Hepatoprotective and antioxidant activity of the methanolic extract of Mucuna pruriens Bak. roots

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Hepatoprotective and antioxidant activity of the methanolic extract of Mucuna pruriens Bak. roots
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  Hepatoprotective and Antioxidant Activity of Linden ( Tilia platyphyllos  L.) Infusion Against Ethanol-Induced OxidativeStress in Rats Yakup Yayalac ı  • Ismail Celik  • Bedia Bat ı Received: 3 October 2013/Accepted: 5 December 2013/Published online: 15 December 2013   Springer Science+Business Media New York 2013 Abstract  The present study was carried out to evaluatethe hepatoprotective effect and antioxidant role of infusionprepared from linden flowers (LF) against ethanol-inducedoxidative stress. The hepatoprotective and antioxidant roleof the plant’s infusion against ethanol-induced oxidativestress was evaluated by measuring liver damage serumbiomarkers, aspartate aminotransferase (AST), alanineaminotransferase, lactate dehydrogenase (LDH), total pro-tein, total albumin, and total cholesterol level; ADS such asGSH, GR, SOD, GST, CAT and GPx, and MDA contentsin various tissues of rats. Rats were divided into fourexperimental groups: I (control), II (20 % ethanol), III(2 % LF), and IV (20 % ethanol  ?  2 % LF). According tothe results, the level of serum marker enzymes, AST andLDH, was significantly increased in group alcohol andgroup LF as compared to control group, whereas decreasedin group IV as compared to ethanol group. With regard toMDA content and ADS constituents, MDA contents of alcohol group in all tissues, except for erythrocytes andheart, and in brain, kidney, and spleen of LF group sig-nificantly increased compared to control group, whereas LFbeverage extract supplementation did not restore theincreased MDA towards close the control level. In addi-tion, while ethanol caused fluctuation in antioxidantdefense system constituents level as a result of oxidativestress condition in the rats, it could have not been deter-mined the healing effects of the LF against these fluctua-tions. The results indicated that LF beverage extract couldnot be as important as diet-derived antioxidants in pre-venting oxidative damage in the tissues by reducing thelipid oxidation or inhibiting the production of ethanol-induced free radicals in rats. Keywords  Linden flowers    Serum biomarkers   Antioxidant defense system    Malondialdehyde    Rats Abbreviation LF Linden flowersAST Aspartate aminotransferaseALT Alanin aminotransferaseLDH Lactate dehydrogenaseTPRO Total proteinTALB Total albuminTCHOL Total cholesterol;ADS Antioxidant defense systemsGSH Reduced glutathioneGR Glutathione reductaseSOD Superoxide dismutaseGST Glutathione-S-transferaseCAT CatalaseGPx Glutathione peroxidaseMDA Malondialdehyde Introduction Many naturally occurring compounds with antioxidativeaction are now known to protect cellular components fromoxidative damage and prevent diseases (Gulcin 2009,2010). A number of such compounds can activate the phaseII detoxification enzymes, which can remove the toxicelements from our system. Intake of such phytochemicalsis therefore beneficial to human health. In addition, manynatural compounds are now known to have a modulatorrole on physiological functions and biotransformation Y. Yayalac ı    I. Celik ( & )    B. Bat ı Department of Biology, Science Faculty, Yu¨zu¨ncu¨ YilUniversity, 65080 Van, Turkeye-mail: icelik@yyu.edu.tr; icelik65@gmail.com  1 3 J Membrane Biol (2014) 247:181–188DOI 10.1007/s00232-013-9622-z  reactions involved in the detoxification process, therebyaffording protection from cytotoxic, genotoxic, and meta-bolic actions of environmental toxicants (Saha and Das2003). Numerous studies demonstrate that a great numberof medicinal and aromatic herbs, as well as fruits andleaves of some berry plants biosynthesize phytochemicalspossessing antioxidant activity and may be used as a nat-ural source of free radical scavenging compounds (Yu et al.2005; Sacchetti et al. 2005). Also, a great number of spices and aromatic herbs contain chemical compounds exhibitingantioxidant properties. These properties are attributed to avariety of active phytochemicals including vitamins,carotenoids, terpenoids, alkaloids, flavonoids, lignans,simple phenols and phenolic acids, and so on (Liu and Ng2000).The Tiliaceae plant  Tilia argentea  (synonym:  T. tom-entosa ; common names: linden or lime), a well-knownOccidental herb, is cultivated in Bulgaria and Albania. Thedried flowers of this plant, which are commonly called‘‘silver linden flowers’’, have been widely used in herbalteas, and as a diuretic, stomachic, antineuralgic, and sed-ative in European countries. By bioassay-guided separa-tion, six flavonol glycosides were isolated from themethanolic extract of linden. On the other hand, they foundthat the methanolic extract from the flowers of   T. argentea (Linden) showed potent protective effects againstd-galactosamine (d-GalN)/lipopolysaccharide-inducedliver injury in mice and on d-GalN cytotoxicity in primarycultured mouse hepatocytes (Matsuda et al. 2002). In folk medicine, the linden flower is used as a diuretic,stomachic,antispasmodic,andsedativeagent(Wichtl2004).Theinflorescenceofthelindencontainsbetween3and10 %polysaccharides which are mucilaginous. Furthermore,condensed tannins (Behrens et al. 2003) (strong antioxi- dants), such as dimers of procyanidin (B-2), were identifiedin this raw material. Other phenolic compounds identified inlinden flowers (LF) are flavonoids, mainly quercetin glyco-sides (rutin, quercitrin, and isoquercitrin), kaempferol gly-cosides (Toker et al. 2001), and phenolic acids (caffeic, p-coumaric, and chlorogenic acids) (Czygan 2007).The reactive oxygen species (ROS) are known to play amajor role in either the initiation or progression of carci-nogenesis by inducing oxidative stress (Gulcin 2006). Peroxides and superoxide anion (  O 2 - ) produce cytotoxi-city/genotoxicity in cellular system (Gulcin et al. 2008, 2010). ROS and nitrogen species are formed in the humanbody, and endogenous antioxidant defenses are not alwayssufficient to counteract them completely. Free radicals andROS have been implicated in many human pathologicalconditions, including rheumatoid arthritis (Kunsch et al.2005), hemorrhagic shock (Childs et al. 2002), cardiomy- opathy (Akhileshwar et al. 2007), cystic fibrosis (Cowley and Linsdell 2002), and gastrointestinal ischemia (Halli-well and Gutteridge 1999; Henrotin et al. 2003; Hogg 1998). A high concentration of ROS leads to the destruc-tion of cell membranes (Mishra 2004), proteins, and nucleic acids (Briganti and Picardo 2003; Bergamini et al. 2004), which is dangerous because it leads to carcinomaformation (Kang 2005). A large number of studies supportthe hypothesis that oxidative damage to DNA, lipids, andproteins may contribute to the development of cardiovas-cular disease, cancer, and neurodegenerative diseases(Halliwell 1996; Gulcin et al. 2006). Diet-derived antiox- idants may therefore be particularly important in protectingagainst chronic diseases (Halliwell 1996; Vendemiale et al. 1999). Plant extracts rich in phenolic compounds withantiradical activity (Gohil and Packer 2002; Sastre et al. 2002; Celik et al. 2009), such as extracts from the linden, could be used as medicines or preventive agents protectinghumans and other animals from the destructive action of free radicals and ROS.Although some studies reported the major flavonolcontents and antioxidant activities in linden varieties in theworld, the literature lacks information on chemopreventiveand antioxidant role of linden grown in Turkey. Therefore,the objective of this study was to determine hepatopro-tective and antioxidant capacity in flowers of linden vari-ety, which are widely grown in C¸ank  ı r ı  province located inInner Anatolia Region of Turkey.There is a growing interest of natural products in humandiet, both due to the possible negative effects of syntheticfood additives on human health and the increased con-sumer perception of this problem in recent years. As far asour literature survey could ascertain, so far no studies havebeen reported on hepatoprotective role and antioxidantcapacity of the LF beverage extract used in this study. Theobjective of this study was to determine healthful potentialsof LF beverage extract against alcohol-induced oxidativestress by evaluating their in vivo hepatoprotective role andantioxidant capacity. Thus, in the present study, we haveextensively studied the antioxidant activity of LF infusionusing in vivo models. For this aim, the treatment of LFinfusion was done orally as food containing 2 % powderedLF infusion because the effect of the functional plantrepresents a well characterized in nutrition and widely usedas consumption by human in our country and worldwide.The serum enzymes were chosen due to their importance asindex of hepatotoxin and hepatoprotective. The antioxidantactivity of LF on some phase II detoxification ADS such asreduced glutathione (GSH), glutathione reductase (GR),superoxide dismutase (SOD), glutathione-S-transferase(GST), catalase (CAT) and glutathione peroxidase (GPx),and malondialdehyde (MDA) contents in the various tis-sues were evaluated during experiment. 182 Y. Yayalac ı  et al.: Hepatoprotective and Antioxidant Activity of Linden ( Tilia platyphyllos  L.)  1 3  Materials and Methods ChemicalsThiobarbituric acid (TBA), butylated hydroxytoluene, tri-chloroacetic acid, ethylenediaminetetraacetic acid (EDTA),GSH, metaphosphoric acid, 5,5 0 dithiobis-(2-nitrobenzoicacid), trihydroxymethyl aminomethane (Tris), 1-chloro-2,4-dinitrobenzene (CDNB), oxidized glutathione,  b -Nic-otinamide adenine dinucleotide phosphate (NADPH),potassium dihydrogen phosphate (KH 2 PO 4 ), and sodiumchloride (NaCl) of technical grade used in this study weresupplied by Sigma Chemical Co. (St. Louis, MO, USA).Kits for antioxidant enzymes analysis were supplied byRandox Laboratories Ltd.AnimalsFour months old Rats (Wistar albino) with an averageweighing 200–250 g were provided from the ExperimentalAnimal Research Center, Yuzuncu Yil University, andwere housed in four groups, each group containing six rats.The animals were housed at 20  ±  2   C in a daily light/dark cycle. All animals were fed a group wheat–soybean meal-based diet and water ad libitum in stainless cages, andreceived humane care according to the criteria outlined inthe ‘‘Guide for the Care and Use of Laboratory Animals’’prepared by the National Academy of Science and pub-lished by the National Institutes of Health. The ethic reg-ulations were followed in accordance with national andinstitutional guidelines for the protection of animal welfareduring experiments. This study was approved by The EthicCommittee of the Yu¨zu¨ncu¨ Y ı l University.Preparation of FoodsBriefly, the natural and organic LF were provided from alocal herbalist in C¸ank  ı r ı , a major linden-producing prov-ince of Turkey. This variety was chosen because theremarkable total production of LF in the region comes fromthe variety.Experimental DesignThe rats were randomly divided into four groups eachcontaining six rats.Group I (Control): the rats received tab water and fedwith standard pellet diet as ad libitum.Group II (Alcohol): the rats received 20 % ethanol waterand fed with standard pellet diet as ad libitum. Dose of ethanol was selected on the basis of a 20 % concentra-tion at which caused oxidative stress when administeredorally (Aykac et al. 1985; Sonde et al. 2000; Yurt and Celik  2011; Dogan and Celik  2012). Group III (2 % LF): the rats received 2 % LF infusionand fed with standard pellet diet as ad libitum.Group IV (2 % LF beverage extract  ?  20 % alcohol):the rats received 2 % LF infusion  ?  20 % alcohol waterand fed with standard pellet diet as ad libitum.Preparation of Tissues Supernatant and ErythrocytePelletsAt the end of the 50 days experiments, the rats wereanesthetized by injection of ketamine (5 mg/100 g bodyweight) intra-peritoneally. The blood samples wereobtained from a cardiac puncture using syringe for thedetermination of serum marker enzyme levels and bio-chemical analysis. The serum samples were obtained bycentrifuging blood samples at 4,000 9 g  for 15 min at 4   C,and enzyme levels were measured in these serum samples.For biochemical analysis, blood samples were put imme-diately into silicon disposable glass tubes with EDTA as ananticoagulant and were centrifuged at 4,000 9 g  for 15 minat 4   C and erythrocyte pellets were obtained. Then, thepellets were washed three times with physiological saline(0.9 % NaCl).The tissues as brain, kidney, spleen, heart, and liverwere dissected and put in petri dishes. After washing thetissues with physiological saline (0.9 % NaCl), sampleswere taken and kept at  - 78   C during the analysis. Thetissues were homogenized for 5 min in 50 mM ice-coldKH 2 PO 4  solution (1:5 w/v) using stainless steel probehomogenizer (20 kHz frequency ultrasonic, Jencons Sci-entific Co.) for 5 min. and then centrifuged at 7,000 9 g  for15 min. All processes were carried out at 4   C. Superna-tants and erythrocyte pellets were used to determine ADSconstituents and MDA contents (Celik et al. 2009; Yurt and Celik  2011; Dogan and Celik  2012). Biochemical AnalysisThe erythrocyte and tissues MDA concentration weredetermined using the method described by Jain et al.(1989) based on TBA reactivity. The erythrocyte and tis- sues GSH concentration were measured using the methoddescribed by Beutler et al. (1963). GST was assayed by following the conjugation of glutathione with CDNB at340 nm as described by Mannervik and Guthenberg(1981). GR activity was assayed according to Carlberg and Mannervik (1975) as the decrease in absorbance of  NADPH at 340 nm. GPx activity was assayed according toPaglia and Valentine (1967) based on that of GPx which catalyzes the oxidation of GSH by cumene hydroperoxide. Y. Yayalac ı  et al.: Hepatoprotective and Antioxidant Activity of Linden ( Tilia platyphyllos  L.) 183  1 3  SOD activity was measured at 505 nm by calculatinginhibition percentage of formazan dye formation (McCordand Fridovich 1969). CAT activity was determined usingthe method described by Aebi (1974), based on that of the rate of H 2 O 2  consumption and as the decrease in absor-bance at 240 nm.Measurement of Enzyme LevelsSerum marker enzyme activities such as AST, ALT, LDH,TPRO, TALB, and TCHOL were measured by an autoanalyzer (BM/HITACHI-911), using the kits.Analysis of DataAll data were expressed as mean  ±  standard deviation(SD). The statistical analyses were made using the Minitab13 for windows packet program. Means and Standarddeviations were calculated according to the standardmethods for all parameters. One-way analysis of variance(ANOVA) statistical test was used to determine the dif-ferences between means of the experimental groupsaccepting the significance level at  p  B  0.05. Results At the end of 50 days of treatment, the hepatoprotectiveeffect of linden infusion against ethanol-induced oxidativestress was evaluated by measuring liver damage via serummarker enzymes AST, ALT, and LDH enzyme levels andTPRO, TALB, and TCHOL levels. On the other hand,antioxidant role of the linden infusion was evaluated bymeasuring CAT, SOD, glutathione peroxidase (GSH-Px),GST, GR enzyme activities and GSH, and MDA contentsin brain, kidney, spleen, erythrocyte, heart, and liver tissuesof rats. The results of experiment showed that the treatmentof rats with alcohol and alcohol  ?  LF beverage extractcontaining diet supplementation caused changes in thelevel of serum biomarkers, MDA content and ADS con-stituents in comparison with control rats. As known, serumAST, ALT, and LDH levels are susceptible to hepatotoxinand serve as markers of liver damage that promote therelease of such aminotransferases from hepatocytes intoblood stream. According to the results, serum AST, ALT,LDH, and TCHOL levels of group II and serum AST, ALT,and LDH levels of group III were significantly increasedcompared with group I. Serum LDH and TCHOL levels of group IV significantly decreased compared with group II(Table 1). With regard to MDA content and ADS constit-uents, MDA contents of II group in all the tissues exceptfor erythrocytes and heart and group III in brain, kidney,and spleen significantly increased compared with group I.On the other hand, while ethanol caused fluctuation inantioxidant defense system constituents level as a result of oxidative stress condition in the rats, it could have not beendetermined the healing effects of the LF against thesefluctuations (Table 2). Discussion Overexposures to oxidative stress caused by environmentalpollutants are thought to increase the risk of cancer. Also, itis known that alcoholic liver disease is a major medicalcomplication of alcohol intake. Oxidative stress plays animportant role in the development of alcohol-related liverdisease (Gulcin 2007; Coban et al. 2008). Hence, efforts are needed to provide effective protection from the dam-aging agents and experimental studies have implicated theinfluence of a functional plant, LF in this regard. The firstaim of this study was to investigate whether the LF infu-sion serum biomarkers could prevent hepatotoxicity of ethanol, and decrease content of the MDA and efficacy onthe antioxidant defense system in rats.The results of the present study demonstrated, for thefirst time, that the treatment of rat with LF effectively didnot protect the rat against alcohol-induced hepatotoxicity, Table 1  Determination hepatoprotective capacity of   Tilia platyphyllos  infusion against ethanol exposed in rats (Mean  ±  SD)Parameters Control X  ±  SD 20 % Ethanol X  ±  SD 2 % LF X  ±  SD 2 % LF  ?  20 % Ethanol X  ±  SDAST (U/L) 146.3  ±  10.90 279.3  ±  22.80 a 184.0  ±  18.30 a 249.3  ±  24.30 a ALT (U/L) 46.2  ±  3.30 76,2  ±  4,60 a 44.3  ±  3.30 61.7  ±  4.50 ab LDH (U/L) 1707.5  ±  12.00 2101.3  ±  127.10 a 2158.8  ±  123.60 a 1711.2  ±  155.00 b TPRO (g/dL) 6.8  ±  0.30 7.0  ±  0.30 7.0  ±  0.30 6.9  ±  0.20TALB (g/dL) 4.3  ±  0.20 4.6  ±  0.30 4.5  ±  0.20 4.2  ±  0.10 b TCHOL (mg/dL) 58.3  ±  4.80 64.3  ±  3.80 a 59.8  ±  4.70 47.2  ±  3.80 ab Each value represents the Mean  ±  SD a Significantly different from control b Significantly different from 20 % ethanol exposed rats at  p \ 0.05 (one way ANOVA)184 Y. Yayalac ı  et al.: Hepatoprotective and Antioxidant Activity of Linden ( Tilia platyphyllos  L.)  1 3  Table 2  Determination antioxidant capacity of   Tilia platyphyllo  infusion against ethanol exposed in ratsTissue Parameters Control X  ±  SD 20 % EthanolX  ±  SD2 % LF X  ±  SD 2 % LF  ?  20 %Ethanol X  ±  SDErythrocyte GSH (mg/ml) 4.15  ±  0.30 4.79  ±  0.26 a 5.19  ±  0.13 a 6.81  ±  0.23 ab MDA (nmol/ml) 1.68  ±  0.2 1.76  ±  0.23 1.63  ±  0.15 2.01  ±  0.21 *a GST (U/ml) 84.75  ±  0.79 92.32  ±  1.56 a 146.92  ±  3.02 a 100.05  ±  3.96 ab GPx (U/ml) 464.09  ±  10.54 446.15  ±  14.73 a 422.00  ±  13.10 a 431.42  ±  11.40 a GR (U/ml) 7.95  ±  0.32 5.48  ±  0.61 a 6.77  ±  0.62 a 5.79  ±  0.54 a SOD (U/ml) 2150.20  ±  56.87 2128.36  ±  28.41 2112.71  ±  27.44 2124.55  ±  53.53CAT (U/ml) 2484.77  ±  131.33 2585.28  ±  156.31 2568.53  ±  156.66 2267.01  ±  187.93 b Liver GSH (mg/g) 21.77  ±  1.37 38.13  ±  0.61 a 38.18  ±  1.47 a 36.94  ±  0.90 ab MDA (nmol/g) 40.48  ±  2.34 47.81  ±  3.24 a 42.74  ±  1.16 45.55  ±  2.08 a GST (U/g) 166.68  ±  8.60 156.33  ±  7.32 a 123.63  ±  6.03 a 200.74  ±  8.98 ab GPx (U/g) 194.65  ±  4.15 158.98  ±  4.41 a 168.25  ±  6.94 a 169.34  ±  6.67 ab GR (U/g) 3.0  ±  0.42 4.13  ±  0.22 4.28  ±  0.64 a 3.60  ±  0.60 ab SOD (U/g) 2232.12  ±  37.21 2065.00  ±  30.15 a 1901.83  ±  57.98 a 1942.90  ±  22.75 ab CAT (U/g) 1831.47  ±  150.59 2233.50  ±  143.50 a 2095.03  ±  186.93 a 2348.53  ±  161.91 a Brain GSH (mg/g) 43.37  ±  1.94 58.78  ±  2.18 a 50.67  ±  1.52 a 47.68  ±  0.94 ab MDA (nmol/g) 20.51  ±  0.78 32.49  ±  1.04 a 29.90  ±  0.95 a 14.25  ±  0.92 ab GST (U/g) 59.65  ±  1.71 60.74  ±  1.83 65.76  ±  1.41 a 74.52  ±  1.40 ab GPx (U/g) 205.15  ±  4.64 199.84  ±  3.35 a 203.43  ±  6.50 221.45  ±  1.36 ab GR (U/g) 0.77  ±  0.09 0.89  ±  0.11 0.85  ±  0.12 0.82  ±  0.12SOD (U/g) 1967.23  ±  67.94 1703.61  ±  150.62 a 1433.54  ±  120.34 a 1650.69  ±  94.62 a CAT (U/g) 234.52  ±  36.70 234.52  ±  56.06 301.52  ±  36.70 a 402.03  ±  59.93 ab Kidney GSH (mg/g) 17.77  ±  0.75 19.71  ±  0.88 a 16.12  ±  0.73 a 17.70  ±  0.74 a MDA (nmol/g) 181.01  ±  17.18 233.15  ±  16.56 a 211.35  ±  15.21 a 128.66  ±  13.51 ab GST (U/g) 68.92  ±  1.67 62.56  ±  1.89 a 58.99  ±  1.49 a 51.95  ±  1.43 ab GPx (U/g) 388.50  ±  6.69 377.13  ±  8.93 a 400.09  ±  9.47 a 404.35  ±  7.55 ab GR (U/g) 1.84  ±  0.28 1.53  ±  0.17 a 1.76  ±  0.28 1.97  ±  0.20 a SOD (U/g) 1886.08  ±  79.28 1784.53  ±  74.13 a 1744.33  ±  132.98 a 1684.05  ±  72.82 ab CAT U/g 1719.80  ±  85.77 1738.78  ±  132.10 2065.99  ±  69.46 a 2017.97  ±  129.75 ab Spleen GSH (mg/g) 51.81  ±  2.48 44.32  ±  3.14 a 44.60  ±  3.48 a 44.81  ±  3.71 a MDA (nmol/g) 82.68  ±  7.15 106.10  ±  6.14 a 125.64  ±  7.55 a 89.70  ±  7.77 b GST (U/g) 41.24  ±  1.23 51.29  ±  1.83 a 45.26  ±  1.53 a 42.99  ±  0.99 ab GPx (U/g) 327.41  ±  9.52 351.71  ±  8.76 a 344.16  ±  9.43 a 342.21  ±  6.87 a GR (U/g) 3.54  ±  0.45 3.83  ±  0.56 2.68  ±  0.54 a 2.89  ±  0.50 ab SOD (U/g) 2118.70  ±  41.90 2040.02  ±  60.06 a 1997.69  ±  49.22 a 1937.72  ±  87.31 ab CAT (U/g) 661.12  ±  53.04 484.67  ±  50.97 a 651.07  ±  71.22 734.82  ±  75.41 b Heart GSH (mg/g) 18.30  ±  0.71 20.17  ±  1.31 a 22.18  ±  1.52 a 21.04  ±  1.23 a MDA (nmol/g) 58.29  ±  2.87 60.55  ±  2.41 59.69  ±  2.70 48.14  ±  4.07 ab GST (U/g) 27.14  ±  1.80 34.83  ±  1.90 a 40.23  ±  1.57 a 39.01  ±  1.88 ab GPx (U/g) 192.85  ±  3.12 191.81  ±  4.60 203.40  ±  5.18 a 207.77  ±  1.81 ab GR (U/g) 0.90  ±  0.13 1.15  ±  0.21 a 0.87  ±  0.07 1.04  ±  0.17SOD (U/g) 2108.39  ±  80.16 2007.39  ±  66.58 a 1982.44  ±  86.55 a 1969.94  ±  69.12 a CAT (U/g) 208.83  ±  37.29 276.95  ±  22.29 a 422.13  ±  42.59 a 422.13  ±  52.76 ab Each value represents the Mean  ±  SD a Significantly different from control b Significantly different from ethanol exposed rats at  p \ 0.05 (one way ANOVA)Y. Yayalac ı  et al.: Hepatoprotective and Antioxidant Activity of Linden ( Tilia platyphyllos  L.) 185  1 3
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