Hesperetin Modifies the Composition of Fecal Microbiota and

Article pubs.acs.org/JAFC

Hesperetin Modifies the Composition of Fecal Microbiota and Increases Cecal Levels of Short-Chain Fatty Acids in Rats Tomonori Unno,*,† Takayoshi Hisada,‡ and Shunsuke Takahashi‡ †

Department of Health and Nutrition, Tokyo Kasei Gakuin University, 22 Sanban-cho, Chiyoda-ku, Tokyo 102-8341, Japan TechnoSuruga Laboratory Company, Ltd., 330 Nagasaki, Shimizu-ku, Shizuoka 424-0065, Japan

Downloaded by UNIV OF NEBRASKA-LINCOLN on September 8, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jafc.5b02649

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ABSTRACT: There has been particular interest in the prebiotic-like effects of commonly consumed polyphenols. This study aimed to evaluate the effects of hesperidin (HD) and its aglycone hesperetin (HT), major flavonoids in citrus fruits, on the structure and activity of gut microbiota in rats. Rats ingested an assigned diet (a control diet, a 0.5% HT diet, or a 1.0% HD diet) for 3 weeks. Terminal restriction fragment length polymorphism analysis revealed that the proportion of Clostridium subcluster XIVa in the feces collected at the third week of feeding was significantly reduced by the HT diet: 19.8 ± 4.3% for the control diet versus 5.3 ± 1.5% for the HT diet (P < 0.01). There was a significant difference in the cecal pool of short-chain fatty acids (SCFA), the sum of acetic, propionic, and butyric acids, between the control diet (212 ± 71 μmol) and the HT diet (310 ± 51 μmol) (P < 0.05), whereas the HD diet exhibited no effects (245 ± 51 μmol). Interestingly, dietary HT resulted in a significant increase in the excretion of starch in the feces. HT, but not HD, might reduce starch digestion, and parts of undigested starch were utilized to produce SCFA by microbial fermentation in the large intestine. KEYWORDS: hesperetin, hesperidin, gut microbiota, short-chain fatty acids, Clostridium subcluster XIVa, starch digestion

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bacteria.11,12 According to an in vitro study by Duda-Chodak,13 HT inhibited the growth of some types of intestinal bacteria in a concentration-dependent manner, but HD could not accomplish such a function; this implies that the influence on intestinal bacteria varies depending on the presence of a rutinoside moiety. However, the comparison of HT and HD on the structure and activity of the gut microbial community in vivo has yet to be realized. The aim was to investigate whether supplementation of these flavonoids in the diet would affect the gut microbiota and colonic fermentation in rats. For this purpose, the microbial diversity in the feces and the amounts of SCFA in the cecum content were analyzed.

INTRODUCTION Flavonoids represent a diverse range of polyphenols that are part of a daily diet. Commonly consumed fruits, vegetables, red wine, and teas contain significant amounts of flavonoids; they are mostly found as glycoside derivatives.1 The mean daily total flavonoid intake for U.S. adults was estimated at 189.7 mg, of which flavan-3-ols account for 83.5%, followed by flavanones (7.6%), flavonols (6.8%), anthocyanidins (1.6%), flavones (0.8%), and isoflavones (0.6%).2 Hesperidin (HD) is a flavanone rutinoside contained in citrus fruits and is abundantly distributed in orange juice and its peel byproduct.3 Hesperetin (HT) is an aglycone form of HD (Figure 1). The absorption and metabolism of HD comprise an active area of research.4,5 After ingestion, a considerable amount of HD reaches the large intestine intact, where colonic bacteria cleave the attached rutinose moiety. The released HT is further broken down into phenolic acids by gut bacteria.6 The large intestine shows the greatest density of bacterial population, with up to 1012 bacteria per gram of gut content. Intestinal bacteria play important roles in metabolic, nutritional, physiological, and immunological processes in the human body.7 Recently, the relationship between gut microbiota and the development of obesity, type 2 diabetes, and cardiovascular diseases has become clearer.8,9 A balanced composition of gut microbiota offers health benefits to the host, whereas microbial imbalances may be associated with the development of metabolic disorders. Certain types of anaerobic intestinal bacteria produce short-chain fatty acids (SCFA), the fermentation end products originated from complex carbohydrates, functioning as regulators of energy intake and energy metabolism.10 There has been a particular interest in the prebiotic-like effects of commonly consumed polyphenols to modulate the microbial balance through the growth stimulation for beneficial bacteria and/or the inhibition for pathogen © XXXX American Chemical Society

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MATERIALS AND METHODS

Chemicals. HT and HD, with purities of >96 and >92%, respectively, were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Thermostable α-amylase and amyloglucosidase were utilized from a commercially available dietary fiber assay kit (Wako Pure Chemical Industries, Ltd.). Soluble starch and porcine pancreatic extract were also from Wako Pure Chemical Industries, Ltd. Dimethyl sulfoxide, methanol, and HPLC grade acetonitrile were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). Animals and Diets. Four-week-old male Wistar rats (n = 21) were purchased from Tokyo Laboratory Animals Science Co., Ltd. (Tokyo, Japan), and were housed individually in stainless steel cages at 22 °C in a room with an automatically controlled 12 h lighting cycle. The rats were fed a commercial chow (type MF, Oriental Yeast Co., Ltd., Tokyo, Japan) for 1 week before being fed the experimental diets. The rats were divided into three groups (n = 7/group): a control diet, a 0.5% HT diet, and a 1.0% HD diet, respectively (Table 1). The experimental diets contained HT or HD at nearly equimolar amounts Received: May 28, 2015 Revised: August 5, 2015 Accepted: August 25, 2015

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DOI: 10.1021/acs.jafc.5b02649 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of (A) hesperetin and (B) hesperidin (hesperetin-7-O-rutinoside).


Table 1. Percentage Composition of Experimental Dietsa casein DL-methionine corn starch sucrose corn oil cellulose powder mineral mix vitamin mix choline bitartrate hesperetin hesperidin

control

0.5% hesperetin

1.0% hesperidin

20 0.3 55 10 5.0 5.0 3.5 1.0 0.2 − −

20 0.3 55 10 5.0 4.5 3.5 1.0 0.2 0.5 −

20 0.3 55 10 5.0 4.0 3.5 1.0 0.2 − 1.0

assigned to categories of operational taxonomic units (OTU). The OTU data were used to identify the phylotypes by matching to those predicted from various phylotypes in the literature.16 Analyses of SCFA in Cecum Content. The SCFA (acetic, propionic, and butyric acids) in the homogenate of cecum content were derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride and were measured by a high-performance liquid chromatography (HPLC) system.17 The derivatives were separated using a reversed-phase column (type FA, 6.0 mm × 250 mm, YMC Co., Ltd., Kyoto, Japan) with an acetonitrile/methanol/water (30:16:54, v/v/v) mobile phase at the flow rate of 1.2 mL/min and were detected with a variable-wavelength monitor with the absorbance set at 400 nm. Analyses of Starch and Proteins in Feces. The concentrations of undigested starch in the feces were determined after hydrolysis with thermostable α-amylase and amyloglucosidase, followed by a determination of the released glucose using an enzymatic colorimetric assay kit (Glucose CII-test, Wako Pure Chemical Industries, Ltd.).18 Results were corrected to a starch basis by multiplying them by 0.9. Total fecal nitrogen was determined by Kjeldahl’s method,19 and results were multiplied by 6.25 to calculate the amount of protein in the feces. Analyses of HP and HD in Feces. The flavonoids were extracted with the mixture of dimethyl sulfoxide and methanol (1:1, v/v).3 One hundred milligrams of the ground fecal samples was sonicated three times with 3 mL of the extraction solvent for 1 min (Branson Ultrasonics, Ltd., Danbury, CT, USA). After centrifugation at 1000g for 10 min, the supernatants were combined, and the volume of combined extracts was adjusted to 10 mL. After a 10-fold dilution, the resulting extract was filtered with a 0.45 μm membrane filter and was injected into the HPLC system. The flavonoids were separated using a reversed-phase column (type Unison UK C18, 4.6 mm × 150 mm, Imtakt Co., Kyoto, Japan) by a two-solvent linear gradient program from initially acetonitrile/water/phosphoric acid (10:90:0.05, v/v/v) to finally acetonitrile/water (80:20, v/v) in 20 min at the flow rate of 0.8 mL/min. The flavonoids were detected at UV 285 nm. In Vitro Pancreatic α-Amylase Inhibition. Porcine pancreatic extract was suspended in 20 mM phosphate buffer (pH 6.9) containing 6 mM sodium chloride. The reaction mixture was composed of 0.1 mL of the various concentrations of test samples, 0.7 mL of 10 mg/mL soluble starch, and 0.1 mL of 0.1 mg/mL porcine pancreatic extract and was incubated at 37 °C for 30 min. The reaction was terminated by adding 1.0 mL of acetonitrile. After centrifugation at 2000g for 10 min, the supernatant was filtered with a 0.45 μm membrane filter. The liberated maltose levels in the filtrate were measured by the HPLC system with a refractive index detector. Separation was carried out by using an amino-bonded silica column (type Unison UK-amino, 4.6 mm × 150 mm, Imtakt Co.) with an acetonitrile/water (80:20, v/v) mobile phase. Statistical Analysis. All data are shown as the mean ± standard deviation (SD). The bacterial 16S rRNA gene copy numbers were converted into logarithm values before the statistical analysis. Significant differences among the groups were determined by oneway ANOVA, followed by Tukey’s test, using a commercial software package (GraphPad Prism 5 for Windows, GraphPad Software, Inc., San Diego, CA, USA). Differences were considered significant at P < 0.05.

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Source of ingredients: casein, corn starch, sucrose, cellulose powder, mineral AIN-76 mix, and vitamin AIN-76 mix (Oriental Yeast Co., Ltd., Tokyo, Japan); DL-methionine and choline bitartrate (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

(16.4 mmol/kg diet). The rats had free access to tap water and the experimental diets for 3 weeks. Food intake and body weight were determined three times weekly. All feces were collected during the third week of the feeding period and stored at −40 °C until further processing. At the end of the experiment, rats were sacrificed by drawing blood from the heart under diethyl ether anesthesia. Cecum and abdominal adipose tissues were immediately excised and weighed. The cecum content was stored at −40 °C until use. This study was approved by the Animal Research Committee of Tokyo Kasei Gakuin University, and all experimental procedures followed the guidelines for the care and use of experimental animals. Determination of Fecal Bacterial 16S rRNA Gene Copy Number. Before analysis, all fecal samples collected for a week were freeze-dried and ground by a grinder mill (type TML161, Tescom Co., Ltd., Tokyo, Japan). DNA was extracted according to the method of Tanigawa et al.14 The bacterial 16S copy number was calculated from the standard curve of known bacterial 16S copy number by quantitative real-time PCR of 16S rRNA gene using 341f (5′CCTACGGGAGGCAGCAG-3′) and 534r (5′-ATTACCGCGGCTGCTGG-3′) primers.15 Amplification reactions were performed by a Rotor-Gene Q apparatus (Qiagen, Germany) using the SYBR Premix Ex TaqII (Tli RNaseH Plus, Takara Bio, Shiga, Japan). Fecal Microbial Composition Analysis. Fecal samples of rats were analyzed by targeting the bacterial 16S rRNA genes using a terminal restriction fragment length polymorphism (T-RFLP) technique according to the procedure described by Nagashima et al.16 The fluorescently labeled primers 516f (5′-TGCCAGCAGCCGCGGTA-3′) and 1510r (5′-GGTTACCTTGTTACGACTT-3′) were used to amplify the 16S rRNA genes. The purified PCR products were digested with the restriction enzyme BslI (New England BioLabs Japan, Inc., Tokyo, Japan). The length of the terminal restriction fragments was determined using an automated sequence analyzer (ABI PRISM 3130xl DNA Sequencer, Applied Biosystems, Carlsbad, CA, USA). The abundance of each terminal restriction fragment was determined on the basis of fluorescence intensity using the software GeneMapper ver. 4 (Applied Biosystems). Fragment sizes were B


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RESULTS Body Weight Gain and Organ Weights. There were no differences among the groups in food intake or body weight gain during the 3 week experiment (Table 2). Averaged daily

Table 4. Effect of Dietary Hesperetin and Hesperidin on the Profile of Microbiota in Feces of Ratsa control


food intake (g) initial body wt (g) final body wt (g) body wt gain (g) liver wt (g) abdominal adipose tissueb (g) mesenteric perirenal epididymal cecum content wt (g)

0.5% hesperetin

1.0% hesperidin

471 ± 3 136 ± 8 291 ± 11 155 ± 7 13.6 ± 3.5 12.8 ± 2.1 b

459 ± 14 136 ± 8 291 ± 7 155 ± 3 12.5 ± 1.4 10.4 ± 1.6 a

464 ± 11 137 ± 8 289 ± 11 152 ± 11 11.4 ± 0.7 12.1 ± 1.5 ab

3.5 4.2 5.1 2.2

± ± ± ±

0.7 0.9 1.1 b 0.6 a

3.1 3.5 3.8 3.4

± ± ± ±

0.2 1.0 0.5 a 0.9 b

3.6 4.2 4.4 2.7

± ± ± ±

0.4 0.7 0.7 ab 0.5 ab

Values are the mean ± SD (n = 7). Treatments with different letters are significantly different (P < 0.05). bSum of the mass of mesenteric, perirenal, and epididymal adipose tissues. a

Values are the mean ± SD (n = 5). Treatments with different letters are significantly different (P < 0.05). bValues are expressed as percentages of the peak area of a particular OTU to the total peak area of all OTUs.

a

intakes of HT and HD were 0.40 g (1.34 mmol) and 0.83 g (1.36 mmol) per kilogram of body weight. The rats fed the HT diet had a lower weight of abdominal adipose tissues (the mass of mesenteric, perirenal, and epididymal adipose tissues) compared with those fed the control diet. The HT diet led to a significant increase in the weight of cecum content, whereas the HD group showed no differences with the control group. Fecal Microbiota. The dry weight of feces collected during the third week of the feeding period did not show statistical significances among the groups (Table 3). Changes in the

their diet presented a remarkable alteration in the proportion of Clostridium. In particular, feeding the HT diet resulted in a significant decrease in the ratio of Clostridium subcluster XIVa in the fecal microbiota compared to other diets. In contrast, the feces of rats fed the control diet displayed very low proportions of Clostridium clusters IV and XVIII, but the HT diet significantly increased their ratios. Treatment with HT in the diet slightly affected the abundance of Bif idobacterium and Lactobacillales, although these results did not reach significance. Amount of Starch and Proteins in Feces. Little starch was excreted in the feces of rats fed the control diet, whereas the feces of rats fed the HT diet contained a significant amount of starch (Table 3). However, the HD diet had little impact on the fecal starch level. Both treatments with HT and HD did not lead to significant differences in protein excretions in the feces as compared with the control. Cecal SCFA Pools. Figure 2 summarizes the cecal SCFA pools in rats fed the different experimental diets. Feeding the HT diet resulted in significant increases in the cecal pools of acetic and butyric acids as compared to the control, but treatment with the HD diet did not yield significantly different results compared with the control. The additions of these flavonoids to the diet had little influence on the cecal pool of propionic acid. Fecal Excretions of HT and HD. Parts of dietary HT and HD were excreted in the feces in unchanged forms. Feces collected during the third week of feeding contained 0.9 mmol of HT in the 0.5% HT diet group and 0.4 mmol of HD in the 1.0% HD diet group (Table 3). Because the rats consumed 2.7 mmol of HT or HD from their respective diet, 33 and 15% were estimated to be excreted in the feces in the unchanged form, respectively. In addition, the feces collected from the rat fed the 1.0% HD diet also contained 1.1 mmol of HT. Inhibition of Pancreatic α-Amylase in Vitro. HT inhibited the enzyme activity of porcine pancreatic α-amylase in a concentration-dependent manner (Figure 3), but HD lacked the inhibitory activity for α-amylase.

Table 3. Fecal Excretions of Starch, Protein, Hesperetin, and Hesperidin in Rats Fed the Experimental Dietsa

dry wtb (g/7 days) fecal nutrient starch (mg/7 days) protein (g/7 days) fecal flavonoid hesperetin (mmol/7 days) hesperidin (mmol/7 days)

control

0.5% hesperetin

1.0% hesperidin

14.4 ± 0.5

14.3 ± 1.1

13.5 ± 1.5

65 ± 2 a 1.4 ± 0.2

91 ± 36 b 1.6 ± 0.2

61 ± 5 a 1.5 ± 0.1

−

0.9 ± 0.3

1.1 ± 0.6

−

−

0.4 ± 0.1

1.0% hesperidin

16S rRNA Gene Copy Number (log10 Copies) all bacteria (per 12.0 ± 0.1 12.2 ± 0.1 12.1 ± 0.1 g dry feces) all bacteria (per 13.2 ± 0.1 13.4 ± 0.1 13.3 ± 0.1 7-day dry feces) Ratio of Peak Area of Each OTUb Bifidobacterium 3.1 ± 3.6 7.1 ± 8.5 3.0 ± 3.5 Lactobacillales 25.0 ± 10.8 29.8 ± 11.3 31.1 ± 13.5 Bacteroides 7.9 ± 3.6 10.7 ± 4.3 11.1 ± 2.9 Prevotella 9.2 ± 7.0 5.0 ± 3.1 3.0 ± 3.2 Clostridium 32.7 ± 3.8 b 20.3 ± 8.7 a 31.5 ± 3.7 b Clostridium cluster IV 0.1 ± 0.3 a 1.2 ± 0.9 b 0.1 ± 0.1 a Clostridium subcluster 19.8 ± 4.3 b 5.3 ± 1.5 a 15.4 ± 5.8 b XIVa Clostridium cluster XI 10.4 ± 1.9 9.1 ± 6.5 14.0 ± 7.1 Clostridium cluster 2.3 ± 0.7 a 4.7 ± 1.9 b 2.1 ± 0.9 a XVIII other 22.1 ± 10.1 25.9 ± 7.2 20.4 ± 12.1

Table 2. Food Intake, Body Weight Gain, and Organ Weights in Rats Fed the Experimental Diet for 3 Weeksa control

0.5% hesperetin

Values are the mean ± SD (n = 7). Treatments with different letters are significantly different (P < 0.05). bCollected during the third week of experimental diet feeding period. a

bacterial 16S rRNA gene copy number and composition in the feces of five randomly selected rats of each group are shown in Table 4. There was no significant difference in the copy numbers of 16S rRNA gene of all bacteria among the groups. After 3 weeks of feeding, the taxonomic composition of the fecal microbiota significantly differed among the groups. In the control rats, the T-RFLP analysis revealed that the order of Clostridium was the most predominant group, followed by Lactobacillales. The fecal microbiota of rats that received HT in C


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of in vitro experiments offers supporting evidence that HT had an inhibitory effect against pancreatic α-amylase in a concentration-dependent fashion. Parts of the starch undigested in the small intestine can then be microbially hydrolyzed to monosaccharides in the cecum (rodents) and colon (humans), where they are further degraded into biologically active metabolites represented by SCFA.22 It is commonly understood that the growth of pathogenic microorganisms could be inhibited by reducing luminal pH with more SCFA productions, eventually increasing the growth and/or activity of beneficial bacteria.23 Feeding the HT diet raised the proportion of Bif idobacterium and the Lactobacillales to a lesser extent, but there were no significant changes. In addition, it is worth noting that HT served to modulate the abundance of Clostridium spp. Particularly, the present study demonstrated that the proportion of Clostridium subcluster XIVa in the feces was significantly decreased by the HT diet as compared to the control diet. Similar results were observed in several previous studies in relation to polyphenols from red wine,24 grape seed extracts,25 and green tea.26 Likewise, feeding a diet abundant in resistant starch can reduce the proportion of Clostridium cluster XIVa in the luminal contents of pig colon.27 Resistant starch is a type of dietary fiber that is highly fermentable by colonic bacteria, resulting in the production of SCFA; an acidified condition for the lumen of the large intestine may be involved in reducing the proportion of Clostridium subcluster XIVa. In the light of increased levels of SCFA in rat cecum content by feeding the HT diet, it seems reasonable to suppose that dietary HT had some degree of influence in modulating the proportion of Clostridium subcluster XIVa through the stimulation of SCFA production. The close associations among the energy harvest from foods, the development of obesity, and the microbial population inhabiting the intestinal tract are becoming clearer. Obese mice had an increased abundance of bacteria from the Firmicutes phylum,28 the main constituents of which were Clostridium cluster IV (mainly Clostridium leptum group) and Clostridium cluster XIVa (mainly Clostridium coccoides group).29 It is known that Clostridium cluster XIVa was substantially increased by high-fat diet feeding, but was reduced by antibiotic treatment.30 There is evidence that an obesity treatment program comprising a calorie-restricted diet and physical activity over 3 months had an impact on the decreased abundance of the Clostridium cluster XIVa on the structure of the fecal microbiota of overweight and obese subjects.31 Furthermore, vegetarians had a decreased relative abundance of Clostridium cluster XIVa bacteria compared with omnivores.32 In this light, alteration of microbial ecology by HT may be of relevance to a significant reduction of abdominal adipose tissue weights of rats. In addition, it is interesting to note that SCFA themselves regulate the balance among fatty acid synthesis, fatty acid oxidation, and lipolysis in the body.10 SCFA formed by colonic bacterial fermentation enter the blood circulation from the intestine and are taken up by tissues, where they function as signal molecules. GPR41 and GPR43 have been identified as SCFA receptors. SCFA activate the sympathetic nervous system by stimulating GPR41 in sympathetic ganglia, leading to an increase in energy expenditure.33 SCFA-mediated activation of GPR43 suppresses insulin signaling in adipocytes and thereby inhibits fat accumulation in adipose tissues.34 Regulation of the energy metabolism by SCFA may also contribute in a practical way to the loss of body fat in rats fed the HT diet. The present study

Figure 2. Effects of dietary supplementations of hesperetin and hesperidin on the cecal SCFA pool. The cecal SCFA pool (μmol) = SCFA concentration (μmol/g) × cecum content (g). Values are the mean ± SD (n = 7). Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. Treatments with different letters are significantly different (P < 0.05).

Figure 3. Dose-dependent inhibition of hesperetin against pancreatic α-amylase activity. Data are the mean ± SD from triplicate measurements. Open circles and solid circles indicate hesperidin and hesperetin, respectively.

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DISCUSSION It is well recognized that the structure and activity of microorganisms inhabiting our gut are influenced by dietary intake. Such diet-induced changes in gut microbial communities could be responsible for the development of metabolic disorders. It is of current interest that dietary polyphenols raise a possibility for modulating the composition of intestinal microorganisms and the formation of fermentation end products, which may be involved in their underlying mechanisms for beneficial health effects. Citrus flavonoids exhibit a range of biological activities and beneficial health effects.20 In a type 2 diabetes animal model, dietary supplementation of 1% (w/w) HD resulted in decreases in blood glucose and serum triacylglycerols.21 This study aimed to evaluate the impact of dietary supplementation with two types of citrus flavonoids on the structure and activity of gut microbiota in rats, in relative relation to the presence of sugar moiety to the flavonoid skeleton. The rats consumed the respective experimental diets that were supplemented with equimolar concentrations of HT and HD. Compared to the control diet, feeding the 0.5% HT diet resulted in a significant increase in starch excretion in the feces. This finding let us understand that dietary HT could interfere with starch digestion in the small intestine, leading to the influx of undigested starch into the large intestine. Indeed, the result D


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(2) Chun, O. K.; Chung, S. J.; Song, W. O. Estimated dietary flavonoid intake and major food sources of U.S. adults. J. Nutr. 2007, 137, 1244−1252. (3) Manthey, J. A.; Grohmann, K. Concentrations of hesperidin and other orange peel flavonoids in citrus processing byproducts. J. Agric. Food Chem. 1996, 44, 811−814. (4) Pereira-Caro, G.; Borges, G.; van der Hooft, J.; Clifford, M. N.; Del Rio, D.; Lean, M. E. J.; Roberts, S. A.; Kellerhals, M. B.; Crozier, A. Orange juice (poly)phenols are highly bioavailable in humans. Am. J. Clin. Nutr. 2014, 100, 1378−1384. (5) Schär, M. Y.; Curtis, P. J.; Hazim, S.; Ostertag, L. M.; Kay, C. D.; Potter, J. F.; Cassidy, A. Orange juice-derived flavanone and phenolic metabolites do not acutely affect cardiovascular risk biomarkers: a randomized, placebo-controlled, crossover trial in men at moderate risk of cardiovascular disease. Am. J. Clin. Nutr. 2015, 101, 931−938. (6) Garg, A.; Garg, S.; Zaneveld, L. J. D.; Singla, A. K. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother. Res. 2001, 15, 655−669. (7) Nicholson, J. K.; Holmes, E.; Kinross, J.; Burcelin, R.; Gibson, G.; Jia, W.; Pettersson, S. Host-gut microbiota metabolic interactions. Science 2012, 336, 1262−1267. (8) Tremaroli, V.; Bäckhed, F. Functional interactions between the gut microbiota and host metabolism. Nature 2012, 489, 242−249. (9) Tagliabue, A.; Elli, M. The role of gut microbiota in human obesity: recent findings and future perspectives. Nutr., Metab. Cardiovasc. Dis. 2013, 23, 160−168. (10) den Besten, G.; van Eunen, K.; Groen, A. K.; Venema, K.; Reijngoud, D. J.; Bakker, B. M. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. J. Lipid Res. 2013, 54, 2325−2340. (11) Etxeberria, U.; Fernández-Quintela, A.; Milagro, F. I.; Aguirre, L.; Martínez, J. A.; Portillo, M. P. Impact of polyphenols and polyphenol-rich dietary sources on gut microbiota composition. J. Agric. Food Chem. 2013, 61, 9517−9533. (12) Cardona, F.; Andrés-Lacueva, C.; Tulipani, S.; Tinahones, F. J.; Queipo-Ortuño, M. I. Benefits of polyphenols on gut microbiota and implications in human health. J. Nutr. Biochem. 2013, 24, 1415−1422. (13) Duda-Chodak, A. The inhibitory effect of polyphenols on human gut microbiota. J. Physiol. Pharmacol. 2012, 63, 497−503. (14) Tanigawa, T.; Watanabe, T.; Otani, K.; Nadatani, Y.; Ohkawa, F.; Sogawa, M.; Yamagami, H.; Shiba, M.; Watanabe, K.; Tominaga, K.; Fujiwara, Y.; Takeuchi, K.; Arakawa, T. Rebamipide inhibits indomethacin-induced small intestinal injury: possible involvement of intestinal microbiota modulation by upregulation of α-defensin 5. Eur. J. Pharmacol. 2013, 704, 64−69. (15) Muyzer, G.; de Waal, E. C.; Uitterlinden, A. G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 1993, 59, 695−700. (16) Nagashima, K.; Mochizuki, J.; Hisada, T.; Suzuki, S.; Shimomura, K. Phylogenetic analysis of 16S ribosomal RNA gene sequences from human fecal microbiota and improved utility of terminal restriction fragment length polymorphism profiling. Biosci. Microflora 2006, 25, 99−107. (17) Miwa, H.; Hiyama, C.; Yamamoto, M. High-performance liquid chromatography of short- and long-chain fatty acids as 2-nitrophenylhydrazides. J. Chromatogr. 1985, 321, 165−174. (18) Unno, T.; Matsumoto, Y.; Yamamoto, Y. Gallated form of tea catechin, not nongallated form, increases fecal starch excretion in rats. J. Nutr. Sci. Vitaminol. 2012, 58, 45−49. (19) Protein (crude) in animal feed. Copper catalyst Kjeldahl method. In Official Method of Analysis, 15th ed.; Helrich, K., Keys, F., Eds.; Association of Official Analytical Chemists, Inc.: Arlington, VA, USA, 1990. (20) Assini, J. M.; Mulvihill, E. E.; Huff, M. W. Citrus flavonoids and lipid metabolism. Curr. Opin. Lipidol. 2013, 24, 34−40. (21) Akiyama, S.; Katsumata, S.; Suzuki, K.; Nakaya, Y.; Ishimi, Y.; Uehara, M. Hypoglycemic and hypolipidemic effects of hesperidin and

also demonstrated that a significant increase in the excretion of starch in the feces was promoted by the dietary addition of HT. Needless to say, an increased energy loss originating from starch in the feces may at least partially influence the body fat reduction by HT. The present study exhibited considerably different results between HT and HD. The HT diet affected the excretion of starch in the feces and the pools of SCFA in the cecum content, but the HD diet did not. The inhibitory effect of HT on starch digestion was stronger than that of HD. That is to say, the attachment of the rutinose moiety to the flavanone structure can diminish the effectiveness. Limiting the pancreatic αamylase activity in the small intestine must be the key to producing SCFA in the large intestine. Although certain types of bacterial strains produced amylolytic enzymes in the intestine,35 it remains to be seen that HT may have an effect on the production and activity of microbial α-amylase. Furthermore, HT also affected the proportion of Clostridium subcluster XIVa in rat feces, but HD did not. Because the feces collected from the rat fed the HD diet contained a significant amount of HT, dietary HD reached the large intestine, where the glycosidic linkage of HD was hydrolyzed to generate HT. Given that both treatments yielded the same compound in the large intestine, it is likely that the modulation of gut microbiota only by the HT diet may be a consequential result of the stimulation of SCFA productions in the large intestine, as is the case with resistant starch. Besides, we must keep constantly in mind the fact that the large intestine is a very active site for the microbial catabolism of HT, leading to further degradation of the flavanone skeleton. Such transformations could be mainly due to the genera of Clostridium spp. in the colon.36 In conclusion, the present study provides detailed insights into the effects of dietary HT on the composition of gut microbiota and the cecal pools of SCFA in rats and may lead to design strategies for functional foods that aim to reduce the risk of obesity in humans. More research also is needed to further elucidate in more depth the underlying molecular mechanisms of dietary polyphenols as well as their microbial metabolites to modulate the composition and activity of gut microbiota and the link to the antiobesity effect.

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AUTHOR INFORMATION

Corresponding Author

*(T.U.) Phone: +81 3 3262 2789. Fax: +81 3 3262 2174. Email: [email protected]. Funding

This work was supported by JSPS KAKENHI Grant 24500998. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Nana Obara, Aki Tanegashima, and Minori Miyoshi for assistance with the animal experiments. ABBREVIATIONS USED HD, hesperidin; HT, hesperetin; SCFA, short-chain fatty acids; T-RFLP, terminal restriction fragment length polymorphism; OTU, operational taxonomic units

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REFERENCES

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Hesperetin Modifies the Composition of Fecal Microbiota and

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