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Effects of phytochemicals on in vitro antiinflammatory activity of Bifidobacterium adolescentis a
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Kyuichi Kawabata , Yuri Kato , Taiken Sakano , Nobuyuki Baba , Kota Hagiwara , Akira b
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Tamura , Seigo Baba , Midori Natsume & Hajime Ohigashi a
Department of Bioscience, Fukui Prefectural University, Fukui, Japan
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Lactic Acid Bacteria Research Department, Food Science Research Laboratories, R&D Division, Meiji Co. Ltd, Odawara, Japan c
Clinical Research and Scientific Information, Product Development Department, Healthcare and Nutritionals Division, Meiji Co. Ltd, Tokyo, Japan d
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Functional Evaluation Department 2, Food Science Research Laboratories, R&D Division, Meiji Co. Ltd, Odawara, Kanagawa, Japan Published online: 27 Feb 2015.
To cite this article: Kyuichi Kawabata, Yuri Kato, Taiken Sakano, Nobuyuki Baba, Kota Hagiwara, Akira Tamura, Seigo Baba, Midori Natsume & Hajime Ohigashi (2015): Effects of phytochemicals on in vitro anti-inflammatory activity of Bifidobacterium adolescentis, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1006566 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1006566
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Bioscience, Biotechnology, and Biochemistry, 2015
Effects of phytochemicals on in vitro anti-inflammatory activity of Bifidobacterium adolescentis Kyuichi Kawabata1,*, Yuri Kato1, Taiken Sakano1,a, Nobuyuki Baba1, Kota Hagiwara1, Akira Tamura2, Seigo Baba3, Midori Natsume4 and Hajime Ohigashi1 1
Department of Bioscience, Fukui Prefectural University, Fukui, Japan; 2Lactic Acid Bacteria Research Department, Food Science Research Laboratories, R&D Division, Meiji Co. Ltd., Odawara, Japan; 3Clinical Research and Scientific Information, Product Development Department, Healthcare and Nutritionals Division, Meiji Co. Ltd, Tokyo, Japan; 4Functional Evaluation Department 2, Food Science Research Laboratories, R&D Division, Meiji Co. Ltd, Odawara, Kanagawa, Japan
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Received May 15, 2014; accepted August 6, 2014 http://dx.doi.org/10.1080/09168451.2015.1006566
Probiotics have been shown to improve the condition of not only the human gastrointestinal tract but also the entire body. We found that quercetin enhances the anti-inflammatory activity of Bifidobacterium adolescentis, which is abundant in human intestines. Here, we assessed whether certain phytochemicals could enhance the anti-inflammatory activity of B. adolescentis. Bifidobacteria were anaerobically cultured with phytochemicals for 3 h, and the anti-inflammatory activity of the supernatants was estimated by testing their ability to inhibit nitric oxide (NO) production by lipopolysaccharidestimulated RAW264 macrophages. Of the 55 phytochemicals tested, phloretin, (+)-taxifolin, and (−)-epigallocatechin gallate as well as quercetin-3-Oglucoside and quercetin-4′-O-glucoside were similar to quercetin in promoting NO suppression by B. adolescentis. In addition, the phytochemicals excluding quercetin increased the concentrations of lactic and acetic acids in the co-culture supernatants. These results suggest that some phytochemicals may activate the anti-inflammatory function of B. adolescentis. Key words:
Bifidobacterium adolescentis; phytochemicals; anti-inflammatory activity; co-culture; functional interaction
The human intestines harbor an estimated 1000 species of micro-organisms, consisting of a total of 100 trillion cells,1,2) which have important and specific
functions in the maintenance of the host homeostasis.3) Commensal anaerobes in the colon ferment non-digestible carbohydrates to short-chain fatty acids, which are the important energy sources to colon epithelial cells as well as protecting these cells from pathogenic bacteria by acidifying the lumen.4,5) The diversity of intestinal microbes contributes to the development of the host innate and adaptive immune systems.6) Recent microbiome studies have shown that an imbalance in microbiota leads to host illnesses, such as obesity, diabetes mellitus, inflammatory bowel diseases, neurodevelopmental disorders, and cancer.7−11) Intestinal microbes can metabolize dietary polyphenols.12) Hydrolyzing enzymes secreted by gut bacteria convert glycosides and conjugates to aglycones, which undergo further modification, such as ring cleavage and dehydroxylation. Daidzein is transformed to the more active compound S-equol by bacteria in the genera Lactobacillus, Eggerthella, and Slackia, but not by Bifidobacterium.13,14) In contrast, the mechanism of bioconversion of flavonoids to more active compounds, while maintaining the flavonoid structure (C6–C3–C6), has not yet been determined. Most polyphenols are converted to phenolics, such as phenylacetic acids, phenylpropionic acids (from the B ring), and phloroglucinol (from the A ring), whereas catechins are metabolized to γ-valerolactones, as well as to phenylacetic and phenylpropionic acids, from the B ring.12) These bacterial metabolites may exhibit health-promoting effects via their anti-inflammatory and anti-oxidative activities, and by their modulation of the diversity of intestinal microbiota.15)
*Corresponding author. Email: [email protected] Abbreviations: ACA, 1′-acetoxychavicol acetate; BA, Bifidobacterium adolescentis; DHPA, 2-(3,4-dihydroxyphenyl)acetic acid; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; DPPA, 3-(3,4-dihydroxyphenyl)propionic acid; EGCG, (−)-Epigallocatechin gallate; FBS, fetal bovine serum; HPA, 2-(3-hydroxyphenyl)acetic acid; NO, nitric oxide; PDA, photodiode array; PHL, phloretin; PPA, 3-phenylpropionic acid; Q3G, quercetin-3-O-glucoside; Q3GA, quercetin-3-O-glucuronide; Q3Gal, quercetin-3-O-galactoside; Q3Rha, quercetin-3-O-rhamnoside; Q4′G, quercetin-4′-O-glucoside, TAX, (+)-taxifolin. a Present address: Laboratory of Biochemistry, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, Graduate Program in Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422–8526, Japan. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Bacteria in the genus Bifidobacterium are grampositive anaerobes and are the predominant component of intestinal microbiota in healthy humans. Some Bifidobacterium species have probiotic functions, including carbohydrate fermentation, vitamin synthesis, modulation of host immune response, carcinogen detoxification, and improvement of gut microbiota ecology.16) These bacteria are among the most important folate producers17) and metabolize indigestible carbohydrate to acetate and lactate, which are further converted to butyrate by butyrate-producing bacteria.18) Besides being the energy source of intestinal epithelial cells, acetate produced by B. longum subsp. longum can prevent lethal infection by enterohemorrhagic Escherichia coli O157:H7.19) In addition, several species of Bifidobacterium have shown anti-inflammatory activity, including Bifidobacterium adolescentis, B. breve, B. bifidum, B. longum subsp. longum, B. catenulatum, and B. longum subsp. infantis, both in vitro20−23) and in vivo.24) Interestingly, the culture supernatant from these cells showed anti-inflammatory activity,20−23) suggesting that bifidobacteria can secrete as yet unidentified anti-inflammatory substance(s). We recently investigated the functional interactions between polyphenols and probiotic bacteria using a coculture model.25) Intestinal anaerobes (e.g. Bacteroides, Lactobacillus, and Bifidobacterium) were incubated with flavonols (galangin, kaempferol, quercetin, myricetin, and fisetin) in cell culture medium under anaerobic conditions for 3 h, and the ability of the conditioned media to suppress the inflammatory activity of lipopolysaccharide-stimulated macrophages was assayed. Interestingly, we have found that quercetin, galangin, and fisetin enhanced the anti-inflammatory activity of B. adolescentis,25) a species of Bifidobacterium present in the large intestines of all age groups, including occasionally in infants.26) In addition, these compounds were not metabolized to other types of bioactive compounds by the bacteria but appeared to increase the production and/or secretion of anti-inflammatory substance(s) from the bacteria.25) These findings suggested that other combinations of probiotic bacteria and dietary phytochemicals could show unique functional interaction. This study therefore evaluated whether other phytochemicals, as well as glycosides and conjugates, could enhance the anti-inflammatory activity of B. adolescentis, similar to quercetin.
Materials and methods Reagents. Nobiletin, auraptene, 1′-acetoxychavicol acetate (ACA), and zerumbone were the kind gifts of Dr. Akira Murakami (Kyoto University, Kyoto, Japan), and 4-hydroyxderricin and xanthoangelol were kindly donated by Dr. Norio Yamamoto (House Wellness Foods Corporation, Itami, Japan). Dulbecco’s modified Eagle’s medium (DMEM, high glucose), penicillin, streptomycin, phloretin, 3′,4′-dihydroxyflavonol, imperatorin, 2-(3-hydroxyphenyl)acetic acid (HPA), 2-(3,4-dihydroxyphenyl)acetic acid (DHPA), 3-phenylpropionic acid (PPA), 3-(3,4-dihydroxyphenyl)propionic acid (DPPA), phloroglucinol, and resorcinol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Thermo
Fisher Scientific (Waltham, MA, USA). Xanthohumol, pinocembrin, (+)-taxifolin, quercetin-3-O-glucoside (Q3G), quercetin-3-O-galactoside (Q3Gal), quercetin-3O-rhamnoside (Q3Rha), quercetin-4′-O-glucoside (Q4′ G), quercetin-3-O-glucuronide (Q3GA), and isorhamnetin were obtained from Extrasynthese (Lyon, France). (−)-Epigallocatechin gallate (EGCG) and quercetin were obtained from Cayman Chemical (Ann Arbor, MI, USA). Naringenin was purchased from ChromaDex (Irvine, CA, USA). 3′,4′-Dihydroxyflavone, 3′,4′,7-trihydroxyflavone, tangeretin, flavanone, and eriodictyol were purchased from Indofine Chemical (Hillsborough, NJ, USA). Biochanin A and formononetin were obtained from LKT Laboratories (St. Paul, MN, USA). Chalcone, butein, isoliquiritigenin, flavone, flavanol, and phlorizin were obtained from Tokyo Chemical Industry (Tokyo, Japan). (R, S)-Equol and (−)-epigallocatechin were purchased from LC Laboratories (Woburn, MA, USA) and Tokiwa Phytochemical (Chiba, Japan), respectively. (−)-Epicatechin and (−)-epicatechin gallate were purchased from Nagara Science (Gifu, Japan). GAM broth was obtained from Nissui Pharmaceutical (Tokyo, Japan). All other reagents were purchased from WAKO Pure Chemicals (Osaka, Japan), unless otherwise specified.
Culture conditions. Murine macrophage RAW264 cells (RCB0535) and B. adolescentis (JCM1275T) were provided by the RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. RAW264 cells were grown in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. B. adolescentis was cultured in GAM broth at 37 °C for 18 h under static and anaerobic conditions using the AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan). Conditioned medium from mono- and co-cultures. Culture conditioned media were prepared as described.25) Briefly, B. adolescentis (108 cfu/mL) and phytochemicals dissolved in dimethylsulfoxide (DMSO) were incubated in serum and antibiotic-free DMEM at 37 °C for 3 h under static and anaerobic conditions [B. adolescentis (with DMSO, final concentration 0.05%) or phytochemical alone as mono-culture; B. adolescentis + phytochemical as co-culture]. The culture media were centrifuged twice, once at 5000 g and once at 15,000 g, each for 3 min at 4 °C, and the supernatants were decanted and supplemented with 100 U/mL of penicillin and 100 μg/mL streptomycin. Control samples consisted of medium containing each phytochemical prior to treatment of RAW264 cells. Determination of NO production. RAW264 cells (2.5 × 105 cells/mL, 0.14 mL) seeded on 96-well plates were incubated with LPS (final concentration, 100 ng/mL) in conditioned medium or in phytochemicalcontaining control medium for further 24 h. Supernatants were mixed with Griess reagent 27) in 96-well plates, and
Activation of Bifidobacteria by Photochemicals
absorbance at 543 nm was measured using SpectraMax M2 (Molecular Devices, Sunnyvale, CA, USA). The concentration of NO was calculated from the standard curve prepared using a sodium nitrate solution. Cell viability was measured by crystal violet staining; none of the tested phytochemicals or conditioned media was cytotoxic to RAW264 cells in any experiment.
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High-performance liquid chromatography (HPLC) analysis of phytochemicals. The content of phytochemicals in the conditioned medium was assessed by HPLC using a photodiode array (PDA) and an Ascentis RP-amide column (100 × 3 mm, 3 μm, SUPELCO, Bellefonte, PA, USA) at 30 °C. The mobile phases for eluting aglycones [quercetin, phloretin, EGCG, and (+)-taxifolin] and quercetin glucoside and conjugates were 80% and 40% acetonitrile, respectively, each containing 0.1% formic acid. Phloretin, EGCG, and (+)-taxifolin were detected at 280 nm, and quercetin aglycone, the glucosides, and conjugates at 254 nm. Measurement of pH and organic acids. The pH of the supernatants was measured by a pH meter (TOA Electronics, Shizuoka, Japan). The determination of lactic acid and acetic acid was performed by the 2-nitrophenylhydrazine derivatization method.28) Organic acids in the supernatant with 2-ethylbutyric acid as an internal standard were coupled to 2-nitrophenylhydr-
3
azine using a labeling kit (YMC, Kyoto, Japan) and then analyzed by HPLC–PDA and an Ascentis C18 column (100 × 3 mm, 3 μm, SUPELCO) at 50 °C. The mobile phase was acetonitrile–methanol–water (23:12:65, v/v, pH 4.5) solution. The derivatives were detected at 400 nm. Statistical analysis. All data are expressed as the mean ± SD from at least three independent experiments. Comparisons between groups were analyzed by either the Tukey–Kramer test or the Student’s t-test. Statistical analysis was performed by SPSS 16.0 J (SPSS Inc., Chicago, IL, USA). Significance was reached at values of p < 0.05.
Results Quercetin dose-dependently enhanced the NO suppressive activity of B. adolescentis. We previously reported that flavonols, including quercetin, galangin, and fisetin, enhanced the anti-inflammatory activity of B. adolescentis.25) Although the conditioned media from B. adolescentis mono-cultures failed to suppress NO production by LPS-treated RAW264 cells, media from bacteria co-cultured with quercetin showed a dose-dependent increase in inhibitory activity Fig. 1A. The initial concentration of quercetin in both monoculture and co-culture media was similarly reduced, and largely disappeared by 1 h Fig. 1B, indicating that,
(A)
(B)
Fig. 1. Quercetin dose-dependently enhanced the NO suppressive activity of B. adolescentis. Notes: (A) B. adolescentis was incubated alone or together with DMSO (final concentration 0.05%, 0 μM quercetin) or quercetin (5–40 μM) in serum-free DMEM at 37 °C for 3 h under static and anaerobic conditions. The conditioned media were collected, and their anti-inflammatory activity was evaluated by assessing their ability to inhibit LPS-induced NO production in RAW264 cells. *p < 0.05 (Tukey–Kramer test). Data are shown as mean ± SD (n = 3). (B) Measurement of quercetin concentrations in the mono- and co-culture conditioned media by HPLC–PDA, as described in the materials and methods section. Data are shown as mean ± SD (n = 3). BA, B. adolescentis; QUE, quercetin; NO, nitric oxide.
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although quercetin enhanced the anti-inflammatory activity of the bifidobacteria, it was not converted to compounds with higher activity than quercetin, a finding similar to that reported previously.25)
Table 1.
Phloretin, (+)-taxifolin, and EGCG enhanced the NO suppressive activity of B. adolescentis . Although we have found that quercetin, galangin, and fisetin enhanced the NO suppressive activity of bifidobacteria,
Screening of phytochemicals capable of increasing the NO suppressive activity of B. adolescentis JCM1275T. NO inhibition (%)a
Conc. (μM)
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B. adolescentis Chalcones Chalcone Butein 4′-Hydroxychalcone Isoliquiritigenin Phloretin Xanthohumol 4-Hydroxyderricin Xanthoangelol Flavones Flavone Chrysin 3′,4′-Dihydroxyflavone Luteolin Diosmetin 7,3′,4′-Trihydroxyflavone Apigenin Nobiletin Tangeretin Flavonols Flavonol 3′,4′-Dihydroxyflavonol (+)-Taxifolin Flavanones Flavanone Pinocembrin Eriodictyol Hesperetin Naringenin Isoflavones Genistein Daidzein (R,S)-Equol Biochanin A Formononetin Catechins (−)-Epicatechin (−)-Epicatechin gallate (−)-Epigallocatechin EGCG Coumarins Auraptene Bergamottin Imperatorin Phenolics HPA DHPA PPA DPPA Phloroglucinol Resorcinol Others ACA Zerumbone Curcumin Resveratrol a
0h
3 h (mo)b
3 h (co)c
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‒
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40 5 10 10 40 5 5 5
29 ± 10 61 ± 6 51 ± 7 35 ± 10 44 ± 10 65 ± 8 60 ± 6 26 ± 9
3±3 26 ± 4 45 ± 5 36 ± 10 17 ± 8 47 ± 8 7± 4 4± 4
1± 2 19 ± 6 15 ± 10* 10 ± 4* 63 ± 10* 5 ± 10* 1± 3 2± 2
20 20 10 10 10 20 10 10 20
17 ± 9 28 ± 2 39 ± 7 31 ± 4 44 ± 10 32 ± 7 42 ± 8 28 ± 7 28 ± 5
12 ± 5 29 ± 7 45 ± 5 39 ± 9 40 ± 9 31 ± 5 36 ± 8 25 ± 3 28 ± 10
14 ± 2 18 ± 3 30 ± 7* 32 ± 6 3 ± 4* 21 ± 3* 34 ± .9 21 ± 3 28 ± 5
20 10 40
30 ± 3 48 ± 7 22 ± 5
4± 2 43 ± 5 19 ± 7
1± 2 51 ± 7 36 ± 7*
40 40 10 40 40
9± 20 ± 40 ± 25 ± 31 ±
5 6 7 8 9
7± 14 ± 34 ± 29 ± 24 ±
8 6 8 8 9
0± 16 ± 16 ± 11 ± 25 ±
10 40 20 20 10
33 ± 58 ± 24 ± 22 ± 19 ±
5 5 6 9 5
33 ± 58 ± 25 ± 29 ± 23 ±
9 8 8 7 7
14 ± 5* 30 ± 7* 3 ± 3* 24 ± 6 19 ± 10
40 20 40 20
3± 1 31 ± 11 13 ± 5 51 ± 7
9± 30 ± 12 ± 31 ±
8 4 5 9
2± 39 ± 3± 49 ±
10 10 40
30 ± 9 26 ± 7 61 ± 5
17 ± 9 10 ± 7 9± 3
40 40 40 40 40 40
0± 8± 2± 6± 4± 7±
1 8 3 9 4 8
0± 3± 1± 6± 0± 2±
0 2 1 7 0 3
3± 0± 3± 0± 3± 5±
4 0 5 0 5 8
56 ± 5 61 ± 10 44 ± 6 61 ± 7
40 ± 44 ± 21 ± 61 ±
9 6 5 2
22 ± 11 ± 0± 25 ±
5* 3* 1* 8*
5 5 20 40
Data are shown as mean SD (n = 3–8). Mono-culture of either phytochemical or B. adolescentis for 3 h. c Co-culture of phytochemical and B. adolescentis for 3 h. *p< 0.05 vs. mono-culture (Student’s t-test). b
0 6 4* 5* 6
4 6* 4* 9*
7± 4 5± 6 3 ± 3*
Activation of Bifidobacteria by Photochemicals
(B) 100
Mono-culture Co-culture
Relative ratio (% %)
Relative ratio (% %)
(A) 100 75 50 25 0
75 50 25
Mono-culture Co-culture
0 0
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(C) 100
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3
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75 50 25 0 0
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Fig. 2. Effect of B. adolescentis on the concentrations of phloretin, (+)-taxifolin, and EGCG. Notes: (A) Phloretin, (B) (+)-taxifolin, and (C) EGCG were incubated in the presence or absence of B. adolescentis for 3 h, and the concentrations of phytochemicals in the conditioned media were analyzed by HPLC–PDA, as described in the materials and methods section. Data are shown as mean ± SD (n = 3).
the effect of other phytochemicals had not been determined. We therefore tested whether 47 phytochemicals could enhance the NO suppressive activity of B. adolescentis JCM1275T. These 47 phytochemicals, including eight chalcones, nine flavones, three flavonols, five flavanones, five isoflavones, four catechins, three coumarins, six phenolics, and four others, were tested at concentrations showing ≤70% inhibition of NO production (Table 1) and having no effect on cell viability (data not shown). Many of the tested compounds had no effect or reduced the anti-inflammatory activity of the conditioned media (Table 1). In contrast, co-culture of B. adolescentis with phloretin, (+)-taxifolin, and EGCG significantly and synergistically enhanced the NO suppressive activity of the bacteria. Their concentrations in the conditioned media from mono- and cocultures were almost the same (Fig. 2). These results suggest that phloretin, (+)-taxifolin, and EGCG may have an effect on B. adolescentis similar to quercetin. In contrast, co-culture with epicatechin gallate also significantly inhibited (39%) the NO production though, this may not result from increasing the anti-inflammatory activity of B. adolescentis by epicatechin gallate but an additive action of them (the inhibition rate of mono-culture media: B. adolescentis, 4%; epicatechin gallate, 30%).
Quercetin glucosides enhanced the NO suppressive activity of B. adolescentis. Because flavonoids generally present as glycosides in plants and undergo conjugation, such as glucuronidation and methylation, in the human intestine after ingestion, we investigated whether quercetin glycosides (Q3G, Q3Gal, Q3Rha,
rutin, and Q4′G) and conjugates (Q3GA and isorhamnetin), as well as phloretin glucoside (phlorizin), similarly enhance the activity of bifidobacteria. At concentrations 100 μM, glycosides and conjugates in mono-culture suppressed NO production by around 20–50%, respectively, after 3 h (Table 2). Intriguingly, co-culture of B. adolescentis JCM1275T with Q3G and Q4′G markedly enhances the NO suppressive activity of the bacteria, with actual inhibition ratios of 33 and 60%, respectively, being greater than the estimated ratio (21 and 25%, respectively). Q3GA also increased the activity in the conditioned medium from the co-culture, but this may be an additive effect (actual, 69%; estimated, 61%). Co-culture of B. adolescentis with Table 2. Effect of glycosides and conjugates on the anti-inflammatory activity of B. adolescentis JCM1275T. NO inhibition (%)a
B. adolescentis Glycosides Q3G Q3Gal Q3Rha Rutin Q4′G Phlorizin Conjugates Q3GA Isorhamnetin a
Conc. (μM)
0h
3 h (mo)b
3 h (co)c
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100 100 100 100 100 100
21 ± 19 ± 15 ± 20 ± 18 ± 11 ±
6 8 9 7 5 1
100 100
56 ± 9 53 ± 10
17 ± 18 ± 20 ± 20 ± 21 ± 15 ±
8 6 9 8 9 3
33 ± 8* 14 ± 8 13 ± 7 15 ± 6 60 ± 10* 15 ± 3
57 ± 8 55 ± 8
69 ± 8* 20 ± 5*
Data are shown as mean SD (n = 4–10). Mono-culture of either phytochemical or B. adolescentis for 3 h. c Co-culture of phytochemical and B. adolescentis for 3 h. *p< 0.05 versus mono-culture (Student’s t-test). b
(A)
125 100 75 50 G3G Q4'G G3GA
25 0
125 100 75 50 G3G + BA Q4'G + BA G3GA + BA
25 0
0
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Quercettin aglycone (µ µM)
(C)
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(B) Relative ratio (% %)
K. Kawabata et al.
Relative ratio (% %)
6
3
0
1 2 Culture (h)
3
10 G3G + BA Q4'G + BA G3GA + BA
7.5 5 2.5 0 0
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Fig. 3. Quercetin glucosides, but not glucuronide, were converted to quercetin aglycone during co-culture with B. adolescentis. Notes: Quercetin glucosides and glucuronide (100 μM) were incubated in the presence or absence of B. adolescentis for 3 h, and their concentrations in the conditioned media were analyzed by HPLC–PDA, as described in the materials and methods section. (A) Glucosides and glucuronide in mono-culture media; (B) glucosides and glucuronide in co-culture media; (C) quercetin aglycone in the co-culture media. Data are shown as mean ± SD (n = 3). BA, B. adolescentis; Q3G, quercetin-3-glucoside; Q4′G, quercetin-4′-glucoside; Q3GA, quercein-3-glucuronide.
(A) other glycosides and conjugates, especially isorhamnetin, reduced the activity when compared with monoculture media. Most of the Q3G, Q4′G, and Q3GA in the mono-cultures were present after 3 h (Fig. 3A), although the amounts were time-dependently reduced during co-culture of Q3G and Q4′G, but not Q3GA, with B. adolescentis. In contrast, the levels of quercetin aglycone were increased Fig. 3B and C, suggesting that the quercetin glucosides may enhance the activity of the bifidobacterium after conversion to aglycones.
(B)
Fig. 4. Effects of phytochemicals on the pH lowering and the organic acids concentration in co-culture supernatants. Notes: B. adolescentis were incubated with DMSO, quercetin (20 μM), (+)-taxifolin (40 μM), phloretin (40 μM), and EGCG (20 μM) for 3 h, and then the change of pH and the concentrations of lactic and acetic acids in the conditioned media were analyzed by a pH meter and HPLC–PDA, respectively, as described in the Materials and Methods section. (A) The pH level, *p < 0.05 (Dunnett test); (B) the concentrations of the organic acids, *p < 0.05 vs. the DMSO group (Dunnett test). Data are shown as mean ± SD (n = 3). QUE, quercetin; TAX, (+)-taxifolin; PHL, phloretin.
Phytochemicals, except for quercetin, increased the concentrations of lactic acid and acetic acid in the coculture medium Since bifidobacterium bacteria is the major producer of lactic acid and acetic acid which were shown to have an anti-inflammatory activity, such as the inhibition of pro-inflammatory cytokine production,22,29) we next investigated the change of pH and organic acids levels in the co-culture medium. After the culture of B. adolescentis with DMSO or phytochemicals [quercetin, (+)-taxifolin, phloretin, and EGCG] for 3 h, the pH of all of the tested supernatants decreased from 7.8 (just before culture) to 7.2 Fig. 4A. On the other hand, phytochemicals excluding quercetin markedly increased the concentrations of either or both lactic and acetic acids in the supernatants as compared to that of the bifidobacteria mono-culture Fig. 4B, suggesting that both the change of pH and the increase of lactic and acetic acids may not correlate with the NO suppressive activity of the co-culture supernatant.
Activation of Bifidobacteria by Photochemicals
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Discussion Intestinal microbiota is very important for the maintenance of human health. Imbalances in their populations have been suggested as a cause of several diseases including obesity, diabetes mellitus, inflammatory bowel diseases, neurodevelopmental disorders, and cancer.7−11) The health-promoting effects of probiotic bifidobacteria and lactic acid bacteria have been associated with vitamin production, carbohydrate fermentation, intestinal epithelial cell protection, immunomodulation, and antipathogen activity.16,30–32) Probiotic anaerobes have also been found to secrete anti-inflammatory agents.25,33,34) Furthermore, our previous study showed that the flavonols quercetin, galangin, and fisetin enhance the production of NO suppressants by B. adolescentis.25) However, whether these effects are distinct to flavonols and evoked by a characteristic structure of these compounds have not yet been clarified. This study therefore investigated the effects of other types of phytochemicals on B. adolescentis, as well as assessing the structureactivity relationships. Of the 55 tested phytochemicals, including 47 aglycones and eight glycosides and conjugates, five compounds, phloretin, (+)-taxifolin, EGCG, Q3G, and Q4′G, enhanced the anti-inflammatory activity of B. adolescentis. The initial levels of phloretin, (+)-taxifolin, and EGCG showed a similar reduction in both mono-culture and co-culture media over 3 h, suggesting that these phytochemicals may, like quercetin, promote the secretion of anti-inflammatory substances from B. adolescentis. Compared with quercetin, (+)-taxifolin lacks the C2–C3 double bond and fisetin lacks the C5 hydroxy group.25) All of the tested phytochemicals containing a catechol structure, including butein, 3′,4′-dihydroxyflavonol, 3′,4′-dihydroxyflavone, 7,3′,4′-trihydroxyflavone, luteolin, eriodictyol, epicatechin, epicatechin gallate, DHPA, and DPPA, were inactive. Compared with fisetin and (+)-taxifolin, 7,3′,4′trihydroxyflavone, eriodictyol, and Q3GA lack the hydroxy group at the C-3 position and are glucuronized. These findings suggest that both the catechol B-ring and the C3 hydroxy group may be the important structural moieties that interact with the bifidobacteria. However, phloretin and EGCG lack these moieties, suggesting that other structures in these compounds may be responsible for their activities. B. adolescentis possesses the genes that encode β-galactosidase35) and β-glucosidase,14) but not those that encode β-glucuronidase and α-L-rhamnosidase. B. adolescentis JCM1275T converted quercetin glucosides to the aglycone forms in a time-dependent manner, but did not convert Q3GA into its aglycone. The level of quercetin aglycone in the co-culture medium was positively correlated with the increase in NO suppressive activity. Because the addition of glucose during co-culture of B. adolescentis and quercetin failed to further increase the anti-inflammatory activity (data not shown), glucose molecules generated by the hydrolysis of quercetin glucoside would be inactive. Intriguingly, Q4′G was more sensitive to bioconversion by B. adolescentis JCM1275T than Q3G, suggesting that the β-glucosidase of this bacterium may prefer Q4′ G as a substrate. This substrate specificity agrees with
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experiments performed using enzymes from rat intestinal tissue.36) Although a large fraction of quercetin glucosides is absorbed by the small intestine,37,38) the glucosides that pass through the small intestine, and Q3GA excreted into the large intestine, may be converted to their aglycones by the β-glucosidase of bifidobacteria and the β-glucuronidase of intestinal microbiota, such as Clostridia and Eubacteria,39) with the aglycones enhancing the activity of B. adolescentis. Alternatively, these compounds may be further converted to inactive phenolics, including phenylacetic and phenylpropionic acids (Table 1). Intestinal bifidobacteria metabolize flavonoids to small compounds, such as phenolics and lactones.12) In our experimental model, some of the tested compounds showed lower anti-inflammatory activity in the co-culture than in mono-culture conditioned media, indicating that these compounds, especially highly hydrophobic compounds, had been degraded by B. adolescentis JCM1275T. Of these highly hydrophobic compounds, xanthohumol, auraptene, bergamottin, and imperatorin have a prenyl group; the sesquiterpene zerumbone lacks an hydroxy group; and butein, 4′-hydroxychalcone, isoliquiritigenin, genistein, equol, curcumin, and resveratrol are also lipophilic (log p > 3).40−42) Daidzein (log p = 2.51) 42) may be degraded but not converted into S-equol.14) In contrast, diosmetin, hesperetin, and isorhamnetin, each of which contains a methoxy group, seem to be more sensitive to bacteria than the polymethoxyflavonoids nobiletin and tangeretin. In addition, flavones, flavonols, and flavanones may have comparatively low sensitivity to B. adolescentis JCM1275T. It is noteworthy that co-culture of B. adolescentis with phloretin, (+)-taxifolin, and EGCG resulted in the increase in lactic acid and acetic acid Fig. 4B, indicating that these phytochemicals may promote the carbohydrate metabolism associated with the production of lactic and acetic acids. Since these organic acids are utilized by butyric acid-producing anaerobes,18) the up-regulation of lactic and acetic acids production by the phytochemicals may lead to the increase of butyric acid production. Moreover, both lactic acid and acetic acid were found to inhibit the production of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-8, from LPS-stimulated neutrophils and colon tumor cells.22,29) However, quercetin had no such effect. Therefore, antiinflammatory substance(s) produced by B. adolescentis during co-culture with the phytochemicals may be neither lactic acid nor acetic acid. In conclusion, our present study found that phloretin, (+)-taxifolin, and EGCG were novel enhancers of the anti-inflammatory activity of B. adolescentis JCM1275T. This implies that fruits and tea containing abundant amounts of quercetin, phloretin, and EGCG can potentiate the anti-inflammatory activity of B. adolescentis in the intestines, and therefore, it is necessary to assess impacts of B. adolescentis administration in combination with these phytochemicals on an animal model of the inflammatory bowel diseases. Furthermore, the mechanisms by which these compounds induce the production/secretion of active substance(s) from B. adolescentis will be the subjects of further investigation.
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Funding This study was supported by a Grant-in-Aid for Young Scientists [22780120] from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K. K.).
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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Effects of phytochemicals on in vitro antiinflammatory activity of Bifidobacterium adolescentis a
a
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a
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Kyuichi Kawabata , Yuri Kato , Taiken Sakano , Nobuyuki Baba , Kota Hagiwara , Akira b
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Tamura , Seigo Baba , Midori Natsume & Hajime Ohigashi a
Department of Bioscience, Fukui Prefectural University, Fukui, Japan
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Lactic Acid Bacteria Research Department, Food Science Research Laboratories, R&D Division, Meiji Co. Ltd, Odawara, Japan c
Clinical Research and Scientific Information, Product Development Department, Healthcare and Nutritionals Division, Meiji Co. Ltd, Tokyo, Japan d
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Functional Evaluation Department 2, Food Science Research Laboratories, R&D Division, Meiji Co. Ltd, Odawara, Kanagawa, Japan Published online: 27 Feb 2015.
To cite this article: Kyuichi Kawabata, Yuri Kato, Taiken Sakano, Nobuyuki Baba, Kota Hagiwara, Akira Tamura, Seigo Baba, Midori Natsume & Hajime Ohigashi (2015): Effects of phytochemicals on in vitro anti-inflammatory activity of Bifidobacterium adolescentis, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1006566 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1006566
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Bioscience, Biotechnology, and Biochemistry, 2015
Effects of phytochemicals on in vitro anti-inflammatory activity of Bifidobacterium adolescentis Kyuichi Kawabata1,*, Yuri Kato1, Taiken Sakano1,a, Nobuyuki Baba1, Kota Hagiwara1, Akira Tamura2, Seigo Baba3, Midori Natsume4 and Hajime Ohigashi1 1
Department of Bioscience, Fukui Prefectural University, Fukui, Japan; 2Lactic Acid Bacteria Research Department, Food Science Research Laboratories, R&D Division, Meiji Co. Ltd., Odawara, Japan; 3Clinical Research and Scientific Information, Product Development Department, Healthcare and Nutritionals Division, Meiji Co. Ltd, Tokyo, Japan; 4Functional Evaluation Department 2, Food Science Research Laboratories, R&D Division, Meiji Co. Ltd, Odawara, Kanagawa, Japan
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Received May 15, 2014; accepted August 6, 2014 http://dx.doi.org/10.1080/09168451.2015.1006566
Probiotics have been shown to improve the condition of not only the human gastrointestinal tract but also the entire body. We found that quercetin enhances the anti-inflammatory activity of Bifidobacterium adolescentis, which is abundant in human intestines. Here, we assessed whether certain phytochemicals could enhance the anti-inflammatory activity of B. adolescentis. Bifidobacteria were anaerobically cultured with phytochemicals for 3 h, and the anti-inflammatory activity of the supernatants was estimated by testing their ability to inhibit nitric oxide (NO) production by lipopolysaccharidestimulated RAW264 macrophages. Of the 55 phytochemicals tested, phloretin, (+)-taxifolin, and (−)-epigallocatechin gallate as well as quercetin-3-Oglucoside and quercetin-4′-O-glucoside were similar to quercetin in promoting NO suppression by B. adolescentis. In addition, the phytochemicals excluding quercetin increased the concentrations of lactic and acetic acids in the co-culture supernatants. These results suggest that some phytochemicals may activate the anti-inflammatory function of B. adolescentis. Key words:
Bifidobacterium adolescentis; phytochemicals; anti-inflammatory activity; co-culture; functional interaction
The human intestines harbor an estimated 1000 species of micro-organisms, consisting of a total of 100 trillion cells,1,2) which have important and specific
functions in the maintenance of the host homeostasis.3) Commensal anaerobes in the colon ferment non-digestible carbohydrates to short-chain fatty acids, which are the important energy sources to colon epithelial cells as well as protecting these cells from pathogenic bacteria by acidifying the lumen.4,5) The diversity of intestinal microbes contributes to the development of the host innate and adaptive immune systems.6) Recent microbiome studies have shown that an imbalance in microbiota leads to host illnesses, such as obesity, diabetes mellitus, inflammatory bowel diseases, neurodevelopmental disorders, and cancer.7−11) Intestinal microbes can metabolize dietary polyphenols.12) Hydrolyzing enzymes secreted by gut bacteria convert glycosides and conjugates to aglycones, which undergo further modification, such as ring cleavage and dehydroxylation. Daidzein is transformed to the more active compound S-equol by bacteria in the genera Lactobacillus, Eggerthella, and Slackia, but not by Bifidobacterium.13,14) In contrast, the mechanism of bioconversion of flavonoids to more active compounds, while maintaining the flavonoid structure (C6–C3–C6), has not yet been determined. Most polyphenols are converted to phenolics, such as phenylacetic acids, phenylpropionic acids (from the B ring), and phloroglucinol (from the A ring), whereas catechins are metabolized to γ-valerolactones, as well as to phenylacetic and phenylpropionic acids, from the B ring.12) These bacterial metabolites may exhibit health-promoting effects via their anti-inflammatory and anti-oxidative activities, and by their modulation of the diversity of intestinal microbiota.15)
*Corresponding author. Email: [email protected] Abbreviations: ACA, 1′-acetoxychavicol acetate; BA, Bifidobacterium adolescentis; DHPA, 2-(3,4-dihydroxyphenyl)acetic acid; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; DPPA, 3-(3,4-dihydroxyphenyl)propionic acid; EGCG, (−)-Epigallocatechin gallate; FBS, fetal bovine serum; HPA, 2-(3-hydroxyphenyl)acetic acid; NO, nitric oxide; PDA, photodiode array; PHL, phloretin; PPA, 3-phenylpropionic acid; Q3G, quercetin-3-O-glucoside; Q3GA, quercetin-3-O-glucuronide; Q3Gal, quercetin-3-O-galactoside; Q3Rha, quercetin-3-O-rhamnoside; Q4′G, quercetin-4′-O-glucoside, TAX, (+)-taxifolin. a Present address: Laboratory of Biochemistry, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, Graduate Program in Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422–8526, Japan. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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Bacteria in the genus Bifidobacterium are grampositive anaerobes and are the predominant component of intestinal microbiota in healthy humans. Some Bifidobacterium species have probiotic functions, including carbohydrate fermentation, vitamin synthesis, modulation of host immune response, carcinogen detoxification, and improvement of gut microbiota ecology.16) These bacteria are among the most important folate producers17) and metabolize indigestible carbohydrate to acetate and lactate, which are further converted to butyrate by butyrate-producing bacteria.18) Besides being the energy source of intestinal epithelial cells, acetate produced by B. longum subsp. longum can prevent lethal infection by enterohemorrhagic Escherichia coli O157:H7.19) In addition, several species of Bifidobacterium have shown anti-inflammatory activity, including Bifidobacterium adolescentis, B. breve, B. bifidum, B. longum subsp. longum, B. catenulatum, and B. longum subsp. infantis, both in vitro20−23) and in vivo.24) Interestingly, the culture supernatant from these cells showed anti-inflammatory activity,20−23) suggesting that bifidobacteria can secrete as yet unidentified anti-inflammatory substance(s). We recently investigated the functional interactions between polyphenols and probiotic bacteria using a coculture model.25) Intestinal anaerobes (e.g. Bacteroides, Lactobacillus, and Bifidobacterium) were incubated with flavonols (galangin, kaempferol, quercetin, myricetin, and fisetin) in cell culture medium under anaerobic conditions for 3 h, and the ability of the conditioned media to suppress the inflammatory activity of lipopolysaccharide-stimulated macrophages was assayed. Interestingly, we have found that quercetin, galangin, and fisetin enhanced the anti-inflammatory activity of B. adolescentis,25) a species of Bifidobacterium present in the large intestines of all age groups, including occasionally in infants.26) In addition, these compounds were not metabolized to other types of bioactive compounds by the bacteria but appeared to increase the production and/or secretion of anti-inflammatory substance(s) from the bacteria.25) These findings suggested that other combinations of probiotic bacteria and dietary phytochemicals could show unique functional interaction. This study therefore evaluated whether other phytochemicals, as well as glycosides and conjugates, could enhance the anti-inflammatory activity of B. adolescentis, similar to quercetin.
Materials and methods Reagents. Nobiletin, auraptene, 1′-acetoxychavicol acetate (ACA), and zerumbone were the kind gifts of Dr. Akira Murakami (Kyoto University, Kyoto, Japan), and 4-hydroyxderricin and xanthoangelol were kindly donated by Dr. Norio Yamamoto (House Wellness Foods Corporation, Itami, Japan). Dulbecco’s modified Eagle’s medium (DMEM, high glucose), penicillin, streptomycin, phloretin, 3′,4′-dihydroxyflavonol, imperatorin, 2-(3-hydroxyphenyl)acetic acid (HPA), 2-(3,4-dihydroxyphenyl)acetic acid (DHPA), 3-phenylpropionic acid (PPA), 3-(3,4-dihydroxyphenyl)propionic acid (DPPA), phloroglucinol, and resorcinol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Thermo
Fisher Scientific (Waltham, MA, USA). Xanthohumol, pinocembrin, (+)-taxifolin, quercetin-3-O-glucoside (Q3G), quercetin-3-O-galactoside (Q3Gal), quercetin-3O-rhamnoside (Q3Rha), quercetin-4′-O-glucoside (Q4′ G), quercetin-3-O-glucuronide (Q3GA), and isorhamnetin were obtained from Extrasynthese (Lyon, France). (−)-Epigallocatechin gallate (EGCG) and quercetin were obtained from Cayman Chemical (Ann Arbor, MI, USA). Naringenin was purchased from ChromaDex (Irvine, CA, USA). 3′,4′-Dihydroxyflavone, 3′,4′,7-trihydroxyflavone, tangeretin, flavanone, and eriodictyol were purchased from Indofine Chemical (Hillsborough, NJ, USA). Biochanin A and formononetin were obtained from LKT Laboratories (St. Paul, MN, USA). Chalcone, butein, isoliquiritigenin, flavone, flavanol, and phlorizin were obtained from Tokyo Chemical Industry (Tokyo, Japan). (R, S)-Equol and (−)-epigallocatechin were purchased from LC Laboratories (Woburn, MA, USA) and Tokiwa Phytochemical (Chiba, Japan), respectively. (−)-Epicatechin and (−)-epicatechin gallate were purchased from Nagara Science (Gifu, Japan). GAM broth was obtained from Nissui Pharmaceutical (Tokyo, Japan). All other reagents were purchased from WAKO Pure Chemicals (Osaka, Japan), unless otherwise specified.
Culture conditions. Murine macrophage RAW264 cells (RCB0535) and B. adolescentis (JCM1275T) were provided by the RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. RAW264 cells were grown in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. B. adolescentis was cultured in GAM broth at 37 °C for 18 h under static and anaerobic conditions using the AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan). Conditioned medium from mono- and co-cultures. Culture conditioned media were prepared as described.25) Briefly, B. adolescentis (108 cfu/mL) and phytochemicals dissolved in dimethylsulfoxide (DMSO) were incubated in serum and antibiotic-free DMEM at 37 °C for 3 h under static and anaerobic conditions [B. adolescentis (with DMSO, final concentration 0.05%) or phytochemical alone as mono-culture; B. adolescentis + phytochemical as co-culture]. The culture media were centrifuged twice, once at 5000 g and once at 15,000 g, each for 3 min at 4 °C, and the supernatants were decanted and supplemented with 100 U/mL of penicillin and 100 μg/mL streptomycin. Control samples consisted of medium containing each phytochemical prior to treatment of RAW264 cells. Determination of NO production. RAW264 cells (2.5 × 105 cells/mL, 0.14 mL) seeded on 96-well plates were incubated with LPS (final concentration, 100 ng/mL) in conditioned medium or in phytochemicalcontaining control medium for further 24 h. Supernatants were mixed with Griess reagent 27) in 96-well plates, and
Activation of Bifidobacteria by Photochemicals
absorbance at 543 nm was measured using SpectraMax M2 (Molecular Devices, Sunnyvale, CA, USA). The concentration of NO was calculated from the standard curve prepared using a sodium nitrate solution. Cell viability was measured by crystal violet staining; none of the tested phytochemicals or conditioned media was cytotoxic to RAW264 cells in any experiment.
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High-performance liquid chromatography (HPLC) analysis of phytochemicals. The content of phytochemicals in the conditioned medium was assessed by HPLC using a photodiode array (PDA) and an Ascentis RP-amide column (100 × 3 mm, 3 μm, SUPELCO, Bellefonte, PA, USA) at 30 °C. The mobile phases for eluting aglycones [quercetin, phloretin, EGCG, and (+)-taxifolin] and quercetin glucoside and conjugates were 80% and 40% acetonitrile, respectively, each containing 0.1% formic acid. Phloretin, EGCG, and (+)-taxifolin were detected at 280 nm, and quercetin aglycone, the glucosides, and conjugates at 254 nm. Measurement of pH and organic acids. The pH of the supernatants was measured by a pH meter (TOA Electronics, Shizuoka, Japan). The determination of lactic acid and acetic acid was performed by the 2-nitrophenylhydrazine derivatization method.28) Organic acids in the supernatant with 2-ethylbutyric acid as an internal standard were coupled to 2-nitrophenylhydr-
3
azine using a labeling kit (YMC, Kyoto, Japan) and then analyzed by HPLC–PDA and an Ascentis C18 column (100 × 3 mm, 3 μm, SUPELCO) at 50 °C. The mobile phase was acetonitrile–methanol–water (23:12:65, v/v, pH 4.5) solution. The derivatives were detected at 400 nm. Statistical analysis. All data are expressed as the mean ± SD from at least three independent experiments. Comparisons between groups were analyzed by either the Tukey–Kramer test or the Student’s t-test. Statistical analysis was performed by SPSS 16.0 J (SPSS Inc., Chicago, IL, USA). Significance was reached at values of p < 0.05.
Results Quercetin dose-dependently enhanced the NO suppressive activity of B. adolescentis. We previously reported that flavonols, including quercetin, galangin, and fisetin, enhanced the anti-inflammatory activity of B. adolescentis.25) Although the conditioned media from B. adolescentis mono-cultures failed to suppress NO production by LPS-treated RAW264 cells, media from bacteria co-cultured with quercetin showed a dose-dependent increase in inhibitory activity Fig. 1A. The initial concentration of quercetin in both monoculture and co-culture media was similarly reduced, and largely disappeared by 1 h Fig. 1B, indicating that,
(A)
(B)
Fig. 1. Quercetin dose-dependently enhanced the NO suppressive activity of B. adolescentis. Notes: (A) B. adolescentis was incubated alone or together with DMSO (final concentration 0.05%, 0 μM quercetin) or quercetin (5–40 μM) in serum-free DMEM at 37 °C for 3 h under static and anaerobic conditions. The conditioned media were collected, and their anti-inflammatory activity was evaluated by assessing their ability to inhibit LPS-induced NO production in RAW264 cells. *p < 0.05 (Tukey–Kramer test). Data are shown as mean ± SD (n = 3). (B) Measurement of quercetin concentrations in the mono- and co-culture conditioned media by HPLC–PDA, as described in the materials and methods section. Data are shown as mean ± SD (n = 3). BA, B. adolescentis; QUE, quercetin; NO, nitric oxide.
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although quercetin enhanced the anti-inflammatory activity of the bifidobacteria, it was not converted to compounds with higher activity than quercetin, a finding similar to that reported previously.25)
Table 1.
Phloretin, (+)-taxifolin, and EGCG enhanced the NO suppressive activity of B. adolescentis . Although we have found that quercetin, galangin, and fisetin enhanced the NO suppressive activity of bifidobacteria,
Screening of phytochemicals capable of increasing the NO suppressive activity of B. adolescentis JCM1275T. NO inhibition (%)a
Conc. (μM)
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B. adolescentis Chalcones Chalcone Butein 4′-Hydroxychalcone Isoliquiritigenin Phloretin Xanthohumol 4-Hydroxyderricin Xanthoangelol Flavones Flavone Chrysin 3′,4′-Dihydroxyflavone Luteolin Diosmetin 7,3′,4′-Trihydroxyflavone Apigenin Nobiletin Tangeretin Flavonols Flavonol 3′,4′-Dihydroxyflavonol (+)-Taxifolin Flavanones Flavanone Pinocembrin Eriodictyol Hesperetin Naringenin Isoflavones Genistein Daidzein (R,S)-Equol Biochanin A Formononetin Catechins (−)-Epicatechin (−)-Epicatechin gallate (−)-Epigallocatechin EGCG Coumarins Auraptene Bergamottin Imperatorin Phenolics HPA DHPA PPA DPPA Phloroglucinol Resorcinol Others ACA Zerumbone Curcumin Resveratrol a
0h
3 h (mo)b
3 h (co)c
‒
‒
4±3
‒
40 5 10 10 40 5 5 5
29 ± 10 61 ± 6 51 ± 7 35 ± 10 44 ± 10 65 ± 8 60 ± 6 26 ± 9
3±3 26 ± 4 45 ± 5 36 ± 10 17 ± 8 47 ± 8 7± 4 4± 4
1± 2 19 ± 6 15 ± 10* 10 ± 4* 63 ± 10* 5 ± 10* 1± 3 2± 2
20 20 10 10 10 20 10 10 20
17 ± 9 28 ± 2 39 ± 7 31 ± 4 44 ± 10 32 ± 7 42 ± 8 28 ± 7 28 ± 5
12 ± 5 29 ± 7 45 ± 5 39 ± 9 40 ± 9 31 ± 5 36 ± 8 25 ± 3 28 ± 10
14 ± 2 18 ± 3 30 ± 7* 32 ± 6 3 ± 4* 21 ± 3* 34 ± .9 21 ± 3 28 ± 5
20 10 40
30 ± 3 48 ± 7 22 ± 5
4± 2 43 ± 5 19 ± 7
1± 2 51 ± 7 36 ± 7*
40 40 10 40 40
9± 20 ± 40 ± 25 ± 31 ±
5 6 7 8 9
7± 14 ± 34 ± 29 ± 24 ±
8 6 8 8 9
0± 16 ± 16 ± 11 ± 25 ±
10 40 20 20 10
33 ± 58 ± 24 ± 22 ± 19 ±
5 5 6 9 5
33 ± 58 ± 25 ± 29 ± 23 ±
9 8 8 7 7
14 ± 5* 30 ± 7* 3 ± 3* 24 ± 6 19 ± 10
40 20 40 20
3± 1 31 ± 11 13 ± 5 51 ± 7
9± 30 ± 12 ± 31 ±
8 4 5 9
2± 39 ± 3± 49 ±
10 10 40
30 ± 9 26 ± 7 61 ± 5
17 ± 9 10 ± 7 9± 3
40 40 40 40 40 40
0± 8± 2± 6± 4± 7±
1 8 3 9 4 8
0± 3± 1± 6± 0± 2±
0 2 1 7 0 3
3± 0± 3± 0± 3± 5±
4 0 5 0 5 8
56 ± 5 61 ± 10 44 ± 6 61 ± 7
40 ± 44 ± 21 ± 61 ±
9 6 5 2
22 ± 11 ± 0± 25 ±
5* 3* 1* 8*
5 5 20 40
Data are shown as mean SD (n = 3–8). Mono-culture of either phytochemical or B. adolescentis for 3 h. c Co-culture of phytochemical and B. adolescentis for 3 h. *p< 0.05 vs. mono-culture (Student’s t-test). b
0 6 4* 5* 6
4 6* 4* 9*
7± 4 5± 6 3 ± 3*
Activation of Bifidobacteria by Photochemicals
(B) 100
Mono-culture Co-culture
Relative ratio (% %)
Relative ratio (% %)
(A) 100 75 50 25 0
75 50 25
Mono-culture Co-culture
0 0
1 2 Culture (h)
3
0
Rela ative ratio (%)
(C) 100
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5
1 2 Culture (h)
3
Mono-culture Co-culture
75 50 25 0 0
1 2 Culture (h)
3
Fig. 2. Effect of B. adolescentis on the concentrations of phloretin, (+)-taxifolin, and EGCG. Notes: (A) Phloretin, (B) (+)-taxifolin, and (C) EGCG were incubated in the presence or absence of B. adolescentis for 3 h, and the concentrations of phytochemicals in the conditioned media were analyzed by HPLC–PDA, as described in the materials and methods section. Data are shown as mean ± SD (n = 3).
the effect of other phytochemicals had not been determined. We therefore tested whether 47 phytochemicals could enhance the NO suppressive activity of B. adolescentis JCM1275T. These 47 phytochemicals, including eight chalcones, nine flavones, three flavonols, five flavanones, five isoflavones, four catechins, three coumarins, six phenolics, and four others, were tested at concentrations showing ≤70% inhibition of NO production (Table 1) and having no effect on cell viability (data not shown). Many of the tested compounds had no effect or reduced the anti-inflammatory activity of the conditioned media (Table 1). In contrast, co-culture of B. adolescentis with phloretin, (+)-taxifolin, and EGCG significantly and synergistically enhanced the NO suppressive activity of the bacteria. Their concentrations in the conditioned media from mono- and cocultures were almost the same (Fig. 2). These results suggest that phloretin, (+)-taxifolin, and EGCG may have an effect on B. adolescentis similar to quercetin. In contrast, co-culture with epicatechin gallate also significantly inhibited (39%) the NO production though, this may not result from increasing the anti-inflammatory activity of B. adolescentis by epicatechin gallate but an additive action of them (the inhibition rate of mono-culture media: B. adolescentis, 4%; epicatechin gallate, 30%).
Quercetin glucosides enhanced the NO suppressive activity of B. adolescentis. Because flavonoids generally present as glycosides in plants and undergo conjugation, such as glucuronidation and methylation, in the human intestine after ingestion, we investigated whether quercetin glycosides (Q3G, Q3Gal, Q3Rha,
rutin, and Q4′G) and conjugates (Q3GA and isorhamnetin), as well as phloretin glucoside (phlorizin), similarly enhance the activity of bifidobacteria. At concentrations 100 μM, glycosides and conjugates in mono-culture suppressed NO production by around 20–50%, respectively, after 3 h (Table 2). Intriguingly, co-culture of B. adolescentis JCM1275T with Q3G and Q4′G markedly enhances the NO suppressive activity of the bacteria, with actual inhibition ratios of 33 and 60%, respectively, being greater than the estimated ratio (21 and 25%, respectively). Q3GA also increased the activity in the conditioned medium from the co-culture, but this may be an additive effect (actual, 69%; estimated, 61%). Co-culture of B. adolescentis with Table 2. Effect of glycosides and conjugates on the anti-inflammatory activity of B. adolescentis JCM1275T. NO inhibition (%)a
B. adolescentis Glycosides Q3G Q3Gal Q3Rha Rutin Q4′G Phlorizin Conjugates Q3GA Isorhamnetin a
Conc. (μM)
0h
3 h (mo)b
3 h (co)c
‒
‒
4± 3
‒
100 100 100 100 100 100
21 ± 19 ± 15 ± 20 ± 18 ± 11 ±
6 8 9 7 5 1
100 100
56 ± 9 53 ± 10
17 ± 18 ± 20 ± 20 ± 21 ± 15 ±
8 6 9 8 9 3
33 ± 8* 14 ± 8 13 ± 7 15 ± 6 60 ± 10* 15 ± 3
57 ± 8 55 ± 8
69 ± 8* 20 ± 5*
Data are shown as mean SD (n = 4–10). Mono-culture of either phytochemical or B. adolescentis for 3 h. c Co-culture of phytochemical and B. adolescentis for 3 h. *p< 0.05 versus mono-culture (Student’s t-test). b
(A)
125 100 75 50 G3G Q4'G G3GA
25 0
125 100 75 50 G3G + BA Q4'G + BA G3GA + BA
25 0
0
1 2 Culture (h)
Quercettin aglycone (µ µM)
(C)
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(B) Relative ratio (% %)
K. Kawabata et al.
Relative ratio (% %)
6
3
0
1 2 Culture (h)
3
10 G3G + BA Q4'G + BA G3GA + BA
7.5 5 2.5 0 0
1 2 Culture (h)
3
Fig. 3. Quercetin glucosides, but not glucuronide, were converted to quercetin aglycone during co-culture with B. adolescentis. Notes: Quercetin glucosides and glucuronide (100 μM) were incubated in the presence or absence of B. adolescentis for 3 h, and their concentrations in the conditioned media were analyzed by HPLC–PDA, as described in the materials and methods section. (A) Glucosides and glucuronide in mono-culture media; (B) glucosides and glucuronide in co-culture media; (C) quercetin aglycone in the co-culture media. Data are shown as mean ± SD (n = 3). BA, B. adolescentis; Q3G, quercetin-3-glucoside; Q4′G, quercetin-4′-glucoside; Q3GA, quercein-3-glucuronide.
(A) other glycosides and conjugates, especially isorhamnetin, reduced the activity when compared with monoculture media. Most of the Q3G, Q4′G, and Q3GA in the mono-cultures were present after 3 h (Fig. 3A), although the amounts were time-dependently reduced during co-culture of Q3G and Q4′G, but not Q3GA, with B. adolescentis. In contrast, the levels of quercetin aglycone were increased Fig. 3B and C, suggesting that the quercetin glucosides may enhance the activity of the bifidobacterium after conversion to aglycones.
(B)
Fig. 4. Effects of phytochemicals on the pH lowering and the organic acids concentration in co-culture supernatants. Notes: B. adolescentis were incubated with DMSO, quercetin (20 μM), (+)-taxifolin (40 μM), phloretin (40 μM), and EGCG (20 μM) for 3 h, and then the change of pH and the concentrations of lactic and acetic acids in the conditioned media were analyzed by a pH meter and HPLC–PDA, respectively, as described in the Materials and Methods section. (A) The pH level, *p < 0.05 (Dunnett test); (B) the concentrations of the organic acids, *p < 0.05 vs. the DMSO group (Dunnett test). Data are shown as mean ± SD (n = 3). QUE, quercetin; TAX, (+)-taxifolin; PHL, phloretin.
Phytochemicals, except for quercetin, increased the concentrations of lactic acid and acetic acid in the coculture medium Since bifidobacterium bacteria is the major producer of lactic acid and acetic acid which were shown to have an anti-inflammatory activity, such as the inhibition of pro-inflammatory cytokine production,22,29) we next investigated the change of pH and organic acids levels in the co-culture medium. After the culture of B. adolescentis with DMSO or phytochemicals [quercetin, (+)-taxifolin, phloretin, and EGCG] for 3 h, the pH of all of the tested supernatants decreased from 7.8 (just before culture) to 7.2 Fig. 4A. On the other hand, phytochemicals excluding quercetin markedly increased the concentrations of either or both lactic and acetic acids in the supernatants as compared to that of the bifidobacteria mono-culture Fig. 4B, suggesting that both the change of pH and the increase of lactic and acetic acids may not correlate with the NO suppressive activity of the co-culture supernatant.
Activation of Bifidobacteria by Photochemicals
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Discussion Intestinal microbiota is very important for the maintenance of human health. Imbalances in their populations have been suggested as a cause of several diseases including obesity, diabetes mellitus, inflammatory bowel diseases, neurodevelopmental disorders, and cancer.7−11) The health-promoting effects of probiotic bifidobacteria and lactic acid bacteria have been associated with vitamin production, carbohydrate fermentation, intestinal epithelial cell protection, immunomodulation, and antipathogen activity.16,30–32) Probiotic anaerobes have also been found to secrete anti-inflammatory agents.25,33,34) Furthermore, our previous study showed that the flavonols quercetin, galangin, and fisetin enhance the production of NO suppressants by B. adolescentis.25) However, whether these effects are distinct to flavonols and evoked by a characteristic structure of these compounds have not yet been clarified. This study therefore investigated the effects of other types of phytochemicals on B. adolescentis, as well as assessing the structureactivity relationships. Of the 55 tested phytochemicals, including 47 aglycones and eight glycosides and conjugates, five compounds, phloretin, (+)-taxifolin, EGCG, Q3G, and Q4′G, enhanced the anti-inflammatory activity of B. adolescentis. The initial levels of phloretin, (+)-taxifolin, and EGCG showed a similar reduction in both mono-culture and co-culture media over 3 h, suggesting that these phytochemicals may, like quercetin, promote the secretion of anti-inflammatory substances from B. adolescentis. Compared with quercetin, (+)-taxifolin lacks the C2–C3 double bond and fisetin lacks the C5 hydroxy group.25) All of the tested phytochemicals containing a catechol structure, including butein, 3′,4′-dihydroxyflavonol, 3′,4′-dihydroxyflavone, 7,3′,4′-trihydroxyflavone, luteolin, eriodictyol, epicatechin, epicatechin gallate, DHPA, and DPPA, were inactive. Compared with fisetin and (+)-taxifolin, 7,3′,4′trihydroxyflavone, eriodictyol, and Q3GA lack the hydroxy group at the C-3 position and are glucuronized. These findings suggest that both the catechol B-ring and the C3 hydroxy group may be the important structural moieties that interact with the bifidobacteria. However, phloretin and EGCG lack these moieties, suggesting that other structures in these compounds may be responsible for their activities. B. adolescentis possesses the genes that encode β-galactosidase35) and β-glucosidase,14) but not those that encode β-glucuronidase and α-L-rhamnosidase. B. adolescentis JCM1275T converted quercetin glucosides to the aglycone forms in a time-dependent manner, but did not convert Q3GA into its aglycone. The level of quercetin aglycone in the co-culture medium was positively correlated with the increase in NO suppressive activity. Because the addition of glucose during co-culture of B. adolescentis and quercetin failed to further increase the anti-inflammatory activity (data not shown), glucose molecules generated by the hydrolysis of quercetin glucoside would be inactive. Intriguingly, Q4′G was more sensitive to bioconversion by B. adolescentis JCM1275T than Q3G, suggesting that the β-glucosidase of this bacterium may prefer Q4′ G as a substrate. This substrate specificity agrees with
7
experiments performed using enzymes from rat intestinal tissue.36) Although a large fraction of quercetin glucosides is absorbed by the small intestine,37,38) the glucosides that pass through the small intestine, and Q3GA excreted into the large intestine, may be converted to their aglycones by the β-glucosidase of bifidobacteria and the β-glucuronidase of intestinal microbiota, such as Clostridia and Eubacteria,39) with the aglycones enhancing the activity of B. adolescentis. Alternatively, these compounds may be further converted to inactive phenolics, including phenylacetic and phenylpropionic acids (Table 1). Intestinal bifidobacteria metabolize flavonoids to small compounds, such as phenolics and lactones.12) In our experimental model, some of the tested compounds showed lower anti-inflammatory activity in the co-culture than in mono-culture conditioned media, indicating that these compounds, especially highly hydrophobic compounds, had been degraded by B. adolescentis JCM1275T. Of these highly hydrophobic compounds, xanthohumol, auraptene, bergamottin, and imperatorin have a prenyl group; the sesquiterpene zerumbone lacks an hydroxy group; and butein, 4′-hydroxychalcone, isoliquiritigenin, genistein, equol, curcumin, and resveratrol are also lipophilic (log p > 3).40−42) Daidzein (log p = 2.51) 42) may be degraded but not converted into S-equol.14) In contrast, diosmetin, hesperetin, and isorhamnetin, each of which contains a methoxy group, seem to be more sensitive to bacteria than the polymethoxyflavonoids nobiletin and tangeretin. In addition, flavones, flavonols, and flavanones may have comparatively low sensitivity to B. adolescentis JCM1275T. It is noteworthy that co-culture of B. adolescentis with phloretin, (+)-taxifolin, and EGCG resulted in the increase in lactic acid and acetic acid Fig. 4B, indicating that these phytochemicals may promote the carbohydrate metabolism associated with the production of lactic and acetic acids. Since these organic acids are utilized by butyric acid-producing anaerobes,18) the up-regulation of lactic and acetic acids production by the phytochemicals may lead to the increase of butyric acid production. Moreover, both lactic acid and acetic acid were found to inhibit the production of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-8, from LPS-stimulated neutrophils and colon tumor cells.22,29) However, quercetin had no such effect. Therefore, antiinflammatory substance(s) produced by B. adolescentis during co-culture with the phytochemicals may be neither lactic acid nor acetic acid. In conclusion, our present study found that phloretin, (+)-taxifolin, and EGCG were novel enhancers of the anti-inflammatory activity of B. adolescentis JCM1275T. This implies that fruits and tea containing abundant amounts of quercetin, phloretin, and EGCG can potentiate the anti-inflammatory activity of B. adolescentis in the intestines, and therefore, it is necessary to assess impacts of B. adolescentis administration in combination with these phytochemicals on an animal model of the inflammatory bowel diseases. Furthermore, the mechanisms by which these compounds induce the production/secretion of active substance(s) from B. adolescentis will be the subjects of further investigation.
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Funding This study was supported by a Grant-in-Aid for Young Scientists [22780120] from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K. K.).
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Activation of Bifidobacteria by Photochemicals
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