Oolong Tea Extract and Citrus Peel Polymethoxyflavones Reduce

Jun 25, 2019 - Liu et al. have demonstrated the antiobesity effect after tea ..... TMAO after false discovery rate adjustment for multiple comparisons...
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Article Cite This: J. Agric. Food Chem. 2019, 67, 7869−7879

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Oolong Tea Extract and Citrus Peel Polymethoxyflavones Reduce Transformation of L‑Carnitine to Trimethylamine‑N‑Oxide and Decrease Vascular Inflammation in L‑Carnitine Feeding Mice Pei-Yu Chen,† Shiming Li,‡ Yen-Chun Koh,† Jia-Ching Wu,† Meei-Ju Yang,∥ Chi-Tang Ho,§ and Min-Hsiung Pan*,‡,†,⊥,# †

Institute of Food Sciences and Technology, National Taiwan University, Taipei 106, Taiwan Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization and Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Huanggang Normal University, Huanggang, Hubei China § Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States ∥ Tea Research and Extension Station, Taoyuan 326, Taiwan ⊥ Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan # Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan

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‡

S Supporting Information *

ABSTRACT: Carnitine, a dietary quaternary amine mainly from red meat, is metabolized to trimethylamine (TMA) by gut microbiota and subsequently oxidized to trimethylamine-N-oxide (TMAO) by host hepatic enzymes, flavin monooxygenases (FMOs). The objective of this study aims to investigate the effects of flavonoids from oolong tea and citrus peels on reducing TMAO formation and protecting vascular inflammation in carnitine-feeding mice. The results showed that mice treated with 1.3% carnitine in drinking water significantly (p < 0.05) increased the plasma levels of TMAO compared to control group, whereas the plasma TMAO was remarkedly reduced by flavonoids used. Meanwhile, these dietary phenolic compounds significantly (p < 0.05) decreased hepatic FMO3 mRNA levels compared to carnitine only group. Additionally, oolong tea extract decreased mRNA levels of vascular inflammatory markers such as tissue necrosis factor-alpha (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Polymethoxyflavones significantly lowered the expression of VCAM-1 and showed a decreasing trend in TNF-α and E-selectin mRNA expression compared to the carnitine group. Genus-level analysis of the gut microbiota in the cecum showed that these dietary phenolic compounds induced an increase in the relative abundances of Bacteroides. Oolong tea extract-treated group up-regulated Lactobacillus genus, compared to the carnitine only group. Administration of polymethoxyflavones increased Akkermansia in mice. KEYWORDS: atherosclerosis, trimethylamine-N-oxide (TMAO), carnitine, gut microbiota, flavin monooxygenases (FMOs), dietary phenolic compounds

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INTRODUCTION

public health measures to limit its impact is expanding worldwide. Studies showed that elevation of TMAO was found to associate with the presence of CVDs in a dose-dependent manner.2,3 In low-density lipoprotein receptor knockout mice, the expression of an inflammatory gene including cyclooxygenase 2 (COX-2), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in aortas could be activated via TMAO injection. Choline feeding mice increased plasma TMAO level and enhanced levels of monocyte chemoattractant protein-1 (MCP-1), tissue necrosis factor-alpha (TNF-α), E-selectin, ICAM-1, and vascular cell adhesion molecule-1 (VCAM-1).6 A recent study demonstrated that early pathological process of atherosclerosis could

It is manifested that carnitine is metabolized to trimethylamine (TMA) by gut microbiota and subsequently oxidized to trimethylamine-N-oxide (TMAO) by host hepatic enzymes, flavin monooxygenases (FMOs).1 In recent years, TMAO was discovered to cause vascular inflammation which highly associated with the increment of cardiovascular disease (CVD) risk.2,3 It is clear that CVDs have become the ubiquitous cause and leading contributor to mortality in developed countries. Among the CVDs, atherosclerosis as a major component has been considered as one of the most concerning public health issues. In the United States alone, atherosclerosis causes approximately 42% of all deaths, affecting one out of four persons.4 Rapid economic growth is accompanied by changing lifestyle and dietary habit such as a high fat rich meat diet and reduced physical activity, and the mortality pattern has switched from predominantly infectious diseases to chronic diseases,5 such as CVDs. Thus, the need for © 2019 American Chemical Society

Received: Revised: Accepted: Published: 7869

May 17, 2019 June 25, 2019 June 25, 2019 June 25, 2019 DOI: 10.1021/acs.jafc.9b03092 J. Agric. Food Chem. 2019, 67, 7869−7879

Article

Journal of Agricultural and Food Chemistry

obtained from Bionovas (Toronto, Canada). Vancomycin, metronidazole, and neomycin sulfate were obtained from Santa Cruz (Dallas, TX, U.S.A.). L-Carnitine was obtained from TCI Chemicals (Chuoku, Tokyo, Japan). Preparation of Oolong Tea Extract and Its Composition. The preparation of oolong tea extract was performed in Tea Research and Extension Station (Taoyuan, Taiwan). Briefly, 1 kg of oolong tea was extracted with 10 L of ddH2O (100 °C, 2 min). The filtrate was directly freeze-dried in an evaporator. The water-extracted pellet was crushed into powder and stored at −20 °C prior to use. The bioactive compounds of oolong tea extract were analyzed on Agilent 1260 infinity HPLC-DAD (U.S.A.), Poroshell 120 EC-C18 (2.7 μm, 150 × 3.0 mm) column. HPLC chromatogram of chemical composition of oolong tea extract is shown in Table 1.

be promoted by TMAO via accelerating endothelial dysfunction through reducing endothelial self-repair capacity and stimulating monocyte adhesion via protein kinase C (PKC)/nuclear factor-κB (NF-κB)/VCAM-1 activation.7 TMAO is known as the catalyzing product of microbiome, and therefore antibiotic administration can markedly decrease TMAO concentration in plasma.1 However, the unfavorable and undesirable side effects of antibiotics and the concern of antibiotic resistance in bacteria have become the consideration of using antibiotics on chronic treatment. Thus, intervention of phytochemicals that has been studied for decades is recently taken as the alternative strategy to suppress elevated TMAO levels and to prevent atherogenesis due to their sight-catching antioxidation, anti-inflammatory, and anticarcinogenesis capabilities.8 It has been widely discussed that diet and nutritional interventions can be one of the possible path for gut microbiota remodeling and composition.8 Some phenolic phytochemicals with poor bioavailability make them potential modulating agents for gut microbiota structural reorganization.9 Recently, oolong tea has become popular and is consumed preferentially in Asia, particularly in Taiwan. Liu et al. have demonstrated the antiobesity effect after tea consumption via gut microbiota recomposition.10 Also, Fenghuang Oolong tea alleviated obesity and prevention of gut dysbiosis in B6 mice.11 Furthermore, the catechins found in tea appear to interfere with the molecular processes underlying the initiation, progression, and rupture of atherosclerotic plaques. A report showed that epigallocatechin-gallate (EGCG) reduced ROS production in the cell model,12 which indicated that catechins in tea can attenuate oxidative stress. Besides, EGCG suppressed the expression of VCAM-1 and ICAM-1 for molecular adhesion in endothelial cells through NF-κB-mediated mechanism.13 Flavonoids are a large family of natural polyphenolic compounds. Among the flavonoids, polymethoxyflavones (PMFs) and demethylated PMFs are the primary bioactive flavonoids abundant in the citrus peel of several species.14,15 In a previous study on colorectal cancer, consumption of PMFs has shown to alter gut microbiota composition by elevating the butyrate-producing probiotics and reducing those species related to colorectal cancer.16 Also, mice treated with PMFs decreased abundance of Gammaproteobacteria,16 which are considered as being TMA-producing bacteria.17 Orange peel extracts enriched with PMFs could down-regulate the expression of a panel of proinflammatory genes, including ICAM-1, NF-κB, IL-1β, and IL-8.18 Also, nobiletin inhibits macrophage foam cell formation, which may prevent atherosclerosis.19 According to the biological activities of oolong tea extract and PMFs, there may be the potential for further utilization of these compounds against TMAO formation and reduce vascular inflammation induced by carnitine feeding through gut microbiota remodeling or other potential mechanisms. This study focuses on the potential for natural dietary compounds (oolong tea extract and polymethoxyflavones) contributing to TMAO reduction and attenuating vascular inflammation and endothelial dysfunction in carnitine-feeding mice.

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Table 1. Chemical Composition of Oolong Tea Extract short form TSC GA GC TSB EGC C TSA EC EGCG GCG ECG CG TF TF3 TF3′ total polyphenols

mean ± SD (mg/g)

chemical names theasinensin C gallic acid gallocatechin theasinensin B epigallocatechin catechin caffeine theasinensin A epicatechin epigallocatechin gallate gallocatechin gallate epicatechin gallate catechin gallate theaflavin theaflavin-3-gallate theaflavin-3′-gallate

1.2 2.1 78.9 0.1 33.8 10.7 54.2 0.3 7.2 36.8 36.8 5.7 4.0 0.2 0.1 0.1 218.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0 0.1 22.1 0.3 0.5 0.3 3.1 0.4 0.4 2.6 3.9 0.4 0.3 0.2 0.0 0.0 31.5

Polymethoxyflavone (PMF) Preparation and Its Composition. PMF was prepared in our laboratory according to the method of Lai et al.20 The chemical composition was determined by HPLC as shown in Table 2. Animal and Treatment Protocol. Six-week-old female C57BL/6 (B6) mice were purchased from BioLASCO (Taipei, Taiwan) and housed acclimatized under a controlled atmosphere (23 ± 2 °C, 50− 70% relative humidity) with a 12 h light/12 h dark cycle. The animal use protocol listed below has been reviewed and approved by the Institutional Animal Care and Use Committee (NTU105-EL-00115). In the experiment, a total of 30 female B6 mice were used and were randomly allotted into five groups: (1) normal diet, (2) 1.3% carnitine water, (3) 1.3% carnitine water with 1% Oolong tea extract diet, (4) 1.3% carnitine water with 1% PMFs diet, and (5) 1.3% carnitine water with antibiotic cocktail for 6 weeks. All mice were fed experimental diets, LabDiet Rodent 5001 diet, and tap water ad libitum at all times. Antibiotic cocktail showed to suppress commensal gut microbiota and included 0.1% ampicillin sodium salt, 0.1% metronidazole, 0.05% vancomycin, and 0.1% neomycin sulfate. The antibiotic cocktail was given via gastric gavage using a 1.5 in. 20 gauge intubation needle every 12 h. At the end of the six-week study, mice were euthanized by CO2 asphyxiation. RNA Extraction and Reverse Transcription. RNA from liver and aorta was extracted using TRIzol reagent. Phase separation is performed by adding 200 μL of chloroform/1 mL TRIzol. Following centrifugation, the aqueous phase (top) was transferred to a new tube with care to not contaminate the solution with the other phases. Isopropanol (500 μL) was added to the new tube and incubated at room temperature for 10 min for RNA precipitation. Ethanol (75%) was used to remove organic reagents and salts. The resulted pellet was resuspended in ddH2O.

MATERIALS AND METHODS

Materials. Oolong tea extract was prepared in Tea Research and Extension Station (Taoyuan, Taiwan). Ampicillin sodium salt was 7870

DOI: 10.1021/acs.jafc.9b03092 J. Agric. Food Chem. 2019, 67, 7869−7879

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Journal of Agricultural and Food Chemistry Table 2. Chemical Composition of PMFsa chemical names

mean ± SE

5,7,3′,4′-tetramethoxyflavone 5,6,7,3′,4′-pentamethoxyflavone 5,4′-dihydroxy-6,7-dimethoxyflavone 3,5,6,7,3′,4′-hexamethoxyflavone 5,6,7,8,3′,4′-hexamethoxylflavone 5,6,7,4′-tetramethoxylflavone 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone 5,4′-dihydroxy-7-methoxyflavone 3,5,6,7,8,3′4′-heptamethoxyflavone 5-hydroxy-6,7,3′,4′-tetramethoxyflavone 5,6,7,8,4′-pentamethoxyflavone 5-hydroxy-3,6,7,3′,4′-pentamethoxyflavone 5-hydroxy-6,7,8,3′,4′-pentamethoxylflavone 5-hydroxy-6,7,4′-trimethoxyflavone 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone 5-hydroxy-6,7,8,4′-tetramethoxyflavone

0.79 ± 0.10 N.D. 6.33 ± 0.00 0.16 ± 0.80 4.91 ± 1.02 0.08 ± 0.30 3.97 ± 0.40 0.64 ± 0.28 64.80 ± 0.68 91.12 ± 2.96 50.15 ± 5.97 46.07 ± 0.82 199.21 ± 1.68 65.52 ± 1.15 170.61 ± 2.17 174.07 ± 5.14 125.2 ± 3.8 750.6 ± 14.3 875.8 ± 18.1

common name sinensetin

nobiletin

5-demethylsinensetin tangeretin 5-demethylnobiletin

5-demethyltangeretin total PMFs total hydroxylated PMFs Total flavones

The values were averages from duplicate samples and were given in mg/g, mean ± SE.

a

Table 3. Body Weight and Daily Intake of Different Treatment Groupsa body weight group ND carnitine Car+Tea Car+PMFs Car+Abs

final (g)

initial (g) 18.47 18.42 18.42 18.47 18.45

± ± ± ± ±

0.72 0.60 0.69 0.69 0.49

a a a a a

20.62 20.29 21.05 19.47 20.97

± ± ± ± ±

0.98 0.62 0.93 0.96 0.68

weight gain (g) a a a a a

2.15 1.87 2.63 0.99 2.39

± ± ± ± ±

1.01 0.76 1.17 0.58 0.52

a a a a

food intake (g/mouse/d) 5.84 6.71 5.23 5.04 8.57

± ± ± ± ±

0.94 0.88 0.92 1.28 1.88

a b c c d

water intake (ml/mouse/d) 4.11 4.59 5.51 3.98 3.91

± ± ± ± ±

0.36 0.36 0.29 0.36 0.85

a b d a a

The significant difference among five groups was analyzed by one-way ANOVA and Duncan’s multiple range test. The values with different letters are significantly different (p < 0.05) between each group. ND indicates normal diet; Car, carnitine; Tea, oolong tea extract; PMFs, polymethoxyflavones; Abs, antibiotic cocktail. a

Quality of RNA and RNA concentrations were then quantified using NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Each RNA sample was then reversetranscribed to cDNA using 1 μg of RNA. Reverse transcription was performed using the SensiFAST cDNA synthesis kit including random hexamers and oligo(dT)s in a 20 μL total reaction volume following manufacturer’s instruction. Briefly, reverse transcription was carried out for 15 min at 42 °C followed by heating at 85 °C for 5 min to inactivate the enzyme. The resulting cDNA was stored at −20 °C for long-term storage and used as a polymerase chain reaction (PCR) template. cDNA Amplification and Visualization. PCR was performed for cDNA amplification of liver and aorta. Amplification of cDNA was achieved using MyTaq Mix which contained all of the reagents (including stabilizers). The reaction cycling was 95 °C for 15 s, 56 °C for 15 s, and 72 °C for 10 s in Biometra TAdvanced thermocycler. The primer sequences used for amplification are listed in Table 3. Amplification of the GAPDH was used as an internal standard. PCR products were electrophoresed on 2% agarose gels stained with SafeGreen Loading Dye. The resultant bands were visualized with MultiGel-21 image system (Top Bio Co., Taiwan). Quantitative Real-Time Polymerase Chain Reaction (qPCR). mRNA of liver and aorta levels were determined by quantitative realtime PCR using StepOnePlus Real-Time PCR system and SYBR Green master mix as compared to constitutively expressed gene (GAPDH) using the relative quantification method (ΔΔCt). Primer sequences (Supplementary Table 1) for SYBR Green reactions were designed in-house and synthesized by Mission Biotech. To quality control the qPCR results, melting curve analysis was performed for all

reactions and some products were separately amplified on a 2% agarose gel for visual inspections. Quantification of Plasma Carnitine and TMAO Levels. Carnitine and TMAO were determined by the Water UPLC system (Acquity, Waters, Mildford, MA, U.S.A.) in a positive multiple reaction monitoring (MRM) mode. The separation was performed using Agilent ZORBAX NH2 Column (5 μm, 4.6 mm × 250 mm) and the column was thermostated at 30 °C. Mobile phase A was composed of 10 mM NaOAc and 0.6% acetic acid in ACN and mobile phase B was 10 mM NaOAc and 0.6% acetic acid in water. The gradient profile is shown in Supplementary Table 2. The MS parameters were set as follows: 100 °C for the source temperature, 650 °C for the desolvation temperature, 2 L/h for the cone gas flow, 600 L/h for the desolvation gas flow, and 1500 V for the capillary voltage. Cone voltage was set at 10 V. Concentrations of each analyte in samples were determined by calibration curves. Microbiota Analysis by Next Generation Sequencing. Microbial community composition was assessed by pyrosequencing 16S rRNA genes derived from mouse cecum (n = 3 for each group). Total DNA was isolated and purified using InnuSPEED Stool DNA kit according to the manufacturer’s instructions. The quality of DNA samples was assessed by NanoDrop 1000 spectrophotometer. DNA samples were sent to Biotools Co., Ltd. for 16S rRNA gene amplification, sequence library construction, and sequencing. The V3−V4 regions of bacterial 16S rDNA were amplified using PCR technology. The library DNA was sequenced by Illumina HiSeq 2500 platform, and paired-end reads (250 bp) were generated. For the data processing, in order to analyze the species diversity in each sample, all effective tags were grouped by 97% DNA sequence similarity into 7871

DOI: 10.1021/acs.jafc.9b03092 J. Agric. Food Chem. 2019, 67, 7869−7879

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Journal of Agricultural and Food Chemistry operational taxonomic units (OTUs). During the construction of OTUs, basic information from different samples had been collected, such as effective tags data, low-frequency tags data, and annotation data of tags. For the veracity of sequencing data analysis, raw data would be merged and filtered to get clean data. The effective data is used to do OTU cluster and species annotation for the respective sequence of each OTU. Statistical Analysis. Quantitative data are presented as mean ± standard deviations (SD). Statistical analysis was conducted with oneway analysis of variance (ANOVA) using SPSS 12.0. A p-value of less than 0.05 was considered as statistically significant, and the Duncan’s multiple range post hoc test was applied if the p-value was less than 0.05. Graphs were created using SigmaPlot 12.5 software.

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RESULTS AND DISCUSSIONS Effects of Treatments on Body Weight, Daily Intake and Organ Weight in C57BL/6 Mice. Six-week-old female C57BL/6 (B6) mice with a similar initial weight were obtained and randomly divided into five experimental groups as follows: normal diet (ND), 1.3% carnitine water (Car), carnitine water with 1% oolong tea extract diet (Car+Tea), carnitine water with 1% polymethoxyflavones (Car+PMFs), and carnitine water with antibiotic cocktail (Car+Abs) as a positive control group. In Car+Abs group, we attempted to deplete mice of their gut microbiota by providing antibiotic cocktail (including 0.1% ampicillin sodium salt, 0.1% metronidazole, 0.05% vancomycin, and 0.1% neomycin sulfate) ad libitum in drinking water according to the previously published protocol.1 However, administration of broad-spectrum antibiotics in drinking water was difficult to reproduce. Mice consistently refrained from drinking the antibiotic concoction, presumably due to the foul taste of metronidazole. There is an observation of weight loss and decrease in water consumption (data not shown). Reikvam et al. also observed the same unwillingness to drink the antibiotic concoction provided ad libitum in BALB/c and B6 mice.21 So we administered antibiotic cocktail by oral gavage every 12 h to ensure a safe and stable delivery of the antibiotics to every mouse subjected to the protocol. After 44 days of the regimen, the mice were euthanized by CO2 asphyxiation for further mechanistic investigation. As shown in Table 3, at the end of the experiment there is no significant difference of the average body weight between each group as well as the weight gain. However, we noticed a transient weight loss of antibiotic treatment group (16.04 ± 0.36 g) as compared to the ND group (18.24 ± 0.69 g) on day 4 (Figure 1). This transient weight loss was believed to be caused by the mice adjusting to receiving antibacterial therapy.21 Within a week, mice receiving the antibiotic therapy regained weight and appeared to be healthy. As depicted in Figure 2, we found that daily food and water intake in the carnitine group were higher compared to the ND group. Meanwhile, daily food intake in the carnitine group was reduced in dietary compound-treated mice and there was no statistically significant difference between the two dietary compound treatment groups (Figure 2). However, oolong tea extract administration up-regulated daily water intake but not in the Car+PMFs group and the antibiotic therapy group (Figure 2). The water intake affected the carnitine consumption as the carnitine was supplemented in the drinking water. Most of the treatment did not affect the weight of the liver, kidney, and spleen in the course of the 6 week experiment except for the antibiotic administration group (Figure 3A). Car +Abs group had lower liver and spleen index as the antibiotic therapy impaired the immune state, which could cause a

Figure 1. Body weight over the course of 44 days. The body weight of female B6 mice was monitored once every 4 days. Values are given as mean ± SD, n = 5−6 for each group as indicated in parentheses. ND indicates normal diet; Car, carnitine; Tea, oolong tea extract; PMFs, polymethoxyflavones; Abs, antibiotic cocktail.

decrease in spleen mass and might affect liver mass through dysbiosis. Oolong Tea Extract and PMFs Inhibited Plasma TMAO Elevation Induced by Chronic L-Carnitine Feeding. Recently, TMAO has been thought to be associated with atherosclerosis and CVD. Gut microbiota metabolism of choline produces TMA and further metabolized to a proatherogenic species, TMAO. In 2013, Koeth et al. demonstrated that L-carnitine, an amine abundant in red meat, also produces TMAO and accelerates atherosclerosis.1 LCarnitine supplementation enhanced TMA and TMAO production in humans and in the mouse model.1,22 In this study, blood samples were immediately obtained from the heart and isolated in plasma for the analysis of carnitine and TMAO by LC-MS/MS after euthanasia. Increasing concentrations of L-carnitine and TMAO standards were used to generate calibration curves for determining plasma concentrations of each analyte (Figure 4A). In the plasma measurement of the concentration of carnitine and TMAO, the carnitine group had a substantial increase in circulating TMAO compared to the ND group, which is consistent with the recent findings. However, plasma TMAO level in the Car+Abs group dramatically decreased to a ND group level, which demonstrated the fact that TMAO production from carnitine is inducible and required mouse gut microbiota metabolism. With antibiotic administration, TMAO producing capacity is almost completely depleted (Figure 4B). Natural phenolic compounds possess many beneficial human health effects. Tea polyphenols suppressed adhesion molecules expression13 and showed a reduced risk of ischemic stroke and cardiovascular events in human adults.23 Citrus peel PMFs prevented atherosclerosis by inhibiting macrophage foam cell formation.19 However, there are no reports on linking the effects of these natural phenolic compounds to the endothelial dysfunction and vascular inflammation caused by TMAO. In this study, plasma TMAO levels in oolong tea extract and PMF-treated mice were significantly (p < 0.05) lower than those in the carnitine group (Figure 4B), indicating that these natural dietary compounds can inhibit plasma TMAO elevation induced by chronic carnitine feeding. 7872

DOI: 10.1021/acs.jafc.9b03092 J. Agric. Food Chem. 2019, 67, 7869−7879

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Journal of Agricultural and Food Chemistry

Figure 2. Daily food and water intake. Food and water intake of female B6 mice were monitored every day. Values are given as mean ± SD. The significant difference among groups was analyzed by oneway ANOVA and Duncan’s multiple range test. The values with different letters are significantly different (p < 0.05) between each group. ND indicates normal diet; Car, carnitine; Tea, oolong tea extract; PMFs, polymethoxyflavones; Abs, antibiotic cocktail.

However, the suppression of TMAO elevation induced by carnitine ingestion of natural phenolic compounds used in this study is not close to that of the normal control mice. By using a higher dose of compounds, the results might show a marked decrease. Bioavailability of L-carnitine varies due to dietary habits. Studies reported that the bioavailability of L-carnitine in red meat eaters who adapted to high-carnitine diets is lower than vegans and vegans produce less TMAO from carnitine.1,22,24 Ingested and unabsorbed L-carnitine is degraded by gut micobiota in the intestine to TMA. Following stable isotope labeled d3-L-carnitine challenge, red meat eaters had less plasma d3-carnitine compared to vegans, which reflected an increase in gut microbial metabolism of carnitine prior to absorption.1 As shown in Figure 4C, plasma carnitine level of the Car+Abs group is the highest among the five groups as there is no gut microbial metabolism of carnitine to TMA in antibiotic-treated mice. Interestingly, circulating carnitine in the carnitine group is similar to the ND group which did not feed with carnitine water (Figure 4C) but an elevation of

Figure 3. Viscera index of mice and the images of organs. The mean index of liver, spleen, and kidney of female B6 mice were presented (A). Values are given as mean ± SD. The significant difference among groups was analyzed by one-way ANOVA and Duncan’s multiple range test. The values with different letters are significantly different (p < 0.05) between each group. The images of organs of each group (B). ND indicates normal diet; Car, carnitine; Tea, oolong tea extract; PMFs, polymethoxyflavones; Abs, antibiotic cocktail. 7873

DOI: 10.1021/acs.jafc.9b03092 J. Agric. Food Chem. 2019, 67, 7869−7879

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Figure 4. Plasma carnitine and TMAO levels. Calibration curves for L-carnitine (m/z = 162) and TMAO (m/z = 76) were used to determine plasma concentrations of each analyte with appropriate m/z and retention time (A). Plasma TMAO (B) and carnitine (C) levels of female B6 mice were measured using LC-MS/MS. Values are given as mean ± SD, n = 3 for each group. The significant difference among groups was analyzed by one-way ANOVA and Duncan’s multiple range test. The values with different letters are significantly different (p < 0.05) between each group. ND indicates normal diet; Car, carnitine; Tea, oolong tea extract; PMFs, polymethoxyflavones; Abs, antibiotic cocktail.

plasma TMAO was observed in the carnitine group and not in the ND group (Figure 4B). These results suggested that prior to absorption carnitine ingested by carnitine-treated mice was utilized to produce TMAO through an intestinal microbiotadependent pathway. Notably, dietary phenolic compounds used in this study significantly increased circulating carnitine and decreased plasma TMAO levels (Figure 4). This result suggests that these compounds might suppress gut microbiota

metabolic activity for metabolizing carnitine and improve carnitine absorption. Oolong Tea Extract and PMFs Decreased Chronic LCarnitine Feeding-Induced Vascular Inflammation in C57BL/6 Mice. Numerous case-control studies have shown a striking association between TMAO levels and atherosclerosis enhancement/CVD risks in a variety of cohorts.1,2 Herein, we demonstrated that carnitine feeding truly increased plasma TMAO levels in the carnitine group; however, natural dietary 7874

DOI: 10.1021/acs.jafc.9b03092 J. Agric. Food Chem. 2019, 67, 7869−7879

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Journal of Agricultural and Food Chemistry

Figure 5. Expression levels of inflammatory genes in aortas.Aortas were harvested and quantitative polymerase chain reaction was used to quantify expression levels of inflammatory genes (TNF-α, E-selectin, and VCAM-1). Melting curve analysis is shown at the right. Values are given as mean ± SE, n = 3 for each group. The significant difference among five groups was analyzed by one-way ANOVA and Duncan’s multiple range test. The values with different letters are significantly different (p < 0.05) between each group. ND indicates normal diet; Car, carnitine; Tea, oolong tea extract; PMFs, polymethoxyflavones; Abs, antibiotic cocktail.

and the effects of oolong tea extract and PMFs supplementations. After 6 weeks of diet, aortas were probed using qPCR for expression level of inflammatory genes. The carnitine group showed significantly enhanced expression of TNF-α, Eselectin, and VCAM-1 compared to the ND group (Figure 5). Furthermore, in natural dietary compound groups, oolong tea extract significantly reduced TNF-α, E-selectin, and VCAM-1 induced by carnitine supplementation. Polymethoxyflavones significantly lowered the expression of VCAM-1 and showed a decreasing trend in TNF-α and E-selectin mRNA

compound-supplemented groups can reduce this elevation (Figure 4B). TMAO was reported to cause vascular inflammation and endothelial dysfunction.6,7,25 TMAO can eventually exacerbate endothelium function, promote atherosclerosis, and increase atherosclerotic plaque size.1,2,26 Mice with chronic carnitine feeding showed significantly enhanced expression of inflammatory genes, including MCP-1, TNF-α, ICAM-1, E-selectin, CD68, and VCAM-1.6 Given the importance of inflammation in atherosclerosis development, we investigated whether chronic carnitine feeding could promote vascular inflammatory gene expression in B6 mice 7875

DOI: 10.1021/acs.jafc.9b03092 J. Agric. Food Chem. 2019, 67, 7869−7879

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Journal of Agricultural and Food Chemistry

Figure 6. Gut microbiota composition analysis. 16S rRNA gene sequencing analysis of cecal content at the phylum level (A). Heat map of 16S rRNA gene sequencing analysis of cecal content at the genus level (top panel) and Erysipelotrichia, whose proportions were significantly changed by carnitine supplementation are shown (bottom panel) (B). PLS-DA demonstrates distinct cecal microbial composition among groups. Each data point represents one sample and each color represents each group (percent variation explained by each PLS is shown in parentheses) (C). Single and double asterisks indicate p < 0.05 and p < 0.01 between groups, respectively. n = 3 for each group. ND indicates normal diet; Car, carnitine; Tea, oolong tea extract; PMFs, polymethoxyflavones; Abs, antibiotic cocktail.

expression compared to the carnitine group. The Car+Abs group had a similar observation with the Car+PMFs group (Figure 5). These results suggested that natural dietary compounds reduced plasma TMAO levels and decreased carnitine feeding-induced vascular inflammation. Seldin et al. demonstrated that three-week choline (1.3%) feeding caused a substantial increase in plasma TMAO and

promoted inflammatory gene expression in cells of the vasculature.6 Reduction on circulating TMAO levels can attenuate aortic lesions.26 In some respects, the reduction of vascular inflammatory gene expression observed in natural dietary compound groups can be explained by reduced plasma TMAO levels (Figure 4B). Our observations support a role for TMAO in the activation of inflammatory pathways in the aorta 7876

DOI: 10.1021/acs.jafc.9b03092 J. Agric. Food Chem. 2019, 67, 7869−7879

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1 and PLS 2 explained 22.68% and 12.17% of variation of gut microbiota composition, respectively. As shown in Figure 6C, the clustering of samples in the carnitine group was isolated from the natural dietary compound groups. This result revealed that the dietary phenolic compound supplementations can modulate gut microbiota to a healthier fashion and subsequently reduce circulating TMAO level. Oolong Tea Extract and PMFs Inhibited TMAO Synthesis by Reducing Hepatic FMO3 mRNA Expression Levels. TMAO is generated through a gut microbiotadependent fashion, via microbiota that can metabolize dietary carnitine to TMA. TMA is then oxidized by enzymes of the flavin monooxygenase (FMO) family in the liver, among which FMO3 is the most active isoform.32 In this study, the carnitine group caused a 2-fold increase of hepatic FMO3 mRNA expression level compared to the ND group. In contrast, significant reductions of hepatic FMO3 mRNA levels were observed in natural dietary compound groups compared to the carnitine group (Figure 7). These

and these compounds can attenuate the vascular inflammation. The report suggested that the activation was mediated, at least in part, by the NF-κB signaling pathway,6 which has been linked previously to reduced vascular responsiveness and inflammation. However, further research is needed to confirm this possibility and the effects of these compounds on vascular inflammatory alleviation. Effects of Oolong Tea Extract and PMFs on Intestinal Microbiota Composition. We next investigated whether natural dietary compound intervention affected the composition of the gut microbiota in the cecum, as the gut microbiota are involved in the metabolism of carnitine and might be associated with the reduction of TMAO after dietary compound intervention. To determine the role of the gut microbiota in the natural dietary compound-induced decrease of TMAO levels, this study explored bacterial populations in the cecum. As shown in Figure 6, analysis of the microbiota at various taxonomic levels revealed diverse patterns in bacterial composition depending on different treatments. In comparison with the ND group, analysis at the phylum level indicated that the carnitine group increased the relative abundance of Proteobacteria and Tenericutes and reduced the relative abundance of Deferribacteres. The abundance of Verrucomicrobia, mucus-degrading bacterium Akkermansia muciniphila and the only identified member of this phylum, was particularly increased in the PMFs-treated mice (Figure 6A). However, the result showed that after antibiotic treatments, the abundance of gut microbiota decreased significantly in all levels. At the phylum level, only Proteobacteria accounted for more than 1% of all the microbiota in mice treated with antibiotics as shown in Figure 6A. Genus-level analysis showed that oolong tea extract and the PMF-treated mice increased the relative abundance of Bacteroides compared to the carnitine group (Figure 6B). The PMF-treated group increased the abundance of Akkermansia, a genus of interest because of its reported links to improvement in obesity and metabolic health, which is consistent with the data from the phylum level (Figure 6A,B). Moreover, abundance of Lactobacillus increased in the Car +Tea group (Figure 6B). The Car+Tea group also observed an increase in the proportions of Bif idobacteriales (data not shown). However, Akkermansia, Lactobacillus, Bacteroides, and Bif idobacterium were reported to be negatively associated with plasma TMAO after false discovery rate adjustment for multiple comparisons in the Chen et al. study.26 Besides, an in vitro study demonstrated that Akkermansia is not a TMAproducer.27 These findings suggested that natural dietary compounds used in this study manipulated gut microbiota altered by carnitine feeding. Notably, there is an increase in relative abundance of Turibacter, which belongs to class Erysipelotrichia, in the carnitine group (Figure 6B). Bacteria from this class were reported to produce TMA from choline in order to mimic a choline deficiency.28,29 In addition, it has been shown that Erysipelotrichia may promote cholesterol accumulation and atherosclerosis via TMAO production.29,30 A study of the impact of TMAO on platelet function also revealed that Erysipelotrichia is associated with both TMAO levels and thrombosis potential.31 This class of bacteria increased in obese people with high CVD risk. The partial least-squares discriminant analysis (PLS-DA) was performed to evaluate the variant of gut microbiota composition among the groups. PLS-DA plot showed that PLS

Figure 7. Expression levels of hepatic FMO3 mRNAExpression levels of hepatic FMO3 mRNAs determined by RT-qPCR and normalized to GAPDH. Values are given as mean ± SD, n = 3. The significant difference among the five groups was analyzed by one-way ANOVA and Duncan’s multiple range test. The values with different letters are significantly different (p < 0.05) between each group. ND indicates normal diet; Car, carnitine; Tea, oolong tea extract; PMFs, polymethoxyflavones; Abs, antibiotic cocktail.

transcriptional changes may explain, in part, the decreased plasma TMAO levels observed in natural dietary compound groups (Figure 4B). Nevertheless, antibiotics administration group showed an increase of FMO3 mRNA level similar to the carnitine group. FMO3 is regulated by a hepatic bile acidactivated nuclear receptor farnesoid X receptor (FXR).32 Specifically, cholic acid stimulates the FMO3 expression.32 The lack of bacteria able to make secondary bile acids after antibiotic administration causes a buildup of primary bile acids like cholic acid. This may explain the up-regulation of hepatic FMO3 mRNA observed in the Car+Abs group. Antisense oligonucleotide (ASO) mediated knockdown of FMO3 levels has been shown to suppress circulating TMAO levels and inhibits atherosclerosis in animal models.33 The study suggested that FMO3 is a negative regulator of nonbiliary RCT.33,34 FMO3 down-regulation not only reduced TMAO formation but also decreased lipogenesis and 7877

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gluconeogenesis through modulating PPARα expression and activity.33 FMO3 ASO-injected mice decreased hepatic lipid and increased daily fecal lipid output.33 Emerging evidence suggested that FMO3, the enzyme that generates TMAO, plays a crucial role in regulating whole body cholesterol balance and atherosclerosis development. The identification of FMO3 substrates and products that play a primary role in promoting nonbiliary RCT of particular relevance to atherosclerosis would be an attractive therapeutic strategy for cholesterol lowering. Recently, TMAO was identified to be associated with atherosclerosis and CVD risk. Broad-spectrum antibiotic administration can effectively deplete TMAO producing capacity. However, the issue of antibiotic resistance remains to be a challenge. Recent reports that used probiotics and nutraceuticals in preventing of TMAO-inducing atherosclerosis and CVD are lacking.35 In this study, based on the beneficial health effects of natural dietary phenolic compounds, we investigated the effects of oolong tea extract and polymethoxyflavones on TMAO levels lowering and decreasing vascular inflammation in carnitine-feeding mice. Recently, we also have demonstrated that nobiletin prevented TMAO-induced vascular inflammation in Sprague−Dawley rats.36 Because of low bioavailability of phenolic phytochemicals, these compounds are possibly developed into therapeutic agents through modulating gut microbiota structure. Our study suggested that dietary phenolic compounds such as oolong tea extract and polymethoxyflavones reduced TMAO formation ability by gut microbiota remodeling and down-regulating carnitine-induced FMO3 elevation and subsequently attenuated carnitinefeeding-induced vascular inflammation. Advancement in our understanding of the effects of natural dietary compounds on gut microbiota composition and hepatic enzyme FMO3 may provide insight into novel therapeutic strategies for the treatment or prevention of TMAO-dependent atherosclerotic CVD.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b03092.

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Article

(Table S1) Sequence of primers used in this study; (Table S2) Gradient profile of HPLC separation used in LC-MS/MS method; (Table S3) Diet composition of each group, in terms of weight and calories provided (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +886 2 33664133. E-mail: [email protected]. ORCID

Shiming Li: 0000-0002-6167-0660 Min-Hsiung Pan: 0000-0002-5188-7030 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Ministry of Science and Technology [105-2320-B-002-031-MY3, 105-2628-B-002-003MY3, 107-2321-B-002-050]. 7878

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