**Improvement of Fatty Acid Profile in Durum Wheat Breads Supplemented with** *Portulaca oleracea* **L. Quality Traits of Purslane-Fortified Bread**

#### **Maria Grazia Melilli 1,\*, Vita Di Stefano 2,\*, Fabiola Sciacca 3, Antonella Pagliaro 1, Rosaria Bognanni 1, Salvatore Scandurra 1, Nino Virzì 3, Carla Gentile <sup>2</sup> and Massimo Palumbo <sup>3</sup>**


Received: 2 May 2020; Accepted: 5 June 2020; Published: 10 June 2020

**Abstract:** The addition of functional ingredients to breads could have effects on preventing cardiovascular diseases, cancers and inflammation. The incorporation of 0–5–10–15% of three populations of dried purslane flour on the rheological, sensorial and nutritional quality of fortified durum wheat breads were evaluated. The increase in dried purslane (up to 15%) caused an increase in the resistance to the mixture and a consequent reduction in its extensibility. The "panel test" gave a largely positive evaluation in 10% of enrichment. The fatty acids in breads resulted higher with the 5% substitution. Contrary to what has been imagined, the increase in percentage of substitution to 10 and 15% did not lead to an increase in linoleic (omega-3) and α-linolenic (omega-6) acid and probably the cause is in the cooking. The total phenols content and the antioxidant potential, evaluated by ferric reducing antioxidant potential (FRAP) and 2,2 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays of the enriched breads increased with the percentage of the dry purslane substitution. The enrichment of the durum wheat flour with 5% purslane resulted in a good compromise to obtain good rheological characteristics of loaves and breads with decreased omega-6/omega-3 ratio and good antioxidant properties.

**Keywords:** durum wheat bread; *Portulaca oleracea* L.; essential fatty acids; omega-6/omega-3 ratio; antioxidants

#### **1. Introduction**

Functional foods can be considered those whole, enriched or enhanced foods, that provide health benefits in addition to basic nutritional functions, when consumed as part of a varied diet on a regular basis.

Recent consumer interest in nutrition and health has increased the commercial demand for functional foods [1,2]. Functional foods can also be obtained by fortifying the product with so called phytochemicals; components with high nutritional value found in nature in different types of plants. Foods fortified with phytochemicals have been associated with the prevention of at least four frequent causes of death: cancer, cardiovascular diseases, high blood pressure, and diabetes [1–4]. Interests in incorporating bioactive ingredients, plant materials, herbs or spices, rich in bio-compounds, into popular

foods such as bread, have grown rapidly due to the increased consumer health awareness [5]. Bread is the staple food of the Mediterranean diet and is appreciated and eaten in countries around the world. The bread produced basically from wheat flour, is rich in carbohydrate and provides more than 50% of the total energy intake. Due its relatively low cost, availability, acceptability, and widespread consumption, bread is an excellent product in which the incorporation of functional ingredients, especially omega-3 fatty acids, is attempted [6]. In recent decades, different research teams have worked on fortifying bread with natural compounds due to the demands for healthier food [7,8]. Thus, whole grains and seeds are commonly used in the production of bread [9,10]. Moreover, a technical challenge for food technologists is the production of breads with greater volume, smoother texture and good shelf life, possessing the quality characteristics derived from the functional properties of fats.

*Portulaca oleracea* L. (purslane) is an annual herbaceous plant with reddish stems and alternate leaves from the family Portulacaceae. Purslane is distributed in many parts of the world and specifically the tropical and subtropical areas. The aerial parts of the plant are somewhat crunchy, have a slight lemony taste and are consumed as salads [11,12]. It is a well known plant in traditional medicine; its medicinal value is evident from its use as purgative, cardiac tonic, emollient, muscle relaxant, anti-inflammatory and diuretic with immune-protective properties [13–15].

Purslane has been described as a power food due its high nutritive and antioxidant properties and activities, mainly acting as a free radical scavenger, metal quencher and lipid peroxidation inhibitor, thanks to its phenolic constituents and several fatty acids. Purslane is abundant in ω-3 fatty acids, particularly in α-linolenic acid (0.83 mg g<sup>−</sup>1), for which it is considered one of the richest plant sources. Apart from α-linolenic acid, which represents nearly up to 30% of purslane oil, other essential fatty acids have also been detected in plant tissues, such as palmitoleic, palmitic, linoleic, oleic and stearic, acids, as well as trace amounts of 20:5ω-3 and 22:6ω-3, namely eicosapentaenoic acid and docosahexaenoic acid, respectively [12,16]. In a number of regions in southern Italy, bread is mainly made from durum wheat, and different varieties have been identified as suitable for both bread and pasta production [17,18]. In this study, the effect of incorporating different amounts of dried purslane flour on the technological, sensorial and nutritional quality of fortified durum wheat breads was evaluated.

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

#### *2.1. Plant Material*

Purslane germplasm was collected in three different sites of eastern Sicily: Caltagirone (Cal; 37◦11 07 N-14◦13 19 W), Cassibile (Cas; 36◦58 33 N-15◦12 18 W) and Santa Venerina (SVen; 37◦40 23 N-15◦19 26 W) during June 2017. In the identified sites of collection, the plant resulted widespread and naturally covered the degraded soils. The biomorfological and chemical characterization of the plant material were reported in a previous study [12]. Linolenicacid resulted as the most abundant (0.86 mg g<sup>−</sup>1), followed by the palmitic (0.76 mg g−1) and oleic acids (0.25 mg g−1) in dried whole plants [7]. The collected parts of the plant were blended thoroughly for homogeneity and washed with deionized water. After draining the excess water, the biomass (leaves + stalks) was dried at 40 ◦C for 3 days and ground into powder (about 300 micron). The final moisture content was less than 8%.

#### *2.2. Bread-Making Test*

Each form of bread was obtained adding to 400 g of commercial durum wheat flour (xg semolina + xg dried purslane) 8 g of sucrose, 8 g of salt, 24 g dehydrated mother yeast and x mL of distilled water, calculated according to the water absorption index (WA) by Brabender farinograph analysis. The obtained mixture was divided into two 200 g shapes and placed in rectangular aluminum pans of 23 × 11 × 5 cm. For each purslane population, different concentrations (5%, 10% and 15% w/w) of the

substitution on the total weight of the flour were studied. Bread without purslane was used as the control (CTRL). The different types of produced bread are reported in Table 1. Three replicates were prepared for each bread sample. The bread loafs were square-shaped in an experimental aluminum box, in leavening conditions of 30 ◦C for 1 h, 75% r.h. and bakedin a Polin mod. Wind Pierre experimental oven (Verona. Italy), at 180 ◦C, for 18 min. The bread samples (Cal5, Cal10, Cal15, Cas5, Cas10, Cas 15, SVen5, SVen10 and SVen15, Table 1) were then subjected to the instrumental measurement for volume (Geass Volumometer, cm3) and the height of the loaf of breads (Vernier Caliper, cm).


**Table 1.** Bread samples.

#### *2.3. Rheological Characteristics*

The dough-mixing properties of the control and the different mix were examined with the Brabender Farinograph (Brabender, Duisburg, West Germany) according to the constant flour weight procedure (AACC n◦ 54–21). Three hundred grams' flour was mixed at the optimum water absorption and the farinograph curve was centered on the 500 BU line. According to the standard procedure, the following farinograph indices were determined: (1) water absorption of blend (WA), (2) development time of dough (DT), (3) stability of dough (S), and (4) the degree of softening of dough (DS) (Table 2). The alveographic test was used to analyze the effect of the additions on the dough rheological behavior performed by Chopin alveograph (Chopin, Villeneuve La Garenne, France) according to the standard alveographic (UNI n◦ 10453 method) (American Association of Cereal Chemists 2000). Each sample was analyzed in triplicate and the deformation energy W (strength) and P/L (tenacity/extensibility ratio) were calculated.

**Table 2.** Alveographic and farinographic indices of the doughs enriched with purslane averaged for concentrations and populations. Different letters indicate differences at *p* < 0.05 (according to the Duncan test).


#### *2.4. Sensorial Analysis of Bread*

Bread samples were submitted to a panel of 10-trained tasters (five men and five women, aged between 27 and 60 years) in order to evaluate the sensory attributes. The panel group was an

on-going panel with prior training. The panelists were selected based on their sensory skills (ability to accurately determine and communicate the sensory attributes as the appearance, odor, flavor and the texture of a product). The samples (CTRL and 5%, 10% and 15% of the fortified bread of all three populations) were served in dishes randomly labeled with three-digit random numbers for all panelists. The beads were sliced (1 cm thick) and were offered in distinct dishes at the same time. Water was provided for rinsing purposes. They were evaluated on crust thickness, elasticity, hardness, friability, apparent softness (force required for compressing the bread slice on a flat surface with a finger, to obtain a deformation about 50% of crust); on crumb elasticity, friability, cohesiveness, humidity, optical evaluation of the average size and homogeneity of the alveoli, cohesiveness to the crust. We asked the panelists to give a bread overall judgment for the overall taste and odor. To this end, a 10-point scale was used: 1—low sensation, 10—high sensation while for overall taste and odor and final overall judgment, 1corresponds to extremely unpleasant and 10 to extremely pleasant. The threshold of acceptability was set at 6.

#### *2.5. Bread Colour Evaluation*

Bread color data were collected with the use of a Minolta colorimeter CR, 400, as previously described by Melilli et al. [19]. The colorimeter was calibrated using the manufacturer's standard white plate (L\* = 96.55; a\* = −0.35; b\* = −0.16), where the L\* value represents light–dark spectrum with a range from 0 (black) to 100 (white), the a\* value represents the green–red spectrum with a range from −60 (green) +60 (red). The b\* value represents the blue–yellow spectrum with a range from −60 (blue) +60 (yellow) (CIELAB color space) [20].

#### *2.6. Fatty Acids Content*

The fatty acids content was determined in the germplasm collected [12], in the CTRL and in the breads enriched with purslane. The fatty acid composition was studied on 0.5 g amounts of samples after the saponification of triglycerides, as described in Melilli et al. [12], after the fatty acids were transformed into corresponding methyl esters (FAMEs) and injected in a gas chromatography–mass spectrometry (GC/MS) (Thermo-Fisher) [21,22]. Using Thermo Scientific Xcalibur Data system software for Windows, the peak areas were determined and identified by the comparison of the retention times with those of a FAMEs standard mix (Supelco CRM18918 SUPELCO FAME Mix C8–C24) separated under the same chromatographic conditions. Triplicate analyses were prepared for each sample, and the analyzed FAMEs were expressed in mg g<sup>−</sup>1.

#### *2.7. Total Phenols Content*

The total phenol content in the bread samples (TPC) was determined using the Folin–Ciocalteu method as reported by Singleton, et al. [23], with some modifications [24]. One gram of fine bread powder was extracted with 10 mL of a solution MeOH:H2O (80:20). After sonication for 40 min, the extracts were filtered and stored in a −20◦C freezer overnight. For the determination of the TPC, 625 μL of the Folin–Ciocalteu reagent (Merck, Darmstadt, Germany, diluted 5 times) and 1.2 mL of Na2CO3 (7% w/v) solution were added to 125 μL of the samples' extracts. The mixtures were vortexed for two minutes and incubated in the dark for 1 h. Absorbance at 760 nm was measured using a spectrophotometer (Eppendorf Bio Spectrometer® basic). The TPC was expressed as mg gallic acid equivalent in 100 g of dried bread samples (mgGAE 100 g<sup>−</sup>1d.m.). For every sample, the protocol was repeated three times.

#### *2.8. Antioxidant Activity*

Bread samples with added purslane at 5 and 10% as well as the CTRL were submitted to analysis. Two aliquots of 5 g of each dried bread sample was extracted three times with 15 mL of 70% ethanol (v/v) distilled water. After a cleanup step via centrifugation (10 min at 10,000× *g*, 4 ◦C), and filtration through a Millex HV 0.45 μm filter (Millipore, Billerica, MA, USA), the supernatants of each extraction

cycle were recovered, combined, and used for the analysis of antioxidant activity. The antioxidant properties of the extracts from bread samples were evaluated using two antioxidant assays: the ABTS (2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay and the ferric reducing antioxidant power (FRAP) assay. The ABTS assay was performed according to Re et al. [25] and the FRAP assay was performed according to Benzie and Strain [26]. The calibration curve was constructed using Trolox, an hydrophilic analogue of vitamin E. The samples were analyzed at five different dilutions, within the linearity range of the assay, as described by Gentile et al. [27]. All the measurements were repeated two times and values expressed as μmol Trolox equivalent (TE) 100 g<sup>−</sup>1.

#### *2.9. Data Analysis*

Data were submitted to the Bartlett's test for the homogeneity of variance and then analyzed using an analysis of variance (ANOVA). Means were statistically separated on the basis of a Student–Newmann–Kewls test, when the 'F' test of the ANOVA for treatment was significant at least at the 0.05 probability.

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

#### *3.1. Rheological Characteristics*

The results of the rheological characteristics (Table 2) show that the origin of the purslane did not result in significant differences in the properties of the dough; on the contrary, the different percentages of substitution of wheat semolina with dried purslane induced highly significant differences both on the parameters detected by alveograph and farinograph.

It is known that gluten is composed of glutenin and gliadin subunits: the first ones give the dough toughness, while the gliadins determine its extensibility. The alveographic indices are normally related to the quality and quantity of gluten; in particular, the *p* value indicates the dough tenacity and the L value, its extensibility. The P/L configuration ratio indicates the balance between the two factors. As shown in previous works, the P/L parameter greatly affects the bread-making quality and a value of this ratio close to unity is favorable to the baking process [17].

Table 2 shows that a partial substitution (from 5 to 10%) of wheat semolina with dried purslane induced significant modifications on the alveographic parameters (P/L and W). By comparing the CTRL sample and the formulation containing 5% dried purslane, the W value decreased by about 50%; on the contrary, the P/L value tripled. In the formulation containing 10% dried purslane, the W value decreased by about 43% and the P/L value increased, indicating inextensible dough. Moreover, when the substitution level increased, up to 15%, the alveographic indices could not be determined, indicating that the high substitution level was opposed to the dough development. The farinographic parameters included the water absorption, development time and the dough stability. Table 2 shows that the value of all the farinographic indices increased with the increase in the percentages of substitution. In particular, it shows the greatest variation of water absorption among farinograph indices, which is in agreement with the observations of Wang et al. [28]. The partial replacement, from 5 to 15% wheat semolina by the dried purslane, led to an increase in water absorption (Table 2). As for the development time, only the mixture with 15% dried purslane showed a significant increase in DT value (7.50 min). Concerning S during mixing, the addition of different percentages of dried purslane caused a progressive increase in the values, in particular in the formulation containing 15% dried purslane. Significantly, S increased from 4.2 min (no addition) to 16.90 min (15%).

#### *3.2. Color, Form and Organoleptic Characteristics of the Fortified Breads*

The color of the food is the first parameter of quality evaluated by consumers. The objective values of CIELAB, on crust and crumb, the height and the volume of the bread samples, subjected to sensory analyses, are reported in Table 3, as the average of the percentage of substitution and of the purslane origin. The color indices were not affected by the type of purslane added both for the crust and crumb, that on average, had L\* values of 41.35 (crust) and 47.87 (crumb).


**Table 3.** L\*, a\*, b\* in the breads enriched with purslane on the average of concentrations and populations. Different letters indicate differences at *p* < 0.05.

The same behavior was noticed for the visual appearance of the loaf of breads (high 5.61 cm with a volume of 263.19 cm<sup>3</sup> as average). Statistical differences in color were recorded in relation to the percentage of purslane substitution for the crust and crumb; in fact, together they resulted darker, increasing from 0 (CTRL) to 15%. The height of the loaf of bread resulted improved by adding 5% purslane flours.

The addition of purslane to bread was expected to influence its structure, altering the organoleptic characteristics. For this reason, the sensory properties of the bread samples were investigated in this work were addressed (Table 4). All the samples had values ranging from 8 to 6 in the overall bread judgment until 10% substitution (Table 4), except for the Cas10 sample (5.3).


**Table 4.** Sensory attributes of the crust and crumb of the fortified breads with purslane and their overall judgment. The threshold of acceptability for the bread's overall judgment is 6.

<sup>a</sup> 1—good feeling and 10—bad feeling; <sup>b</sup> 1—extremely unpleasant, 10—extremely pleasant. CTRL—bread without purslane.

For crumb, the addition of purslane led to a darker color than for the CTRL. The thickness resulted higher than 4.0 in all the samples than in the CTRL (3.0), while the hardness sensation was higher until 10% substitution only for the Cas and Cal bread samples. SVen breads, on average of purslane fortification, gave the similar results (5.1) with respect to the CTRL (5.0).

The elasticity of crumb resulted improved in the Cas5 and Cas10 breads, while the other samples gave values lower than the CTRL (5.0). The alveoli average size and distribution were not affected by the dry purslane addition.

In general, the addition of purslane improved the darkness, as can be expected, with an increase in the percentage of substitution. With the exception of the 15% substitution, all the bread samples had good elasticity, thickness, friability and apparent softness with very small variations between the purslane accessions. The general judgment was always close to 6 (acceptability threshold) in the bread samples (means Cas 5.8, Cal 5.8 and SVen 6.2).

#### *3.3. Chemical Characterization and Antioxidant Activity*

Table 5 shows the FAMEs content detected in the CTRL bread and the fortified breads. The literature data of the FAMEs of dry purslane were also reported [12]. The content of the fatty acids in breads was influenced by both the percentage of substitution and the type of purslane used to enrich the bread samples. As it is possible to observe, the content in linolenic acid, linoleic acid and oleic acid is strongly influenced by the percentage of substitution. In fact, the initial content of FAMEs detected in dry purslane influenced the final fatty acid content of the fortified breads. The CTRL bread sample showed good levels of total FA (fatty acids 221.2 mg 100 g−1). The results obtained showed a 54% increase in Cas5, 23% in SVen5 and 10% in Cas10. On the contrary, no increase in content was observed in all the other bread samples.

**Table 5.** Fatty acid methyl esters (FAME) content (mg 100 g<sup>−</sup>1) in the fortified breads. Different letters indicate statistical differences at *p* < 0.05 among percentage of substitution into the same purslane population.


\* [12]; (LA: linoleic acid; ALA: α-linolenic acid; PUFA: polyunsaturated fatty acid; SFA: saturated fatty acid).

Regarding α-linolenic acid (ALA), 8.4 mg 100 g−<sup>1</sup> was the concentration of this fatty acid in the CTRL, but its content was higher in the all samples analyzed, in particular in the Cal5 (13.2 mg 100 g<sup>−</sup>1) Cas5 (16.6 mg 100 g−1) and SVen5 (13.2 mg 100 g−1). The linoleic acid concentration in CTRL was 132.3 mg 100 g<sup>−</sup>1, and increased considerably in Cas5 (203.0 mg 100 g−1) and SVen5 (143.8 mg 100 g−1). In general, for both Cas5 and SVen5, there was an increased concentration of PUFA compared to the CTRL (140.7 mg 100 g<sup>−</sup>1) of 219.6 and 156.9 mg 100 g−1, respectively. Focusing the contents of ALA and LA in dry purslane [7], we would have expected an increase with higher purslane substitution, which instead was not observed. We hypothesized that the baking process may have altered the structure of the PUFAs. Regarding the ω6/ω3 ratio, which in the CTRL was 15.7 mg g−1, this decrease was observed in all the samples and was within a range of 8.6 and 11.2 mg g<sup>−</sup>1.

These data agree with Giaretta et al. [29], that found the addition of kinako flour and chia seed to bread resulted in a significant increase in the content of polyunsaturated fatty acid (PUFA) with a lower ω6/ω3 ratio. The effect of the baking process was mainly directed on the ω6/ω3 ratio.

The importance of the ratio of the ω6/ω3 essential FA is well examined by Simopoulos [30]. According to the author, western diets are deficient in ω3 fatty acids, and have excessive amounts of ω-6 fatty acids, promoting the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases, whereas increased levels of omega-3 PUFA (a low ω-6/ω-3 ratio) exert suppressive effects.

On the contrary of FA, the TPC content, reported in Figure 1, increased with the percentage of dry purslane substitution. Common bread presented a TPC content of 27 mgGAE 100 g<sup>−</sup>1. On the average of the three types of purslane, adding 5% dry purslane the TPC content was double (56 mgGAE100 g<sup>−</sup>1). At 10 and 15% substitution, the effect of the dry purslane added resulted statistically different. The increase in TPC, using Cal purslane resulted 75 (Cal 10) and 91 mgGAE100 g−<sup>1</sup> (Cal 15), while using Cas or SVen purslane, the increase in TPC was smaller (73 mgGAE100 g<sup>−</sup>1, value averaged at 15% of Cas and SVen substitution).

**Figure 1.** The total phenol content (TPC) of the fortified breads, obtained with durum wheat flour and purslane at three different concentrations. (ns) indicates not statistically different. (\*\*\*) significant at 0.001 probability level. LSD (Least Significant Difference) (*p* < 0.05) Cas = 15.3; LSD (*p* < 0.05) Cal = 12.8; LSD (*p* < 0.05) SVen = 14.2.

Considering the results of the sensory properties of the fortified breads, the antioxidant properties, in terms of FRAP and ABTS, were analyzed until 10% purslane substitution. The FRAP (ferric reducing antioxidant power) assay was based on the reduction of ferric ion by phenols and the ABTS assay with 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) was based on free radical scavenging [16–18]. Results are graphed in Figure 2. An increase in antioxidant activity is one of the main aims of food supplementation. Both assays (FRAP and ABTS assays) highlighted the capacity of purslane to increase the antioxidant potential of enriched samples vs. the CTRL. We decided to focus the interest on the samples with the lower substitution, also in view of the best values of ALA content, TPC and antioxidant potential activity.

Regarding the ABTS results: breads with 5% purslane had an antioxidant activity of 244.7 μmol TE 100 g−<sup>1</sup> vs. 159.9 μmol TE 100 g−<sup>1</sup> in CTRL. The results of the FRAP assay had the same trend: also in this case, values did not result statistically different among the types of purslane, that on average had an antioxidant potential of 8921 vs. 580 μmol TE 100 g−<sup>1</sup> in CTRL.

**Figure 2.** Trolox equivalent (TE) antioxidant activity (FRAP assay and ABTS assay) of the fortified breads obtained with durum wheat flour and the three populations of purslane at 5 and 10% substitution. Different letters among bars indicate significant differences at 0.05 probability level. FRAP: ferric reducing antioxidant power); ABTS 2,2 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid).

#### **4. Conclusions**

The rheological characteristics of the wheat flour dough enriched with dried purslane provided useful information on the modifying effect of these additions on the behavior of the dough during its development, mixing and testing. However, the fundamental difference in the rheological effect of the additions was that the increase in the dose of dried purslane caused a reduction in its extensibility.

The inclusion in the wheat flour of plants rich in bioactive compounds certainly gave fortified bread good potential in relation to health benefits. Even though the increasing interest of consumers in these products, the food development and food design have to find a good compromise between the percentage of substitution of plant/herbs or spice and the sensorial properties. In our work, the enrichment of durum wheat flour by up to 5% purslane resulted in a good compromise to obtain the good rheological characteristics of loaves and breads with a decrease inomega-6/omega-3 essential FA ratio and good antioxidant properties. Considering in the Sicilian and Mediterranean tradition, common bread is made mainly using durum wheat, the addition of only 5% dry purslane could be a useful strategy to increase the bioactive compounds with potential health benefitsin bread. Trials concerning the shelf life of the products are ongoing.

**Author Contributions:** Conceptualization, M.G.M.; data curation, M.G.M., F.S., V.D.S., C.G. and M.P.; formal analysis, M.G.M., F.S., A.P., R.B. and V.D.S.; funding acquisition, M.G.M.; investigation, F.S., A.P., S.S., N.V. and C.G.; methodology, M.G.M., C.G. and M.P.; project administration, M.G.M.; resources, M.G.M.; software, N.V., C.G. and M.P.; supervision, M.G.M.; V.D.S. writing—original draft, M.G.M., V.D.S., C.G. and M.P.; writing—review and editing, M.G.M. and V.D.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work has been supported by National Research Council, CNR—DISBA project NutrAge (project nr. 7022).

**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/).

### *Review* **Health Benefits and Molecular Mechanisms of Resveratrol: A Narrative Review**

#### **Xiao Meng 1, Jing Zhou 2, Cai-Ning Zhao 3, Ren-You Gan 4,\* and Hua-Bin Li <sup>1</sup>**


Received: 5 February 2020; Accepted: 12 March 2020; Published: 14 March 2020

**Abstract:** Resveratrol is a bioactive compound in many foods. Since its anticancer activity was reported in 1997, its health benefits have been intensively investigated. Resveratrol has antioxidant, anti-inflammatory, immunomodulatory, glucose and lipid regulatory, neuroprotective, and cardiovascular protective effects, therefore, can protect against diverse chronic diseases, such as cardiovascular diseases (CVDs), cancer, liver diseases, obesity, diabetes, Alzheimer's disease, and Parkinson's disease. This review summarizes the main findings of resveratrol-related health benefits in recent epidemiological surveys, experimental studies, and clinical trials, highlighting its related molecular mechanisms. Resveratrol, therefore, has been regarded as a potent candidate for the development of nutraceuticals and pharmaceuticals to prevent and treat certain chronic diseases.

**Keywords:** resveratrol; bioactivities; anticancer; anti-obesity; antidiabetes; molecular mechanisms

#### **1. Introduction**

Many foodstuffs and their bioactive components are beneficial to certain diseases including cardiovascular diseases (CVDs) and cancer [1,2]. Resveratrol is a polyphenol, which naturally occurs in numerous foods, such as blueberries and peanuts, as well as grapes and their derived products like red wine [3]. Since resveratrol was reported to possess strong anticancer properties in tumor initiation, promotion, and progression stages in the well-regarded journal *Science* in 1997 [4], its bioactivities and health benefits have been intensively investigated. Many epidemiologic studies demonstrated that resveratrol is effective in the prevention of some diseases, such as CVDs and cancer [5,6]. Additionally, numerous experimental studies have illustrated that resveratrol is beneficial to a broad range of diseases, including CVDs, diabetes, obesity, cancer, liver diseases, Alzheimer's disease, and Parkinson's disease through redox/inflammatory/immune signaling pathways as well as the interplay between lipid and glucose metabolism [7–14]. Driven by the promising results from experimental research, many clinical trials have also elicited the efficacy of resveratrol against certain diseases [15,16].

This review summarizes the main findings regarding the bioactivities and health impacts of resveratrol, based on systematically searching English literature from Web of Science Core Collection and PubMed in the last five years, using the key word "resveratrol". The literature was categorized into epidemiological surveys, experimental studies, and clinical trials. In particular, this review highlights the health benefits of resveratrol on chronic diseases including CVDs, cancer, liver diseases, obesity, diabetes, Alzheimer's disease, and Parkinson's disease, and its related molecular mechanisms. We hope

that this narrative review paper can provide updated information about resveratrol and can attract more attention to its health benefits.

#### **2. Observational Studies**

Since resveratrol was illustrated to be one of the key factors in wine contributing towards the French paradox (high intake of saturated fat but low mortality from coronary heart disease), it has attracted overwhelming interest worldwide, and many epidemiological studies have investigated the relationship between resveratrol consumption and human health [17–20]. Specifically, dietary patterns rich in resveratrol were demonstrated to significantly reduce all-cause mortality [21,22]. Resveratrol also showed its potential to improve CVD risk factors, presenting significantly decreased fasting blood glucose, triglycerides (TGs), and heart rate [6]. In addition, a case-control study reported a significant inverse association between resveratrol from grapes (but not from wine) and breast cancer risk [17]. Furthermore, a lower risk of esophageal cancer was linked with higher resveratrol intake [5]. However, some null results or even harmful effects on health have also been reported. For instance, in a cross-sectional study in the Iranian population, resveratrol intake (top quantile, 0.054 mg/day and more) was positively associated with high blood pressure (hazard ratios, HR: 1.52; 95% confidence intervals, 95% CI: 1.02–2.27), without an significant association with waist circumference, TG, high-density lipoprotein (HDL), blood glucose, and metabolic syndrome [19]. Similar null outcomes were found in other studies [18,23]. In fact, the findings from different epidemiological studies are often inconsistent, because their validity depends on many factors like the study design, sample size, resveratrol dose, follow-up duration, as well as the participants' race, health status, eating patterns, and their food preference (Table 1). Nevertheless, the positive results support further exploration of what other health effects resveratrol might provide and how it achieves them.



#### *Foods* **2020**, *9*, 340

necrosis factor α; WC, waist

circumference.

#### **3. Experimental Studies**

Given the observed benefits based on resveratrol consumption, a great deal of research has explored more health outcomes of resveratrol as well as the underlying molecular mechanisms.

#### *3.1. Antioxidative Activities*

Resveratrol has shown strong antioxidant properties in many studies [24,25]. Resveratrol protects against oxidative stress, one of the primary contributors to many diseases, through various redox-associated molecular pathways (Figure 1 and Table 2). For instance, resveratrol upregulated the phosphatase and tensin homolog (PTEN), which decreased Akt phosphorylation, leading to an upregulation of antioxidant enzyme mRNA levels such as catalase (CAT) and superoxide dismutase (SOD) [26]. Resveratrol could also improve the antioxidant defense system by modulating antioxidant enzymes through downregulation of extracellular signal-regulated kinase (ERK) activated by reactive oxygen species (ROS) [27]. Meanwhile, resveratrol reduced the ischemia-reperfusion injury-induced oxidative stress by inhibiting the activation of the p38 mitogen-activated protein kinase (MAPK) pathway, thus the levels of antioxidants like glutathione (GSH) increased, and free radicals were directly scavenged [28]. Furthermore, resveratrol activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) to maintain the structural stability of forkhead box O1 (FoxO1), facilitate its translocation, and accomplish its transcriptional function [25]. Moreover, resveratrol was demonstrated to improve systemic oxidative and nitrosative stress by activating AMPK, then sirtuin 1 (SIRT1) and the nuclear factor erythroid-2-related factor 2 (Nrf2) associated antioxidant defense pathways, as Nrf2 acts as the master regulator of numerous genes encoding antioxidants and phase II-detoxifying enzymes and molecules [29,30]. Additionally, resveratrol exhibited antioxidant bioactivities by regulating antioxidant gene expression via the Kelch-like ECH-associated protein 1 (Keap1)/Nrf2 pathway and SIRT1 [31]. Recently, resveratrol was reported to attenuate oxidative injury owing to the induced autophagy via the AMPK-mediated inhibition of mammalian target of rapamycin (mTOR) signaling or via the activation of transcription factor EB (TFEB), which promoted the formation of autophagosomes and lysosomes as well as their fusion into an autolysosome [32,33]. Generally, resveratrol protects against oxidative stress mainly by (i) reducing ROS/reactive nitrogen species (RNS) generation; (ii) directly scavenging free radicals; (iii) improving endogenous antioxidant enzymes (e.g., SOD, CAT, and GSH); (iv) promoting antioxidant molecules and the expression of related genes involved in mitochondrial energy biogenesis, mainly through AMPK/SIRT1/Nrf2, ERK/p38 MAPK, and PTEN/Akt signaling pathways; (v) inducing autophagy via mTOR-dependent or TFEB-dependent pathway.

**Figure 1.** The antioxidant molecular mechanisms of resveratrol. -1 Resveratrol unanchors Nrf2 in the cytoplasm, disrupting its Keap1-dependent ubiquitination and degradation. The built-up Nrf2 translocates into the nucleus, binds to ARE, and initiates the transcription of many antioxidative genes such as SOD and CAT to reduce oxidative stress. -2 Resveratrol promotes the transcriptional functions of FoxOs in the nucleus to facilitate the transcription of many antioxidative genes like HO-1, contributing to the reduction of oxidative stress. -3 Resveratrol upregulated PTEN, a major antagonist of PI3K, blocking the Akt activation. Consequently, the activated Akt reduces, leading to decreased FoxOs phosphorylation. Therefore, less *p*-FoxOs translocate from the nucleus to the cytoplasm, and more FoxOs remain in the nucleus to act as transcriptional factors. -4 Resveratrol activates AMPK to maintain the structural stability of FoxOs, facilitate its translocation, and accomplish its transcriptional function. In addition, the activated AMPK phosphorylates PGC-1α, which can translocate into the nucleus, and be deacetylated by SIRT1. Then PGC-1α promotes Nrf2, leading to increased antioxidative gene expression and then reduced oxidative stress. Resveratrol activates AMPK, leading to SIRT1 activation, which inhibits MAPK signaling pathways and results in autophagy. -5 Resveratrol induces autophagy by activating TFEB, which promotes the formation of autophagosome and lysosome as well as their fusion into an autolysosome, leading to reduced oxidative stress. Abbreviations: AC, acetyl; Akt, protein kinase B; AMPK, AMP-activated protein kinase; ARE, antioxidant response element; CAT, catalase; ERK, extracellular signal-regulated kinase; FoxO, forkhead box protein O; GPx, glutathione peroxidase; GβL, G protein β subunit-like; HO-1, heme oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; LKB1, liver kinase B1; MAP2K, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; mSIN1, mammalian stress-activated protein kinase interacting protein 1; mTOR, mammalian target of rapamycin; mTORC2, mTOR Complex 2; NAD, nicotinamide adenine dinucleotide; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; Nrf2, nuclear factor (erythroid-derived 2)-like 2; P, phosphorylation; p53, phosphoprotein p53; PDK1, phosphoinositide dependent kinase 1; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1α; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PTEN, phosphatase and tensin homolog; Rictor, the rapamycin-insensitive companion of mTOR; SIRT1, sirtuin 1; SOD, superoxide dismutase; STAT, signal transducer and activator of transcription; TF, transcription factor; TFEB, transcription factor EB.

#### *3.2. Anti-Inflammatory Activities*

Resveratrol has been illustrated to have potent anti-inflammatory activities in many studies (Table 2) [34–37]. Resveratrol protected from inflammation not only by inhibiting the production of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), but also by inducing anti-inflammatory heme oxygenase-1 (HO-1) in RAW264.7 macrophages [38]. Additionally, resveratrol suppressed IL-6 transcription, modulating the inflammatory responses as an estrogen receptor α (ERα) ligand mediated by SIRT1, which functions as an ER coregulator [39]. Resveratrol could also inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling independent of SIRT1 [40]. Moreover, resveratrol attenuated inflammation by downregulating high mobility group box 1 (HMGB1) as well as suppressing NF-κB and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways [41,42]. In addition, both in vitro and in vivo, the anti-inflammatory effects of resveratrol were associated with its inhibition of the toll-like receptor 4 (TLR4)/NF-κB signaling cascade [43,44]. Furthermore, resveratrol attenuated inflammation by inhibiting the activation of NACHT, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome and inducing autophagy via SIRT1 upregulation and AMPK activation [34,35]. In a study on osteoarthritis, resveratrol interrupted an inflammatory amplification loop [45]. Specifically, the resveratrol-induced NF-κB inhibition resulted in decreased IL-6 secretion, leading to suppressed signal transducer and activator of transcription 3 (STAT3) activation in macrophages. Since STAT3 was responsible for the positive regulation of IL-6 secretion, inhibition of STAT3 made IL-6 levels even lower. Resveratrol could also block ERK1/2 activation, consequently upregulating MyD88 Short, a negative regulator of inflammation [36]. Altogether, resveratrol was able to regulate the pro- and anti-inflammatory cytokines and chemokines to protect against inflammation, mainly by upregulating SIRT1, suppressing NF-κB, and the associated cascades, as well as inhibiting NALP3 inflammasome activation.

#### *3.3. Immunomodulating E*ff*ects*

Resveratrol has exerted immunomodulating effects in various studies (Table 2) [46–48]. Resveratrol modulates immune response against pathogens like viruses, bacteria, and some toxic materials. For instance, resveratrol upregulated immune responses and reduced immunocyte apoptosis in chickens receiving conventional vaccinations and improved the growth of young chickens [49]. Resveratrol also reduced the activity of respiratory syncytial virus and inhibited the toll/IL-1 receptor domain-containing adaptor inducing β interferon (TRIF) expression through upregulating sterile α and armadillo motif protein (SARM) [50]. In addition, resveratrol prevented enterovirus 71 (EV71) replication and reduced the virus-induced elevated IL-6 and TNF-α secretion in rhabdosarcoma cells via suppressing IκB kinase (IKK)/NF-κB signaling pathway [48]. Moreover, resveratrol inhibited human rhinoviruses-16 replication and normalized virus-induced IL-6, IL-8 and regulated on activation normal T cell expressed and secreted (RANTES) as well as the expression of intercellular adhesion molecule-1 (ICAM-1) [47]. Additionally, resveratrol maintained the immune function in rotavirus-infected piglets, resulting in attenuated diarrhea and inflammation [51]. Moreover, resveratrol triggered an immune response to protect against non-typeable *Haemophilus influenzae* (a respiratory bacterium) without developing resistance in vitro [46]. Resveratrol also decreased bacterial viability and reduced infectious airway inflammation without noticeable host toxicity in vivo [46,52]. In addition, resveratrol showed immunomodulatory properties via reducing bacterial and inflammatory biomarkers in lipopolysaccharides (LPS)-challenged primary Atlantic salmon macrophages [52]. Resveratrol also modulated immunity caused by some toxic materials like concanavalin A (Con A), showing upregulation of SIRT1 and reduction of cytokines such as TNF-α, interferon γ (IFN-γ), IL-6, and monocyte chemoattractant protein-1 (MCP-1) [53]. Interestingly, resveratrol was found to strongly enhance immune activity in immunosuppressive mice, showing a bidirectional regulatory effect on immunity [54]. Specifically, resveratrol improved spleen lymphocyte proliferation, enhanced the function of peritoneal macrophages, and increased the CD4<sup>+</sup> cells in peripheral blood. Furthermore, some cytokines in the serum were upregulated, such as IL-1α/β, IL-2, and TNF-α. Based on a statistical analysis of human microarray data, a recent study revealed that resveratrol regulated many immune response pathways including peroxisome proliferator-activated receptor α (PPAR-α)/retinoid X receptor α (RXRα) activation, IL-10 signaling, natural killer cell signaling, leucocyte extravasation

signaling, and IL-6 signaling [55]. Recently, it was revealed that resveratrol could suppress the aryl hydrocarbon receptor (AhR) pathway, resulting in the reversal of imbalanced Th17/Treg, the main characteristic of immune thrombocytopenic purpura [56].In short, resveratrol could modulate both cellular and humoral immunity to reduce replication and the viability of pathogens, and bidirectionally regulate the related cytokine/chemokine production through the canonical immune response pathways as mentioned above.

#### *3.4. Cardiovascular Diseases*

Resveratrol has been reported to protect against CVDs in certain research (Table 2) [57–59]. Resveratrol prevented the pathological progression of hypertension, a major risk factor of CVDs, through Nrf2 activation, owing to its antioxidant and anti-inflammatory capacity [60]. Resveratrol could also lower blood pressure in hypertensive mice by inducing oxidative activation of cyclic guanosine monophosphate (cGMP)-dependent protein kinase 1α (PKG1α) [61]. Atherosclerosis is another main contributor to CVDs. Resveratrol was able to block atherosclerotic plaque progression by acting against pro-atherogenic oxysterol signaling in M1 (inflammation-encouraging) and M2 (inflammation-decreasing) macrophages [57]. Meanwhile, resveratrol prevented the activation of inflammasome, a contributor to the vascular inflammatory injury and atherosclerosis, via downregulating NF-κB p65 and p38 MAPK expression, and upregulating SIRT1 expression [62]. In addition, resveratrol ameliorated atherosclerosis partially through restoring intracellular GSH via AMPK-α activation, resulting in inhibited monocyte differentiation and reduced pro-inflammatory cytokine production [59]. Moreover, resveratrol regulated the band 4.1, ezrin, radixin, and moesin (FERM)-kinase and Nrf2 interaction, leading to decreased expression of ICAM-1 and then the inhibition of monocyte adhesion [63]. Resveratrol also exhibited antithrombotic effects via decreasing the tissue factors like TNF-α, and such action could be facilitated by aortic endothelial cells that could deconjugate resveratrol metabolites to free resveratrol [64]. Furthermore, one of the atherosclerosis consequences, high fat/sucrose diet-induced central arterial wall stiffening, was improved by resveratrol based on its protective activities against oxidative stress and inflammation [7]. Resveratrol also effectively prevented CVD by improving the cardiac and vascular autonomic function, protecting the erythrocytes via interacting with hemoglobin and reducing heme-iron oxidation [65,66]. In a heart failure model, resveratrol mitigated atrial fibrillation by upregulating PI3K and endothelial NOS (eNOS) [8]. In summary, the cardiovascular protective effects of resveratrol mainly depend on the capabilities of reducing oxidative stress and alleviating inflammation through Nrf2 and/or SIRT1 activation, PI3K/eNOS upregulation, and NF-κB downregulation.

#### *3.5. Cancers*

Resveratrol has exhibited protective impacts on various cancers, like colorectal cancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, liver cancer, and gastric cancer (Table 2) [14,67–71]. For instance, resveratrol was reported to inhibit the formation and growth of colorectal cancer by downregulating oncogenic KRAS expression [68]. Resveratrol also prevented tumorigenesis and progression of non-small cell lung cancer (NSCLC) cells by interrupting glycolysis via inhibition of hexokinase II expression, which was mediated by downregulation of the epidermal growth factor receptor (EGFR)/Akt/ERK1/2 signaling pathway [69]. Moreover, resveratrol showed pro-apoptotic/anti-proliferative effects in LNCaP cells (human prostate adenocarcinoma cells) through inducing the expression of cyclooxygenase (COX)-2, promoting ERK1/2 activation, and facilitating p53-dependent anti-proliferation gene expression [14]. In addition, resveratrol could decrease the efficiency of ovarian cancer cells adhering to peritoneal mesothelium in vitro by downregulating the production of α5β1 integrins and upregulating the release of soluble hyaluronic acid [70]. Resveratrol was also reported to inhibit the expression of phospholipid scramblase 1 (PLSCR1), leading to the growth inhibition of HeLa cells [71]. Furthermore, resveratrol showed proliferation-inhibitory and apoptosis-inducing effects in HepG2 cells by activating caspase-3 and caspase-9, upregulating the Bax/Bcl-2 ratio, and inducing p53 expression [72]. Resveratrol also inhibited the invasion and migration of human gastric cancer cells by blocking the epithelial-to-mesenchymal transition, which was mediated by metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) [73]. Additionally, resveratrol protected against breast cancer metastasis by promoting antitumor immune responses via blunting STAT3, leading to inhibited generation and function of tumor-evoked regulatory B cells (tBregs) as well as decreased production of transforming growth factor β (TGF-β) (a downstream target of STAT3), which was required by the tBregs to convert resting CD4<sup>+</sup> T cells to the metastasis-promoting FoxP3<sup>+</sup> regulatory T cells (Tregs) [67]. Although some research showed that resveratrol may be beneficial in breast cancer chemoprevention due to its non-estrogen function [74], different doses of resveratrol showed controversial effects due to its interaction with estrogens, which induce cellular proliferation and play a key role in breast cancer development and progression. Specifically, it was found that high concentrations of resveratrol could inhibit the proliferation of estrogen receptor alpha positive (ERα+) breast cancer, while low concentrations increased the growth of ERα+ cells [75,76]. It was reported that at low concentrations, resveratrol not only bound to ERs due to its structural similarity with E2, but also increased the formation of estrogen precursor steroids and inhibited the inactivation of active steroids, resulting in elevated active estrogen levels, leading to breast cancer cell growth and progression [77,78]. However, "more resveratrol is better" was challenged in some cases [79]. For instance, in terms of colorectal cancer chemoprevention, lower doses of resveratrol from dietary exposures exerted a better efficacy than high doses (200 times higher previously used in phase I clinical trials) due to its pro-oxidant activity at high doses and AMPK signaling upregulation.

Collectively, resveratrol has shown its anticancer bioactivities by impairing glycolysis, inhibiting cancer cell growth and proliferation, inducing apoptosis, promoting antitumor immune responses, and preventing adhesion, migration and invasion of cancer cells by modulating related molecules and gene expression through various signaling pathways. Of note, different doses may lead to very different effects, which sometimes could be opposite. Therefore, consideration regarding doses and matrix should be paid more attention in future studies.

#### *3.6. Liver Diseases*

Resveratrol has shown its protective impacts on several liver diseases in some studies (Table 2) [9,80–82]. Specifically, resveratrol alleviated non-alcoholic fatty liver disease (NAFLD) by upregulating the low-density lipoprotein receptor (LDLR) and scavenger receptor class B type I (SRB1) gene expressions in the liver [83], or by regulating autophagy and decreasing the activity of NF-κB, resulting from restoring its inhibitor, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) [84]. Resveratrol also improved high-fat diet (HFD)-induced fatty liver by downregulating adipose differentiation-related proteins and increasing the numbers of CD68<sup>+</sup> Kupffer cells [9]. As for chemical-induced liver diseases, resveratrol could markedly restore the morphology and function of alcohol-injured liver through inducing autophagy, or downregulating hypoxia-inducible factor 1α (HIF-1α) expression [85,86]. In addition, resveratrol ameliorated CCl4-induced liver injury by blocking the Notch signaling pathway [82]. Furthermore, resveratrol attenuated N -Nitrosodimethylamine-induced hepatic fibrosis by restoring the architecture and normalizing collagen deposition, mainly due to its antioxidative activities and downregulation of smooth muscle actin (α-SMA), which suppressed hepatic stellate cell activation [80,81]. Moreover, resveratrol pretreatment mitigated liver cirrhosis by improving the homing of bone marrow-derived mesenchymal stem cells [87]. In summary, resveratrol could improve NAFLD, chemical-induced liver injuries, fibrosis, and cirrhosis by modulating redox status, regulating lipid metabolism, ameliorating inflammation, and inducing autophagy with various cytokines, chemokines, and transcription factors involved.

#### *3.7. Diabetes*

Resveratrol has been elicited to attenuate diabetes and its relevant complications in many studies (Table 2) [88–91]. Resveratrol was observed to significantly reduce blood glucose levels, plasma lipids, and free fatty acids in diabetic mice, and it inhibited the expression of inflammatory mediators (e.g., ICAM-1, vascular cell adhesion molecule-1, and MCP-1) both in the aorta and in the blood, by inhibiting the NF-κB pathway [92]. In addition, resveratrol could relieve diabetes via increasing insulin action and glucose utilization due to visfatin expression restoration, SIRT1 activation, and glucose transporter modulation [89]. Moreover, resveratrol increased glucose uptake to improve insulin resistance in the muscle by decreasing diacylglycerol (DAG) accumulation and protein kinase C θ (PKC-θ) translocation, and preventing lipolysis under the condition of adipose hypoxia, because resveratrol could preserve phosphodiesterase 3B expression (PDE 3B) to downregulate cyclic adenosine monophosphate (cAMP), leading to the inhibition of protein kinase A (PKA)/hormone-sensitive lipase (HSL) activation [90]. Moreover, resveratrol showed protective effects on adipose tissue in diabetic mice by preventing ROS-mediated mitochondrial fission via AMPK-dependent upregulation of Drp1 phosphorylation, and by blocking the activation of NALP3 inflammasome via inhibition of endoplasmic reticulum stress (ERS) [93]. Resveratrol also protected against diabetic complications such as myocardial fibrosis, diabetic nephropathy, and erectile dysfunction [11,88,94]. Furthermore, maternal resveratrol administration to the rats was evidenced to prevent the offspring's glucose intolerance and islet dysfunction, which were associated with gestational diabetes [91]. In summary, resveratrol could effectively regulate glucose metabolism, improve insulin resistance, improve diabetic complications, and restore the function of multiple systems via modulating SIRT1/NF-κB/AMPK signaling pathways and some associated molecules like NALP3 inflammasome, as well as the expressions of relevant genes.

#### *3.8. Obesity*

Obesity has become a severe health issue globally. Resveratrol significantly decreased the body weight and fat mass in mice with HFD-induced obesity, showing reduced leptin and lipids in plasma, modulated metabolism of glucose and insulin, and restored immune dysfunction, via the activation of PI3K/SIRT1 and Nrf2 signaling pathways, and the inhibition of transcriptional regulators (e.g., EP300 gene), which are involved in the differentiation of adipocytes as well as lipid storage and metabolism [95,96]. Moreover, besides a significant dose-dependent decrease of weight gain and lipid deposition in the liver and adipose tissues of HFD-induced obese mice, low concentrations of resveratrol (1–10 μM) suppressed adipogenic differentiation in pre-adipocytes, downregulated the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) and perilipin protein in differentiated adipocytes, and inhibited TNF-α-induced lipolysis in mature adipocytes [97]. Additionally, resveratrol prevented against obesity through markedly enhancing the catecholamine production, accompanied by suppressing the pro-inflammatory M1 macrophages and activating anti-inflammatory M2 macrophages in white adipose tissue, which play a pivotal role in the trans-differentiation of white adipocytes into beige adipocytes [98]. Furthermore, resveratrol administrated to the pregnant and lactating mice led to promoted white adipose browning and thermogenesis in the male descendants, and these health benefits persisted and prevented obesity in their future life [99]. In addition, resveratrol protected against sarcopenic obesity by improving mitochondrial function and reducing oxidative stress through the PKA/liver kinase B1 (LKB1)/AMPK pathway [100]. Resveratrol also showed positive impacts on obesity-related complications, such as reproductive dysfunction like infertility and endocrine disorders [101,102]. To summarize, resveratrol has been illustrated to decrease body weight, regulate lipid deposition, modulate adipocyte gene expression, and promote white adipose browning, via PI3K/SIRT1, Nrf2, PPAR-γ, TNF-α, and PKA/LKB1/AMPK signaling pathways (Table 2).

#### *3.9. Alzheimer's Disease and Parkinson's Disease*

Alzheimer's disease and Parkinson's disease are neurodegenerative disorders, seriously decreasing life quality, while resveratrol may have the potential to improve these diseases. For instance, resveratrol inhibited the aggregation of amyloid β (Aβ), a key factor in Alzheimer's disease, by modulating specific proteins such as ubiquitin-like protein (UBL)/X-box binding protein 1 (XBP-1) involved in proteostasis [103]. Furthermore, resveratrol prevented memory loss in Alzheimer's disease by decreasing elevated levels of mitochondrial complex IV protein in the mouse brain via the activation of SIRT1 and AMPK pathways [104,105]. In terms of Parkinson's disease, resveratrol ameliorated ERS by downregulating the gene expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), inhibiting caspase-3 activity in the rat brain, and ameliorating oxidative damage via suppressing xanthine oxidase activity and protein carbonyl formation as well as activating the glutathione peroxidase and Nrf2 signaling pathway [10]. Resveratrol also alleviated Parkinson's disease through elevating miR-214 expression, leading to decreased mRNA expression of α-synuclein [106]. Taken together, resveratrol ameliorated Alzheimer's disease and Parkinson's disease by activating the SIRT1, AMPK, and Nrf2 signaling pathways and modulating the associated gene expressions (Table 2).

#### *3.10. Sex-Dependent E*ff*ects of Resveratrol*

Acting as an estrogen agonist, resveratrol showed sex-dependent effects on some diseases, which causes increasing concerns (Table 2). CVD risk increases with increasing age, gradually in men while disproportionately in women, and such a lower risk was in association with estrogen's cardioprotective properties [107,108]. In a recent in vivo study, sex differences were observed in rats with surgically-induced myocardial infarction (MI) due to the resveratrol treatment (2.5 mg/kg/d) [109]. Superior improvements were observed in females in terms of interventricular septal wall dimension at systole (IVSDs), end-systolic volume (ESV), ejection fraction (EF), fractional shortening (FS), and isovolumic relaxation time (IVRT), among which IVRT was purely sex-dependent. In another study, long-term resveratrol treatment in rats (50 mg/L in drinking water, 21 days) increased the relaxations to estrogen in aortae, more potently in males, probably due to the effects of resveratrol on promoting nitric oxide and/or suppressing superoxides [110]. Alongside this, it was revealed that resveratrol (20 mg/kg) significantly increased dopamine transporter (DAT) in the striatum in female but not in male mice [111]. The in vitro study in the same research indicated that resveratrol upregulated DAT in the dopaminergic cells by inducing its gene transcription. Additionally, sex differences in the effect of resveratrol were also found in a mouse model of dextran sulfate sodium-induced colitis [112]. Adverse effects were observed in females but not in males, regarding weight loss, stool consistency, and discomfort. Such results indicate that special attention should be paid to the application of resveratrol, a phytoestrogen, which can interact with hormone receptors and result in sex-dependent effects that can be beneficial or harmful.



*Foods* **2020** , *9*, 340


#### *Foods* **2020** , *9*, 340



**Table 2.***Cont.*


**Table2.***Cont.*

