In vitro and in vivo protective effects of fermented preparations of dietary herbs against lipopolysaccharide insult

Food Chemistry 134 (2012) 758–765

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In vitro and in vivo protective effects of fermented preparations of dietary herbs against lipopolysaccharide insult Shambhunath Bose a, Mi-Young Song b, Jong-Kyoung Nam c, Myeong-Jong Lee c, Hojun Kim c,⇑ a

Institute of Medical Research, College of Medicine, Dongguk University Seoul, Republic of Korea Graduate School of Oriental Medicine, Dongguk University Gyeongju, Republic of Korea c Graduate School of Oriental Medicine, Dongguk University Seoul, Republic of Korea b

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Article history: Received 17 August 2011 Received in revised form 27 December 2011 Accepted 28 February 2012 Available online 6 March 2012 Keywords: Lipopolysaccharide Fermented herbs RAW264.7 cells Probiotics Gut permeability

a b s t r a c t Lipopolysaccharide (LPS) is known to produce endotoxic shock by triggering systemic inflammatory responses. Here, we evaluated the protective effects of three fermented/re-fermented herbs, Rhizoma Atractylodis Macrocephalae, Massa Medicata Fermentata, and Dolichoris Semen, in an LPS-mediated inflammatory insult, either individually in vitro using RAW264.7 cells or in combination in in vivo using rats. In general, each of the fermented herbs showed appreciable in vitro anti-inflammatory activity, although the degree of this activity varied with the herb used. Moreover, a mixture of fermented herbal extracts in combination with probiotics significantly attenuated the blood endotoxin and CRP levels, as well as the gut permeability, and significantly augmented the intestinal Lactobacillus spp. colonisation in LPS-treated rats. However, these effects were not observed following the administration of the corresponding mixture of unfermented herbal extracts. Thus, our results highlight the beneficial impacts of the use of fermented herb products with probiotics to combat LPS-mediated inflammatory insults. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction LPS, or endotoxin, is an integral component of gram-negative bacteria and is one of the major causative agents of systemic inflammatory responses that lead to a number of adverse effects, including: endotoxic shock, tissue injury, sepsis, multiple organ failure, and death. Activated macrophages are known to be the dominant proinflammatory cells involved in the onset of the systemic inflammatory responses. More specifically, these cells are responsible for most of the cellular and molecular pathophysiology of sepsis due to their production of a number of cytokines, such as tumour necrosis factor alpha (TNF-a), interleukin (IL)-1, IL-6, IL-8, and IL-12 and other proinflammatory molecules, including platelet-activating factor, prostaglandins, enzymes, and free radicals, such as nitric oxide (NO). Endotoxin has been shown to destabilize gut permeability, permit bacterial translocation, and alter the host immune defence mechanisms, which, as a result, collectively enhance the risk of developing infections from the constituents of the enteric microbiota (Deitch, Berg, & Specian, 1987; Deitch et al., 1989).

⇑ Corresponding author. Address: Department of Oriental Rehabilitation Medicine, Dongguk University-Seoul, Graduate School of Oriental Medicine, 814 Siksadong, Goyang, Gyeonggi-do, Republic of Korea. Tel.: +82 31 961 9111; fax: +82 31 961 9009. E-mail address: [email protected] (H. Kim). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.02.175

Substantial evidence has indicated the anti-inflammatory effects of dietary herbs, which manifest through a number of mechanisms (Kaplan et al., 2007). Accordingly, Rhizoma Atractylodis Macrocephalae (RAM), Massa Medicata Fermentata (MMF), and Dolichoris Semen (DS, also known as hyacinth bean), which are utilised in various dietary preparations in China, Korea, Japan, and India, are frequently used either alone or in mixed herbal formulations for the treatment of a number of inflammatory disorders (Fan et al., 2005; Kim et al., 2007; Ryu et al., 2011). Fermentation is frequently used to break down or convert certain undesirable substrate components into compatible ones and this process can also increase the activities of the biological substrates by modifying naturally occurring molecules such as isoflavones, saponins, phytosterols, and phenols. The beneficial health effects and the anti-inflammatory activities of fermented herbal or food products, either alone or in combination, are well documented (Deiana et al., 2002; Parvez, Malik, Ah Kang, & Kim, 2006; Telekes et al., 2007). Additionally, the probiotics used for fermentation may also exert health-promoting effects (Parvez et al., 2006), and the anti-inflammatory properties of probiotics have also been reported by a number of studies (Grimoud et al., 2010; Parvez et al., 2006). These findings prompted us to evaluate the protective effects of the fermented/re-fermented extracts of RAM, MMF, and DS on an LPS-driven inflammatory-insult. Accordingly, we examined the anti-inflammatory effects of the fermented/re-fermented herb products (along with the associated probiotics) individually on

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LPS-treated RAW264.7 cells in vitro, and in combination (fermented herbal formulation, FHF) in rats exposed to LPS in vivo, where a commercial colostrum preparation was used as the standard biological-in-origin anti-inflammatory agent. The re-fermentation of MMF was performed using Leuconostoc mesenteroides, whereas Bacillus licheniformis was employed for the fermentation of both RAM and DS. Leuconostoc spp. are Gram-positive, non-motile and asporogenous bacteria (Hemme & Foucaud-Scheunemann, 2004). They are facultative anaerobic cocci, usually occurring in pairs or short chains (Hemme & Foucaud-Scheunemann, 2004), although the cell morphology can vary with the growth conditions. On the otherhand, Bacillus spp. are rod-shaped, sporulating, obligate or facultative anaerobic bacteria which test positive for the enzyme catalase (Turnbull, 1996). The selection of the above two bacterial spp. for the herbal-specific fermentation in our study was based on the research outcome of a local herbal pharmaceutical company (Korea Medicine Biofermentation Co., Ltd., Seoul, Korea) in collaboration with us, which revealed that the herbal fermentation/re-fermentation was optimum when mediated by L. mesenteroides (for MMF) or by B. licheniformis (for RAM and DS). This is also in agreement with earlier studies which have shown that Leuconostoc spp. play an important role in the fermentation of a wide range of products including foods, such as dairy products, meat, fish, cereals, vegetables, and fruits as well as serve as probiotics (Hemme & FoucaudScheunemann, 2004), suggesting that these bacteria are of worthy choices as a starter to mediate the re-fermentation of MMF, which is composed of a mixture of several fermented herbs and grains. On the other hand, B. licheniformis, which is listed in the third edition of the Food Chemicals Codex (1981) as a source of carbohydrase and protease enzymes, has been safely used for large-scale industrial fermentation as well as in commercial human and animal probiotic products (de Boer, Priest, & Diderichsen, 1994; Hong, Duc, & Cutting, 2005). Substantial evidence has revealed that Bacillus spp. play an important role in the fermentation of different legume products like beans (Allagheny, Obanu, Campbell-Platt, & Owens, 1996), as well as herbs (Hsu & Chiang, 2009; Li, Shen, Liu, & Zhang, 2006; Li, Zhang, & Shen, 2006), suggesting that these bacteria are of suitable options as starter for the fermentation of RAM and DS. Finally, we also studied the anti-inflammatory activity of a combination of laboratory-unfermented herbs (unfermented herbal formulation, UHF) in LPS-treated rats to compare the in vivo effectiveness of this combination to that of corresponding FHF.

(L. mesenteroides for MMF, and B. licheniformis for both RAM and DS) and were fermented for 24 h either at 35.4 °C (L. mesenteroides) or 31 °C (B. licheniformis). The corresponding unfermented samples were prepared in a similar way, except that they received 2% (v/v) of the respective sterile bacterial culture medium instead of the bacterial inoculum. For the in vivo studies, the corresponding laboratoryunfermented or fermented/re-fermented herbal extracts were combined in equal volumes and mixed vigorously to produce the UHF and FHF, respectively. Finally, each of the preparations was subjected to low speed centrifugation to sediment the particles, and the supernatant phase of the resultant products was used for the experiment.

2. Materials and methods

After three to four to 4 cycles of sub-culturing, RAW264.7 cells were seeded at a density of 8  105 cells/well in 6-well plates. Following an overnight culture, the cells were treated for 24 h with an individual fermented herb at doses that corresponded to 50 and 100 ll of the extracted herbal preparation per ml of culture medium. The control cells and cells that were assigned to get treatment with LPS alone received sterile water instead of the herbal extract. Following this treatment, LPS (from Pseudomonas aeruginosa, Sigma–Aldrich, St. Louis, MO, USA), which was diluted in sterile PBS at pH 7.4, was added to each non-control well (control received PBS only) at a final concentration of 10 lg/ml. The control cells received PBS only. The cells were incubated under these conditions for an additional 24 h before they were used in the gene expression experiments. The total cellular RNA was extracted using a commercial TrizolÒ reagent kit (Invitrogen, Carsbad, CA, USA) according to the kit manufacturer’s instructions. The purity and concentration of the extracted RNA was determined by spectrophotometry. An equal amount of the RNA preparation (2 lg) was reverse transcribed for the generation of cDNA using a SprintTM RT Complete Oligo-(dT)18 cDNA synthesis kit (Clontech, Mountain View, CA, USA) according

2.1. Herbal extraction and fermentation/re-fermentation Dried herbs were purchased from the Department of Medicine, Dongguk International Hospital (Goyang, Korea). The extraction and the fermentation/re-fermentation of the herbs were performed according to our laboratory-optimised procedure. Briefly, 20 g of dried powder from each herb was mixed with 200 ml of boiled Milli-Q water, and this mixture was subjected to ultrasonication at 70 °C to disperse the particles and then incubated at 70 °C for 3 h in a water bath under continuous shaking. Following this procedure, the samples dedicated for fermentation/re-fermentation were either supplemented with glucose (2% w/v, for RAM and MMF) or Luria–Bertani broth powder (2.5% w/v, for DS). The resultant products were then autoclaved for 20 min at 121 °C, which in addition to sterilization of the samples and killing the already existing microbes that were involved in the natural fermentation of MMF also served to decoct the samples. After cooling the preparations to room temperature, the samples dedicated for fermentation/re-fermentation were inoculated with a fresh subculture (2% v/v) of bacteria

2.2. Cell culture The RAW264.7 murine macrophage cell line was suspended in DMEM supplemented with 10% heat inactivated faetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin. The cells were cultured in this medium at 37 °C in a humidified atmosphere containing 5% CO2 in air. 2.3. Cytotoxicity assessment The cell viability was determined colorimetrically using 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) as the chromophore. After three to four cycles of sub-culturing, the RAW264.7 cells were seeded into 24-well plates at a density of 2  105 cells/well and were incubated overnight. Subsequently, the cells were treated for 24 h with an individual fermented herb at doses that corresponded to 5, 10, 25, 50, and 100 ll of the extracted herbal preparation per ml of culture medium. The control cells were treated with sterile water instead of an herbal extract. MTT was added to the cells 3 h prior to the end of the treatment schedule, at a final concentration of 0.5 mg/ml. After the completion of the MTT reaction, the culture media were removed carefully from the wells, and DMSO was added to the cells in order to liberate and dissolve the formazan crystal products. Following this, absorbance was read at 570 nm using a microplate reader (Spectramax Plus, Molecular Devices, Sunnyvale, CA, USA). The viability of the control cells, in terms of their absorbance, was considered to be 100%. 2.4. Determination of the expression of key inflammatory genes in RAW 264.7 cells by quantitative real-time PCR (qRT-PCR)

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to the instructions of the kit manufacturer. The qRT-PCR of the samples was performed in a LightCycler instrument (Roche Applied Science, Indianapolis, ID, USA) using a LightCyclerÒ FastStart DNA Master SYBR Green kit (Roche Applied Science). The amplification reactions were conducted according to the kit manufacturer’s instructions, in a total reaction volume of 20 ll that contained the PCR mix, 1 ll of cDNA, and gene-specific primers (10 pmol for each). The sequences of the primers (Bioneer, Daejeon, Korea) used were as follows: cyclooxygenase-2 (COX-2)-forward: 50 -AGAAGGAAATGGC TGCAGAA-30 , and COX-2-reverse: 50 -GCTCGGCTTCCAGTATTGAG-30 (Liu et al., 2006); glyceraldehyde 3-phosphate dehydrogenase (GA PDH)-forward: 50 -TGATGACATCAAGAAGGTGGTGAAG-30 , and GAPDH-reverse: 50 -TCCTTGGAGGCCATGTAGGCCAT-30 (Puthalakath et al., 2001); inducible nitric oxide synthase (iNOS)-forward: 50 -A GCCCAACAATACAAGATGACCCTA-30 , and iNOS-reverse: 50 -TTCCTGT TGTTTCTATTTCCTTTGT-30 (Kang et al., 2007). The optimised annealing temperatures of the primers for the PCR reactions were 53 °C for COX-2 and 56 °C for GAPDH and iNOS. The conditions used for the PCR amplifications were as follows: an initial incubation step at 95 °C for 10 min followed by 30 amplification cycles, each one consisting of a denaturation step at 95 °C for 10 s (COX-2) or 30 s (GAPDH and iNOS), an annealing step at the corresponding optimised temperature for 10 s (COX-2) or 30 s (GAPDH and iNOS), and an extension step at 72 °C for 15 s (COX-2) or 90 s (GAPDH and iNOS). This reaction was followed by a melting curve analysis to verify the specificity of the amplicon. The resultant data were processed and analysed using the LightCycler software provided by the instrument manufacturer (Roche Applied Science). The relative gene expression was quantitated following the standard 2DCt calculation using the housekeeping gene GAPDH for normalisation, where Ct is the crossing threshold value calculated by the software and DCt = (Ct-target gene – Ct-GAPDH). 2.5. Measurement of NO production by RAW 264.7 cells The generation of NO, in terms of nitrite production, was determined colorimetrically using the Griess reagent (Promega, Madison, WI, USA). For this assay, the cell culture media collected from the cells that were employed for the previously described inflammatory gene analysis were uses as the samples. Briefly, following the desired treatments, 100 ll of culture medium from each well was mixed with an equal volume of the Griess reagent and incubated at room temperature for 10 min. The absorbance was read at 540 nm using a microplate reader (Spectramax Plus), and the nitrite concentration of each sample was determined using a freshly prepared sodium nitrite standard curve. 2.6. Animals and treatments Male 8-week-old Sprague–Dawley rats (Orient Bio, Seongnamsi, Korea) weighing 200 ± 20 g were housed at a controlled temperature (20 ± 2 °C) with relative humidity (40–60%), and were maintained in a 12-h light–dark cycle (lights on at 7:00 a.m.). The animals were provided ad libitum access to water and a standard normal chow diet (Soyagreentec, Hwaseong-Si, Korea), which contained 20% protein, 4.5% fat, and 63% calories from carbohydrate. All experimental procedures, including the care and handling of the animals, were performed according to the international guidelines (Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, USA; National Academy Press: Washington D.C., 1996). Prior to the study, the rationale, design, and protocols of the experiments were approved by the Institutional Animal Ethical Committee, Dongguk University. Following acclimatisation for 7 days, the animals were randomly divided into different experimental groups as follows: (1) control; (2) LPS + water

treated; (3) LPS + UHF-treated; (4) LPS + FHF-treated; and (5) LPS + colostrum-treated. At 48 h prior to sacrifice, the LPS (from Escherichia coli 055:B5, Sigma–Aldrich), which was dissolved in a physiological saline solution, was administered by IP injection to the animals in groups two through five at a dose of 1 mg/kg, while the animals in group 1 received injections of saline without LPS. The rats in groups 3 and 4 were orally administered the UHF and FHF formulations, respectively, at a volume (on a per kg body weight basis) representing the decoction-extracted product from 200 mg of each herb. Instead of herbs, the animals in groups 1 and 2 were given water, and those in group 5 were given colostrum (1 ml/rat using a 10% v/v solution of a commercial product, Colostrum Technologies GmbH, Königsbrunn, Germany). The water, herbal and colostrum dosing were performed once daily for five consecutive days prior to LPS treatment. Twelve hours after treatment with LPS, the animals were transferred to individual metabolic cages and were deprived of food for 12 h, but provided free access to water. Subsequently, 1 ml of aqueous test solution containing 66 mg/ml lactulose and 50 mg/ml mannitol was orally administered to each rat. The animals were then maintained for 24 h with access to water, but without access to food, and after this period, samples of blood, stool, and urine were collected for further analysis. The serum was separated by centrifuging the blood at 1000g for 15 min at 4 °C. Each of the samples collected were stored at 70 °C until the analyses were performed. 2.7. Measurement of serum endotoxin and CRP levels The endotoxin levels in the serum samples were measured by an Endo-Check TM analyzer (Diatech Korea, Seoul, Korea) using a Limulus Ameobocyte Lyasate (LAL)-based kit (Diatech Korea). The assay was conducted in accordance with the kit manufacturer’s instructions. The serum CRP concentrations were determined by ELISA using a rat-specific commercial kit from BD Biosciences (San Diego, CA, USA). 2.8. Determination of lactulose and mannitol in the urine as markers of gut permeability The levels of lactulose (L) and mannitol (M) in the urine were measured using K-Lactul and K-Manol kits, respectively, from Megazyme (Bray, Co., Wicklow, Ireland). The analysis was performed according to the instructions of the kit manufacturer. The urine lactulose and mannitol levels were quantitated in terms of the recovery percentage of the ingested dose, and the L/M ratios were then calculated. 2.9. Determination of Lactobacillus spp. and universal bacterial DNA content in rat stool by qRT-PCR The extraction of DNA from the stool samples was performed using a DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) according to the instructions of the kit manufacturer. The purity and concentration of DNA from the extracts was determined by spectrophotometry. The qRT-PCR assays were performed, as previously described for the cDNA of RAW264.7 cells, in a final reaction volume of 20 ll that contained the PCR mix, the template DNA (100 ng), the primers (10 pmol for each), and bovine serum albumin (2.1 lg). The sequences of the primers (Bioneer, Daejeon, Korea) used to target the 16S rRNA gene of the bacteria were as follows: Lactobacillus spp.-forward: 50 -GAGGCAGCAGTAGGGAATCTT C-30 , and Lactobacillus spp. -reverse: 50 -GGCCAGTTACTACCTCTATC CTTCTTC-30 (Cani et al., 2008); universal bacteria-forward: 50 -CCTA CGGGAGGCAGCAG-30 , and universal bacteria-reverse: 50 -ATACCGC GGTGCTGG-3’ (Nakamura et al., 2009). The optimised PCR annealing temperature for each primer was 60 °C. The conditions used for

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Fig. 1. The effect of different fermented herbal extracts (FHE) on the viability of cells. The RAW264.7 cells were treated for 24 h with sterile water (W) or with increasing doses of FMMF, FRAM, or FDS extracts following which the cell viability was measured by MTT assay. The detailed treatment regimen and experimental conditions are described in the Section 2. The viability of the cells that received the sterile water treatment was set to 100%. The data are represented as the means ± SEM, n = 4–6. No statistically significant differences were observed between the treatment groups.

the PCR amplifications were as follows: an initial incubation step at 95 °C for 10 min followed by 40 amplification cycles, each one consisting of a denaturation step at 95 °C (10 s for universal bacteria and 15 s for Lactobacillus spp.), an annealing step at 60 °C (10 s for universal bacteria and 20 s for Lactobacillus spp.), and an extension step at 72 °C (15 s for universal bacteria and 45 s for Lactobacillus spp.) This procedure was followed by melting curve analysis to verify the specificity of the amplicon. The resultant data were processed and analysed using the LightCycler software (Roche Applied Science). The DNA levels were approximated as 2Ct, where Ct was the crossing threshold value calculated by the software. The abundance of Lactobacillus spp. in the samples was quantitated as the ratio of the 2Ct value of the Lactobacillus spp. to that of the universal bacteria.

2.10. Statistical analyses The values are expressed as the means ± SEM. The statistical package for social science (SPSS) software program (version 17.0; SPSS, Chicago, IL, USA) was used for the analysis of the data. A one-way ANOVA followed by Bonferroni’s post hoc test was performed to determine the significant differences between the study groups. The post hoc analyses were carried out only when the means were significantly different by one-way ANOVA. When the error variance was found to be heterogeneous using Levene’s test, a logarithmic transformation of raw data was performed and is indicated accordingly in the results section. The differences were considered significant when p < 0.05.

3. Results and discussion First, we judged the anti-inflammatory activity of each of the three fermented/re-fermented herbal preparations in RAW264.7 cells treated with LPS. To determine the optimal non-lethal dose for the administration of the fermented/re-fermented herbal formulations for evaluating their in vitro anti-inflammatory activities, a dose-dependent cytotoxicity study was conducted. As compared to the control treatment, none of the fermented herbs produced a significant change in the viability of the treated cells, and this was true at doses as high as 100 ll of the extracted preparation per ml of the cell culture medium (Fig. 1). Accordingly, the 50 and 100 ll/

ml doses of each fermented herb were used for the evaluation of anti-inflammatory activity. As an inflammatory mediator, NO plays a vital role in almost every stage of the development of inflammation. The production of NO is enzymatically catalysed by inducible nitric oxide synthase (iNOS), the expression of which is induced following LPS treatment in many cell types including macrophages (Kleinert, Schwarz, & Förstermann, 2003). In this study, exposure to LPS consistently caused a marked increase in iNOS transcription and NO production (Figs. 2A and B), which was significantly suppressed when the cells were co-treated each of the fermented herbs at both experimental doses. However, this suppression was at a maximum following treatment with fermented RAM (FRAM), as treatment with the smaller dose of this herb led to a 90% inhibition in LPS-induced iNOS transcription and – NO production and treatment with the larger dose of FRAM inhibited LPS-induced iNOS expression by 98% and LPS-induced NO production was below the limit of detection. COX-2, the rate-limiting enzyme that catalyzes the synthesis of prostaglandin E2 (PGE2), is also known to play a vital role in the development of inflammation. Similar to iNOS, the expression of this protein in macrophages is also triggered by LPS, which has been shown to be under the control of Tpl2-dependent CREB activation signals (Eliopoulos, Dumitru, Wang, Cho, & Tsichlis, 2002). In the present study, LPS treatment led to a profound increase in COX-2 transcription (Fig. 2C), and this level was significantly reduced following treatment with either FRAM or fermented DS (FDS) at each experimental dose. The degree of COX-2 inhibition was 66% (lower dose) and 48% (higher dose) following treatment with FRAM and 35% (lower dose) and 48% (higher dose) following treatment with FDS. However, neither the lower nor the higher dose of fermented MMF (FMMF) treatment produced any significant changes in the LPS-induced COX-2 expression. Taken together, the above study in general revealed the appreciable in vitro antiinflammatory activities of the fermented formulations, although the degree of this activity varied between the herbs. These findings support the well-documented anti-inflammatory effects of fermented herbal preparations (Deiana et al., 2002; Parvez et al., 2006; Telekes et al., 2007) and the associated probiotics. The results from our in vitro studies prompted us to scrutinise the in vivo protective effect of the combination of the above mentioned fermented (vs. unfermented) herbal extracts against an LPS-mediated inflammatory insult. First, we judged the ability of the mixed laboratory-unfermented or -fermented herbal formulations (UHF

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LPS Fig. 2. The effect of different fermented herbal extracts (FHE) on the expression of iNOS (A) and COX-2 (C), and on nitrite production (B) in cells. The RAW264.7 cells were treated for 24 h with the indicated doses of FMMF, FRAM, or FDS extracts. The control cells (N) and the non-control cells that were assigned to get treatment with LPS alone (W) received sterile water instead of FHE. After this treatment, the non-control and control cells were treated for 24 h with LPS and PBS, respectively, following which the level of expression of iNOS and COX-2 genes, and the production of nitrite were determined. The detailed treatment regimen and experimental conditions are described in the Section 2. The level of gene expression in the control cells was set to 100%. The data are represented as the means ± SEM, n = 3. The data were log-transformed prior to analysis by ANOVA, and the means without a common letter differed when p < 0.05.

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and FHF, respectively) to remove endotoxin from the blood of rats treated with LPS. As expected, a very low concentration of endotoxin, which was below the detection limit of the assay, was evident in the sera of control rats (Fig. 3). However, the 48 h treatment with LPS led to a 310-fold increase in the serum endotoxin level, which suggested a state of endotoxemia in the LPS-treated rats. The level of endotoxin in the sera of the LPS-treated animals was reduced, by 58% and 60%, following the treatment with UHF and colostrum, respectively. However, these reductions were found to be statistically insignificant. In contrast, there was a significant attenuation (73%) in the endotoxin level of the LPS-treated rats when they were co-treated with FHF, which indicates the advantage of using fermented herbal products in combination with probiotics to eliminate endotoxin from the blood. Our results are also in alignment with those from a clinical study which demonstrated that the administration of probiotics decreased the serum endotoxin levels in infants (Urao, Fujimoto, Lane, Seo, & Miyano, 1999). It has been suggested that the activation of the intestinal innate immune system by probiotics could be responsible for the enhanced clearance of endotoxin (Schiffrin et al., 2009). C-reactive protein (CRP), an acute phase serum protein, plays a significant role in establishing protection against bacterial pathogens and regulating inflammatory responses. The level of this protein is increased to varying degrees in response to infection,

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Fig. 3. The effect of herbal extracts or colostrun on the serum endotoxin level of LPS-treated rats. The rats were treated with water (for control and LPS + water groups), UHF, FHF, or colostrum once daily for five consecutive days. The rats were then treated with saline (for control group) or LPS (for non-control groups) for 48 h. Following the desired treatment schedule, blood was collected and the endotoxin levels in the serum samples were measured. The detailed treatment regimen and experimental conditions are described in the Section 2. The values are represented as the means ± SEM, n = 5. The data were log-transformed prior to analysis by ANOVA, and the means without a common letter differed when p < 0.05.

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trauma and immunomodulatory stimuli. In this study, LPS treatment resulted in a significant augmentation (4.42-fold) of the serum level of this protein (Fig. 4), which is in agreement with an earlier report from clinical studies (Poloyac, Tosheva, Gardner, Shedlofsky, & Blouin, 1999). The CRP level in the LPS-treated animals was reduced by 57% upon co-treatment with UHF. However, this reduction was found to be statistically insignificant. In contrast, the CRP level significantly declined in the LPS-treated animals when they were co-treated with either colostrum (79% decrease) or FHF (78% decrease), which highlights the beneficial effects of fermentation of herbs and the use of probiotics in response to LPS-mediated inflammation. In an earlier study, the anti-inflammatory activity and the reduction in serum CRP level associated with probiotics were shown (Kekkonen et al., 2008), and these effects were found to be strain-specific. A previous report found a considerable accumulation of endotoxin in the intestines of rats that had been intravenously injected with LPS (Ge, Ezzell, & Warren, 2000). Endotoxin has been shown to augment gut permeability (Deitch et al., 1989), which can allow bacteria and toxins to cross the mucosal barrier to result in the release of inflammatory mediators that, in turn, may cause further damage to the gut. In our study, rats exposed to LPS had a significantly greater (60%) urine L/M ratio in comparison to that of the control animals (Fig. 5), which indicated that the onset of leaky gut syndrome was a consequence of LPS treatment. Although statistically insignificant, a 39% decrease in the urine L/M ratio was observed in LPS-treated animals when they were co-exposed to UHF. In contrast, a significant decrease in the urine L/M ratio was seen in LPS-treated animals when they were co-exposed to either colostrum (40% depletion) or FHF (51% decrease). These results suggest that the use of the proposed mixture of fermented herbs (rather than unfermented ones) in combination with probiotics is advantageous for the reduction of LPS-induced gut permeability. The mammalian system is colonised by trillions of microbes, the majority of which live in gastrointestinal tract (GIT), predominantly by maintaining a symbiotic relationship with their host. Microbial colonisation of gastrointestinal tract (GIT) plays a vital role in the protection of the epithelial barrier by maintaining expression of the tight junction proteins (Sharma, Young, & Neu, 2010; Wlodarska & Finlay, 2010). Besides, it has been shown that

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Fig. 4. The effect of herbal extracts or colostrum on the serum CRP level of LPStreated rats. The rats were treated with water (for control and LPS + water groups), UHF, FHF, or colostrum once daily for five consecutive days. The rats were then treated with saline (for control group) or LPS (for non-control groups) for 48 h. Following the desired treatment schedule, the blood was collected and the CRP levels in the serum samples were measured. The detailed treatment regimen and experimental conditions are described in the Section 2. The values are expressed as the means ± SEM, n = 5. The data were log-transformed prior to analysis by ANOVA, and the means without a common letter differed when p < 0.05.

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Lactulose/mannitol ratio

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Fig. 5. The effect of herbal extracts or colostrum on the gut permeability of LPStreated rats. The rats were treated with water (for control and LPS + water groups), UHF, FHF, or colostrum once daily for five consecutive days. The rats were then treated with saline (for control group) or LPS (for non-control groups) for 48 h. Twelve hours after treatment with LPS, the animals were deprived of food for 12 h following which they received oral treatment of lactulose and mannitol. The animals were then maintained for 24 h without access to food. Following the desired treatment schedule, the urine samples were collected and the lactulose (L) and mannitol (M) levels in urine were quantitated in terms of the recovery percentage of the ingested dose, and the L/M ratios were calculated. The detailed treatment regimen and experimental conditions are described in the Section 2. The values are expressed as the means ± SEM, n = 5, and the means without a common letter differed when p < 0.05.

prior treatment with probiotics reduces circulating endotoxin levels and bacterial translocation and also preserves the gut barrier integrity in response to hemorrhagic shock (Luyer et al., 2005). Accordingly, to explore the possible link between the proposed herbal formulations and the intestinal microbes (particularly the commensal bacteria) for the protection of the gut from LPS toxicity, we quantified the intestinal population of Lactobacillus spp. by their relative abundance in the stool. It has been shown that lactic acid bacteria (LAB), including Lactobacillus spp. inhibit proinflammatory cytokine production in experimental colitic mice (Lee et al., 2008). Moreover, Lactobacilli were found to attenuate endotoxemia and bacteremia in rats with severe intra-abdominal infections (Thorlacius et al., 2003). In our study, LPS treatment resulted in a definitive, but insignificant, reduction in the relative abundance of Lactobacillus spp. (24% decrease) in the stool of rats (Fig. 6). Notably, the faecal populations of Lactobacillus spp. in the LPS-treated rats were insignificantly augmented upon co-exposure to either UHF or colostrum, but were increased significantly upon co-treatment with FHF. These results support the beneficial impact of herbal fermentation and probiotics on the gut commensal microbial environment and it is conceivable that in addition to its anti-inflammatory action, an augmentation in the intestinal population of LAB, such as Lactobacillus, might be one of the mechanisms by which FHF combats LPS-induced destabilization of gut permeability. In summary, our results demonstrate significant in vitro antiinflammatory activities of the fermented products of the proposed herbs. These activities were shown to act in part through the attenuation of COX-2 and iNOS gene expression as well as the suppression of NO production. However, the extent of these anti-inflammatory effects varied between the herbs studied. Although the unfermented herb mixture attenuated the serum endotoxin and CRP levels, reduced intestinal permeability, and augmented the gut commensal bacteria in rats challenged with LPS, each of these effects were found to be statistically insignificant. In contrast, however, the fermented herb mixture in combination with the respective probiotics used for fermentation demonstrated significant in vivo anti-inflammatory

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Relative DNA level of Lactobacillus spp. (% of control)

200

160

Control LPS + water LPS + UHF LPS + FHF LPS + colostrum

a,c a,b

a,b 120

a,b

80

b

40

0 Fig. 6. The effect of herbal extracts or colostrum on the population of Lactobacillus spp. in relation to that of universal bacteria in the stool of LPS-treated rats. The rats were treated with water (for control and LPS + water groups), UHF, FHF, or colostrum once daily for five consecutive days. The rats were then treated with saline (for control group) or LPS (for non-control groups) for 48 h. Following the desired treatment schedule, the stool was collected and the content of Lactobacillus spp. and universal bacterial DNA (the gene-encoding 16S rRNA) in the stool samples were determined by qRT-PCR. The detailed treatment regimen and experimental conditions are described in the Section 2. The relative DNA content of the Lactobacillus spp. in the stool of the control group was set to 100%. The values are expressed as the means ± SEM, n = 3. The data were log-transformed prior to analysis by ANOVA, and the means without a common letter differed when p < 0.05.

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