Metabolomics Reveals that Dietary Ferulic Acid ... - ACS Publications

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Metabolomics Reveals that Dietary Ferulic Acid and Quercetin Modulate Metabolic Homeostasis in Rats Limin Zhang, Manyuan Dong, Guangyong Xu, Yuan Tian, Huiru Tang, and Yulan Wang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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Journal of Agricultural and Food Chemistry

Metabolomics Reveals that Dietary Ferulic Acid and Quercetin Modulate Metabolic Homeostasis in Rats Limin Zhang,*,† Manyuan Dong,†Guangyong Xu,†,§Yuan Tian,† Huiru Tang,*,‡ Yulan Wang*,†,ζ

†

CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory

of Magnetic Resonance and Atomic and Molecular Physics, National Centre for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences (CAS), Wuhan 430071, China ‡

State Key Laboratory of Genetic Engineering, Collaborative Innovation Centre for Genetics

and Development, Shanghai International Centre for Molecular Phenomics, Zhongshan Hospital, School of Life Sciences, Fudan University, Shanghai 200433, PR China §

School of Environmental and Safety Engineering, Changzhou University, Jiangsu, 213164,

China. ζ

Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,

Zhejiang University, Hangzhou 310058, PR China. *To

whom

correspondence

should

be

addressed.

Dr.

Limin

Zhang,

E-mail:

[email protected]; Telephone: +86-27-87198430, Fax: +86-27-87199291; and Dr. Huiru Tang,

Email:

[email protected];

Telephone:

+86-27-87197104,

Fax:

+86-27-87199291; and Dr. Yulan Wang, E-mail: [email protected]; Telephone: +86-27-87197143, Fax: +86-27-87199291;

1

ACS Paragon Plus Environment


Abstract: Phenolic compounds ingestion has been shown to have potential preventive and therapeutic effects against various metabolic diseases such as obesity and cancer. To provide a better understanding of these potential benefit effects, we investigated the metabolic alterations in urine and feces of rat ingested ferulic acid (FA) and quercetin (Qu) using NMR-based metabolomics approach. Our results suggested that dietary FA and/or Qu significantly decreased short chain fatty acids and elevated oligosaccharides in the feces, implying that dietary FA and Qu may modulate gut microbial community with inhibition of bacterial fermentation of dietary fibers. We also found that dietary FA and/or Qu regulated several host metabolic pathways including TCA cycle and energy metabolism, bile acid, amino acid and nucleic acid metabolism. These biological effects suggest that FA and Qu display outstanding bioavailability and bioactivity and could be used for treatment of some metabolic syndromes, such as inflammatory bowel diseases and obesity.

Keywords: ferulic acid; quercetin; bioavailability; bioactivity; metabolomics;

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Introduction Phenolic compounds widely occur in the plant kingdom, such as fruits, vegetables, herbs and beverages and therefore an improtant constituent of the human diet.1,2 Although the phenolics consumption fluctuates significantly between cultures, ethnic groups, and even within a narrow geological location, it is estimated that the daily ingestion for humans ranges from about 20 mg to 1 g, which is reported to be higher than vitamin E.3 These compounds are currently receiving much attention because of their beneficial health effects such as anti-inflammation, hepatoprotection and particularly anti-oxidation.4-7 Phenolic compounds are mainly identified in accordance to the characteristics of their carbon skeleton, including flavonoids, phenolic acids and the less encountered stilbenes and lignans. Amongst many phenolic acids, the most common are caffeic acid and, to a lesser extent, ferulic acid (FA, Figure 1A). FA exists in seeds and leaves ussually covalently linked to lignin and other biopolymers. For example, in wheat, FA is ester-linked to cell wall carbohydrates and occurs in higher concentration in the alcurone, pericarp and embryo cell walls.8 Human studies showed that FA ingestion can be metabolized and excreted in the form of conjugates such as 3-hydroxyphenyl and 3-methoxy-4-hydroxy phenyl derivatives of phenyl propionic acid and excreted in urine.9 Previous feeding animals studies also indicted that FA is metabolied to the same hydroxy methoxy derivatives as in the human studies observed, with FA conjugation of glucuronide partly excreted in urine.10 FA intraperitoneal administrated to rats is metabolized mainly as its free form and 3-hydroxy phenyl propionic acid.11 Flavonoids are also widely distributed in plant foods and sub-divided into some 3



families based on their structural characteristics: flavonols, flavanols, flavones, flavanones isoflavones and anthocyanidins. The average daily flavonoids ingestion is around 25 mg in humans.12 In the flavonol sub-category, quercetin (Qu, Figure 1B) is the most common component in vegetables and fruits. Qu is incorporated from foods into the intestinal cells in its glycosylated forms, primarily as β-glycosides, and glycosylation markedly influence its efficiency of Qu absorption.13,14 After hydrolysis to the aglycones, these glycosides are conjugated with glucuronides and/or sulfates in the small intestinal cells and most complex substances are metaboliezed and get into the intestinal lumen, and only a small part is transported into the blood stream where its concentration is of < 1.5 µM.15 Finally it is excreted into urine within a short term (̴ 25 h) after ingestion.16 Both Qu and FA exhibit many biological activities such as anti-oxidation and anti-inflammation. The best-described property is the capacity of antioxidation by scavenging reactive oxygen species (ROS) and inhibiting free radical related lipid oxidation.17 Most of previous studies mainly focused on the absorption, transformation and metabolism of phenolic compounds ingestion in the intestinal cells and other organs, such as liver and kidney.8,18 However, less research has been performed for the biochemical effects of them on the endogenous metabolic profiling in animals or humans, which is directly or indirectly related to the prevention of various diseases. Increasing evidence shows that NMR-based metabolomics is a vigorous tool in analyzing the metabolic responses to a range of toxic insult, drug action and disease.19-21 Statistical data analysis including pattern recognition (PR) methods, such as principal components analysis (PCA) and partial least-squares (PLS), can facilitate the NMR data 4


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and provide assessment of toxic lesion processes, and characterization of drug action mechanism.22,23 This approch has been successfully employed to investigate the biochemical effects of the bioactive compounds such as epicatechin in a rat model, which showed a demonstration of the overall endogenous metabolic effects of epicatechin consumption.24 Moreover, the biochemical effects of soya isoflavones ingestion in humans has also been invesigated by using NMR spectra on biofluids.25 In the current study, 1H NMR-based metabolomics coupled with multivariate statitistical analysis were employed to analyze the biological effects of FA and Qu consumption on the endogenous metabolic profiling in urine and fecal extracts of rats. The aim of this study is to enrich our understanding in bioavailability and bioactivity of phenolic compounds such as FA and Qu.

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Materials and Methods Tested Reagents and Diet Ferulic acid (98%) and quercetin (98%) were purchased from Advanced Technique Industry Co. (HongKong, China). The diet containing FA and Qu was maken by mixing the powders of FA, Qu and chow diet with the method of step-by-step magnification, thus the mixed diet were uniform and FA/Qu were well-distributed physically in the chow diet. Animal Treatment The procedures of animal raising and treatment were carried out based on the National Guidelines for Experimental Animal Welfare (MOST of People’s Republic of China, 2006) and were approved by the Local Animal Welfare Committee with permission No. SYXK (E) 2009-0051. A total of fourty one 7-week-old male Sprague-Dawley (SD) rats were obtained from Tongji Medical School, Huazhong University of Science and Technology, and raised in groups of three to five per cage in a condition-well controlled room (12 h light/dark cycle and constant temperature of 22 ± 1 ℃) and given water and food ad libitum. After acclimatization for one week, rats were randomly divided into three groups as control fed with normal chow diet (n=13), treatment groups fed wth FA-containing (n=14) and Qu-containing chow diet (n=14) for one week, respectively. Daily food intake was recorded for rats and the average FA or Qu consumption was around 0.4 mg/kg body weight/day. Average daily wet mass of feces was around 6 g. Feces and 24-h pooled urine (daily volume of 0.8-1.0 mL) samples were collected individually by using metabolic cages at day 3 and day 6 over the one-week experimental period. After collection, the samples were qucikly snap frozen in liquid nitrogen and then stored at -80 ℃. 6


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Sample Preparation for NMR Spectroscopy Urine samples were prepared by mixing 550 µL of each urine and 55 µL phosphate buffer (K2HPO4/NaH2PO4, 1.5 M, pH 7.4, 50% v/v D2O)26 containing internal standard (0.005% TSP-d4) as a chemical shift reference (δ 0.00). The supernatant was transfered into NMR tubes for analysis following centrifugation at 16099 g for 10 min. Fecal metabolites were extracted as previously described.31 Briefly, fecal pellets (~50 mg) were homogenized in 750 µL of a phosphate buffer solution (0.1 M sodium phosphate buffer and pH 7.4) containing 30% D2O, 0.2% NaN3 and 0.005% TSP-d4. The sample was mixed vigorously using a vortex mixer for 1 min. The homogenates were then snap frozen in liquid nitrogen and subjected to three freeze-thaw cycles with rapid and vigorous manual mixing between each cycle. After the third cycle, the sample was further comminuted using a tissue-lyzer (Qiagen TissueLyser, Retsch GmBH, Germany). Such fecal extraction procedure was repeated twice. The obtained supernatants were combined together following with centrifugation for 10 min (11180 g and 4 ℃) and 550 µL supernatants were used for NMR analysis. 1

H NMR Spectra of Urine and Fecal Extraction 1H NMR spectra of urine and aqueous

fecal extraction were performed on a Bruker Avance III 600 MHz spectrometer equipped with an inverse detection cryogenic probe operating at 600.13 MHz for 1H (298 K). A water-suppressed one-dimensional NMR spectrum was acquired employing the typical NOESY pulse sequence (recycle delay-90º-t1-90º-tm-90º-acquisition) with optimized NMR parameters including a recycle delay, T1 of 2 s, t1 of 3 µs and a mixing time, tm, of 100 ms. Other parameters such 90o pulse (̴ 10.0 µs), data points (32 K) for each spectrum and 7



spectral width (20 ppm) were also adjusted and optimized according to the experimental requirements. In order to identify NMR signals, a range of 2D NMR spectra including 1

H-1H correlation spectroscopy (COSY), 1H-1H total correlation spectroscopy (TOCSY),

1

H-13C heteronuclear single quantum correlation (HSQC), and 1H-13C heteronuclear

multiple bond correlation (HMBC) were performed and processed for selected samples as described previously.27-29 Data Processing and Statistical Analysis All free induction decays were processed by Fourier transformation after multiplying an exponential function with a 1 Hz line broadening factor. The spectra were calibrated to TSP-d4 at δ 0.00. Following NMR spectral phase and baseline correction, 1H NMR spectrum of urine (δ 0.5-9.5) and fecal extraction (δ 0.6-9.0) was decomposed into regions with equal width 0.004 ppm (2.4 Hz) and integrated using AMIX software package (V3.8, Bruker-Biospin, Germany). The signals located at δ 4.7-5.3 and δ 4.5-5.3 in the spectra of fecal extraction and urine were were discarded for imperfect water suppression and the region δ 5.5-6.0 in the spectra of urine was also removed for urea signal. After normalization of each bucketed region to the total integration of NMR signals, multivariate analysis was performed on the data sets with the SIMCA-P+ software (version 12.0, Umetrics, Sweden). Principal Component Analysis (PCA) with mean centering was initially conducted on each type of NMR data separately to generate an overview for the change tendency. To obtain information on the significantly changed metabolites contributed to classifications, Orthogonal Projection to Latent Structure with Discriminant Analysis (OPLS-DA) was subsequently performed on the unit variance scaling (UV) NMR data and cross-validated with P values of CV-ANOVA. The 8


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loadings with color-coded correlation coefficient plots were processed according to the procedure described by previous study.30 Here a cutoff value of |r| > 0.514 (r > 0.514 and r < -0.514) was used for correlation coefficient as significant based on the sample number and discrimination significance (p < 0.05). Further, relative quantifications of the significantly changed metabolites to the internal standard (TSP-d4) were also obtained in urine and fecal extraction of FA and Qu-treated rats. Graphical illustrations and statistical analysis were employed with GraphPad Prism version 6.0 (GraphPad). Multiple group comparisons were performed by two-tailed Student’s t-test or Mann-Whitney test and P-values < 0.05 were regarded as significance. Results NMR-based Metabolic Profiling Figure 2 shows the typical 1H NMR spectra for urine collected from a control rat (A), FA-treated rat at day 3 (B) and day 6 (C), Qu-treated rat at day 3 (D) and day 6 (E). The NMR signals of metabolites were identified in accordance to previous publications and further confirmed with 2D NMR spectra.26,31 The detailed information such as chemical shifts and peak multiplicities are shown in Table S1. The endogenous metabolites were mainly comprised of a number of organic acids including butyrate, methylmalonate, lactate, acetate, taurine, and, hippurate, tricarboxylic acid (TCA) cycle intermediates (citrate, 2-oxoglutarate and succinate), and alanine, dimethylamine, dimethylglycine, creatinine, urea and allantoin. Visually, the NMR spectra showed remarkable differences in the whole metabolic profiles between urine samples obtained from control and dietary FA or Qu rats. Dietary FA rat (Figures 2B and C) had relatively higher levels of urinary hippurate and fumarate but lower levels of 2-oxoglutarate, 9



creatinine and butyrate than control (Figure 2A). Similarly, comparison of the urinary NMR spectra from control and dietary Qu rats (Figures 2D and E) indicated that the levels of taurine, 2-oxoglutarate, hippurate, and phenylacetylglycine (PAG) were changed as consequences of Qu intake. Figure 3 shows the typical 1H NMR spectra of fecal extracts obtained from a control (A), FA-treated rat at day 3 (B) and day 6 (C), Qu-treated rat at day 3 (D) and day 6 (E). The main peaks observed belong to bile acids, various organic acids, such as acetate, propionate and lactate, some amino acids (tyrosine, glycine and phenylalanine), nucleotide metabolites, such as uracil, adenine and hypoxanthine, monosaccharides (glucose, arabinose, galactose and xylose) and oligosaccharides, as well as TCA cycle intermediates (fumarate and succinate), which are all shown in Table S1 with their NMR parameters. Statistical data analysis of these NMR metaboplic profiles were employed to obtain the detailed information about metabolic alterations of rats after FA or Qu consumption. Metabolic Alterations Induced by Dietary FA and Qu PCA of the normalized 1H NMR data from urine and fecal extracts was carried out to produce an overview of the variations between the controls and those treatments with dietary FA or Qu. Subsequently, pair-wise OPLS-DA was conducted comparing the NMR data from control rats with the FA and Qu treatment at matched time points for the biological smples (Figures 4-7). In urine, rats after dietary FA igestion exhibited significant differences from controls in the 1H NMR profiles, which were verified with the validated model parameters at both time points. Relative quantification of the significantly changed metabolites was obtained from the urine of rats after FA and Qu treatment for 3 days and 6 days (Figure 4C). 10


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Compared to the control, animals after FA consumption have marked higher levels of urinary hippurate and fumarate but lower levels of TCA cycle intermediates (2-oxoglutarate and citrate), creatinine, and allantoin at day 3. In addition, FA treatment also caused lower levels

of

butyrate,

dimethylamine

(DMA),

phenylacetylglycine

(PAG)

and

indoleacetylglycine (IAG) compared to controls at day 6 (Figure 4C). Qu treatment significantly induced increases in the levels of betaine, fumarate and hippurate and reduction in the levels of creatinine over the experimental period. Furthermore, Qu-treated rats have lower levels of butyrate, PAG and IAG but higher level of taurine at day 6, and lower level of allantoin at day 3 (Figure 5C) than those in urine samples of controls. The metabolic profiles of fecal extracts were also markedly altered after FA and Qu ingestion as indicated by the OPLS-DA coefficient loading plots (Figures 6 and 7). Dietary FA significantly resulted in the lower levels of butyrate, phenylalanine and some nucleotide metabolites, such as uracil, adenine and hypoxanthine at day 3 of post treatment compared to normal dietary group. At day 6 of post treatment, FA-treated rats showed lower levels of bile acids, propionate and some amino acids including leucine, isoleucine, valine, alanine, lysine, arginine, glutamine, aspartate, asparagine and tyrosine, which were not observed at day 3. Further, FA caused higher levels of histidine, 3-4-dihydroxyphenylacetate and oligosaccharides in feces compared to those of controls (Figure 6C). Dietary Qu caused lower levels of fecal butyrate, 3-4-dihydroxyphenylacetate and some amino acids including glutamine, aspartate, histidine and tyrosine from day 3 onward. At day 6, Qu consumption also induced lower levels of fecal bile acid, leucine, isoleucine, valine, phenylalanine, and some nucleotide metabolites and higher levels of oligosaccharides (Figure 7C). 11



Discussion NMR resonances of FA were clearly observed here in urine sample, suggesting that FA could be excreted in part as conjugated FA-glucuronide form or as its free form in urine.34 Previous studies showed that most phenolic compounds were transformed in the colon by the intestinal microbiota before absorption and metabolized mainly in the liver, metabolism however took place first at the intestinal mucosa32,33 when smaller dosages were used. Both Free FA and conjugated FA can enter the systemic circulation and reach many tissues such as kidney, where a fraction of the FA is excreted into urine.34 This is contrast to Qu, where free or conjugated form of Qu was not observed in urine in this study. Previous research showed that most of Qu enter into the intestinal lumen, and only a small part is transported into the circulation of the blood at concentrations of

Metabolomics Reveals that Dietary Ferulic Acid ... - ACS Publications

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