Article pubs.acs.org/JAFC
Rice Hull Smoke Extract Protects Mice against a Salmonella Lipopolysaccharide-Induced Endotoxemia Sung Phil Kim,† Seok Hyun Nam,*,† and Mendel Friedman*,‡ †
Department of Biological Science, Ajou University, Suwon, 443-749, Republic of Korea Western Regional Research Center, Agricultural Research Service, U.S Department of Agriculture, 800 Buchanan Street, Albany, California 94710, United States
‡
ABSTRACT: Endotoxemia (sepsis, septic shock) is an inflammatory, virulent disease that results mainly from infection by Gram-negative bacteria. The present study investigates the inhibitory effects of a rice hull smoke extract (RHSE) against murine endotoxemia induced by Salmonella lipopolysaccharide and D-galactosamine (LPS/GalN). Pretreatment of the mice with RHSE via dietary administration for 2 weeks resulted in the suppression (in %) of LPS/GalN-induced catalase by 70.7, superoxide dismutase (SOD) by 54.6, and transaminase (GOT/GPT) liver enzymes by 40.6/62.5, the amelioration of necrotic liver lesions, and the reduction of tumor necrosis factor-α (TNF-α) by 61.1 and nitrite serum level by 83.4, as well as myeloperoxidase (MPO) enzyme associated with necrotic injury of the lung and kidney by 65.7 and 63.3, respectively. The RHSE also extended the lifespan of the toxemic mice. The results using inflammation biomarkers and from the lifespan studies suggest that the RHSE can protect mice against LPS/GalN-induced liver, lung, and kidney injuries and inflammation by blocking oxidative stress and TNF-α production, thereby increasing the survival of the toxic-shock-induced mice. These beneficial effects and previous studies on the antimicrobial effects against Salmonella Typhimurium in culture and in mice suggest that the smoke extract also has the potential to serve as a new multifunctional resource in human food and animal feeds. Possible mechanisms of the beneficial effects at the cellular and molecular levels and suggested food uses are discussed. KEYWORDS: rice hull liquid smoke, mice, endotoxemia prevention, mechanism, food preservative, Salmonella lipopolysaccharide, cytokines, myeloperoxidase, oxidative enzymes, histopathology, liver/kidney injuries prevention
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flavor enhancer in dairy, meat, and seafood products.17−20 In the United States, liquid smoke has been granted generally recognized-as-safe (GRAS) status as a natural flavoring.21 In a previous study,22 we described the production and composition of a new liquid rice hull liquid smoke extract (RHSE) with a smoky aroma and sugar-like odor prepared by pyrolysis of rice hulls followed by liquefaction of the resulting smoke. The liquid smoke contained 161 compounds, as characterized by gas chromatography and mass spectrometry. In vitro, ex vivo, and in vivo mouse assays showed that the extract has strong antioxidative, antiallergic, and anti-inflammatory properties. In two other studies, we determined the protective effect of RHSE against type 2 diabetes in mice induced by a high-fat diet. Feeding the supplemented diet with 0.5% or 1% RHSE for 7 weeks resulted in significantly reduced blood glucose and triglyceride and cholesterol concentrations, higher serum insulin levels, and improved glucose tolerance compared with the control of mice on a high-fat diet.23,24 It seems that RHSEsupplemented food could protect insulin-producing islet cells against damage triggered by oxidative stress and local inflammation associated with diabetes. In a follow-up study, RHSE was also tested for bactericidal activity against Salmonella Typhimurium using the disc-
INTRODUCTION Endotoxemia (sepsis, septic shock) is a systemic rapidly progressive infectious disease characterized by an overwhelming activation of the immune system and release into the bloodstream of microbial endogenous mediators of inflammation.1−11 No effective therapeutic treatment seems to be available to protect patients from the tissue damage and organ failure induced Salmonella lipopolysaccharide (LPS) and other glycolipid cell wall components of Gram-negative bacteria that are associated with endotoxemia and can result in high mortality.12,13 A review of 27 published studies by Hurley and Opal2 revealed the surprising result that endotoxemia detection can predict mortality among patients bacteremic with non-Escherichia coli Enterobacteriaceae but not with E. coli. It is also relevant to note that dietary fructose induced hepatic injury and endotoxemia in nonhuman primates, suggesting that fructose might also contribute to the metabolic disease.14 These considerations stimulated a worldwide investigation to discover the potential of natural compounds to inhibit the pathogenesis of endotoxemia. Rice hulls, which account for 20% of the rice crop, are a byproduct of postharvest rice processing.15 Rice hulls consist mainly of lignin, hemicellulose, cellulose, and hydrated silica.16 A byproduct of the combustion of rice hulls is the smoke that is generated. Liquid smoke has gained widespread acceptance in the food industry, replacing traditional smoking practices. Liquid smoke has the potential for use as an all-natural antimicrobial agent against E. coli, Listeria, Salmonella, and Staphylococcus and is now used widely as a preservative and © 2014 American Chemical Society
Received: Revised: Accepted: Published: 7753
March 31, 2014 July 8, 2014 July 19, 2014 July 28, 2014 dx.doi.org/10.1021/jf501533s | J. Agric. Food Chem. 2014, 62, 7753−7759
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diffusion method.25 The minimum inhibitory concentration (MIC) value of RHSE was 0.822% (v/v). The in vivo antibacterial activity of RHSE (1.0%, v/v) was also examined in a Salmonella-infected Balb/c mouse model. Mice infected with a sublethal dose of the pathogens were administered intraperitoneally a 1.0% solution of RHSE at four 12 h intervals during the 48 h experimental period. The results showed that RHSE antimicrobial potency approached that of the standard medical antibiotic vancomycin and that the combination of RHSE and vancomycin acted synergistically against the foodborne bacteria. The inclusion of RHSE (1.0% v/w) as part of a standard mouse diet fed for 2 weeks decreased mortality of 10 mice infected with lethal doses of Salmonella. Photomicrographs of histological changes in liver tissues show that RHSE also protected the liver against Salmonella-induced pathological necrosis lesions. These beneficial results suggest that RHSE has the potential to complement wood-derived smokes as an antimicrobial flavor formulation for use in human food and animal feeds. Because RHSE inhibited Salmonella in culture and in vivo, expectations are that it might protect the mice against Salmonella LPS-induced murine endotoxemia (sepsis). The objective of this study was to determine whether this is the case.
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for 60 h after the LPS and GalN challenge. Apart from the determination of the survival rate, the other experiments were carried out 4 h after challenge of a sublethal dose of the LPS and GalN (5 μg/ kg and 700 mg/kg, respectively). The mice were sacrificed by CO2 asphyxia, and livers and kidneys from each group were excised and weighed. The blood from the three groups of mice was collected by cardiac puncture and allowed to clot at room temperature for 30 min. Subsequently, the blood was subjected to centrifugation at 2000g for 30 min. The separated serum was stored at −20 °C until use. Histology of Liver Tissue. For histological analysis, the liver tissue of the mice was fixed with paraformaldehyde (4%) in phosphate buffer (0.5 M; pH 7.4). The tissue was rinsed with water, dehydrated with ethanol, and embedded in paraffin. The tissues were sectioned into samples (4 μm) and mounted onto glass slides. The sections were then dewaxed using xylene and ethanol and stained with hematoxylin and eosin Y (H&E) to reveal the hemorrhagic necrosis in the liver. Histological changes were observed under a light microscope at ×100 magnification. Serum Nitrite and Nitrate Levels. Serum nitrite and nitrate levels were measured primarily following the method of Misko et al.,27 with some modification. Briefly, serum was filtered through an Ultrafree-MC microcentrifuge filter unit (Millipore, Bedford, MA, USA) for 1 h at 14000 rpm to remove the hemoglobin released by cell lysis. The serum (50 μL) was incubated in a reaction mixture [40 mM Tris Cl, 40 μM reduced β-nicotinamide adenine dinucleotide phosphate, 40 μM flavin adenine dinucleotide, and nitrate reductase 0.05 U/mL at pH 7.9] at 37 °C for 15 min. Reduced samples were incubated with an equal volume of Griess reagent [1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid] at room temperature for 15 min. The absorbance at 570 nm was recorded using a microplate reader (model 550, BioRad). The total nitrate/nitrite concentration was determined by comparison with a reduced sodium nitrate standard curve. TNF-α and Serum Transaminase. TNF-α level was determined using an enzyme-linked immunosorbent assay (ELISA) kit specific for murine TNF-α according to the manufacturer’s instruction. After the termination of all reactions, the absorbance of the chromophore at 420 nm was measured using a microplate reader. The activities of serum enzymes glutamic-oxaloacetic transaminase (GOT) and glutaminepyruvic transaminase (GPT) were estimated using a commercial colorimetric kit. Briefly, appropriately diluted serum (200 μL) was added to the reaction solution (0.1 mL; L-asparaginic acid and αketoglutaric acid mixture). The resultant mixture was incubated at 37 °C for 30 min (GPT) or 60 min (GOT), and 2,4-dinitrophenylhydrazine (1 mL) was added. After incubation at room temperature for 20 min, 0.4 N sodium hydroxide (10 mL) was added, and the absorbance of the solution was measured at 505 nm using a microplate reader. Antioxidant Enzyme Activities in Mouse Liver. The liver tissue was homogenized in 10 volumes of phosphate-buffered saline (PBS) on ice for 30 s using a power-driven Polytron homogenizer (Pro Scientific, Monroe, CT, USA). The homogenate was centrifuged at 9000g at 4 °C for 20 min. The supernatant was recovered and used to measure two antioxidant enzyme activities, catalase and superoxide dismutase. For the catalase activity assay at time zero, each supernatant (1.8 mL) was mixed with PBS (0.2 mL) containing hydrogen peroxide (10 mM). The reaction mixture (1 mL) was immediately added to a cuvette and placed into a UV−vis spectrophotometer (model V-550, Jasco, Tokyo, Japan). The enzyme activity was observed via degradation of hydrogen peroxide as determined by a decrease in UV light absorbance at 240 nm over time. The measurement of absorbance was taken at 15 s intervals after the addition of the homogenate to the hydrogen peroxide buffer. Units of catalase activity per milligram of protein were calculated following the method of Nandi et al.28 For the superoxide dismutase (SOD)-like activity assay, the supernatant (100 μL) was mixed with a homogenizing buffer (1.5 mL; 50 mM Tris, 10 mM EDTA, pH 8.5), then pyrogallol (100 μL; 7.2 mM) was added, and the reaction mixture was incubated at 25 °C for 10 min. The reaction was terminated by adding 1 M HCl (50 μL), and the absorbance was then measured at 420 nm. One unit was
MATERIALS AND METHODS
Test Compounds. Salmonella enterica LPS and D-galactosamine (GalN) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents were of analytical grade and used without further purification. The RPMI 1640 medium, Hank’s Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and other cell culture reagents were obtained from Hyclone Laboratories (Logan, UT, USA). The enzyme kit for glutamic-oxaloacetic transaminase/glutamic-pyruvic transaminase (GOT/GPT) was the product of Asan Pharmaceutical Co. (Seoul, Korea). The ELISA kit for the quantification of tumor necrosis factorα (TNF-α) was obtained from Biosource USA (Camarillo, CA, USA). Chemicals. The rice hull liquid smoke extract (RHSE) was obtained from Daewon GSI (Waegwan, Korea). The industrial production of the commercially available RHSE, characterized chemical components, and beneficial bioactivities in chemical and cell assays were described in our previous publication in this journal.22 GC/MS analysis showed that the rice hull extract contained 161 characterized aliphatic, aromatic, and heterocyclic compounds. The aqueous extract was acidic, had a specific gravity of 1.007, and contained 0.026% solids. The in vitro antioxidative activity of the extract as determined by the DPPH assay was similar to that of the widely used food antioxidant BHT (butylated hydroxytolune). Animals and Treatment. The protocol for the mice studies was approved by the Ethics Committee for Animal Care and Use, Ajou University, Republic of Korea. All experiments were performed in compliance with the relevant laws and institutional guidelines. Fiveweek-old female BALB/c mice were purchased from Orient Bio Inc. (Seoul, Korea) and were hosted under a 12 h light/dark cycle at 20− 22 °C and relative humidity of 50 ± 10%. The mice were fed freely a pelletized commercial chow diet obtained from Orient Bio Inc. (catalogue no. 5L79) and sterile tap water during the entire period. After acclimation for 1 week, the mice were then divided into three groups (n = 10), avoiding any intergroup difference in body weight. The endotoxemic shock assay in a murine model was carried out following the method of Yun et al.,26 with modification. Briefly, two groups of mice each (PBS-treated control and RHSE-treated experimental group) were fed a pelletized commercial chow diet without and with RHSE (1.0%, v/w) during the entire experimental period. After 14 consecutive days of RHSE administration, the control and experimental mice were injected with LPS and GalN (5 μg/kg and 700 mg/kg, respectively) via an intraperitoneal (ip) route. To determine the survival rate, lethal dose of LPS and GalN (20 μg/kg and 700 mg/kg, respectively) were employed. The mice were observed 7754
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defined as the amount of enzyme that inhibited the oxidation of pyrogallol by 50%. The activity was expressed as units per milligram of protein.29 Myeloperoxidase (MPO) Activity. MPO activity was assayed according to the method of Bralley et al.30 Briefly, the supernatants recovered from lung and kidney tissue homogenates (30 μL) were added to the reaction mixture (200 μL) consisting of 100 μL of PBS (80 mM phosphate, 140 mM NaCl, pH 5.4), 85 μL of PBS2 (220 mM phosphate, 140 mM NaCl, pH 5.4), and 0.017% H2O2 (15 μL). The reaction was started by adding tetramethylbenzidine (18.4 μM) in aqueous dimethylformamide (8%; 20 μL) and was left standing for 3 min at 37 °C. The reaction was stopped by the addition of sodium acetate buffer (30 μL; 1.46 M; pH 3.0). The absorbance (OD) of the supernatant was then read in a microplate reader at 655 nm for 30 min. The reaction rate (change in OD/time) was derived from an initial slope of the curve. A calculation curve was used to show the rate of reaction plotted against the concentration of a standard human MPO preparation. Serum Urea Nitrogen and Creatinine. The concentration of urea nitrogen and creatinine were determined using the commercial QuantiChrom creatinine assay kit and QuantiChrom urea assay kit, respectively (Bioassay System, Hayward, CA, USA). Statistical Analysis. Results are expressed as the mean ± SD of three independent experiments. Significant differences between means were determined by ANOVA test using the Statistical Analysis Software package SAS (Cary, NC, USA). P < 0.05 is regarded as significant.
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RESULTS AND DISCUSSION Dose Selection. The reason why we did not assess the dose-related mortality in mice is because (a) in previous studies, we found that 1% to be the optimal dose for expressing maximum levels of anti-inflammatory activities, including inhibition of NO production and release of anti-inflammatory cytokines and protection of cell viabilities without any side effects both in vitro and in mice; and (b) we also used 1% of the rice hull extract to protect Salmonella-infected mice against mortality. The present complementary study shows that the 1% concentrations also protected the mice against endotoxemia. Effects of RHSE on Weights and Histopathology of Mouse Liver. Figure 1 shows that the induction of endotoxemia by LPS/GalN resulted in a significant increase in liver weight as well as in the formation of hemorrhagic lesions in the liver tissues. By contrast, no apparent hepatic injury was observed in the vehicle-treated control group. As anticipated, RHSE treatment through dietary administration (1%, v/w) markedly blocked the hemorrhagic liver injury as well as hepatomegaly induced by a lethal dose of the LPS/GalN challenge. These results demonstrate strongly that RHSE protected the mice against injury of a major organ associated with endotoxemia. Effects of RHSE on Antioxidant Enzyme Activities in Mouse Liver. To find out whether reactive oxygen species (ROS) level is a crucial factor for the incidence of liver injury associated with LPS/GalN-induced endotoxemia, the activity of hepatic antioxidant enzymes including catalase and SOD-like enzymes was evaluated in the control and experimental mice groups. Table 1 shows that in the liver of LPS/GalN-treated mice, the dietary administration of RHSE induced a significant suppression of the catalase and SOD-like enzyme activities from 24.53 to 7.18 U/mg protein (∼71% change) and from 18.65 to 8.49 U/mg protein (54% change), respectively. These results show that dietary administration of RHSE reduced
Figure 1. Modulation of liver injury in LPS/GalN-induced endotoxemic mice by administration of diet supplemented with RHSE (1.0%, v/w). (A) Liver sections from LPS/GalN (5 μg/kg/700 mg/kg)-administered mice show disorganized hepatic architecture, intense cellular necrosis, and marked hemorrhagic lesions compared with the liver section of vehicle-treated mice. Dietary administration of RHSE markedly ameliorated LPS/GalN-induced liver damage. (B) Increase in liver weight by LPS/GalN injection was reversed by dietary administration of RHSE. Mice were sacrificed 4 h after LPS/GalN treatment, and the livers were weighed. Each liver specimen was fixed with 4% paraformaldehyde, and sections were stained with hematoxylin and eosin (H&E). Magnification, ×100 for each of the three treatments, and ×200 for the magnified sections. Circular areas indicate local hemorrhage and necrosis regions. Bars not sharing a common letter are significantly different between groups at p < 0.05.
Table 1. Effects of RHSE on Antioxidant Catalase and Superoxide Dismutase (SOD)-Like Enzyme Activities in LPS/GalN-Induced Endotoxemic Mouse Livera sample vehicle LPS/GalN (5 μg/kg/700 mg/kg) RHSE (1.0%, v/w) dietary administration
catalase activity (U/mg protein)
SOD-like activity (U/mg protein)
3.12 ± 0.28 a 24.5 ± 2.0 b
4.77 ± 0.32 a 18.7 ± 1.5 b
7.18 ± 0.43 c
8.49 ± 0.69 c
Data are expressed as the mean ± SD (n = 10). Values in each column with the same letter are not significantly different between groups at p < 0.05. a
oxidative damage in the liver induced by the LPS/GalN challenge. Effects of RHSE on Liver Transaminase Enzyme Activities in Mouse Serum. Figure 2 shows that, compared 7755
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the normal control mice group (∼7.8- and 20.2-fold increases, respectively). The dietary administration of RHSE significantly decreased the TNF-α and nitrite/nitrate concentrations from 401.6 to 156.2 pg/mL (∼61% change) and from 19.3 to 3.2 μM (∼83% change), respectively. These results show that RHSE protected the treated mice against increased production of nitrite/nitrate, a source of bioactive nitric oxide (NO), and TNF-α, a critical factor associated with the cause of the endotoxemia syndrome. Effects of RHSE on Lung and Kidney Injuries in Endotoxemic Mouse. The evidence of necrotic cell injuries in the lung and kidney in LPS/GalN challenged endotoxemia was further confirmed by the observed decreases in the MPO enzyme activity induced by RHSE (Table 3). The results in Table 3 show that RHSE treatment via the oral route markedly decreased the MPO activities from 12.6 to 4.3 U/g protein (∼66% change) in the lung and from 6.8 to 2.5 U/g protein (∼63% change) in the kidney, respectively. The protective effect of RHSE treatment against LPS/GalNinduced renal injury was also evaluated by measuring creatinine and urea nitrogen concentrations in the serum. Table 3 also shows that creatinine and urea nitrogen levels in the serum of endotoxemic mice were significantly greater than those in normal mice group (∼1.7- and ∼4-fold increases, respectively), indicating the association of endotoxemia with the release of these amino acid metabolites from the damaged kidney. By contrast, RHSE administration via the oral route significantly lowered LPS/GalN-triggered increases in creatinine and urea nitrogen levels by up to 35.6% and 62.0%, respectively. The present study shows that RHSE neutralized the toxic effect of the LPS-induced endotoxin by reducing the expression of the cytokine TNF-α and production of ROS. Effects of RHSE on Mortality of Endotoxemic Mice. To determine the therapeutic effects of RHSE on life expectancy, a lethal dose of LPS/GalN was used to induce sepsis and to determine the protection by RHSE against mortality. Each group of 10 mice was fed on a diet supplemented with RHSE (1%, v/w) during the entire experimental period. Figure 3 shows that mice treated with a lethal dose of LPS/GalN all died after 50 h. By contrast, the group treated with RHSE through dietary administration survived >60 h. These observations demonstrate strikingly the potential of RHSE to protect mice against sepsis-induced lethality, presumably via down-regulating the production of the pro-inflammatory cytotoxic factors such as reactive oxygen radicals, cytokines, and nitric oxide. Mechanistic Aspects. We previously reported22−25 that the extract elicited changes in the expression of seven genes and two biomarkers [(prostaglandin E2 (PGE2) and leukotriene-4 (LTB4)] associated with inflammation. The extract also inhibited generation of NO through inhibition of inducible nitric oxide synthase (iNOS) gene expression and suppressed
Figure 2. Effect of dietary administration of RHSE (1.0%, v/w) on glutamic-oxaloacetic/glutamic-pyruvic transaminases (GOP/GTP) in LPS/GalN-induced endotoxemic mice. The mice were pretreated with RHSE for 2 weeks through dietary administration before the LPS/ GalN treatment. Mice were sacrificed 4 h after LPS/GalN (5 μg/kg/ 700 mg/kg) treatment, and blood was collected to produce serum. Bars not sharing a common letter are significantly different between groups at p < 0.05.
with the normal control group, the liver enzyme levels of the transaminase GOT and GPT, primary enzymes that serve as an index of hepatic injury, increased significantly in the serum of the LPS/GalN-treated mice. In the mice fed an RHSEsupplemented diet (1.0% v/w), GOT and GPT activities decreased substantially from 231.7 to 137.3 U/mL (∼41% change) and from 167.3 to 62.8 U/mL (∼63% change), respectively. These results show that RHSE protected the liver against necrotic injury associated with endotoxemia. Effects of RHSE on TNF-α and Nitrite Concentrations in Mouse Serum. Table 2 shows that TNF-α and nitrite/ Table 2. Effects of RHSE on Tumor Necrosis Factor-α (TNF-α) and Nitrite Concentrations in Mouse Seruma sample vehicle LPS/GalN (5 μg/kg/700 mg/kg) RHSE (1.0%, v/w) dietary administration
TNF-α (pg/mL)
nitrite (μM)
51.3 ± 3.9 a 401 ± 26 b 156 ± 14 c
0.951 ± 0.021 a 19.3 ± 1.1 b 3.205 ± 0.053 c
Data are expressed as the mean ± SD (n = 10). Values in each column with the same letter are not significantly different between groups at p < 0.05. a
nitrate levels in the serum of LPS/GalN-induced endotoxemic mice were markedly increased compared with those observed in
Table 3. Effects of RHSE on Lung and Kidney Injuries in LPS/GalN-Induced Endotoxemic Mice Assessed by Changes in Myeloperoxidase, Creatinine, and Blood Urea Nitrogen (BUN) Concentrationsa myeloperoxidase (U/g protein) sample
lung
kidney
BUN (mg/dL)
creatinine (mg/dL)
vehicle LPS/GalN (5 μg/kg/700 mg/kg) RHSE (1.0%, v/w) dietary administration
2.922 ± 0.093 a 12.59 ± 0.89 b 4.32 ± 0.12 c
1.86 ± 0.11 a 6.83 ± 0.23 b 2.51 ± 0.18 c
0.334 ± 0.018 a 0.582 ± 0.033 b 0.375 ± 0.021 c
14.2 ± 1.1 a 56.9 ± 4.4 b 21.6 ± 1.5 c
Data are expressed as the mean ± SD (n = 10). Values in each column with the same letter are not significantly different between groups at p < 0.05. a
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immune cell apoptosis, and enhanced bacterial elimination without apparent adverse effects, suggesting the need for the evaluation of this and other plant antioxidants as potential therapeutic agents.10 Similar promising results have been reported with the phenolic antimicrobial carvacrol, a natural compound present in oregano oil, which is used as a salad dressing.33 It has also been shown that the antimicrobial and antiinflammatory natural phenolic compound 3,4,5-trihydroxybenzoic acid protected mice against LPS-induced endotoxemia and associated symptoms and biomarkers.34 Interestingly, cocoa (Theobroma cacao), a polyphenol-rich food added to a high-fat diet reduced metabolic endotoxemia and adipose tissue inflammation in mice by modulating eicosanoid metabolism.35 Another murine study showed that a bactericidal peptide derived from zebra fish phosvitin inhibited LPS-induced tumor necrosis factor (TNF-α) and interleukin (IL-1β) in mice and increased the survival of endotoxemic mice and thus has the potential for the clinical treatment of LPS-induced sepsis.36 The antioxidant ascorbic acid (vitamin C) inhibited alcoholinduced endotoxemia in guinea pigs, suggesting that the vitamin has the potential to decrease endotoxemia symptoms by reducing small intestinal bacterial overgrowth (SIBO) and the activation of NF-kB and the synthesis of cytokines.37 Acute and chronic consumption of red wine, a rich source of antioxidant and antimicrobial flavonoids,38 by middle-aged men increased the Bifidobacterium and Prevotella gut microbiota that correlated negatively with LPS concentration.39 A similar beneficial effect was observed with the wine polyphenolic compounds resveratrol40 and quercetin.41 Gossypol, an anti-inflammatory compound present in cotton seeds, seems to be a promising therapeutic agent for the treatment of lung injury, owing to its ability to protect mice against LPS-induced acute lung injury and to inhibit the NF-kB and MAPKs signaling pathways.42 The antiendotoxemia efficacy of plant phenolic antioxidants mentioned above at the cellular level might be due to their ability to counteract the adverse effects associated with the reduction in the LPS-induced murine hepatic level of the glutathione (GSH) antioxidant that protect liver tissues against injury.43 It needs to be emphasized, however, that other properties of some proposed antiendotoxemia compounds such as those of gossypol, which is reported to inhibit male spermatogenesis,44 might reduce their potential value in endotoxemia therapy. Other nonfood related proposed compounds and plant extracts also need to be evaluated for possible toxicity. These considerations suggest that food-related compounds that are considered generally accepted as safe (GRAS) might have advantages over others that need to be evaluated further for possible safety. In summary, the results of the present and cited studies show that, because the commercially available rice hull liquid smoke extract exhibits antiallergic, antidiabetic, antiendotoxemia, and antimicrobial properties, it has the potential to serve as an antimicrobial food preservative and might contribute to the prevention and therapy of several chronic diseases. Because the chemical nature of the large number of individual compounds in the rice hull smoke extract varied widely, we do not know which individual compound or combination of compounds might be responsible for the observed bioactivity. Future studies should determine the bioactivities of individual compounds isolated from the liquid rice hull extract and the
Figure 3. Histogram showing the effect of dietary administration of RHSE (1.0%, v/w) on LPS/GalN-induced endotoxemia induced lethality. BALB/c mice were fed RHSE-supplemented diet for 2 weeks and then challenged ip with a lethal dose of LPS/GalN (20 μg and 700 mg per kg body weight). RHSE was then again administered through the same route during the entire experimental period. Plotted values are mean values of triplicate determinations.
the inflammatory reaction through inhibition of pro-inflammatory gene expressions, including tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6) and increased the production of interferon-γ (IFN-γ) produced by the spleen, suggesting that it stimulated the immune system. In the present study, we report that the extract suppressed the production of TNF-α, confirming its anti-inflammatory properties as well as the antioxidative enzymes catalase and superoxide dismutase, suggesting that the beneficial effects might also be associated with inhibition of cell-damaging reactive oxygen species (ROS). On the basis of the mentioned biomarkers, it seems that the antiendotoxemia mechanism might involve suppression of production of NO, cytokines TNF-α, IL-β, and IL-6, and the eicasinoids PGE2 and LTB4, and the inhibition of ROS, thus blocking the observed organ injuries caused by endotoxininduced acute inflammation. Related Studies. There have been other recent reports of natural compounds that have antiendotoxemia properties and they are described briefly here, in context of the present study. We found that a polysaccharide isolated from the liquid culture of Lentinus edodes (Shiitake) mushroom mycelia containing black rice bran did not inhibit the growth of Salmonella in vitro but did protect mice against LPS-GalNinduced liver, lung, and kidney injuries and inflammation and enhanced the survival of the toxic shock-induced mice by blocking oxidative stress and TNF-α production, suggesting that the novel polysaccharide has the potential to serve as a new functional food.31 Other researchers recently reported that flucoidan, a sulfated polysaccharide from the brown alga Fucus evanescens, increased resistance to LPS, increased the survival time, and prevented endotoxin-induced damage in mice through the inhibition of pro-inflammatory cytokines (TNF-α and IL-6), attenuation of microcirculatory disorders, and regulation of the immune system.32 The administration of the plant phenolic antioxidant chlorogenic acid is reported to prevent mortality of mice induced by endotoxemia and polymicrobial sepsis through modulation of cytokine and chemokine release, suppression of 7757
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possible synergism of combinations of the extract, compounds isolated from the extract, and other bioactive food compounds and extracts that have been shown to exhibit beneficial in vivo effects. Such dietary combinations, in which the individual components might exert their effect by different mechanisms, might act as multifunctional foods with therapeutic potential and might make it possible to use lower amounts of each component. It is also necessary to demonstrate that the liquid rice hull smoke is safe. Our in vitro, ex vivo, and in vivo previous and present studies are designed to contribute to this assessment.
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AUTHOR INFORMATION
Corresponding Authors
*For S.H.N.: phone, 82-31-219-2619; fax, 82-31-219-1615; Email,
[email protected]. *For M.F.: phone, 01-510-559-5615; fax, 01-510-559-6162; Email,
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Carol E. Levin for assistance with the preparation of the manuscript. ABBREVIATONS USED BHT, butylated hydroxytoluene; BPP, pro-processed polymer; BUN, blood urea nitrogen; ELISA, enzyme-linked immunoassay; FBS, fetal bovine serum; GalN, D-galactosamine; GOT, glutamic-oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase; GRAS, generally accepted-as-safe; GSH, reduced glutathione; HBSS, Hank’s Balanced Salt Solution; HMGB1, high mobility group box-1 protein; HPAEC-PAD, high performance liquid chromatography with pulsed amperometric detection; IFN-γ, interferon-γ; IL-6, interleukin-6; LPS, lipopolysaccharide; MAPK, mitogen activated protein kinase; MIC, minimum inhibitory concentration; MPO, myeloperoxidase; p-NPP, p-nitrophenyl phosphate; PBS, phosphate saline buffer; RHSE, rice hull smoke extract; SIBO, small intestinal bacterial overgrowth; SOD, superoxide dismutase; TNF-α, tumor necrosis factor-α
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