**First-Pass Metabolism of Polyphenols from Selected Berries: A High-Throughput Bioanalytical Approach**

**Francisco J. Olivas-Aguirre 1,\*, Sandra Mendoza 2, Emilio Alvarez-Parrilla 3, Gustavo A. Gonzalez-Aguilar 4, Monica A. Villegas-Ochoa 4, Jael T.J. Quintero-Vargas <sup>1</sup> and Abraham Wall-Medrano 3,\***


Received: 29 March 2020; Accepted: 11 April 2020; Published: 13 April 2020

**Abstract:** Small berries are rich in polyphenols whose first-pass metabolism may alter their ultimate physiological effects. The antioxidant capacity and polyphenol profile of three freeze-dried berries (blackberry, raspberry, Red Globe grape) were measured and their apparent permeability (Papp) and first-pass biotransformation were tracked with an ex vivo bioanalytical system [everted gut sac (rat) + three detection methods: spectrophotometry, HPLC-ESI-QTOF-MS, differential pulse voltammetry (DPV)]. Total polyphenol (ratio 0.07-0.14-1.0) and molecular diversity (anthocyanins > flavan-3-ols), antioxidant capacity (DPPH, FRAP), anodic current *maxima* and Papp (efflux > uptake) were in the following order: blackberry > raspberry > Red Globe grape. Epicatechin, pelargonidin & cyanin (all), callistephin (raspberry/blackberry), catechin (grape), cyanidin glycosides (blackberry) and their derived metabolites [quinic acid, epicatechin, cyanidin/malvidin glucosides, and chlorogenic/caffeic acids] were fruit-specific and concentration-dependent. Time-trend DPV kinetic data revealed concurrent epithelial permeability & biotransformation processes. Regular permeability and high-biotransformation of berry polyphenols suggest fruit-specific health effects apparently at the intestinal level.

**Keywords:** anthocyanins; berries; polyphenols; bioaccesibility; differential pulse voltammetry; first-pass metabolism; HPLC-ESI-QTOF-MS; apparent permeability

### **1. Introduction**

Regular consumption of small berries has been associated with several health benefits. Epidemiological studies and controlled clinical trials indicate that their acute-chronic consumption exerts synergistic and independent effects on lowering several pathophysiological markers including hyperglycemia, hyperinsulinemia, dyslipidemia, pro-inflammatory cytokines, hypertensive factors and oxidative stressors [1]. In fact, there is also an inverse association between berry consumption and many risk factors for cardiovascular disease and type-2 diabetes [2]; most, if not all, of these health benefits, are related to the amount and phytochemical diversity present in each berry, from which those with antioxidant activity (e.g., polyphenols) have been the most studied [3]. However, the heterogeneity in physiological response after their intake can hinder their beneficial effects in specific subpopulations [1,4].

To exert their physiological effects, berry polyphenols must be present in sufficient amounts in raw or prepared foods, be both bioaccessible (the fraction released from the food matrix during gastrointestinal digestion) and bioavailable (the fraction that reaches systemic circulation as the parent compound or a metabolite). The bioaccessibility of polyphenols is closely related to their physicochemical structure, the food matrix that contains them and the presence of anti-nutritional factors that could interfere with their release ability and intestinal absorption [5]. In this sense, the bioaccessibility of polyphenols from berry fruits is higher compared to other fruits, due to a concomitant effect between their natural higher level [6] and their low content of non-digestible carbohydrates and protein, both associated with an efficient gastrointestinal (GI) delivery that enhances their bioavailability [2,7].

On the other hand, the absorption, pharmacokinetics and systemic metabolism of polyphenols and their and biotransformation by the GI microbiota, have been extensively studied in the last decade [2,5]; however, the first-pass metabolism (a.k.a. pre-systemic metabolism) of bioaccessible polyphenols also modifies their ultimate health effects [1]. Polyphenols present in plant foods are commonly biotransformed (e.g., conjugation, de-glycation) before and during their pre-systemic passage, a phenomenon that involves several brush-border enzymes and a tightly-regulated influx/efflux interchange that sustain their cellular homeostasis [8,9]. These events are not normally considered when evaluating the pharmacokinetics of polyphenols, partially due to the absence of high sensitivity methods [10,11] to record such metabolic changes.

The evaluation of bioaccessible and bioavailable fractions of polyphenols has been proposed recently as a "quality" parameter in berry breeding programs. However, studies reporting the extent to which bioaccessible polyphenols are bio-transformed during their first-pass metabolism and colonic fermentation are still very scarce [2,9]. In this study, we used a high-throughput ex vivo bioanalytical system [everted gut sac (rat) + three detection methods: spectrophotometry, HPLC-ESI-QTOF-MS, differential pulse voltammetry (DPV)] to evaluate the apparent permeability (*Papp*) and enteral biotransformation of polyphenols from three berry fruits with graded levels of polyphenols (blackberry > raspberry > Red Globe grape); to the best of our knowledge, this ex vivo bioanalytical approach is reported for the first time.

### **2. Materials and Methods**

### *2.1. Chemicals and Standards*

Pure (≥ 93%) chemical standards were purchased from Cayman Chemicals (Ann Arbor, MI, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fluorescein, Folin–Ciocalteu (FC) phenol reagent, ACS-grade salts & acids and all enzymes and chemicals used for the *in vitro* digestion and *ex vivo* apparent permeability assays, were purchased from Sigma-Aldrich Fluka (St. Louis, MO, USA). Analytical and HPLC-MS grade solvents were obtained from JT-Baker (Avantor Performance Materials S.A. de C.V., Ecatepec de Morelos, Estado de Mexico, Mexico); sodium pentobarbital (Pisabental®) was acquired from PISA Agropecuaria (Guadalajara, Jalisco, Mexico).

### *2.2. Samples and Extracts*

Fully ripe Red Globe grape (*Vitis vinifera* L., 18◦ Brix, pH 4), raspberry (*Rubus idaeus L,* 10◦ Brix, pH 3) and blackberry (*Rubus* spp., 10◦ Brix, pH 3) were purchased locally (Ciudad Juarez, Chihuahua, Mexico; 31◦44 22"N, 106◦29 13"O), transported immediately under cooling conditions (2–4 ◦C), frozen (−80 ◦C), freeze-dried [−42 ◦C, 48 h; light-protected vessels (LabconcoTM Freezone 6, Labconco Co., Kansas City, MO, USA)], grounded to a fine powder (≤0.40 μm) and kept at −20 ◦C until use. Organic extracts (80% methanol) from all three freeze-dried samples (1:20 w/v; three batches per sample) were obtained by ultra-sonication (10 min; Fisher Scientific FS220H, Thermo Fisher Scientific, Waltham, MA, USA), centrifugation (4 ◦C, 15 min, 1650<sup>×</sup> *<sup>g</sup>*; Eppendorff® centrifuge, mod. AG 5810R, Hamburg, Germany) and rotoevaporation (40 ◦C; Büchi® R-114 evaporator, Büchi Labortechnik AG, Flawil, Switzerland). Freeze-dried samples were further dissolved in HPLC grade or Milli-Q water for analysis.

### *2.3. High-Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (HPLC-ESI-QTOF-MS)*

Identification of individual polyphenols in organic extracts (methanol 80%) was carried on an HPLC-ESI-QTOF-MS instrument according to Torres-Aguirre et al. [12]. Chromatographic separation was performed on an Agilent 1200 series system (Agilent Technologies, Palo Alto, CA, USA). The equipment was equipped with a vacuum degasser, an auto-sampler, and a quaternary pump. Extracts (sugar-free) were firstly separated using a reverse phase C18 analytical column (2.1 mm × 50 mm × 1.8 μm particle size; ZORBAX Eclipse Plus), protected with a guard cartridge of the same packing and maintained at 25 ◦C. The mobile phase [formic acid (0.1%) in Milli-Q water (A) and acetonitrile (B)] was pumped at 0.4 mL/min into the HPLC System. Two microliters were injected, and the sample was eluted following the gradient elution program: 0–4 min (90% A), 4–6 min (70% A), 6–8 min (62% A), 8–8.5min (40% A), 8.5–9.5 min (90% A) and the column was further re-equilibrated for 3 min. The quadrupole time-of-flight mass spectrometer (QTOF-MS; Agilent Technologies, Palo Alto, CA, USA) was coupled to a dual electrospray ionization (ESI) interface. The ESI-QTOF-MS operating conditions were: Source temperature (120 ◦C), gas desolvation temperature (340 ◦C), drying, nebulizing and collision gas (nitrogen; 13 L/min), capillary voltage (4.5 kV) and mass scan (100–1000 *m*/*z*).

Before analysis, all samples (three batches x triplicate, *n* = 9) were filtered and concentrated by solid-phase extraction (Oasis HLB micro Elution plates, 96-well, 30 μm; Waters, Milford, MA, USA). Individual polyphenol identification was done by comparing the exact mass and molecular composition of the pseudo-molecular ion and/or quantification was performed by comparing with retention times (*rt*), UV-Vis spectra and molecular ion mass [*m*/*z* <sup>±</sup> 0.1, M−H<sup>+</sup> (anthocyanins and rutin) or M−H<sup>−</sup> (all other polyphenols) mode] of pure phenolic standards (freshly prepared from stock solutions for each measurement), using the Mass Hunter Workstation Data Acquisition Software (ver. B.07.00; Agilent Technologies, Inc.) and an open-access MS-library (MassBank; https://massbank.eu/MassBank). The individual concentration of phenolic compounds was expressed in μg/g extract. Three different batches by triplicate (*n* = 9) from each fruit were evaluated.

### *2.4. Total Antioxidant Capacity*

Trolox equivalent antioxidant capacity (TEAC) of organic extracts (pure methanol; 1:20 w/v) per sample (three batches by quadruplicate, *n* = 12) was evaluated by the DPPH method (515 nm), the ferric ion reducing antioxidant power assay (FRAP, 630 nm) and the oxygen radical absorbance capacity [ORAC; fluorescein: 10 nM, (excitation (485 nm)/emission (520 nm), AAPH (240 mM)], as previously described [13], using a FLUOstar™ OMEGA spectrophotometer (BMG LABTECH; Chicago, IL, USA) in UV/VIS (DPPH, FRAP) and fluorescence (ORAC) modes. For all three assays a trolox standard curve [0.006–0.2 <sup>μ</sup>mol/mL, R2 <sup>≥</sup> 0.95] was used. Values were expressed as mg or <sup>μ</sup>mol of trolox equivalents (TE) per g (DPPH, FRAP) or <sup>μ</sup>mole 1 <sup>×</sup> 1010 (ORAC) per g of freeze-dried sample <sup>±</sup> standard deviation (*n* = 12) and as percentage considering blackberry antioxidant titers as 100% (sample with the highest total polyphenol content) [7].

### *2.5. In Vitro Digestion*

The method reported by Campos-Vega et al. [14] with minor modifications was used. For the oral stage, three otherwise healthy subjects were invited to participate in the study, providing written informed consent prior to participation. In fasting conditions and after brushing their teeth without toothpaste, each subject chewed each freeze-dried fruit (1 g × three batches, *n* = 3) 15 times for approximately 15 s. Chewed samples were collected into a beaker containing 5 mL of distilled water and subjects rinsed their mouths with another 5 mL of distilled water for 60 s. The volume of saliva +

water was considered for data correction. For the gastric stage, pooled salivary samples per subject and sample were re-mixed per participant (*n* = 3) in an aseptic vessel and an aliquot (10 mL) was adjusted to pH 2 using HCl solution (2 N). Pepsin from porcine gastric mucosa (55 mg ≥ 250 units/mg protein, Sigma-Aldrich) dissolved in 0.94 mL of 20 mM hydrochloric acid was added to each sample and incubated for 2 h at 37 ◦C with constant agitation. For intestinal stage, a simulated intestinal extract was prepared 30 min before use by dissolving gall Ox (3 mg of bovine bile; CAS: 8008-63-7, Sigma-Aldrich) and porcine pancreatin (2.6 mg, 8 × USP, Sigma-Aldrich, St. Louis, MO, USA) in 5 mL Krebs–Ringer buffer (118 mM NaCl, 25 mM NaHCO3, 11 mM glucose, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4; pH 6.8]. Five mL of this solution were added to each sample coming from the gastric stage, pH adjusted to 7.2–7.4 with NaOH (2 M) and incubated for 2 h at 37 ◦C with constant agitation. All digested samples [three (pooled samples from gastric-to-intestinal phases per berry fruit) × triplicate, *n* = 9], were immediately transferred to the ex vivo bioanalytical system.

### *2.6. Rat Everted Gut Sacs*

Six small intestinal gut sacs were obtained from six young male albino rats (~300 g BW) which were fasted overnight (16–20 h) and anesthetized with an intraperitoneal injection of sodium pentobarbital (70 mg/kg BW, Pisabental, Guadalajara, Jalisco, Mexico), before surgical procedures and euthanasia, as suggested by Campos-Vega et al. [14]. Briefly, the intestine was exposed by a midline abdominal incision and 20–25 cm of the jejunal section was excised and placed in a gasified (CO2) Krebs-Ringer buffer solution at 37 ◦C. Each gut sac was gently washed externally with the same buffer and everted over a glass rod, re-excised into 6 cm-segments, filled (basolateral side) with 1 mL of Krebs-Ringer buffer (to avoid tissue denaturation) and fastened with braided silk sutures to a final length of approximately 4 cm [15]. Experiments were performed by triplicate and a blank was prepared using distilled water instead of in vitro digested sample. The experimental protocol was approved by the Animal Experimentation Ethics Committee of the Autonomous University of Ciudad Juarez (Code FO-CIP-01/254063; 30 July 2016) and animals cared according to the corresponding Mexican regulations (NOM-062-ZOO-1999) and the National Institutes of Health (NIH) guide for the care and use of laboratory animals.

### *2.7. Real-Time Monitoring of Phenolic First-Pass Metabolism*

The ex vivo first-pass metabolism biosystem (Figure 1) consisted of a 15 mL of pre-digested (oral-gastric-intestinal) samples (*n* = 3 per dried berry) and three 4 cm-closed everted duodenal sacs incubated in an oscillating (60–80 cycles/ min) water bath at 37 ◦C for 2 h, in an anaerobic chamber. Bioaccesible (from Section 2.6) and biotransformed polyphenols, withdrawn from the apical side (Figure 1, "out"), were tracked by three independent analytical methods:

#### 2.7.1. Spectrophotometry (Method 1)

Total polyphenols were quantified spectrophotometrically (765 nm) with the FC method (TPFC) at the end of the experiment (120 min; *t120*); values were expressed as mean ± SD values [3 independent samples x triplicate, *n* = 9; mg of gallic acid equivalents (GAE)/ mL] as previously reported [7]. The apparent permeability coefficient (Papp; Equation (1), efflux (ER; Equation (2) and uptake (UR; Equation (3)) ratios were calculated as follows, using the concentration of total polyphenols (Method 1) inside (basolateral; B) and outside (apical; A) the everted sacs at 120 min (*t120*):

$$P\_{\rm app} = \left(\Delta Q \!/ \Delta t\right) \times \left(1 - A C\_0\right)^{-1} \tag{1}$$

$$\mathbf{ER} = (\mathbf{B} \to \mathbf{A}) \times (\mathbf{A} \to \mathbf{B})^{-1} \tag{2}$$

$$\text{UIR} = \left(\text{A} \rightarrow \text{B}\right) \times \left(\text{B} \rightarrow \text{A}\right)^{-1} \tag{3}$$

where <sup>Δ</sup>*Q*/Δ*t* is the steady-state flux (mg·s−1) of polyphenols transported across the membrane per second, *A* (cm2) is the surface area available for permeation and *C0* (mg/mL) represents the initial concentration of total polyphenols in the donor chamber (apical side of everted sacs; Figure 1). *Papp* (mean <sup>±</sup> SD) values were calculated and expressed in 10−<sup>5</sup> cms<sup>−</sup>1.

**Figure 1.** Real-time ex vivo monitoring of first-pass phenolic metabolism using the everted gut sac technique. The apparent permeability (P*app*) and biotransformation of bioaccesible phenolic compounds (PC) and their associated first-pass metabolites were followed by spectrophotometry (Folin–Ciocalteau; 120 min), HPLC-ESI-QTOF-MS (120 min) and, differential pulse voltammetry (DPV; 0 to 120 min). Diffusion from apical (A; out) to basolateral (B; in) and B-A were considered permeability and efflux, respectively.

### 2.7.2. Differential Pulse Voltammetry (DPV; Method 2)

Differential pulse voltammetry (DPV) real-time (0–120 min) measurements of mixed polyphenols (parent + metabolites) were monitored (by triplicate) using a potentiostat (BASi® EC Epsilon potentiostat/galvanostat; West Lafayette, IN, USA) and voltammetric measurements were carried out with a standard three-electrode electrochemical cell [working (glassy carbon, carefully polished with diamond spray, particle size 1 μM), counter (platinum wire) and reference (Ag|AgCl|KCl; 3M) electrodes]. Experimental conditions were: room temperature, pH, (7.2–7.4), scan range (0–600 mV) and rate (5 mVs<sup>−</sup>1), pulse width (70 ms) and amplitude (50 mV); these conditions were selected to avoid the interference of electrochemical species other than polyphenols and the current density (μ<sup>A</sup> <sup>×</sup> <sup>10</sup><sup>−</sup>5) from the first (t0) oxidation peak (current *maxima*; mean = 203 mV, range 180 to 216 mV) was chosen as the reference value to estimate total polyphenols by DPV (TPDPV)

### 2.7.3. HPLC-ESI-QTOF-MS (Method 3)

Non-targeted mass spectral identification (MassBank; https://massbank.eu/MassBank) and semi-quantification [as ion abundance (IA)] at*t0* and *t120* of parent polyphenols and their metabolites was performed by HPLC-ESI-QTOF-MS as reported above, following Koistinen et al. [10] recommendations. To avoid phytochemical loss by direct drying, individual samples (1 mL) were cleanup and concentrated by solid-phase extraction in Oasis HLB micro Elution 96-well plates (30 μm; Waters). Considering that real-time oxidation/reduction reactions readily occur within the ex vivo bioanalytical system used in this study, molecular ion identification was performed under the following considerations: *m*/*z* ± 0.3, <sup>M</sup>−H<sup>+</sup> (anthocyanins and rutin) or M−H<sup>−</sup> (all other polyphenols) mode].

### *2.8. Statistical Analysis*

Results were expressed as mean ± standard deviation (SD) obtained from at least by triplicate. Inter-group (Red Globe grape, raspberry, blackberry) comparisons were performed by one-way-ANOVA followed by Tukey's post hoc test and the statistical significance was defined at *p* < 0.05. When needed, Pearson's product-moment correlation (*r*) was used to establish any possible correlation between response variables. Quadratic/cubic regression curves were constructed to explain electrochemical data (DPV). All statistics were performed using the statistical program NCSS 2007 (NCSS, Statistical Software, Kaysville, UT, USA).

### **3. Results and Discussion**

### *3.1. Phenolic Profile of Berry Samples*

Edible berries are rich in flavan-3-ols and anthocyanins that are barely affected during processing [16], although their content and molecular diversity is cultivar dependent [6]. In a preceding paper [7] we reported the spectrophotometric estimation (per g DW) of polyphenol subgroups in freeze-dried Red Globe grape, raspberry, and blackberry as follows: total polyphenols 9.4, 17.6 and 22.7 mg GAE, flavonoids 7.0, 13.1 and 35.3 mg quercetin equivalents (QE), monomeric anthocyanins (0.01, 0.49 and 0.67 mg cyanidin-3-*O*-glycoside equivalents), proanthocyanidins (0.22, 0.23 and 0.06 mg QE) and hydrolysable phenols (3.7, 7.2, 11.5 mg GAE). In the present study, we confirmed that these polyphenol subgroup titers correlate (*r* ≥ 0.76) with the overall content (ratio 0.07-0.14-1.0) and molecular diversity of flavan-3-ols and anthocyanins in the same fruits (blackberry> raspberry > Red Globe grape; Table 1).

**Table 1.** HPLC-ESI-q-TOF-MS and cheminformatics of polyphenols from three edible berries 1,2.


<sup>1</sup> Results are expressed as mean (*n* ≥ 9) ± standard deviation (μg /g dry weight basis); different superscript letters between samples for a same compound means statistical differences (*p* < 0.05); retention time (*rt*, min), molecular ion [*m*/*<sup>z</sup>* ± 0.3, positive (anthocyanins) or negative (flavan-3-ols) mode], below quantification limit (–). <sup>2</sup> Total polar surface area (TPSA. Å2) and octanol/water partition coefficient (LogP) values were retrieved from Molinspiration chemoinformatics (https://www.molinspiration.com/), using each compound's canonical SMILE sequence retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/.

Our data also indicate that epicatechin, pelargonidin, and cyanidin-3,5-*O*-diglucoside (cyanin) were present in the evaluated samples, but catechin (Red Globe grape), kuromanin (cyanidin-3-*O*-β-glucoside) and cyanidin-3-*O*-arabinoside (blackberry) and callistephin (pelargonidin-3-*O*glycoside; raspberry and blackberry) were fruit-specific.

Several anthocyanidins (aglycones) and related anthocyanins (3-*O*-glycosides and acyl glycosides) have been identified in grapes and shrubby berries. Colombo et al. [17] identified several flavan-3-ols (e.g., catechin, epicatechin, proanthocyanidin di/trimers), flavanols (quercetin and derivates), anthocyanins (all but pelargonidin glycosides), *cis*-resveratrol and caftaric acid (esterified phenolic acid) in Red Globe grape. However, pelargonidin (3,5,7,4'-tetrahydroxyflavium) and its 3-O-glycoside (callistephin) were not reported by these authors although they have been reported, in trace amounts, in certain grape varieties [18]. Also, blackberry and raspberry were better sources of polyphenols as compared to Red Globe grape, particularly in anthocyanin content. It is well-known that these berries are good sources of flavones (e.g., apigenin, chrysin), flavonols (e.g., kaempferol), phenolic acids (e.g., ellagic acid, caffeic acid), ellagitannins (e.g., sanguiin H-6, lambertianin C), anthocyanidins (all but peonidin) and anthocyanins such as cyanidin, delphinidin and pelargonidin glycosides [3,16,19].

The amount and natural occurrence of anthocyanidins (aglycone) and derived anthocyanins (glycosylated forms) are influenced by many factors including the type of cultivar and pre/postharvest handling of grapes [18], blackberries and raspberries [6]. Besides this, biosynthesis of anthocyanins in berry fruits is tightly controlled during the transcription of several genes involved in the flavan-3-ol proanthocyanidin pathway, in a fruit-specific manner [18,20]. Taking this into consideration, blackberry and raspberry are more valuable than Red Globe grape from a nutraceutical standpoint [1], even if their parent polyphenols are biotransformed into other ones [20,21]; the specific phenolic fingerprint of these two berries may be related to different yet complementary effects on preventing several non-communicable chronic diseases including certain types of cancers, cardiovascular disease, type II diabetes, inflammation and oxidative stress [2].

### *3.2. Antioxidant Capacity of Berry Samples*

The antioxidant capacity of a given molecule (or a complex antioxidant mixture) is defined by its ability to reduce free reactive species (pro-oxidants or free radicals). The evaluation of the antioxidant capacity of plant-based foods by simultaneously using more than one method is a recommended practice in food science and technology [13].In this study, we used a single electron-transfer (SET; FRAP), a hydrogen atom transfer (HAT; ORAC) and a combined SET/HAT (DPPH) method to assay the antioxidant capacity in the studied samples (Figure 2).

**Figure 2.** Antioxidant capacity of hydroalcoholic extracts from commercial *Red Globe* grape, raspberry and blackberry. Values were expressed as mean [*n* <sup>≥</sup> 9; mg TE/ gDW (DPPH, FRAP) or 1 <sup>×</sup> 10<sup>1</sup> <sup>μ</sup>mol TE/g DW (ORAC)]; percentages above bars indicate differences between samples considering blackberry antioxidant titers as 100%.

FRAP and DPPH values ranged between 5 (Red Globe grape)-19 (blackberry) and 10 (Red Globe grape)-20 (blackberry) mg TE/ g DW and from 11 (raspberry) to 16 (Red Globe grape) <sup>×</sup> 10<sup>1</sup> <sup>μ</sup>mol TE/g DW with the ORAC method; similar trends in antioxidant capacity have been reported by other authors for the same berry fruits [3,16]. Also, the antioxidant capacity trend (blackberry > raspberry > Red Globe grape) was directly proportional to their phenolic content (Table 1), as measured by FRAP (100%-71%-26%; *r* = 0.86) and DPPH (100%-80%-49%; *r* = 0.84) methods but not with ORAC (100%-95%-140%; *r* = −0.42). DPPH and FRAP titers also correlate (*r* ≥ 0.95) with all polyphenol subgroups (total polyphenols, flavonoids, and anthocyanins) reported in our previous study [7] and same results have been reported for other polyphenol-rich fruits [6].

Conventionally, the higher the content of polyphenols in berry fruits, the higher their antioxidant capacity. It is important to point out that the radical scavenging capacity of most polyphenols is mediated by HAT rather than SET mechanisms. However, the antioxidant capacity is also related to the number and position of hydroxyl groups, the O–H bond dissociation enthalpy and conjugation/resonance effects [21]. Although the main antioxidant capacity mechanism in complex phytochemical mixtures is difficult to establish, the observed antioxidant capacity pattern (mostly blackberry > raspberry > Red Globe grape) apparently is the result of synergism between flavonoid species, the number of their available hydroxyl groups (particularly *O*-hydroxyls inA&B rings) and their level of glycosylation [21,22]. In support of this, Rice-Evans et al. [23] reported the following trend in antioxidant capacity with the ABTS radical (mixed SET/HAT mechanism): cyaniding > epicatechin/catechin > oenin > pelargonidin, and so, major drivers of the overall antioxidant capacity in the studied samples seem to be catechin (Red Globe grape), epicatechin (raspberry, blackberry) and cyanidin glycosides (blackberry). Lastly, cyanidin has a higher antioxidant capacity than its derived glycosides in the ORAC assay [24] and the catechin content shows a better lineal relationship with ORAC values than that observed with other flavan-3-ols [25]; whether these arguments justify Red Globe grape's antioxidant activity in the ORAC method (Figure 2 merits future study.

### *3.3. Apparent Permeability of Berry Polyphenols*

The net bioaccesibility (TPFC = oral + gastric + intestinal) of polyphenols (as mg GAE per g DW) from Red Globe grape, raspberry, and blackberry was 2.0, 3.6 and 4.2 (A*t0*, Table 2) which represents 21.3, 20.4 and 18.6% of their original content [7].


**Table 2.** Apparent permeability of phenolic compounds from selected berries.1,2.

<sup>1</sup> Results are expressed as mean ± standard deviation (*n* ≥ 9; mg GAE /g dry weight; Folin-Ciocalteu method), different superscript letters within a same row means statistical differences (*p* < 0.05). <sup>2</sup> Total polyphenol content by the Folin-Ciocalteu method (TPFC), basal (*t0*) and final (*t120*) apical (A) or basolateral (B) concentration. Apparent permeability coefficient (Papp; cm\*s-1 x 10-5). Statistical difference between efflux (ER) vs. uptake (UR) ratios as determined by t-student test (*p* < 0.05).

In our preceding study we also reported that anthocyanins, but no other flavonoids were pH-unstable under simulated intestinal conditions (pH 7.0); similar results have been reported for strawberry [26], maqui berry [27] and blueberry [28]. After intestinal digestion (Table 2), 1.3, 1.5 and 2.4 mg GAE per g DW remain in the apical side (At120) suggesting 35%, 58% and, 43% of net polyphenol absorption; however, the basolateral (serosal) concentration of polyphenols at 2h (Bt120) was blackberry (0.13) > Red Globe grape/ raspberry (~0.095) and the absorptive (At120→Bt120) and secretory (Bt120→At120) Papp (cm·s−<sup>1</sup> <sup>×</sup> <sup>10</sup><sup>−</sup>5) were Red Globe grape (1.20)<sup>&</sup>gt; blackberry (0.06)/ raspberry (0.07) and Red Globe grape (1.55)/blackberry (1.38) > raspberry (0.98), respectively. In consequence, efflux (19.1, 16.1, 1.3) were higher than uptake (0.05, 0.06, 0.78) ratios were fruit-specific (*p* ≤ 0.02) and concentration-dependent (blackberry > raspberry > Red Globe grape).

Many transport mechanisms seem to be involved in the uptake/efflux behavior of polyphenols. Dixit et al. [29] used a standardized everted sac-based biosystem to evaluate the permeability behavior of atenolol (Pubchem CID: 2249; XLogP3 = 0.2, simple paracellular transport), metoprolol (Pubchem CID: 4171; XLogP3 = 1.9, transcellular transport) and propranolol (Pubchem CID: 4946; XLogP3 = 3.0; passive diffusion), reporting absorptive (A→B) *Papp* values of 0.054, 0.84 and 1.64 cms−<sup>1</sup> <sup>×</sup> 10−5, respectively. Considering miLogP values reported in Table 1, and the fact that anthocyanins and anthocyanidins cannot cross cell membranes passively, passive (Red Globe grape) and paracellular transport (blackberry/raspberry) may be major transport mechanisms in the studied samples.

Molecular bioinformatics provided information on the capability of each berry polyphenol to be absorbed by the intestinal epithelia. Most polyphenols listed in Table 1 had a topological polar surface area [TPSA; 92.1 (pelargonidin) to 270.6 (cyanin)] and octanol/ water partition coefficient [LogP; −4.61 (cyanin) to 1.37 (catechin/epicatechin)]. It is known that phytochemicals with a TPSA > 140 Å2 or <sup>≤</sup> 60 Å2 have low and high permeability, respectively and those neutral or with a LogP <sup>&</sup>gt; 2.0 easily permeate by passive diffusion [30]. The "pH partition" hypothesis postulates that non-ionized

(neutral) molecules are passively transported through the lipid membranes [30] and according to the modified *theoretical transcellular permeability* model proposed by Farrell et al. [31], the *Papp* of monomeric polyphenols is well explained by *logP* and molecular weight. Based on this, active more than passive transport and regular permeability of polyphenols from the assayed berries should be expected [8,9].

However, P-glycoprotein (*P-gp* or MDR-1) and breast cancer resistance protein (BCRP) can simultaneously reduce the first-pass bioavailability of certain polyphenols by acting as efflux modulators [5,32], as it has been proposed for polyphenols coming from spent coffee [14] and mango bagasse [33]. In fact, certain polyphenols may also act as competitive inhibitors of *P-gp* since it has a high affinity toward molecules with a planar ring system ranging from 200 to 1900 Da [32]. However, uptake/efflux behavior is also an asymmetric function that depends on the chemical structure of a particular polyphenol to be absorbed and the rate to which it is bioaccesible during *in vivo* or *in vitro* gastrointestinal conditions [14,33,34]; but such dynamic changes in transport behavior could not be observed with our endpoint assay (Method 1).

### *3.4. Ex Vivo Biotransformation of Berry Polyphenols*

As previously discussed, several in vitro and ex vivo permeability models have been developed to mimic specific aspects of gastrointestinal metabolism, each one with advantages and disadvantages [15,29]. Particularly, the sensitivity and reliability of the everted gut sac technique increase when combined with high-throughput detection methods such as electrochemical and HPLC/MS-based methods [10,34]. On the other hand, experimental data such as retention time (*rt*), mass-spectra (*m*/*z*+/−) of a particular standard molecule is often used in targeted metabolomics (Level 1) to objectively identify and quantify the same molecule in a given biological sample that presumably contains it. Conversely, untargeted metabolomics (Level 2) focuses on the global detection and relative quantitation of metabolites of unknown chemical nature, and their putative identification and semi-quantification rely upon spectral matching with databases (e.g., MassBank) or as suggested in earlier publications [10]. In this study, a level 2 strategy was used to partially identify (HPLC-MS-QTOF-MS) and semi-quantify (DPV current *maxima* and ion counts) of major [effluxed (B-A)] metabolites coming from the *ex vivo* biotransformation of parent polyphenols (Table 1).

### 3.4.1. DPV

Voltammetric methods are useful to predict the antioxidant activity of plant foods [11] and biological samples. Particularly, *maxima* DPV anodic current highly correlates with the antioxidant capacity (FRAP, ABTS and, DPPH) and concentration (HPLC and spectrophotometry) of pure polyphenols [35,36] but it is more sensitive (μM vs. mM) to quantify total polyphenols (TPDPV) in grapes, raspberries and red wines [11,19]. DPV has been also used to evaluate the *Papp* of specific drugs in vitro using intestinal cell monolayers or ex vivo using intestinal reperfusion models. However, to our knowledge, reports on the electrodynamics of polyphenols during their first-pass metabolism using an everted gut sac have not been reported before. DPV voltammograms (vs. Ag|AgCl|KCl reference electrode) of pre-digested berries at neutral pH (Figure 3) showed the following trend [potential peak (mV)/ anodic current density (μA <sup>×</sup> 10−5)]: 180/0.24 (*Red Globe* grape) > 216/0.34 (raspberry) > 212/0.44 (blackberry); the estimated TPDPV ratio was 0.6:0.8:1.0 which linearly correlated (r ≥ 0.95) with TPFC (Table 2) and antioxidant capacity (Figure 2; DPPH and FRAP).

The oxidation potential of monomeric polyphenols depends on the amount and position (*ortho* or *para* > *meta*) of reactive hydroxyl groups in benzene ring(s) and the *ortho*-effect between two hydroxyl groups or hydroxyl/carbonyl groups [35]. Alcalde et al. [36] evaluated the relationship between the molecular structure and electrochemical behavior of fifteen polyphenols by DPV, demonstrating that polyphenols with higher sensitivities (lower DPV potential) are strong antioxidants and that flavonoids are more electroactive (lower potentials) than phenolic acids (higher potentials); they also found that catechin exhibits two oxidation peaks at ~200 (A ring) and ~600 (B ring) mV (vs. Ag|AgCl|KCl reference electrode) at pH 5.0, while at pH 7.5 [37] and 3.6 [11] it exhibits both oxidation peaks at 148/537 mV and ~450/750 mV, respectively.

**Figure 3.** Differential pulse voltammograms of post-digested berry samples, during their *ex vivo* apparent permeability and biotransformation. Polarization rate 5mV\*s−<sup>1</sup> (abscissa values <sup>×</sup> 10<sup>−</sup>3); phase I ("absorption"; grey rectangle), phase II ("biotransformation" open white), pH = 7.2–7.4.

Kuromanin and cyanin are major anthocyanins in berry fruits [3,16] both exhibiting the same oxidation potential in their catechol group [P1 (current peak): B-ring; SET mechanism, reversible reduced] but different oxidation behavior at their resorcinol moiety (P2: A-ring, SET mechanism, irreversible) associated with the additional glucosylation (shift right: +95 (pH 3.5–4.5), +130 (pH 7.0) mV] and P1 shifts to lower potentials (vs. Ag|AgCl|KCl reference electrode) when increasing pH [20–22].

The aforementioned inverse relationship between pH and oxidation potential has been also demonstrated for several anthocyanins from *Vitis vinifera* [22]. Together, this evidence supports the idea that the observed DPV current peak *maxima* at *t0* in all three voltammograms (Figure 3) may be partially explained by a berry-specific amount of polyprotic species at neutral pH (7.2–7.4), which is the case of the not-methylated anthocyanidins and anthocyanins reported in Table 1. It is worth mentioning that ellagic acid, the most representative phenolic acid in berries, exhibits a very low oxidation potential nearby neutral pH [38] and so, it seems that it does not contribute to the observed current peak *maxima* at *t0* in all three samples (Figure 3).

Since redox processes and apparent permeability of polyphenols were concurrent events in the *ex vivo* bioanalytical system used here, we used time-trend kinetic data to gain more insights on such events. For this purpose, the following procedure was followed: *A)* The real-time electrodynamic behavior exhibited by each pre-digested sample (Figure 3) from 0 (basal) to 120 min was arbitrarily divided into two segments, before and after current *maxima* [Red Globe grape (180 mV), raspberry (216 mV), blackberry (212 mV)], and labeled as "absorption" (stage 1) and "metabolite production" (stages 2); here we postulate that a time-trend reduction of current density before *maxima* current peak is mainly due to an apparent permeability and/or structural modification of parental polyphenols while increments after peak *maxima* are more likely to be due to new chemicals species with reduced electroactivity. *B)* Goodness-of-fit regression models for stage 1 (quadratic) and stage 2 (cubic) were then obtained and, *C)* theoretical current density values (TCD; <sup>μ</sup>A <sup>×</sup> 10<sup>−</sup>5) were calculated using the mean potential peak value (203 mV) at each time point (Table S1 and Figure S1).

During stage 1, a time-trend lineal reduction of TCD for Red Globe grape (R<sup>2</sup> = 0.91) but not for raspberry or blackberry was documented (Table S1 and Figure S1); this seems to be related to the lower amount and less diverse polyphenol profile (high catechin) in this table grape (Table 1), to higher electrochemical stability and apparent permeability and to a lower efflux of polyphenols (Table 2) than that observed for raspberry and blackberry. It is known, that anthocyanins>flavan-3-ols are pH-sensitive under simulated intestinal conditions [7,28], that anthocyanidins are more electroactive than their corresponding anthocyanins [24], that anthocyanin di-glycosides are more stable than mono-glycosides [21,22] and that GLUT-2 and SGLT-1 are efficient transporters for anthocyanins and flavan-3-ols at lower apical concentrations while *P-gp*/MCRP efflux systems are activated upon GLUT-2/SGLT-1 saturation [33,39,40].

During stage 2, less electroactive (~400–600 mV) species were erratically produced (X3-behavior) in a berry-specific trend (Figure 3, Table S1 and Figure S1): raspberry (X<sup>3</sup> range = 3 to 10)> Red Globe grape (X3 range= <sup>−</sup>2 to 0.2)> blackberry (X<sup>3</sup> range= <sup>−</sup>2 to <sup>−</sup>9). DPV is also useful to study the real-time redox phenomena in vitro (particularly under acidic conditions) such as the time-course photo-degradation of 4-acetamidophenol (a.k.a. acetaminophen) [41] or the electro-Fenton degradation of 3-methyl phenol (*m*-cresol) [42] in which new molecules with higher (acetaminophen) or lower (*m*-cresol) potentials are produced from these synthetic phenolics.

However, to our knowledge, the real-time degradation of natural polyphenols under neutral pH has not been reported yet, nor the use of DPV for monitoring their time-trend enteral biotransformation. Although the evidence points out to a berry-specific ex vivo biotransformation of parental polyphenols, the chemical nature of phenolic metabolites could not be evidenced by this method. Nonetheless, partially oxidized flavan-3-ols and anthocyanins > phenolic acids seem to be predominant antioxidant species at neutral pH within the narrowed potential range used in this study (0−600 mV), a fact previously reported by other authors [35,37].

### 3.4.2. HPLC-ESI-QTOF-MS

The European Cooperation in Science and Technology Commission (COST; FA-1403 POSITIVe action) recommends the use of high through-output analytical platforms in untargeted metabolomics to evaluate the inter-individual variability in the physiological response to phytochemical intakes [4,10]. Particularly, HPLC-ESI-MSn is widely used in untargeted polyphenol metabolomics [19,28]; such a platform was used here to track the ex vivo small gut biotransformation (2 h; end-point assay) of parent polyphenols from three berries with graded levels of phenolic compounds [7]. Table 3 shows the chemical nature and apparent content of bio-accessible polyphenols (released by in vitro digestion) before their ex vivo biotransformation (t0) that substantially differed from those identified in the assayed fruits who were chemically extracted (Table 1).


**Table 3.** First-pass metabolism of phenolic compounds from selected berries: HPLC-ESI-q-TOF-MS 1,2.

<sup>1</sup> Results are expressed as mean (*n* ≥ 9; ion counts) ± standard deviation of selected phenolic compounds detected with a signal-to-noise ratio ≥10:1. <sup>2</sup> Retention time (*rt*, min), mass-to-charge ratio [*m*/*<sup>z</sup>* ± 0.1, positive (anthocyanins) or negative (all other polyphenol) ion mode], cyanidin (Cy3G; kuromanin) or malvidin (Ma3G; oenin)-3-*O*-glucosides; initial (A*t0*), final (A*t120*) and change (Δ (%) = [1-( *t120*/ *t0*)] × 100), apical ion abundance; reduced (−), increased (+).

Such a difference is even more evident when considering the HPLC-ESI-QTOF-MS profile at a signal-to-noise ratio < 10:1 (Table S2; values at *t0*). The same has been reported for black and green currants [43] and strawberries [26] when comparing the polyphenolic profile of these berries before and after *in vitro* digestion, it was observed that not only the quantity of parental polyphenols but also their chemical nature differed (also observed in Figure 4). Many biological and analytical factors are involved in this phenomenon, including the pH-instability (neutral > acidic) of polyphenols, particularly anthocyanins [8,20], the REDOX status of parent/metabolites (*m*/*z* ± 0.3), HPLC-ESI-QTOF-MS limit of detection (10:1 signal-to-noise) and their reversible interaction with digestive enzymes and mucin [5,10,44].

**Figure 4.** First-pass metabolism of polyphenols from blackberry, raspberry and *Red Globe* grape. Stepwise metabolite production from parental polyphenols (Table 1) detected by HPLC-ESI-QTOF-MS (both, below (Table 3) and over (Table S2) a signal-to-noise 10:1 ratio results from a concerted action of epithelial enzyme machinery and jejunal microflora. Triangle magnitude and direction (A<~>B) is derived from Table 2. Apical (A), basolateral (B), catechol-*O*-methyltransferase (COMT), 3' hydroxyl removal (3'DOH), deglycosylation (DGL).

Steinert et al. [45] using a CaCO2 monolayer transport system showed that the apical-to-intracellular transport of blackcurrant anthocyanins occur faster than their translocation across the basolateral membrane and that ~11% of all anthocyanins disappeared from the apical chamber within the first twenty minutes; the authors concluded that cell metabolism rather than apparent permeability was involved in the first-pass metabolism of black currant anthocyanin. Kuntz et al. [39] also studied the apparent and apical bioavailability of anthocyanins from grape/blueberry juice and smoothie permeability in transwell chambers with and without Caco-2 cell (ATCC© HTB37TM) monolayers, showing that both specific and absolute anthocyanin concentration decreased overtime in apical chambers without cells at neutral (7.4) but not acidic (2.0) pH and that total anthocyanin disappearance were even more evident with cells than without them. Both research groups also documented a structure-specific disappearance rate of anthocyanins due to concurrent absorption and biotransformation processes.

Another plausible explanation comes from microbial biotransformation. As previously mentioned, the absorptive behavior [Papp (A*t120*→B*t120*)] and uptake ratio [(A→B) <sup>×</sup> (B→A)−1] was inversely related to the fruit-specific polyphenol-richness and luminal biotransformation (Figure 4). This implies that the resident time of parent polyphenols, particularly those from blackberry and raspberry, in the apical side was long enough to be used as substrates for brush border enzymes and possibly by the resident microbiota including but not restricted to *Lactobacillus* sp., *Actinobacterium* sp. and *Clostridium* sp. which together represents ~70% of normal rat duodenal microbiota [46] and whose substrate preference include flavonoids, anthocyanins, and ellagitannins [47]. However, depending

on the composition of the microbiota, different metabolites may be produced from the microbial biotransformation (postbiotics) of berry anthocyanins, despite the fact that certain phenolic acids and flavonoids may also act as prebiotics [2]; this double effect of polyphenols enlarges their recognized health benefits [1,3–5].

The 2 h (*t0* vs*. t120*) ex vivo exposure to the intestinal epithelium reflected both the epithelial in/out interchange discussed above (see dotted triangles in Figure 4) and a great biotransformation phenomenon characterized by a low or no detection of parental anthocyanins and anthocyanidins and higher production of small molecular weight (<sup>≤</sup> 354 gmol<sup>−</sup>1) polyphenols (Table 3, Table S2). Extensive and rapid deglycosylation of anthocyanins occurs in vivo and ex vivo releasing anthocyanidins with a reduced polarity (less TPSA). The resulting anthocyanidins may be either absorbed by passive (paracellular) diffusion or subject to microbial breakdown (particularly on B ring) producing phenolic acids (e.g., protocatechuic, chlorogenic and caffeic acids) and polyols (e.g., quinic acid; Figure 4) [2,9] and C6-C3-C6-derived intermediates [44]. Chen et al. [8] followed the in vitro bioaccessibility and biotransformation of kuromanin under simulated GI conditions showing that this anthocyanin rapidly disappears but a wide range of metabolites (namely protocatechuic acid and derivates, cyanidin, caffeic and ferulic acids) were produced instead, all of them showing different permeability behaviors.

Lastly, since cyanin just have and additional glucose moiety at 5' when compared to kuromanin, its metabolic fate may be the same after enzymatic deglycosylation [9]. Kuromanin (and possibly cyanin) is partially deglycosylated by β-glucosidase (EC 3.2.1.21) and lactase-phlorizin hydrolase (EC 3.2.1.108) but it undergoes extensive *in vivo* biotransformation to low molecular weight breakdown metabolites and, a wide range of phase II metabolites including anthocyanin methylation [20]; since some of these metabolites are either reported in Table 3 or Table S2, this partially supports an extensive kuromanin biotransformation from blackberry and raspberry (Figure 4). Although a straightforward identification of C6-C3-C6 compounds derived from kuromanin, cyanin or callistephin has not been reported yet, the removal of functional groups *ex vivo* (as hypothesized in this study) may interconvert anthocyanidins (e.g., the loss of a hydroxyl group from the B-ring of cyanidin gives rise to pelargonidin) as it has been shown in vivo [44]. The biotransformation of chlorogenic (Table 3) and protocatechuic (Table S2) acids, two of the most abundant phenolic acids in edible fruits, gives quinic and caffeic acid (Figure 4) whose further methylation gives ferulic and isoferulic acids [48]; however, whether the intestinal or microbial catechol-*O*-methyltransferase (COMT; EC 2.1.1.6) activity is involved in the transformation of kuromanin into callistephin (from blackberry), deserves further study.

### **4. Conclusions**

A moderate permeability (~20%) and a high ex vivo biotransformation of parent polyphenols (molecular breakdown and isomerized anthocyanin products) from the assayed berries were found in this study. This partially suggests fruit-specific health effects, most likely at the intestinal level due to a higher efflux phenomenon. The ex vivo high-through output bioanalytical approached used here [everted gut sac (rat) + three detection methods: spectrophotometry, HPLC-ESI-QTOF-MS, differential pulse voltammetry (DPV)], provided important information on berry polyphenol biotransformation during their ex vivo first-pass metabolism that may help to understand the metabolic fate and effects of the studied berries; however, further studies are needed to understand the biological activities of biotransformed phenolics and not only their parental molecules.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/4/311/s1, Table S1 and Figure S1: Voltamperometric behavior of mixed antioxidants from three berry fruits, Table S2: First-pass metabolism of polyphenols from selected fruits- HPLC-ESI-q-TOF-MS (signal-to-noise ratio < 10:1).

**Author Contributions:** Conceptualization, A.W.-M.; Data curation, S.M., G.A.G.-A. and M.A.V.-O.; Formal analysis, F.J.O.-A., S.M., G.A.G.-A., M.A.V.-O., J.T.J.Q.-V. and A.W.-M.; Funding acquisition, A.W.-M.; Investigation, F.J.O.-A., E.A.-P. and J.T.J.Q.-V.; Methodology, F.J.O.-A., E.A.-P., M.A.V.-O. and J.T.J.Q.-V.; Project administration, F.J.O.-A. and A.W.-M.; Resources, G.A.G.-A.; Writing – original draft, F.J.O.A. and A.W.-M.; Writing – review & editing, S.M., E.A.-P., G.A.G.-A., M.A.V.-O. and A.W.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** National Council of Science and Technology (CONACyT: Basic science project (CB-2015-1/ 254063) and a doctorate scholarship for FJOA

**Acknowledgments:** All authors acknowledge the financial support from the National Council of Science and Technology (CONACyT) through a granted basic science project (CB-2015-1/ 254063) and a doctorate scholarship for FJOA, is gratefully acknowledged. All authors are indebted to all academic authorities (UACJ, UAQ, and CIAD) and to PROMEP for their institutional support.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **Interaction of Polyphenols as Antioxidant and Anti-Inflammatory Compounds in Brain–Liver–Gut Axis**

### **Amritpal Singh, Yu Fung Yau, Kin Sum Leung, Hani El-Nezami and Jetty Chung-Yung Lee \***

School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China; amritpal@connect.hku.hk (A.S.); hyfyau@connect.hku.hk (Y.F.Y.); sam612@connect.hku.hk (K.S.L.); elnezami@hku.hk (H.E.-N.)

**\*** Correspondence: jettylee@hku.hk; Tel.: +852-2299-0318

Received: 2 July 2020; Accepted: 24 July 2020; Published: 26 July 2020

**Abstract:** Oxidative stress plays an important role in the onset as well as the progression of inflammation. Without proper intervention, acute inflammation could progress to chronic inflammation, resulting in the development of inflammatory diseases. Antioxidants, such as polyphenols, have been known to possess anti-oxidative properties which promote redox homeostasis. This has encouraged research on polyphenols as potential therapeutics for inflammation through anti-oxidative and anti-inflammatory pathways. In this review, the ability of polyphenols to modulate the activation of major pathways of inflammation and oxidative stress, and their potential to regulate the activity of immune cells are examined. In addition, in this review, special emphasis has been placed on the effects of polyphenols on inflammation in the brain–liver–gut axis. The data derived from in vitro cell studies, animal models and human intervention studies are discussed.

**Keywords:** oxidative stress; inflammation; polyphenols; antioxidant

### **1. Introduction**

One of the main innate responses of the immune system is inflammation, which is an important non-specific response to any kind of injury and infection, such as physical wounds, toxins and tissue damage. It is a crucial response to the alteration of tissue integrity, to initiate healing and restore tissue homeostasis [1]. Several types of white blood cells, such as neutrophils and macrophages, and cytokines are involved in the inflammatory process. Cytokines play an enormous part in the inflammatory response and are mainly produced by helper T cells and macrophages [2]. They can be classified into pro-inflammatory cytokines, such as interleukin (IL)-1β and the tumor necrosis factor (TNF)-α, and anti-inflammatory cytokines, such as IL-4 and IL-10 [2]. The regulation and balance between the two types of cytokines is crucial for the immune system. An overproduction of pro-inflammatory cytokines could lead to autoimmune diseases and chronic inflammatory diseases, thus highlighting the need for anti-inflammatory cytokines to prevent chronic inflammatory conditions [2].

The inflammatory response is a multi-stage process which involves a triggering system, a sensor mechanism, signal transmission and the production of inflammatory mediators [1]. The inflammatory response could be triggered by various danger signals, which could be from exogenous, such as invasion by microorganisms, or endogenous sources, such as tissue damage. The exogenous and endogenous signaling molecules are termed pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) respectively [1]. PAMPs and DAMPs are sensed by a variety of pattern recognition receptors (PRRs) which include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectins and receptors for advanced glycation end-products (RAGE) [1]. The activation of PRR triggers intracellular signaling

cascades, including kinases, such as mitogen-activated protein kinases (MAPKs), adaptors, the myeloid differentiation primary response protein 88 (MyD88), and transcription factors, such as the nuclear factor kappa B (NF-κB). Furthermore, the activation of NLR could trigger cytokine maturation, which are key to inflammation development, through inflammasomes. For instance, activated NLRP3 are associated to the adaptor protein ASC (apoptosis associated speck-like containing a CARD domain) and caspase-1 to form inflammasome, which promotes the conversion of pro-IL-1β and pro-IL-18 to mature IL-1β and IL-18, respectively [1]. Upon DAMP binding, TLR interacts with MyD88 which activates downstream signaling, resulting in NF-κB and activator protein-1 (AP-1) activation [3]. The signaling pathways mentioned above upregulate the expression of inflammatory mediators like cytokines for inflammation development.

It has been long known that significant oxidative stress could cause cellular damage and modification of genes, which triggers the inflammatory signaling cascade for the onset of inflammation in various inflammatory diseases [4]. As part of the inflammatory response, large amounts of reactive oxygen species (ROS) are generated, which could further promote oxidative stress and chronic inflammation if produced for lengthened periods [1,4]. Besides, several studies have pointed out that oxidants have a significant part in the activation of TLRs [5,6]. The studies have shown that the translocation of TLR4 to the cell membrane was upregulated after exposure to oxidants [5,6]. This enhances the responsiveness of cells to a danger signal for the onset of pro-inflammatory signaling pathways. One of the most discussed pathways in inflammation is the NF-κB pathway. It is a key regulator of inflammation due to its sensitivity to ROS. The activation of NF-κB could result from two pathways, the canonical and alternative pathways [1,7]. In both pathways, NF-κB is freed from its inhibitor, IκB, resulting in NF-κB translocation to the nucleus for the expression of target genes [1].

Under oxidative stress, the expression of antioxidant genes is upregulated, which is modulated by the nuclear factor erythroid 2-related factor 2 (Nrf2) [8]. Nrf2 activation is induced by ROS by the removal of its inhibitor, Kelch-like erythroid CNC homolog-associated protein 1 (Keap1), allowing the translocation of Nrf2 to the nucleus for the expression of genes involved in the antioxidant response [8].

As mentioned earlier, significant oxidative stress results in the propagation of inflammation, which illustrates the importance of redox balance in the resolution, and prevention of inflammation. Redox homeostasis is maintained by antioxidants, which could be from endogenous or exogenous (natural or synthetic) sources. The endogenous sources of antioxidants consist of enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) [4]. Minerals such as zinc, selenium and copper are essential for the activation of the antioxidant enzymes as they are co-factors for these enzymes [9]. The natural exogenous antioxidants include ascorbic acid (vitamin C), α-tocopherol (vitamin E), carotenoids and flavonoids [4,9]. Antioxidants can scavenge free radicals which terminates the chain reaction of oxidation. Besides, they could prevent the initiation of a chain reaction by binding to transition metal ions that catalyze ROS generation [4,9]. As a result, antioxidants are able to reduce oxidative stress.

Polyphenols have been long known to be potent antioxidants. Polyphenols are found in various types of food, including fruits and vegetables, and can be classified into flavonoids and non-flavonoids [10]. Examples of flavonoids include anthocyanins, epigallocatechin gallate (EGCG) and curcumin, while an example of a non-flavonoid is resveratrol (RES) [10–12]. Because of their effects on oxidative stress, researchers have studied the effects of polyphenols in conditions with common underlying factors such as oxidative stress and inflammation. Polyphenols have been studied as potential anti-inflammatory agents in various inflammatory diseases, such as non-alcoholic fatty liver disease (NAFLD), inflammatory bowel disease and neurodegenerative diseases [10–12]. As illustrated in Figure 1, it is hypothesized that polyphenols would modulate the inflammatory signaling pathway via an antioxidant-based mechanism. It is expected that polyphenols would reduce oxidative stress, which would inhibit signal transduction for the production of pro-inflammatory mediators. The aim of this review will be to discuss the effects of polyphenol intervention in experimental and clinical

settings on inflammation in specific organs, namely the brain, liver and gut, with reference to their antioxidant and anti-inflammatory properties.

**Figure 1.** Potential mechanism of action of polyphenols in inflammation inhibition. Polyphenols may target the reactive oxygen species (ROS) to reduce oxidative stress. (1) ROS reduction could reduce the amount of damage-associated molecular patterns (DAMPs); (2) ROS reduction could also arrest phosphorylation of IκB kinase (IKK), which would block the dissociation of IκB from NF-κB; (3) ROS could directly phosphorylate IκB, which could be prevented by polyphenols. These pathways would inhibit nuclear translocation of NF-κB.

### **2. Polyphenol Intervention in Brain Inflammation**

Some of the most common brain disorders include neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease [10–13]. Despite the exact causes of these diseases being unclear, these disorders have been linked to common underlying factors which are high levels of oxidative stress and inflammation [10–13]. Due to these reasons, the use of antioxidants as potential therapeutic agents has emerged. For instance, the effects of polyphenols have been studied on factors that are involved in disease progression. The findings of these studies in the last 15 years (2004–2019) are summarized in Table 1.



#### *Antioxidants* **2020** , *9*, 669

TNF-α: tumor necrosis factor alpha.

### *2.1. In Vitro Models of Polyphenol Treatment in Brain Inflammation*

Among several in vitro studies, one group reported the inhibition of TLR4 signaling in cells treated with quercetin [14]. Besides, the expressions of TLR2 and TLR4 being reduced by quercetin, it further decreased the production of pro-inflammatory cytokines [15]. Furthermore, quercetin has been shown to attenuate the activity of inflammatory enzymes, including inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX) [15]. In studies that tested anthocyanin-rich extracts in response to lipopolysaccharide (LPS) stimulation, a significant reduction in the production of TNF-α and IL-1β, and expressions of iNOS and COX-2 was reported [17–21]. A study on rat highly aggressively proliferating immortalized (HAPI) cells demonstrated similar results, but with no significant effect on iNOS expression [23]. In mouse microglial cells, anthocyanins reduced NO and TNF-α release and iNOS expression [22]. The reduced levels of pro-inflammatory mediators have been attributed by the modulation of inflammatory signaling pathways, since decreased levels of active p38 MAPK and NF-κB have been observed [17–21]. Besides microglial cells, anthocyanins testing on astrocytes has also revealed similar findings. For instance, rat astrocytes treated with anthocyanin-rich lingonberry extract lowered ROS production, suggesting an anti-oxidative mechanism [24]. In a human astrocyte study, the secretion of IL-6 was reduced at low doses of LPS and anthocyanins [16]. However, in the absence of LPS, IL-6 secretion increased when treated with high concentration of anthocyanin, suggesting antioxidant toxicity with a single compound supplementation [16]. Some studies have highlighted that the synergistic effects of mixed polyphenols provided better outcomes than single compound supplementation. Moreover, it was reported that the anti-inflammatory effects were more significant when the cells were treated with high concentrations of anthocyanins.

### *2.2. In Vivo Models of Polyphenol Treatment in Brain Inflammation*

In a mouse model of Parkinson's disease, the neuroprotective effects of grape seed and skin extract (GSSE), which is a mixture of polyphenolic compounds that is mostly comprised of flavonoids namely catechins, gallic acid, vanillin and 2,5-dihydroxybenzoic acid, were studied [25]. A reduction in ROS production, nuclear translocation of NF-κB p65 subunit and loss of SOD was observed [25]. RES inhibited TLR4, NF-κB and cytokine secretion in LPS and β-amyloid (Aβ)-mediated microglia neuroinflammation [26]. EGCG could attenuate TLR4 signaling in LPS-impaired adult hippocampal neurogenesis [27]. In addition, findings from anthocyanin treatment in LPS-induced mouse models have reported a decrease in several markers of oxidative stress and pro-inflammation. For example, levels of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, were attenuated [28–31]. Furthermore, NF-κB activation was inhibited [28–30]. In one study, the expression of anti-inflammatory cytokines, such as IL-10, increased when the mice were pre-treated with anthocyanins [31]. Purple sweet potato extract showed anti-inflammatory effects in mice fed a high-fat diet (HFD), in which the expressions of iNOS, COX-2, TNF-α, IL-6 and IL-1β attenuated, and the levels of IL-10 increased [32]. Furthermore, the activation of p38 MAPK and NF-κB was inhibited [32]. Similarly, anthocyanin treatment in a rat middle cerebral artery occlusion/reperfusion (MCAO/R) model demonstrated decreased expressions of NF-κB, NLRP3 and pro-inflammatory cytokines [33].

For human studies in relation to polyphenol intervention, a few have been conducted. One study on the administration of RES was shown to decrease neuroinflammation in patients with Alzheimer's disease [34]. The findings from in vitro and in vivo animal studies have demonstrated the benefits of polyphenols on brain inflammation. However, more human studies are required to validate the beneficial effects of polyphenols in brain-related inflammation.

### **3. Polyphenol Intervention in Liver Inflammation**

One of most common liver diseases is NAFLD [35,36]. This disease could further progress to non-alcoholic steatohepatitis (NASH), and can eventually develop into hepatocellular carcinoma [35]. Similar to brain inflammation, the effects of polyphenol interventions in liver inflammation have been

extensively researched. The findings of the effects of polyphenols on liver health in the last 15 years (2004–2019) are summarized in Table 2.


**Table 2.** Summary of the effects of polyphenols on liver inflammation in the last 15 years (2004–2019).


↑: increase; ↓: decrease; ALT: alanine aminotransferase; AST: aspartate transaminase; C3G: cyanidin-3-glucoside; CAT; catalase; CK: cytokeratin; COX: cyclooxygenase; EGCG: epigallocatechin gallate; FGF: fibroblast growth factor; GCL: glutamate-cysteine ligase; GPx: glutathione peroxidase; GSH: reduced glutathione; GSSG: oxidized glutathione; GST: glutathione s-transferase; HFD: high fat diet; HO: heme oxygenase; hs-CRP: high sensitivity C-reactive protein; ICAM: intercellular adhesion molecule; IFN-γ: interferon gamma; IL: interleukin; iNOS: inducible nitric oxide synthase; MCD: methionine-choline deficient; MCDHFD: methionine-choline deficient high fat diet; MCP: monocyte chemoattractant protein; MDA: malondialdehyde; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; NF-κB: nuclear factor kappa B; Nrf: nuclear factor erythroid 2-related factor; RES: resveratrol; ROS: reactive oxygen species; SOD: superoxide dismutase; TBARS: thiobarbituric acid reactive substances; TG: triglyceride; TLR: toll-like receptor; TNF-α: tumour necrosis factor alpha.

### *3.1. In Vitro Models of Polyphenol Treatment in Liver Inflammation*

A few in vitro studies have illustrated the beneficial outcomes of polyphenols on oxidative stress and inflammation. Mouse macrophages pre-treated with rutin attenuated oxidative stress and *MCP-1*, *TNF-*α, *IL-6*, *IFN-*γ, *IL-1*β genes expressions [40]. In a study on human hepatocyte HepG2 cells, the glutamate cysteine ligase (GCL) activity and GSH level were improved with cyanidin-3-*O*-β-glucoside (C3G) treatment [39]. Moreover, theaflavins could reduce ROS generation in the same type of cell line [38]. Curcumin treatment also increased GCL activity and GSH level, and attenuated ROS production in glucose-induced hepatic stellate cells [37].

### *3.2. In Vivo Models of Polyphenol Treatment in Liver Inflammation*

Much like the in vitro studies, several animal in vivo studies have demonstrated encouraging effects of polyphenol interventions. Quercetin has shown to improve thiobarbituric acid reactive substances (TBARS), GPx and CAT levels, and down-regulate TNF-α, IL-6 and COX-2 mRNA expressions in two mouse models [41,42]. Moreover, it was reported that the TLR4 protein concentration also decreased [42].

In mice fed with HFD, the expressions of TNF-α and MCP-1 genes were attenuated by rutin [40]. In another study, troxerutin increased GPx, SOD and GSH levels, which subsequently reduced ROS production [43]. Theaflavins supplementation lowered TBARS, ROS and TNF-α production in mice fed with a methionine and choline deficient high fat diet (HFD) [44]. Furthermore, the levels of MCP-1 and TNF-α were decreased with baicalein supplementation in HFD mice [45]. Oxidative stress could be reduced through the inhibition of macrophage infiltration when HFD mice are treated with RES [46]. In a few studies in which mice were given a methionine and choline-deficient diet, oxidative stress and inflammation were reduced with curcumin [47,48]. One of the studies reported the attenuation of ICAM-1, COX-2 and MCP-1 expressions and NF-κB signaling [47]. Another phenolic compound, silibinin, was shown to reduce iNOS expression, ROS production and NF-κB activation in mice fed with methionine and choline-deficient diet [49]. In addition, a similar model reported that silibinin supplementation improved GSH levels, and down-regulated IL-6 and TNF-α expressions [50].

Besides mouse models, polyphenol intervention has been studied in rat models. In rats fed with HFD, quercetin supplementation upregulated the expressions of Nrf2 and heme oxygenase 1 (HO-1), and down-regulated the expression of NF-κB [51]. In a similar model, mice supplemented with rutin had higher GPx expression and lower plasma MDA levels [52]. One study showed that, with EGCG supplementation, the GSH level was improved, whereas the plasma and liver MDA levels were reduced in rats fed with HFD [53]. Furthermore, EGCG could increase GPx and CAT activity, and attenuate iNOS, COX-2 and TNF-α expressions [54]. Similarly, genistein treatment decreased TNF-α, and plasma and liver MDA levels in HFD-fed rats [55]. In rats fed with a high cholesterol diet and naringenin, the attenuation of the production of pro-inflammatory cytokines, namely TNF-α, IL-6 and IL-1β, through the inhibition of the NF-κB pathway was also reported [56]. In fructose fed rats, RES increased SOD activity and Nrf2 and GSH levels [59]. Furthermore, RES reduced lipid peroxidation in the same model. The effects of coffee polyphenols have also been studied in a few rat models. The supplementation of coffee polyphenols ameliorated GSH/glutathione disulphide (GSSG) ratio, and attenuated TNF-α and IFN-γ expressions [57]. Besides, coffee polyphenols could reduce hepatic oxidative stress and steatosis in rats fed with HFD [58]. It was also reported that coffee polyphenols suppressed not only the expressions of pro-inflammatory cytokines, but also suppressed the expressions of anti-inflammatory cytokines, such as IL-4 and IL-10 [57].

A few of the polyphenols have also been subject to clinical testing, with the target group being the patients with NAFLD. For instance, supplementation of catechins decreased urinary F2t-isoprostane excretion in the treatment group that was given a higher dose [60]. Moreover, silymarin supplementation ameliorated the NASH score and serum oxidative stress [63]. A few studies have tested the effects of RES in NAFLD patients and have produced mixed results. Several studies observed an improvement in the inflammatory markers, such as TNF-α, cytokeratin 18 (CK-18), IL-6 and NF-κB [61,62]. On the other hand, one study reported the harmful effects of a high concentration of RES, in which the levels of enzymes, like alanine aminotransferase (ALT) and aspartate transaminase (AST) increased [64]. These findings suggest that polyphenols could prevent the progression of NAFLD to NASH. However, supplementation of a high dose could have adverse effects on liver health.

### **4. Polyphenol Intervention in Gut Inflammation**

Compared to the other organs that have been discussed in this review, polyphenol intervention in gut inflammation had not been a subject of profound research until recently. Most of the research has been on berry polyphenols and RES [65,66]. The interventions have targeted the models of inflammatory bowel disease, which is a term used to describe inflammatory disorders in the gut [66]. Some common examples of inflammatory bowel disease include Crohn's disease and ulcerative colitis (UC) [66]. The findings of polyphenol interventions in vitro and in vivo studies in the last 15 years (2004–2019) are summarized in Table 3.


effects of polyphenols on gut inflammation in the last 15 years (2004-2019).




MCP: monocyte factor; PBMC: peripheral blood mononuclear

species; ROS: reactive oxygen species; SOD: superoxide dismutase; TBARS:

TNF-α: tumour necrosis factor alpha; UC: ulcerative colitis; VCAM: vascular cell adhesion molecule.

chemoattractant

 protein; MDA:

 cell; PG-PS:

malondialdehyde;

 MPO: peptidoglycan-polysaccharide;

myeloperoxidase;

 NF-κB: nuclear factor kappa B; NO: nitric oxide; Nrf: nuclear factor erythroid 2-related

 PGD2:

thiobarbituric

 acid reactive substances; TLR: toll-like receptor; TNBS: 2, 4,

prostaglandin

 D2; PGE2:

prostaglandin

 E2; RES: resveratrol; RNS: reactive nitrogen

6-Trinitrobenzenesulfonic

 acid;

### *4.1. In Vitro Models of Polyphenol Treatment in Gut Inflammation*

Much like the brain and liver, quite a few studies have been conducted to evaluate the effects of polyphenols on gut health. The amelioration of oxidative stress and inflammatory markers has been observed in several in vitro studies on human intestinal cell lines. For example, the expressions of TNF-α, IFN-γ-induced protein 10 (IP-10) and IFN-γ receptor 2 were inhibited by anthocyanins [67,68]. One study demonstrated the beneficial effects of RES in human intestinal Caco-2 cells treated with LPS. It was reported that RES pre-treatment suppressed COX-2 expression, prostaglandin E2 (PGE2) release and NF-κB activation [69]. In a similar study, iNOS and TLR4 expression, NF-κB activation and NO release were reduced but only in high RES concentration treatment groups [70]. The same results were obtained from RES treatment in human colon SW480 cells [70]. Moreover, RES upregulated HO-1 and GCL expression through the activation of the Nrf2 pathway and improved the GSH/GSSG ratio in human colon epithelial HT-29 cells [73]. Besides, ROS production, iNOS and COX-2 expression, and NO and PGE2 release could be attenuated with RES. However, it had no significant effect on the activation of NF-κB [72]. In Caco-2 cells stimulated by IL-1β or TNF-α, the phosphorylation of IκB could not be inhibited by RES, which allowed the activation of NF-κB [71]. This shows that stimulation by pro-inflammatory cytokines could trigger the inflammatory cascade even in the presence of an anti-inflammatory agent like RES.

### *4.2. In Vivo Models of Polyphenol Treatment in Gut Inflammation*

In vivo animal models have also been used to better understand the impact of polyphenols. In a rat model of colitis induced by dextran sodium sulphate (DSS), blueberry powder reduced myeloperoxidase (MPO) activity and MDA concentration, but had an insignificant effect on MCP-1 level [90]. MPO activity is used to determine neutrophil infiltration, where a high activity would represent higher oxidative stress [65]. In another rat model of DSS-induced colitis, RES treatment decreased COX-2, PGE2 and NO levels. However, no significant change occurred in the TBARS level [98]. Another substance used to induce colitis in animal models is 2, 4, 6-trinitrobenzenesulfonic acid (TNBS). One study observed the attenuation of MPO activity, and VCAM-1, ICAM-1, MDA, NO and GSH levels with RES administration [91]. In addition to these effects, RES administration suppressed the expressions of MCP-1, cytokine-induced neutrophil chemoattractant 1 (CINC-1), TNF-α, IL-1β, IL-6 and IL-12 in rats with TNBS-induced colitis [92]. Pre-treatment of rats with RES lowered MPO activity, and IL-1β, PGE2 and prostaglandin D2 (PGD2) levels from TNBS-induced colitis [93]. In one study, even though the expression of certain inflammatory mediators was downregulated with RES treatment, the level PGE2 was increased [94]. Moreover, RES reduced colon MDA level and promoted GPx activity. Despite that, the MPO, SOD and CAT activities were unaffected [95]. In rats with methotrexate-induced or oxazolone-induced colitis, MPO activity was suppressed by RES administration [96,97]. Furthermore, the expression of pro-inflammatory cytokines, like IL-6, TNF-α and IL-1β, was decreased in a peptidoglycan-polysaccharide-induced colitis rat model [99].

Similar effects were demonstrated in mice models. Anthocyanins inhibited MPO activity, and the increase of pro-inflammatory cytokines in mice with TNBS-induced colitis. At the same time, the expression of the anti-inflammatory cytokine, IL-10, was upregulated [74]. Same effects were observed in both acute and chronic inflammatory conditions in a different study [77]. One study reported the suppression of inflammatory mediators and neutrophil infiltration, and the increase of CAT and SOD activity with blueberry extract. However, these effects were more prominent in the pre-treated group than the post-treated group [75]. Likewise, black raspberry extract attenuated TNF-α and IL-1β expressions, and NF-κB and COX-2 activity in mice with DSS-induced colitis. Yet, it had no significant effect on MDA and inflammatory cells infiltration [76]. Another study showed the inhibition of macrophages and neutrophils infiltration, and NF-κB nuclear translocation [78]. In a study on cranberry extract and dried cranberries treatment, dried cranberries attenuated MPO activity and pro-inflammatory cytokines production [79]. In mice that were given an HFD, cranberry extract downregulated the expression of inflammatory mediators but had an insignificant effect on MDA and

SOD levels [80]. RES intervention has also been studied in various DSS-induced colitis mouse models. One study recorded a decrease in protein levels of iNOS, COX-2 and TNF-α [81]. Similar effects were observed in another study alongside an increase in IL-10 level [82]. In addition, a few studies showed an attenuation in the levels of pro-inflammatory cytokines and inflammatory enzymes with RES administration [83,88]. Moreover, RES supplementation reduced iNOS protein levels and NF-κB activation in colons of mice with DSS-induced colitis [87]. In mice with spontaneous chronic colitis, RES administration for 28 weeks decreased the levels of pro-inflammatory cytokines in the colon and serum [89]. On the other hand, RES had no significant impact on MPO activity and levels of TNF-α, PGE2, IL-6 and IL-10 in mouse models of DSS-induced colitis [84,86]. Although the MPO activity and the expressions of pro-inflammatory cytokines were downregulated, Yao et al. found the SOD and GPx activities to be suppressed as well [85].

Several studies have been conducted on polyphenol intervention in patients with UC. For instance, anthocyanins were reported to reduce TNF-α, IFN-γ and MCP-1 levels and NF-κB activation. In the same study, the levels of IL-22, IL-10 and IL-17A were elevated [68]. One study on subjects with mild to moderate UC showed a reduction in fecal calprotectin level with anthocyanins treatment, which suggests that neutrophil migration was lowered. However, an increase in disease activity was observed after the termination of the treatment [100]. Besides anthocyanins, RES has also been tested in subjects with UC. One study reported a decrease in high-sensitivity CRP and TNF-α levels, and the suppression of NF-κB activation [101]. Additionally, RES administration in patients with mild to moderate UC ameliorated plasma SOD activity and lessened plasma MDA level [102]. Even though there have been encouraging results from some clinical interventions, more clinical trials are required to consider polyphenols as potential therapeutics for gut inflammatory diseases.

### **5. Research Gap**

Although polyphenols have demonstrated anti-inflammatory properties in vitro and in vivo animal studies, there is inconclusive evidence of their effects in humans. Currently, there is insufficient evidence to support the use of polyphenols as therapeutics in subjects with inflammatory diseases. There is a need for more human trials on polyphenol intervention to gain more conclusive evidence. Moreover, it is worth noting that the available human studies have only demonstrated symptom amelioration in subjects with inflammatory diseases. For instance, RES administration reduced inflammation in Alzheimer's disease patients [34]. Other studies also reported the amelioration of symptoms with polyphenol supplementation in subjects with liver inflammatory diseases [60–63]. Furthermore, remission was observed from clinical interventions related to polyphenol administration in gut inflammatory disease patients [68,100–102]. Albeit these trials demonstrated beneficial effects, they could not demonstrate complete resolution of inflammation. As a result, it is rather premature to use polyphenols to treat inflammatory diseases.

In addition, a few studies have shown that there could be harmful effects associated with polyphenol treatment. For example, studies have reported an increase in pro-inflammatory cytokine secretion and NF-κB activation [16,71,94]. A study also reported the downregulation of antioxidant enzymes [85]. In another report, it showed RES supplementation resulted in the deterioration of liver health in patients with NAFLD [64]. Most of these adverse effects have been related to the administered dose of polyphenols. Therefore, further research on the appropriate dosage of polyphenols to produce beneficial effects and prevent adverse effects is required to reach a consensus.

### **6. Conclusions**

Several in vitro and in vivo animal studies have demonstrated the antioxidant and anti-inflammatory effects of polyphenols in the brain–liver–gut axis. Polyphenols have been shown to target different stages of the inflammatory cascade to reduce the severity of inflammation. In general, the natural antioxidants seem to be more useful in the prevention of inflammation rather than in resolution. Although some antioxidants have had promising effects in vitro and animal studies, those results could not be extrapolated to human studies. As a result, further research is needed on polyphenol intervention in human trials, and on ways to improve the bioavailability and efficacy of polyphenols in subjects with inflammatory diseases.

**Author Contributions:** A.S. wrote the manuscript. Y.F.Y., K.S.L., H.E.-N. and J.C.-Y.L. conceptualized the manuscript. J.C.-Y.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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