD), hexane fraction (HF), and water fraction (WF) were prepared as previously described.7
2. Preparation of the Aqueous Extract and Protein Fractions
Freeze-dried fruit bodies dissolved in distilled water at a ratio of 1:10 (w/v) were stirred overnight at 4°C and then filtered to remove residue. The super-natant was freeze-dried to yield the crude aqueous extract (CA). CA (20 g) was dissolved in 400 mL distilled water. Protein fractions were prepared by an ammonium sulfate precipitation method8 to achieve saturation of 30%, 60%, and 90% (w/v). The protein fractions denoted as PF30, PF60, and PF90 were freeze-dried and maintained at −20°C.
3. Preparation of CWH and PP
Freeze-dried fruit bodies were blended and soaked in absolute ethanol for 24 h to remove organic com-pounds of low molecular weight. Next, the mixture was filtered, and the fruit body residue was collected and dried in an oven at 60°C for 12 h. This residue was then boiled in water at a ratio 1:20 (w/v) for 3 h at 100°C. The mixture was filtered, and 200 mL of the filtrate was collected and freeze-dried to yield the crude hot water extract (CHW). Absolute etha-nol was added to the remaining filtrate at a ratio of 1:5 (v/v). The polysaccharide precipitate was col-lected by centrifugation at 9000 rpm for 15 min at 25°C, followed by deproteination using Sevag reagent (n-butanol/chloroform, 1:4 v/v).9 Finally, the supernatant was lyophilized to yield the partially purified polysaccharide fraction (PP). Both CHW and PP were maintained at −20°C.
B. Antioxidant Assays
1. Folin-Ciocalteu Antioxidant Assay
The Folin-Ciocalteu assay used to determine the total antioxidant capacity was the modified method of Abdullah et al.,10 and the results are expressed as mg/mL gallic acid equivalents (GAE). Briefly, 250 L (100 µg/mL) of each extract was individ-ually added to 250 L of a solution of 10% (v/v) Folin-Ciocalteu reagent and incubated for 2 min at room temperature. Aqueous sodium carbonate [10% (w/v), 500 L] was added, and each mixture
Volume 18, Number 2, 2016
Protective Effect of Pleurotus pulmonarius Against Human Low-Density Lipoprotein Oxidation 111
was kept in the dark for 1 h, after which its A750 was measured using a UV-Vis spectrophotometer (Shimadzu, Japan). A calibration curve using gallic acid concentrations between 2 and 10 mg/mL was prepared, and the results were expressed as mg/mL GAE. The extract with the greatest antioxidant capacity was then subjected to an analysis of dose dependence.
2. DPPH Radical-Scavenging Assay
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activities of the extracts were evaluated according to the method of Kuda and Yano with minor modifications.11 Briefly, 5 L of each extract (100 g/mL) was mixed with 195 L of 1 mM DPPH. Each mixture was placed into a well of a 96-well microplate and incubated for 30 min at room tem-perature in the dark. Next, the A515 was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader (Sunrise, TECAN). Methanol served as the blank to replaced extract. The 50% inhibitory concentration (IC50) was determined for quercetin and the extract that showed the greatest inhibition. The percentage of radical scavenging activity was calculated as:
where A0 is the absorbance of the blank, and A1 is the absorbance of the sample.
3. Metal Chelating Assay
The metal chelating capability of each extract was evaluated as described12 with minor modifications. Extract (1 mL, 100 g/mL) was mixed with 100 L of 2 mM FeCl2. After 1 min, 200 mL of 5 mM ferrozine (3-[2-pyridyl]-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate) was added into the solution mixture. Distilled water (3.7 mL) was added to a final volume of 5 mL. Each reaction mixture was incubated for 20 min at room temperature in the dark before the A562 was measured using a UV-Vis spectrophotometer. The blank was distilled water. The IC50 was determined
for quercetin and the extract with the greatest inhibi-tion. Activity was calculated as:
Chelating activity = 100 × [(A0 – A1)/A0]
where A0 is the absorbance of the blank, and A1 is the absorbance of the sample.
4. CUPRAC Assay
The cupric ion reducing antioxidant capacity (CUPRAC) assay was that of Öztürk et al.12 with minor modifications. Briefly, 1 mL of each extract (100 g/mL) was added into the reaction mixture containing 1 mL of 1.0 × 10–2 M copper, 1 mL of 7.5 × 10–3 neocuproine, and 1 mL of 1 M ammo-nium acetate (pH 7). The solution was mixed and incubated for 30 min at room temperature. A459 was measured using a UV-Vis spectrophotometer. Water served as the blank. The extract with the greatest activity was subjected to dose-dependent analysis.
5. Inhibition of Lipid Peroxidation Using the Buffered Egg Yolk Assay
The ability of each extract to inhibit lipid peroxida-tion was determined using the method of Abdullah et al.10 The formation of thiobarbituric acid reac-tive substances (TBARS) was measured at 532 nm using an ELISA microplate reader. The buffered egg yolk/Fe2+ mixture served as the control. The IC50 was determined for quercetin and the extract that showed the greatest degree of inhibition. The inhibition of lipid peroxidation was calculated as:
% Inhibition = 100 × [(A0 – A1)/A0]
where A0 is the absorbance of the control and A1 is the absorbance of the sample.
C. Chemical Compositions of Potent Antioxidant Extracts
Carbohydrate, protein, and metal-ion compositions of the extracts were measured as follows. First, total carbohydrate content was determined using the phenol–sulfuric acid colorimetric assay.13 Second,
International Journal of Medicinal Mushrooms
M.H.Z. Abidin, Abdullah, & N.Z. Abidin112
protein content was determined using Coomassie Plus (Bradford) Protein Assay kit reagents (Thermo Scientific). Finally, the total amounts of the anti-oxidative ions of manganese (Mn), zinc (Zn), and copper (Cu) were determined photometrically using the Spectroquant zinc, copper, and manganese test methods (Merck Millipore).
MD, WF, CA, and CHW were subjected to liq-uid chromatography–tandem mass spectrometry (LC/MS/MS), and MD and HF were analyzed by gas chromatography/mass spectrometry (GC/MS) as described previously.7
D. Inhibition of Human LDL Oxidation
1. Measurement of Conjugated Diene Formation
Conjugated diene (CD) formation was measured following the modified method of Ahmadvand et al.14 Human LDL was adjusted to a final concen-tration of 150 µg protein/mL, and the oxidation reaction of LDL was initiated by adding freshly prepared 0.1 mM FeSO4 solution. The reaction mixture was then incubated at 37°C with P. pul-monarius extracts or α-tocopherol (Calbiochem) positive control (10 µg/mL). Distilled water served as the blank. The formation of CD was determined by UV spectrometry at A234 for every 5 min in the 2-h incubation.
2. Measurement of TBARS
To determine the formation of TBARS, LDL was adjusted to a final concentration 150 µg protein/mL, and the oxidation reaction of LDL was initiated by adding freshly prepared 1 M FeSO4 solution. The reaction mixture was then incubated at 37°C for 3 h with P. pulmonarius extracts (1000 µg/mL) or α-tocopherol, positive control (100 µg/mL). After 3-h incubation, 500 µL of 15% trichloroacetic acid and 1000 µL of 1% thiobarbituric acid were added to the reaction mixture and incubated at 100°C for 15 min. The blank was distilled water. Finally, the formation of TBARS was measured using an ELISA plate reader at A532. A calibration curve using a malondialdehyde (MDA) bis-(dimethyl acetal)
concentration from 0 to 100 µM was prepared as a standard, and the result was expressed as µmol MDA/mg protein-LDL.
E. Cell Culture
Human aortic endothelial cells (HAECs) were purchased from Lonza (USA) and maintained in endothelial growth basal medium (EBM)-2 complete medium supplemented with hydrocortisone (0.2 mL), hFGF-B (2 mL), VEGF (0.5 mL), R3-IGF-1 (0.5 mL), ascorbic acid (0.5 mL), hEGF (0.5 mL), GA-1000 (0.5 mL), heparin (0.5 mL), and FBS growth factor (10 mL). The cells were seeded (2500–5000 cells/cm2) into T-flasks (Corning, USA) coated with 1% gelatin and incubated at 37°C in humidified 5% CO2. The growth medium was changed the day after seeding and every 2 days thereafter.
1. Cell Viability Measurement
Cell viability was measured using the 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA) assay. HAECs (10 × 103 cells/well) were seeded in 96-well plates (Corning, USA) and incubated at 37°C in humidi-fied 5% CO2 for 24 h. The extracts were added the day after and incubated for 24 h. Next, the super-natant was replaced with 100 µL of fresh EBM-2 medium followed by addition of 20 µL of MTT (5 mg/mL) and incubated at 37°C for 4 h. The MTT solution was removed and subsequently added with 100 µL of DMSO (100%). The absorbance was then measured at 570 nm. The percentage of cell viability was calculated relative to the untreated cells as the control group.
2. Protective Effect Against Hydrogen Peroxide–Induced Endothelial Damage
Hydrogen peroxide (H2O2; 500 µM) was induced before treatment with extracts for 6 h. Next, the cells were treated with extracts at a final concentration of 200 µg/mL in the well. The cell viability was determined the day after treatment.
Volume 18, Number 2, 2016
Protective Effect of Pleurotus pulmonarius Against Human Low-Density Lipoprotein Oxidation 113
F. Statistical Analysis
All results are the mean ± standard deviation of triplicates and were subjected to one-way analysis of variance. Significance differences were determined using the Duncan test at the 95% confidence level (p < .05). Analyses were performed with STATGRAPHICS Plus for Windows 3.0. Correlation except the regression analysis was performed using Microsoft EXCEL 2010 statistical analysis.
III. RESULTS AND DISCUSSION
A. Folin-Ciocalteu Assay
In the presence of phenolic compounds and alka-line pH, the Folin-Ciocalteu reagent is reduced to a blue complex. Because the Folin-Ciocalteu reagent is reduced by many different substances, includ-ing ascorbic acid,15 this assay measures the total reducing capacity of a sample. MD possessed the greatest value, 4.94 mg/mL GAE (Table 1). The
values for the other extracts ranged from 1.08 to 3.61 mg/mL GAE. Quercetin, the positive control, had a slightly greater value (5.26 mg/mL GAE) than MD. The reducing capacity of MD was dose-dependent (Fig. 1).
B. DPPH Assay
The DPPH assay is based on the principle that antioxidant compounds act as hydrogen donors or free radical scavengers. It reports the free rad-ical scavenging activity of a sample as a color change from purple to yellow. Thus, activity is measured as a decrease in A515. Quercetin (10 g/mL) exhibited the greatest activity 22.15% inhibi-tion, followed by WF (20.06%), DCMF (18.27%), and MD (17.93%) (Table 1). The result clearly showed that the most polar fraction, WF, is better than nonpolar fractions such as DCMF and HF. This is supported by Tomsone et al.16; the increase in DPPH free radical scavenging activity is cor-related with increased solvent polarity. The IC50
TABLE 1: The Antioxidant Capacities of Extracts from Fruiting Bodies of Pleurotus pulmonarius
value for WF was 8.0 mg/mL and that for quer-cetin was 0.099 mg/mL.
C. Metal Chelating Ability Assay
For this assay, the Fe2+-FerroZine complex is dis-rupted by other metal ions, and the extent to which the complex is destroyed is measured as a reduction in the red color of the solution. The greatest metal-chelating activity was exhibited by CHW (29.68%), followed by PP (21.71%) and WF (16.28%) (Table 1). The metal chelator, EDTA, caused 14.3% inhibi-tion at 10 g/mL. In contrast, Alam et al.17 reported that at 1 mg/mL the strongest metal-chelating ability (87.50% inhibition) was from the methanol extract of Lentinus lepideus, and that a hot water extract had the weakest chelating activity (75.91%).17 Our results may differ from those of Alam et al.17 because two different mushroom species were tested. In comparison with the IC50 value found for EDTA (0.052 mg/mL), the metal chelating capacity of CHW (9.5 mg/mL) is weak.
D. CUPRAC Assay
CUPRAC was evaluated by measuring the forma-tion of the stable Cu(I)-neocuproine complex that
is the product of a redox reaction between an anti-oxidant and the Cu(II)-neocuproine reagent, as the chromogenic oxidizing agent. The formation of the complex is accompanied by an increase in the orange-yellow color of the solution, and the absor-bance was measured at 450 nm. CA (100 g/mL) and quercetin (10 g/mL) had CUPRAC values of 0.16 followed by DCMF, PF60, and CHW (100 µg/mL) at 0.15, which is not significantly different from those of CA and quercetin (Table 1). Further analy-sis of CA, DCMF, PF60, and CHW showed that their CUPRACs are dose-dependent (Fig. 2) and that the CUPRAC value for CA was highest (A459, 2.37) at a concentration of 5 mg/mL. We therefore consider CA to have the most potent CUPRAC.
E. Inhibition of Lipid Peroxidation of Buffered Egg Yolk
Peroxidation of polyunsaturated fatty acids induced by free radicals produces several by-products, which can cause oxidative damage in humans.10 Among these by-products, MDA has been most widely used as an indicator of lipid peroxidation. In this assay, MDA reacts with thiobarbituric acid to form TBARS.18 At 1000 g/mL, HF exerted the greatest inhibition of lipid peroxidation (33.62%), followed by WF (28.47%) and PP (25.50%) (Table 1), with quercetin exhibiting 11.7% inhibition at 10 g/mL. However, inhibition by HF (IC50 = 2.4 mg/mL) is very weak compared with that of quercetin (IC50 = 0.015 mg/mL).
FIG. 1. Dose-dependent antioxidant activity of the methanol/dichloromethane extract of Pleurotus pulmo-narius fruiting bodies measured by the Folin-Ciocalteu assay. Values are expressed as the mean ± standard deviation of three replicates.
FIG. 2. Dose-dependent antioxidant activity of Pleurotus pulmonarius fruiting bodies extracts on cupric-ion- reducing antioxidant capacity. Values are expressed as the mean ± standard deviation of three replicates.
Volume 18, Number 2, 2016
Protective Effect of Pleurotus pulmonarius Against Human Low-Density Lipoprotein Oxidation 115
F. Characterization of Extracts Considered Potent by LC/MS/MS and GC/MS
MD contained 10.86% (w/w) carbohydrate, 23.19% (w/w) protein, 1.31 mg/g Mn, 0.97 mg/g Zn, and 0.93 mg/g Cu (Table 2). LC/MS/MS of MD iden-tified flavonoids, benzenoid, and chalcone-based compounds (Table 3). GC/MS of MD found 9,12- octadecadienoic acid (Z,Z)-, 13-docosenamide, (Z)-, and ergosterol to be the most abundant compounds (Table 4). Linoleic acid (9,12-octadecadienoic acid) is an unsaturated essential fatty acid. Conjugated linoleic acid exhibits potent antioxidant activity when compared with α-tocopherol (vitamin E), a known antioxidant.19 Ergosterol may act as plant phytosterols do to inhibit cholesterol absorption by
removing ergosterol from dietary mixed micelles formed during digestion.20,21
WF contained 13.19% (w/w) carbohydrate, 5.05% (w/w) protein, 0.32 mg/g Mn, 0.20 mg/g Zn, and 0.17 mg/g Cu (Table 2). LC/MS/MS analysis showed that WF contained leucine, cinnamic acid, ergothioneine, tryptophan, 3,3-O-di- O-methylellagic acid, and phenolic compounds (Table 5). Phenolic compounds are known natural antioxidants, and DPPH radical scavenging activity has been significantly correlated to total phenolic content tested by principal component statistical analysis.22 Leucine is an essential amino acid and is classified as a hydrophobic amino acid (nonpolar). Hydrophobic amino acids showed great antioxidant activity, as mentioned previously.23,24
TABLE 2: Carbohydrate, Protein, and Metal Ion Concentrations in Selected Extracts and Fractions
Most Potent Extract or Fraction According to the Bioassay (in Parentheses)
CHW contained 55% (w/w) carbohydrate, 27.71% (w/w) protein, 2.07 mg/g Mn, 0.20 mg/g Zn, and 0.21 mg/g Cu (Table 2). Several different flavonoids were identified by LC/MS/MS (Table 6). Flavonoids are known to have metal chelating activ-ities.25 CA contained ergothioneine, tryptophan, and methylellagic acid (Table 7). CA contained 43.81% (w/w) carbohydrate, 30.57% (w/w) protein, 0.59 mg/g Mn, 0.43 mg/g Zn, and 0.59 mg/g Cu (Table 2).
The most potent extracts for DPPH and CUPRAC assays, WF and CA, respectively, each possess ergothioneine and tryptophan. Ergothioneine is an amino acid antioxidant commonly found in mushrooms. Several species of mushrooms con-tain large amounts of ergothioneine, especially L. edodes, Pleurotus ostreatus, P. eryngii, and Grifola frondosa.26 Ours is the first report of the presence of ergothioneine in P. pulmonarius. Free tryptophan
may act as an antioxidant.27 Methylellagic acid is a derivative of ellagic acid, a natural phenolic antioxidant. Ellagic acid was found to be the most potent antioxidant as measured by several antioxi-dant assays including DPPH.28 Thus, ergothioneine, tryptophan, and methylellagic acid may be the antioxidants reported by the DPPH and CUPRAC assays of WF and CA.
HF contained 6.19% (w/w) carbohydrate, 29.33% (w/w) protein, 2.13 mg/g Mn, 0.85 mg/g Zn, and 0.30 mg/g Cu (Table 2). The relatively large amount of protein present in HF, a nonpolar solvent, is interesting because the proteins possibly have a substantial greater than average nonpolar/hydro- phobic amino acid content. Nonpolar or hydropho-bic amino acids have been shown to have stronger antioxidant activity than do polar or hydrophilic amino acids.23 This makes hydrophobic amino acids containing a large proportion of nonpolar residues
TABLE 4: Three Most Abundant Compounds Identified in the Crude Methanol-Dichloromethane Extract by GC/MS
more efficient antioxidants under lipid-based condi-tions.29 The three most abundant compounds found in HF using GC/MS are 9,12-octadecadienoic acid (Z,Z)-, ergosterol, and n-hexadecanoic acid (Table 8). Similar to MD, HF contains 9,12-octadecadi-enoic and ergosterol, which have demonstrated antioxidant capability.19–21,30
G. Inhibition of Human LDL Oxidation
This study shows that differential potencies of different metabolites in different assays. The five most potent extracts, MD, WF, CHW, CA, HF, were selected and further investigated for the inhibition of human LDL oxidation by quantifying conjugated diene (CD) formation (the initial stage of LDL oxi-dation) and thiobarbituric acid reactive substances (TBARS) formation (the late stage of LDL oxida-tion) assays.
1. Measurement of CD Formation
CD formation is the initial stage of LDL oxidation. It is thought that by preventing the formation of CD would inhibit the initial oxidation of LDL. The level of CD can be measured directly in the solution at 234 nm. The increase of 234 nm CD absorption indicates the higher rate of LDL oxidation. Figure 3 shows that at 10 µg/mL, CHW and CA increased
the lag phase and inhibited the formation of CD bet-ter than α-tocopherol, the positive control. On the other hand, WF, HF, and MD enhanced the forma-tion of CD. CHW and CA therefore have potential in preventing the initial LDL oxidation.
2. Measurement of TBARS
The TBARS assay was used to investigate the final stage of LDL oxidation, by measuring the reac-tion of MDA and thiobarbituric acid reagent. The final product of this reaction is known as TBARS, which can be measured at 532 nm. In this assay, α-tocopherol was the positive control at 100 µg/mL, and extract concentration was 1000 µg/mL. Referring to Figure 4, the highest inhibition of MDA formation (LDL oxidation) was exhibited by α-tocopherol, followed by the most potent extract CA with 10.89 µmol MDA/mg protein-LDL and 18.32 µmol MDA/mg protein-LDL respectively compared to negative control (LDL and FeSO4) with 34.97 µmol MDA/mg protein-LDL. It is inter-esting to point out that HF is the most potent for lipid peroxidation by egg yolk assay but enhanced the formation of CD and weak inhibitor for oxi-dation of human LDL. This might be due to the different mechanisms involved different source of lipids.31 CHW and CA were the best extracts to pre-vent the oxidation of LDL at early and late stage of
International Journal of Medicinal Mushrooms
M.H.Z. Abidin, Abdullah, & N.Z. Abidin118
FIG. 3. The effect of the potent antioxidant extracts (MD, HF, WF, CA, CHW) and α-tocopherol at concentration 10 µg/mL on the formation of CD from human LDL oxi-dation. Values were expressed as means ± standard deviation of three replicate measurements.
FIG. 4. The ability of the potent antioxidant extracts (MD, HF, WF, CA, CHW) and α-tocopherol in inhibiting human LDL oxidation at concentration 1000 µg/mL and 100 µg/mL, respectively. Values were expressed as means ± standard deviation of three replicate measurements.
TABLE 8: Three Most Abundant Compounds Identified in the Hexane Fraction Extract by GC/MS
oxidation (Figs. 3 and 4). This indicates that water is the best solvent in extracting potential antioxi-dant compounds for protection of human LDL. As mentioned earlier, CHW was shown to have flavo-noid compounds through LCMS/MS analysis. Vaya et al.32 reported about 20 types of selected flavonoid compounds were shown to have inhibitory effects toward human LDL oxidation. They found that fla-vonoids with two hydroxyl groups at ring B in a mutual ortho position, showed the most effective inhibitory effect for both initial (CD formation) and final (MDA formation) stage of oxidation. As discussed previously, CA was identified to have known antioxidant ergothioneine that is commonly found in mushrooms. CA was also found to have tryptophan and methylellagic acid, which are also reported to have antioxidant properties.27,28
H. Protective Effects of Extracts Against H2O2-Induced Cytotoxic Injury in HAEC
1. Cytotoxicity of the Extracts on HAEC
Based on the results (Fig. 5), cell viability was not less than 80% when treated with up to 200 µg/mL concentration of the extracts. The extracts possessed IC50 values higher than 200 µg/mL. It is proven that HF, WF, CA, and CHW extracts were not toxic to the HAEC. At 200 µg/mL, WF showed the lowest cell viability of 84.74%. At 12.5 µg/mL, CA pos-sessed the highest cell viability of 95.60%. All these extracts were then further investigated for their protective effects against H2O2-induced cytotoxic injury in HAEC analysis.
Volume 18, Number 2, 2016
Protective Effect of Pleurotus pulmonarius Against Human Low-Density Lipoprotein Oxidation 119
2. Protective Effect of the Extracts (MD, HF, WF, CA, CHW) Against H2O2-Induced HAEC Death
According to Figure 6, CA exhibited protection maintaining cell viability of 81.65% followed by α-tocopherol (79.63%) and WF (74.579%) compared to negative control (cells + H2O2) with 63.468%. However, based on statistical analysis, there was no significant different between CA and α-tocopherol. CHW and HF offered weak protec-tive effect, maintaining 66.162% and 64.310% cell viability, respectively. As previously mentioned, CA consists of ergothioneine, tryptophan, and methylellagic acid. Ergothioneine was proven to be absorbed into endothelial cells membrane via organic cation transporter and hence protect the cells from oxidative stress.33 A review by Paul and Snyder has emphasized that the lack of ergothio-neine in cells would lead to the DNA damage, and protein and lipid oxidation induced by oxidative stress.34 Therefore, ergothioneine might be the key compound that protects HAEC cells from H2O2.
IV. CONCLUSIONS
This study revealed that MD, WF, CHW, CA, and HF exhibited potent antioxidant activities in
the Folin-Ciocalteu, DPPH radical scavenging, metal chelating, CUPRAC, and lipid peroxidation assays, respectively, and that different extraction methods produce extracts with varying antioxidant capacities and also different chemical composi-tions. The known antioxidant compounds found in extracts of P. pulmonarius are flavonoids (MD, CHW), ergosterol (MD, HF), ergothioneine (WF, CA), and phenolic acid derivatives (WF, CA). CA exhibited the most potent extract in protective effect against human LDL oxidation and HAEC damage from H2O2-induced oxidative stress. Ergothioneine might be the compound responsible for the activities based on the previous studies. Given these results, P. pulmonarius may be a food that promotes good health. Our findings should be of use for the food manufacturing industry and for the preparation of functional food or alternative therapeutics for oxida-tive stress–related diseases.
ACKNOWLEDGMENTS
The authors give special thanks to the Mushroom Research Centre, University Malaya, for the facili-ties and for financial support (University Malaya Research Grant RP014A-13AFR and PPP No. PG142-2012B).
FIG. 5. The cytotoxicity of the extracts (MD, HF, WF, CA, CHW) on HAEC cells. The final concentration of DMSO used was less than 0.1% (v/v) and it did not affect cell viability. Values were expressed as means ± standard deviation of three replicate measurements.
FIG. 6. The protective effect of the extracts (MD, HF, WF, CA, CHW) against H2O2-induced HAEC death. The final concentration of DMSO used was less than 0.1% (v/v) and it did not affect cell viability. Values were expressed as means ± standard deviation of three repli-cate measurements.
International Journal of Medicinal Mushrooms
M.H.Z. Abidin, Abdullah, & N.Z. Abidin120
REFERENCES
1. Chang ST, Wasser SP. The role of culinary-medicinal mushrooms on human welfare with pyramid model for human health. Int J Med Mushrooms.2012;14(2):95–134.
2. Banik S, Nandi R. Effect of supplementation of rice straw with biogas residual slurry manure on the yield, protein and mineral contents of oyster mushroom. Ind Crop Prod. 2004;20(3):311–9.
3. Gunde-Cimerman N. Medicinal value of the genus Pleurotus (Fr.) P. Karst. (Agaricales s.l., Basidiomycetes). Int J Med Mushrooms. 1999;1(1):69–80.
4. Niki E, Shimaski H, Mino M. Antioxidantism-free radical and biological defence. Tokyo: Gakkai Syuppan Center; 1994.
5. Laguerre M, Bayrasy C, Panya A, Weiss J, McClements DJ, Lecomte J, Decker EA, Villeneuve P. What makes good antioxidants in lipid-based systems? The next theo-ries beyond the polar paradox. Crit Rev Food Sci Nutr. 2015;55(2):183–201.
6. Botterweck A, Verhagen H, Goldbohm R, Kleinjans J, Van Den Brandt P. Intake of butylated hydroxyanisole and butylated hydroxytoluene and stomach cancer risk: results from analyses in the Netherlands cohort study. Food Chem Toxicol. 2000;38(7):599–605.
7. Mayakrishnan V, Abdullah N, Abidin MHZ, Fadzil NHM, Johari NMK, Aminudin N, Abidin NZ. Investigation of the antioxidative potential of various solvent fractions from fruiting bodies of Schizophyllum commune (Fr.) mush-rooms and characterization of phytoconstituents. J Agricult Sci. 2013;5(6):58–69.
8. Lau C-C, Abdullah N, Shuib AS, Aminudin N. Proteomic analysis of antihypertensive proteins in edible mushrooms. J Agr Food Chem. 2012;60(50):12341–8.
10. Abdullah N, Ismail SM, Aminudin N, Shuib AS, Lau BF. Evaluation of selected culinary-medicinal mushrooms for antioxidant and ACE inhibitory activities. Evid Based Complement Alternat Med. 2012:1–12.
11. Kuda T, Yano T. Changes of radical-scavenging capacity and ferrous reducing power in chub mackerel Scomber japoni-cus and Pacific saury Cololabis saira during 4° C storage and retorting. LWT Food Sci Technol. 2009;42(6):1070–5.
12. Öztürk M, Aydoğmuş-Öztürk F, Duru ME, Topçu G. Antioxidant activity of stem and root extracts of rhubarb (Rheum ribes): an edible medicinal plant. Food Chem. 2007;103(2):623–30.
13. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Calorimetric method for determination of sugars and related substances. Anal Chem. 1956;28:350–6.
14. Ahmadvand H, Khosrowbeygi A, Ghasemi M. Inhibitory effect of Allium ascalonicom hydroalcoholic extract on low-density lipoprotein (LDL) oxidation induced by CuSO4 in vitro. J Med Plants Res. 2011;5(6):1012–7.
15. Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 2005;91(3):571–7.
16. Tomsone L, Kruma Z, Lepse L. Influence of genotype and harvest time on the phenolic content of horseradish (Armoracia rusticana L.) roots. Res Rural Dev. 2012:124–30.
17. Alam N, Yoon KN, Lee TS. Evaluation of the antioxi-dant and antityrosinase activities of three extracts from Pleurotus nebrodensis fruiting bodies. Afr J Biotechnol. 2011;10(15):2978–86.
18. Moon JK, Shibamoto T. Antioxidant assays for plant and food components. J Agr Food Chem. 2009;57(5):1655–66.
19. Ha YL, Storkson J, Pariza MW. Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conju-gated dienoic derivatives of linoleic-acid. Cancer Res. 1990;50(4):1097–101.
20. Jasinghe VJ, Perera CO. Distribution of ergosterol in different tissues of mushrooms and its effect on the con-version of ergosterol to vitamin D-2 by UV irradiation. Food Chem. 2005;92(3):541–6.
21. Teichmann A, Dutta PC, Staffas A, Jagerstad M. Sterol and vitamin D-2 concentrations in cultivated and wild grown mushrooms: effects of UV irradiation. LWT-Food Sci Technol. 2007;40(5):815–22.
22. Keleş A, Koca I, Gençcelep H. Antioxidant properties of wild edible mushrooms. J Food Proc Technol. 2011;2:130.
23. Mohamed T, Issoufou A, Zhou H. Antioxidant activity of fractionated foxtail millet protein hydrolysate. Int Food Res J. 2012;19(1):207–13.
24. Kunio S, Hiroyuk IU, Hirotomo O. Isolation and charac-terization of free radical scavenging activities peptides derived from casein. J Nutr Biochem. 2000;11:128–31.
25. Barros L, Pereira C, Ferreira ICFR. Optimised analysis of organic acids in edible mushrooms from Portugal by ultra-fast liquid chromatography and photodiode array detection. Food Anal Method. 2013;6:309–16.
26. Dubost NJ, Beelman RB, Peterson DG, Royse DJ. Identification and quantification of ergothioneine in cultivated mushrooms by liquid chromatography mass spectroscopy. Abstr Pap Am Chem Soc. 2005;230:U83.
27. Elias RJ, McClements DJ, Decker EA. Antioxidant activ-ity of cysteine, tryptophan, and methionine residues in continuous phase beta-lactoglobulin in oil-in-water emul-sions. J Agr Food Chem. 2005;53(26):10248–53.
28. Hayes JE, Allen P, Brunton N, O’Grady MN, Kerry JP. Phenolic composition and in vitro antioxidant capacity of four commercial phytochemical products: olive leaf extract (Olea europaea L.), lutein, sesamol and ellagic acid. Food Chem. 2011;126(3):948–55.
29. Amadou I, Le G-W, Shi Y-H, Jin S. Reducing, radical scav-enging, and chelation properties of fermented soy protein meal hydrolysate by Lactobacillus plantarum Lp6. Int J Food Propert. 2011;14(3):654–65.
30. Wiseman H. Vitamin-D is a membrane antioxidant - ability to inhibit iron-dependent lipid-peroxidation in liposomes
Volume 18, Number 2, 2016
Protective Effect of Pleurotus pulmonarius Against Human Low-Density Lipoprotein Oxidation 121
compared to cholesterol, ergosterol and tamoxifen and rele-vance to anticancer action. Febs Lett. 1993;326(1–3):285–8.
31. Tokumura A, Toujima M, Yoshioka Y, Fukuzawa K. Lipid peroxidation in low density lipoproteins from human plasma and egg yolk promotes accumulation of 1-acyl analogues of platelet-activating factor-like lipids. Lipids. 1996;31(12): 1251–8.
32. Vaya J, Mahmood S, Goldblum A, Aviram M, Volkova N, Shaalan A, Musa R, Tamir S. Inhibition of LDL oxidation
by flavonoids in relation to their structure and calculated enthalpy. Phytochemistry. 2003;62(1):89–99.
33. Sit ASM, Ho EYW, Li RWS, Man RWK, Kwan YW, Vanhoutte PM, Leung GPH. Ergothioneine shows protec-tive effect on endothelial cells in oxidative stress. FASEB J. 2011;25:630.3.
34. Paul BD, Snyder SH. The unusual amino acid l-ergothi-oneine is a physiologic cytoprotectant. Cell Death Differ. 2010;17(7):1134–40.
![Effect of Aqueous Extract and Polyphenol Fraction Derived ...plasmalevelsofTC,TGs,andLDL-Cinanimalmodels[5, 6]. On the other hand, oxidative stress is involved in the pathogenesis](https://static.documents.pub/doc/80x56/60c0cea6864d2108c631b920/effect-of-aqueous-extract-and-polyphenol-fraction-derived-plasmalevelsoftctgsandldl-cinanimalmodels5.jpg)
