Regarding cancer, it is known that the proteolytic activity which contributes to the degradation of ECM and basal membrane represents a key step in cell invasion, metastasis and angiogenesis [4]. Moreover, several studies have posed evidence of the crucial role of gelatinases in numerous invasive cancers, such as breast cancer [5,6]. Recent observations also provide evidence that MMPs modulate various aspects of inflammation, some of which seem to be essential for the suppression of innate immune response against tumor cells; in this context, MMPs may exert an immune regulatory function in tumor microenvironment, helping cancer cells to escape immune surveillance [7]. It is easy to understand that the identification of MMP inhibitors (MMPi) today represents an important opportunity for the treatment and prevention of numerous chronic and life-threatening diseases. For these reasons, MMPs inhibitor drug discovery has emerged as an important area of research in many fields of medical sciences as also attested by the discovery or repositioning of several MMPs inhibitors [8–10].

In the last decade, great interest was generated in the characterization and use of natural compounds credited with high bioactive potential, including anti-inflammatory, antibacterial, antioxidant, anti-cancer and anti-diabetic properties [11–13]. Into this scenario fits the interest in the identification of natural compounds capable of controlling the expression and activity of MMPs. Specifically, flavonoids have been found to influence MMPs levels in different ways. In many cell types, flavonoids have been described to down-regulate MMPs biosynthesis. Quercetin, for instance, was reported to inhibit the invasivity of murine melanoma cells by decreasing pro-MMP-9 via PKC pathway [14]. It is also known that certain MMPs can be activated by oxidative stress and the antioxidative effect of flavonoids may influence this mechanism [15].

*Salvia miltiorrhiza*, also known as red sage or Chinese sage, is one of the most widely used traditional herb medicines recommended for the treatment of a variety of diseases, such as cardiovascular diseases, hepatitis, hepatocirrhosis, chronic renal failure and dysmenorrhea. In *S. miltiorrhiza,* two pharmacologically active fractions were identified: lipophilic diterpenoids transhinones and water-soluble phenolic acids [16]. In recent years, research has been focused on the phenolic acid fraction where twenty-five phenolic acid compounds have been isolated and identified. Specifically, Salvianolic acid B (SalB) represents the most abundant compound, accounting for 3–5% of total dried weight [17]. To date, SalB has been identified as an agent which may be useful in controlling the expression of MMP-2 and MMP-9 in pathological conditions involving the cardiovascular system. In this context, we should cite the inhibition of MMP-2 upregulation in human aortic smooth muscle cells via suppression of NAD(P)H oxidase-derived reactive oxygen species [18], the prevention of the infarct-induced cardiac remodeling through competitive inhibition of MMP-9 [19] and the attenuation of cardiac fibroblast migration, collagen and cytokine secretion through the in vitro inhibition of the catalytic domain of MMP-9 [20]. However, little attention has been paid to the role of SalB in tumor cell lines characterized by high invasivity potential, in which MMPs play a leading role. The aim of this work is therefore to verify the effect of SalB in regulating the in vitro function of MMP-9 in MDA-MB-231 human breast cancer cells. The only studies that involved the treatment of MDA-MB-231 cells with SalB focused attention on different biochemical and molecular aspects. For example, the work published by Sha et al. [21] analyzed the effects induced by the natural compound on the cell viability, cell cycle and apoptosis of triple-negative MDA-MB-231 cells with the hormone receptorpositive MCF-7 cells as the control. The main finding of the work was specifically related to

SalB ability to enhance the cell apoptosis and decrease cell proliferation by regulating the ceramide glycosylation enzymes. Overall, most of the studies performed on human breast cancer cells involved the use of non-invasive phenotypes (MCF-7 cells), confirming the role of SalB in cell proliferation, migration and invasion abilities, without specific assessments on the gelatinases function [22].

#### **2. Results**

*2.1. In Silico Evaluations and In Vitro Analysis of the Interaction between Salvianolic Acid B and MMP-9*

The SwissADME (absorption, distribution, metabolism and excretion) web tool was exploited with the aim to obtain a prediction concerning the pharmacokinetic properties of SalB. The main information of the analysis is reported in Table 1, and indicate low absorption from the gastrointestinal (GI) tract and the inability to permeate the blood–brain barrier (BBB). Furthermore, SalB seems unable to inhibit the activity of cytochrome P450 isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4), whose function is associated with drug elimination through metabolic biotransformation. In Figure 1, the bioavailability radar that gives a first glance on the drug-likeness of the compound is instead reported, considering a total of six physicochemical properties: lipophilicity, size, polarity, solubility, flexibility and saturation. From this point of view, SalB showed itself to be a good candidate with reference to lipophilicity and solubility, while the report highlighted values outside the physicochemical range in the case of size, polarity, flexibility and saturation.

In order to predict the most probable binding conformations between SalB and MMP-9, a preliminary in silico study was performed. The most favorable docking is associated with a ΔG value equal to −14.786 kcal/mol; in this condition (Figure 2A), SalB is able to approach the active site of the enzyme, placing its carboxylic group close to the catalytic zinc ion. In addition to this, the distance between the O6 SalB carboxylic oxygen and the catalytic zinc ion is 2.2 Å (Figure 2B), suggesting the plausible existence of a non-covalent interaction.

**Table 1.** SwissADME (absorption, distribution, metabolism and excretion) parameters attributed to SalB.

MW = molecular weight; HBA = H-bond acceptors; HBD = H-bond donors; TPSA = Topological Polar Surface Area; GI = gastrointestinal; BBB = blood–brain barrier; P-gp = permeability glycoprotein; iLOGP, XLOGP3, WLOGP, MLOGP, SILICOS-IT represent lipophilicity predictive models [Daina et al. 2017]; CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4 are isoforms of cytochrome P450.

**Figure 1.** Bioavailability radar chart of SalB. The bioavailability radar gives a first glance on the drug-likeness of the compound, considering a total of six physicochemical properties: lipophilicity, size, polarity, solubility, flexibility and saturation. The pink zone represents the physicochemical space for oral bioavailability, and the red line represents the specific oral bioavailability properties associated with the analyzed compound.

**Figure 2.** Molecular Docking analysis of the interaction between SalB and MMP-9. (**A**) SalB ability to approach the MMP-9 catalytic pocket. (**B**) Molecular docking of SalB in the catalytic domain of MMP-9 (PDB code: 1GKC). SalB seems able to interact with the catalytic Zn2+ ion which is coordinated by three histidines (HIS401, HIS405, HIS411). The analysis specifically suggests the existence of a coordinated bond between the catalytic Zn2+ atom and a carboxyl oxygen of SalB structure.

To confirm the computational results, an enzymatic inhibition assay was performed. For this purpose, a recombinant active human MMP-9, composed of the catalytic domain, the gelatin binding domain and the metal binding domain (amino acids 107–457), was used. Increasing the concentrations of SalB ranging from 0 μM to 100 μM, in the presence of different concentrations of substrate (0.375–1.5 μM), suggested a competitive inhibition of the activity performed by the MMP-9 catalytic domain (CDMMP-9) (Figure 3A), and allowed to define a Ki value equal to 57.37 ± 3.96 μM (Figure 3B).

**Figure 3.** Kinetic analysis of SalB against the catalytic domain of MMP-9. (**A**) Double reciprocal plots of 1/V versus 1/[S] suggest the competitive inhibition of the activity performed by the catalytic domain of MMP-9 (CDMMP-9). (**B**) Secondary plot of KMapp versus different SalB concentrations. The Ki value of SalB against CDMMP-9 was calculated to be equal to 57.37 μM.

#### *2.2. Evaluation of Gelatinolytic Activity in MDA-MB-231 Cells*

The zymographic analysis was performed on conditioned media obtained from MDA-MB-231 cells treated for 24 h and 48 h with SalB 5 μM (SalB-5) and 50 μM (SalB-50). Such analysis was effective in highlighting the gelatinolytic potential attributable to MMP-2 and MMP-9.

As shown in Figure 4A, SalB was able to influence the activity of MMP-9, which was significantly reduced in the presence of the higher concentration of the natural compound (50 μM), both after 24 h and 48 h of treatment (*p* < 0.05). The analysis also highlighted the activity of the active form of the enzyme, which follows the same pattern observed for the zymogen, with lower ability to hydrolyze gelatin in SalB-50 samples at both treatment times (*p* < 0.05).

**Figure 4.** Gelatin Zymography for the evaluation of MMP-9 and MMP-2 activity. Zymographic evaluation of MMP-9 (**A**) and MMP-2 (**B**) activity in conditioned media obtained from MDA-MB-231 cells treated for 24 h and 48 h with SalB 5 and 50 μM.

With regard to MMP-2 (Figure 4B), no significant variations were evidenced, both in relation to the dosage and the timing of the treatment (*p* > 0.05).

#### *2.3. Salvianolic Acid B Had no Effects on TIMP-1 Expression in MDA-MB-231 Cells*

The potential role of SalB in influencing the endogenous regulation of MMP-9 activity was evaluated by focusing the attention on TIMP-1. Figure 5 shows the results obtained from reverse zymography and Western blot performed on conditioned culture media, and RT-PCR applied on mRNA purified from the corresponding cells. By combining these different approaches, no significant changes (*p* > 0.05) were found in the expression and in the release of TIMP-1 in the extracellular environment.

**Figure 5.** Analysis of TIMP-1 through reverse zymography, Western blot and RT-PCR. The role of the tissue inhibitor of metalloproteinases-1 (TIMP-1) has been evaluated through reverse zymography and Western blot that were performed on conditioned media obtained from MDA-MB-231 cells treated for 48 h with SalB 5 and 50 μM. In the same sampling were also collected cells for transcripts evaluation by RT-PCR.

#### *2.4. MMP-9 Expression Was Affected in Treated MDA-MB-231 Cells*

The samples previously used for the zymographic evaluations, were also subjected to Western blot analysis to verify the relative amount of the enzyme in the conditioned culture media obtained from the various treatments (Figure 6A). In this case, SalB significantly affected the amount of MMP-9 in the conditioned culture medium (*p* < 0.01) only when it was administered at the highest concentration (50 μM) during 48 h treatment.

**Figure 6.** Analysis of TIMP-1 through reverse zymography, Western blot and RT-PCR. The Western blot analysis and RT-PCR of MMP-9. (**A**) The MMP-9 expression was firstly evaluated through Western blot on conditioned media obtained from MDA-MB-231 cells treated for 24 h and 48 h with SalB 5 and 50 μM. Different lower case letters (a,b) indicate significant differences (*p* < 0.05) for the proMMP-9, while different upper case letters indicate significant differences (*p* < 0.05) for the active form of MMP-9. (**B**) The collection of MDA-MB-231 cells was useful to perform the analysis on MMP-9 transcripts after 48 h treatment with SalB 5 and 50 μM.

In order to verify any changes at the molecular level, the MMP-9 transcripts in cells subjected to 48 h treatment were purified and analyzed. As shown in Figure 6B, a significant reduction in MMP-9 transcript expression (*p* < 0.05) was effectively noted following the treatment with 50 μM SalB, while no variations were evidenced in SalB-5 samples (*p* > 0.05).

#### **3. Discussion**

Several studies have been conducted to evaluate the ability of SalB to regulate the activity of gelatinases, particularly of MMP-9. To our knowledge this is the first study in which this aspect is evaluated on human MDA-MB-321 breast cancer cells, a highly invasive cellular model characterized by a marked constitutive expression of MMP-9.

Before the evaluation of SalB effect on the selected cell line, a preliminary in silico study was performed for the prediction of the drug-likeness of the natural compound and the most probable binding conformations between SalB and MMP-9. The SwissADME web tool represents an informative approach in order to obtain a prediction about the biotransformation of specific compounds into drugs. First of all, this evaluation evidenced for SalB low absorption from the gastrointestinal tract and the inability to permeate the blood–brain barrier (BBB). In particular, this last aspect argues in favor of the fact that SalB should not induce adverse effects at the level of the central nervous system. Besides this, SalB does not represent a substrate of the permeability glycoprotein (P-gp) that was suggested to be one of the most relevant members among the ATP-binding transporters, responsible for limiting the oral bioavailability of drugs that act as its substrates [23]. In the sphere of pharmacokinetic parameters, the finding concerning the interaction of the natural compound with cytochromes P450 is also relevant, which is actively involved in the processes of drug elimination through metabolic biotransformation [24]. SalB does not represent an inhibitor of these factors, an aspect of considerable importance considering that the inhibition of these isoenzymes was reported to be one of the main causes of pharmacokinetics-related drug–drug interactions, leading to toxic or other unwanted adverse effects due to the lower clearance and accumulation of the drug or its metabolites [25]. The bioavailability radar generated by the web tool, that gives a first glance on the drug-likeness of the compound, showed overall good attitudes for SalB with reference to lipophilicity and solubility, while conditions outside the physicochemical range were evidenced in the case of size, polarity, flexibility and saturation. This finding testifies to the fact that in the pharmaceutical field it could be more plausible for the use of specific structural domains of the natural compound, rather than the molecule in its entirety.

With reference to the specific interaction between SalB and MMP-9, molecular docking evaluations were performed. Specifically, the most favorable docking is associated with a ΔG value equal to −14.786 kcal/mol; in this condition SalB is able to approach the active site of the enzyme, placing its carboxylic group close to the catalytic zinc ion. In this conformation, a distance of 2.2 Å was calculated between the SalB carboxylic oxygen and the catalytic zinc atom, suggesting the possible onset of a non-covalent interaction between SalB and MMP-9, in addition to the three coordination interactions between zinc and the catalytic histidines (HIS401, HIS405 and HIS411). Such geometrical constraints are therefore compatible with a tetracoordinated chelation model of the catalytic zinc ion as also suggested by Jacobsen et al. [9] who inserted the carboxylic residue in the list of the "zinc binding domains".

In order to confirm the computational results, an enzymatic inhibition assay was performed by using a recombinant active human MMP-9. The calculated Ki value was equal to 57.37 ± 3.96 μM, a finding in full agreement with what has been previously reported by Jiang et al. [19], who found a Ki value of 79.2 μM, by using a similar analytical strategy.

The encouraging data obtained by the in silico analysis confirmed by the enzymatic inhibition assay, provided a rationale for the in vitro investigation on human MDA-MB-231 breast cancer cells, characterized by a marked invasive phenotype, supported by a high constitutive expression of several MMPs, especially MMP-9 [26].

In order to define the best experimental conditions for cell treatment with SalB, an MTT cell viability assay was performed, allowing us to define the range of concentrations of SalB and the time of exposure in which the cytotoxic effect does not exceed 10% of cell mortality with respect to untreated cells. In our intention, this was performed to make sure that the treated cells maintain the same vitality as the control cells, in order to ensure that any variations in the expression of MMP-9 were to be totally attributed to the effect of SalB and not simply to a reduction in cell viability. Through this preliminary approach, we believe we have given greater strength to the considerations made on the specific effect of the natural compound on the MMP-9 function. Cells were then treated with two concentrations of SalB, 5 μM (SalB-5) and 50 μM (SalB-50), for 24 h and 48 h, respectively.

The zymographic analysis of the conditioned media showed the ability of SalB to inhibit the activity of MMP-9. The analysis also highlighted the activity of the active form of the enzyme, which follows the same pattern observed for the zymogen. This observation suggests that the relation between the two forms remains unchanged under all the experimental conditions and testifies to the fact that SalB had no effect on the proenzyme activation mechanism. The observed reduction in activity may therefore depend on a competitive inhibition model based on the prevalent interaction of SalB with the catalytic domain of the enzyme. This hypothesis finds support in the results obtained from the in silico investigation and is in agreement with what was previously observed by Jiang et al. [19] who hypothesized a competitive inhibition of MMP-9 by the SalB. In the same zymograms, the activity of MMP-2 was also visualized. In this case, the SalB did not induce significant variations; a behavior already highlighted in a previous study in which no significant regulation of SalB on MMP-2 activity was found, suggesting that the natural compound was more specific on MMP-9 than on MMP-2 [19]. However, it must be reported that in other studies conducted on murine models or alternative cell lines, a drastic reduction in MMP-2 activity was reported as a consequence of the ability of SalB to interact with mediators of gelatinase expression. One of the best characterized mechanisms concern the inhibition of the tumor necrosis factor-α (TNF-α)-induced MMP-2 upregulation in human aortic smooth muscle cells via suppression of the NAD(P)H oxidase-derived reactive oxygen species [18,27].

It is known that the MMP activity in the extracellular environment is finely regulated by specific endogenous inhibitors (TIMPs) which interact with the enzyme in a stoichiometric 1:1 ratio. For this reason, in the next phase of the study, the role of TIMP-1, which possesses a prevailing selectivity for gelatinases, was evaluated in order to better understand the effect of SalB treatment on MDA-MB-231 cells, and in particular on MMP-9 function. By combining different approaches, no significant changes were found in the expression and in the release of TIMP-1 in the extracellular environment, a fact that confers additional strength to the SalB ability to act as an MMP-9 inhibitor. Concerning the relationship between SalB and TIMPs, no specific studies have been conducted. However, the study performed by Dai et al. [28] must be taken into consideration. They investigated the antifibrotic activity of the active compounds of *Salviae miltiorrhizae* on mice oral mucosal fibroblasts and reported the ability of tanshinone IIA, salvianolic acid A and SalB to reduce TIMP-1 and TIMP-2 expression, a phenomenon that predisposes an increase in gelatinases activity. In agreement with what has been described, it should be also reported that other studies highlighted that flavonoids generally have the ability to influence the expression of TIMPs, with even quite marked effects on the ECM remodeling processes. These are evidence observed in cell lines and in vivo models in which, contextually to the effect on TIMPs, perturbations were also monitored for the expression and activity of other factors involved in crucial signaling pathways such as focal adhesion kinase (FAK), phosphatidylinositol-3-kinase (PI3K)-Akt, signal transducer and activator of transcription 3 (STAT3), nuclear factor κB (NFκB), and mitogen-activated protein kinase (MAPK) [29].

The same samples used for the zymographic evaluations were also subjected to Western blot analysis to verify the amount of the enzyme in the conditioned culture media obtained from the various treatments. In this case, SalB seems to be able to affect the amount of MMP-9 in the conditioned culture medium only if administered at the highest concentration for relatively longer time intervals. This may be due both to a direct effect of SalB in the reduction of gene expression, but also to the ability of the natural compound to interfere with the release of the zymogen from the cytosol to the extracellular environment. For this reason, the MMP-9 transcripts from cells subjected to 48 h treatment with SalB were purified and analyzed in order to highlight any changes in their expression. A significant reduction in MMP-9 expression was effectively noted following the treatment with 50 μM SalB. This therefore confirmed SalB tendency to reach the cytoplasm and interfere with the biochemical mechanisms responsible for regulating the gene expression. In this regard, Lin et al. [27] discussed the ability of SalB to inhibit MMP-2 and MMP-9 expression in LPS-stimulated human aortic smooth muscle cells (HASMCs) advancing the hypothesis of a mechanism based on the suppression of JNK and ERK phosphorylation. This is the first time, in our knowledge, that this aspect is highlighted in invasive tumor cells. Therefore, the specific biochemical mechanisms involved require further and more specific evaluations.

#### **4. Materials and Methods**

#### *4.1. Reagents*

The Salvianolic acid B (SalB), dimethyl sulfoxide (DMSO), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], trypan blue, Triton X-100, Tween 20 and type B gelatin were purchased from Sigma-Aldrich Chemical Co. (Milan, Italy). Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), penicillin, streptomycin, glutamine and trypsin were purchased from Euroclone S.p.A. (Milan, Italy). Human recombinant catalytic domain of MMP-9 was obtained from Vinci Biochem S.r.l. (Firenze, Italy). All other chemicals were reagent grade. Stock solution of SalB (50 mM) was prepared in DMSO and stored in the dark at −20 ◦C.

#### *4.2. ADME Analysis and Modelling of the Enzyme-Inhibitor Interaction*

When the condition of developing a new drug is expected, it is essential to carry out evaluations that can give preliminary information on the compound being studied, with particular regard to absorption, distribution, metabolism and excretion (ADME). For this purpose, we used the web service SwissADME (http://www.swissadme.ch/index.php, accessed on 16 November 2022) [30,31] which was made available by the Swiss Institute of Bioinformatics.

The evaluation of the interactions between SalB and MMP-9 was performed by using the web service (SwissDock) developed by the Swiss Institute of Bioinformatics in order to predict the most favorable binding modes that may occur between a target protein and a small molecule. This web tool exploits the docking software EADock DSS [32] and the CHARMM force field method for calculation [33]. As a model was used, the crystal structure of MMP-9 complexed with a reverse hydroxamate inhibitor (PDB code: 1GKC), and the docking clusters related to the most favorable interactions with SalB (ZINC entry: 49538628) were visualized and analyzed by using PyMOL Molecular Graphics System.

#### *4.3. Fluorometric Inhibition Assay of MMP-9 Activity*

The in vitro ability of SalB to influence MMP-9 activity was performed through a fluorometric inhibition assay. For this purpose, we used the recombinant catalytic domain of MMP-9 (CDMMP-9) which represents the 39 kDa active site of the protein (aa 107–457 + NT His Tag). The enzymatic residue was supplied as lyophilized powder which was reconstituted following manufacturer's instructions with pre-chilled 30% glycerol solution to 10 U/μL. The rapid and sensitive determination of CDMMP-9 proteolytic potential in the presence of increasing concentrations of SalB (from 5 μM to 100 μM) was evaluated with a spectrofluorimetric method, as previously reported [10]. Briefly, the methodology is based on monitoring the ability of the enzyme to specifically cleave the self-quenched synthetic substrate MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 (Peptide Institute INC, Osaka, Japan).

The apparent Michaelis constant (KMapp) values for the interaction between the enzyme and different SalB concentrations were calculated by using the double reciprocal plots of 1/V versus 1/[S]. Such parameters were then used to draw a secondary plot for the determination of the apparent inhibition constant (Ki).

#### *4.4. Identification of the Experimental Conditions and Cell Culture*

The The MDA-MB-231 human breast cancer cells were obtained from the American Type Culture Collection (ATCC) and maintained in exponential growth in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM glutamine, and kept in a humidified atmosphere with 5% CO2 at 37 ◦C. Cellular viability was determined by trypan blue exclusion assay.

The concentration range and the timing of the cellular treatment with the SalB have been defined through the MTT colorimetric approach which measures viable cells by assessing the conversion of MTT into formazan crystals by mitochondrial activity [34]. Exponentially growing cells were plated in 96-well plates and after 24 h, the medium was

replaced with fresh medium and cells were treated with SalB in the range of concentration from 5 μM to 100 μM for 12 h, 24 h, 48 h and 72 h. Negative controls received the same amount of DMSO (in the ratio 1:1000 in the culture medium) used to solubilize the SalB administered in the other wells. At the end of each incubation period, MTT was added to each well at the final concentration of 0.5 mg/mL and incubated at 37 ◦C for 3 h. The reaction responsible for the formazan release was stopped by the addition of 0.04 N isopropanol, the absorbance was measured at 570 nm in a microplate reader (Biorad, Hercules, CA, USA).

After defining the parameters for cell treatment with SalB, cells were seeded in six well culture dishes at a density of 5 × <sup>10</sup><sup>4</sup> cells/well. After 24 h, the medium was replaced with fresh medium and cells were treated with 5 μM (Sal-5) and 50 μM (Sal-50) of SalB. Control cells (CTR) received DMSO in a concentration equal to that used for the solubilization of Sal-5 and Sal-50. After 24 h and 48 h, conditioned media were collected, aliquoted and stored at −20 ◦C until analysis.

#### *4.5. Evaluation of Gelatinases Activity by Zymography and Determination of TIMPs Expression by Reverse Zymography*

Protein concentration in conditioned media was determined by Bradford protein concentration assay. Volumes of each sample corresponding to 10 μg of total proteins were diluted in a non-reducing sample buffer without heating and resolved by 8% SDS-PAGE containing 0.3 mg/mL type B gelatin. The gels were then incubated for 45 min in a renaturation buffer (50 mM Tris-HCl pH 8.0, containing 2.5% Triton X-100) to remove SDS. Subsequently, a 24 h incubation in the developing buffer (50 mM Tris-HCl pH 8.0, containing 5 mM CaCl2, 200 mM NaCl and 0.02% Brij 35) was performed to allow enzyme renaturation and activity. Gels were then stained in a 0.1% solution of Coomassie Blue R250 in 40% (*v/v*) methanol and 10% (*v/v*) acetic acid. This analysis was preceded by preliminary evaluations, in which the supernatant of HT1080 fibrosarcoma cells was used as a reference standard for both MMP-2 and MMP-9, as recommended by Toth et al. [35]. Furthermore, in order to verify the metallo-protease nature of the activity displayed, were carried out tests in which the incubation of the zymographic gels was performed in a developing buffer in which a chelating agent (EDTA 100 mM) was added (in this condition no degradation of the substrate was found). Quantitative analysis of visualized spots was performed by using ImageJ software [36].

To reveal the presence of TIMPs in collected samples, reverse zymography was performed as previously described with a modification [37]. Briefly, a cell conditioned medium rich in gelatinases able to degrade gelatin during the incubation in the developer buffer was added to the gel. The presence of TIMPs was visualized as dark bands in which the TIMP bound to the enzyme inhibits its gelatinolytic activity.

#### *4.6. Western Blotting Analysis of MMP-9 and TIMP-1*

Samples of conditioned media containing 40 μg of total proteins, were mixed with a reducing sample buffer and subjected to 10% SDS-PAGE. Separated proteins were then trans-blotted onto polyvinilidene difluoride (PVDF) transfer membranes. The non-specific protein binding site on membranes was blocked by incubation in a solution containing 5% non-fat dry milk (Biorad, Milan, Italy) in TBS 0.1% Tween 20 (TBS-T) for 1 h, and then incubated overnight at 4 ◦C with the primary antibodies for human MMP-9 and TIMP-1 diluted in 1% non-fat dry milk in TBS-T. Membranes were then washed in TBS-T, incubated for 1 h with secondary HRP-conjugated antibody diluted in blocking solution, and the immunoreactive bands were detected by inducing a chemiluminescence reaction through the ECL chemiluminescent reagent (GE Healthcare, Little Chalfont, England). Quantitative analysis of immunoreactive spots was performed by using ImageJ software [36].

#### *4.7. RNA Purification and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)*

Total RNA was extracted from treated cells with EUROGOLD Total RNA Mini kit (EuroClone, Milan, Italy) and 1 μg of each sample was subjected to retrotranscriptase reaction and PCR amplification in the same tube using the HyperscriptTM One-step RT-PCR Premix (TEMA RICERCA, Bologna, Italy) and following manufacturer's indications. Expression levels of MMP-9 and TIMP-1 were normalized to the expression levels of the housekeeping β-actin gene. PCR was performed by using the following oligonucleotides: MMP-9 (sense 5 -CGC AGA CAT CGT CAT CCA GT-3 , anti-sense 5 -GGA TTG GCG TTG GAA GAT GA-3 ), TIMP-1 (sense 5 -CTG TTG TTG CTG TGG CTG ATA-3 , antisense 5 -CCG TCC ACA AGC AAT GAG T-3 ) and β-actin (sense 5 -ATG ATG ATA TCG CCG CGC TCG-3 , antisense 5 -GCG CTC GGT GAG GAT CTT CA-3 ). The RT-PCR products were resolved by 1.5% agarose gel electrophoresis.

#### *4.8. Statistical Analysis*

Data were statistically analyzed by using SigmaPlot 12.0 software (Systat software, Inc., San Jose, CA, USA) for Windows operating system. Differences between means were evaluated by Student's t-test with confidence levels set at 95% (*p* < 0.05) and 99% (*p* < 0.01).

#### **5. Conclusions**

In the present research, for the first time to our knowledge, the ability of salvianolic acid B, an active compound of *Salviae mitiorrhizae*, to modulate the function of MMP-9 in human MDA-MB-231 breast cancer cells was demonstrated in an in vitro model characterized by a high invasive potential. Particularly, we found a marked tendency of the natural compound to interact with the catalytic domain of MMP-9 with the consequent inhibition of its activity, presumably reducing the ability of the enzyme to interact with the substrate. Furthermore, an inhibitory effect on the enzyme expression was observed. From this point of view, more specific studies will have to be conducted in order to characterize the biochemical mechanisms that oversee this phenomenon.

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

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

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

**Acknowledgments:** Authors are grateful to Dr. Mariolina Angelini for the kind cooperation.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**

### *Review* **Recent Advances in Natural Polyphenol Research**

**Irene Dini \* and Lucia Grumetto \***

Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano 49, 80131 Napoli, Italy **\*** Correspondence<EMAIL_ADDRESS>(I.D<EMAIL_ADDRESS>(L.G.)

**Abstract:** Polyphenols are secondary metabolites produced by plants, which contribute to the plant's defense against abiotic stress conditions (e.g., UV radiation and precipitation), the aggression of herbivores, and plant pathogens. Epidemiological studies suggest that long-term consumption of plant polyphenols protects against cardiovascular disease, cancer, osteoporosis, diabetes, and neurodegenerative diseases. Their structural diversity has fascinated and confronted analytical chemists on how to carry out unambiguous identification, exhaustive recovery from plants and organic waste, and define their nutritional and biological potential. The food, cosmetic, and pharmaceutical industries employ polyphenols from fruits and vegetables to produce additives, additional foods, and supplements. In some cases, nanocarriers have been used to protect polyphenols during food processing, to solve the issues related to low water solubility, to transport them to the site of action, and improve their bioavailability. This review summarizes the structure-bioactivity relationships, processing parameters that impact polyphenol stability and bioavailability, the research progress in nanocarrier delivery, and the most innovative methodologies for the exhaustive recovery of polyphenols from plant and agri-waste materials.

**Keywords:** antioxidant; circular economy; agri-food wastes; sustainability; flavonoids; polyphenols bioavailability; polyphenols activity; functional food; nutraceuticals; cosmeceuticals; nano-delivery; bioavailability; health

**Citation:** Dini, I.; Grumetto, L. Recent Advances in Natural Polyphenol Research. *Molecules* **2022**, *27*, 8777. https://doi.org/10.3390/ molecules27248777

Academic Editor: Nour Eddine Es-Safi

Received: 12 November 2022 Accepted: 9 December 2022 Published: 11 December 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Natural polyphenols are secondary metabolites of plants, vegetables, cereals, fruits, coffee, tea, and other plants. The exceptional functionality and biocompatibility of the polyphenols have stimulated the interest of researchers to use them as building blocks in functional foods, supplements, cosmetics, and drugs [1,2]. They have a phenolic ring, a basic monomer responsible for the protective action against oxidative injury [3]. Polyphenolic compounds can moderate oxidative stress and prevent or even inhibit oxidation by chelating iron and scavenging reactive radicals [4,5]. Dietary polyphenols can act as antioxidants, anti-inflammatory, and antiallergic compounds, decrease and prevent age-related diseases, can help against cardiovascular events (i.e., through their hypocholesterolemic, anti-thrombotic, antihypertensive, and anti-atherogenic), cancer, osteoporosis, diabetes, and neurodegenerative diseases [6]. The dietary polyphenols' bioavailability depends on the chemical and physical characteristics of the natural matrix that contains them, the stability during the digestive process, the intestinal enzymes' metabolization, and intestinal microbiota [7]. The gut microflora can modify the polyphenols' bioactivity and bioavailability [8]. Their bioaccessibility can be affected by preservation and processing methods, the interaction with the matrix components, and the fluids and enzymes secreted during digestion [9]. Physical, chemical, and enzymatic treatments can alter their properties. The preservation and processing can determine damage to the native polyphenol molecules and the production of new "process-derived" compounds [10]. Nanodelivery technology can improve polyphenols' absorption, bioavailability, functional quality, and performance [11–15].

This review summarizes the natural polyphenols classes, the extraction and methods performed to isolate them from natural sources and agro-waste, the factors that affect their bioavailability, and the application and development of nanodelivery systems.

#### **2. Polyphenols in Nature**

Polyphenols are involved in plant defense against pathogens and ultraviolet radiation [16]. The plants' outer layers contain higher phenolics [17]. Insoluble phenolics are in cell walls, while soluble phenolics are in the plant cell vacuoles [18]. The degree of ripeness during harvest time, pedoclimatic conditions, infections, processing, and storage can affect the polyphenolic content [19]. The phenolic acids (e.g., derivatives of cinnamic acid and benzoic acid), flavonoids (e.g., flavonols, flavanones, flavones, flavanols, isoflavones, and anthocyanins), lignans, and stilbenes are the most naturally occurring classes of compounds (Figure 1). The shikimate pathway produces the phenolic acids. The phenylpropanoids pathway forms lignans, lignins, flavonoids, and stilbenes [20,21]. The biosynthesis of complex polyphenols is linked to primary metabolism: the flavonoids' ring B and the chromane ring originate from the amino acid phenylalanine, obtained from the shikimate pathway, whereas ring A is formed from three malonyl-CoA units added through sequential decarboxylation condensation reactions [22]. In food, polyphenolic compounds can impact astringency, bitterness, flavor, color, and oxidative stability [19].

**Figure 1.** Chemical structures of the different polyphenol classes. The colors indicate the sub-class. The numbers indicate the positions in the nomenclature.

#### **3. Polyphenols Bioavailability**

There is no relation between the concentration of polyphenols in food and their bioavailability in the human body. The polyphenols, after ingestion, pass through the gastrointestinal epithelium and enter the circulatory vessels to reach the site of action. In food, polyphenols can exist as aglycon, glycosides, esters, or polymers.

The polyphenols' chemical structure limits the rate, absorption, and metabolites circulating in the plasma. The polyphenolic compounds with hydroxyl groups can be modified by methylation, glucuronidation, or sulfation enzymatic reactions. The 5–10% of total polyphenolic compounds may be metabolized in the small intestine. The rest of the polyphenols accumulate in the large intestine and are evacuated in the feces [23]. The conjugated polyphenols must be hydrolyzed by colonic microflora or intestinal enzymes (i.e., β-glucosidases and lactase-phlorizin hydrolase) before absorption [24]. During the absorption process, they are transformed into oligomeric phenols by gastric acid in the stomach, and glycosidic polyphenols are cleaved by cytosolic glucosidase and lactase in the small intestine into aglycon and glycoside(s) (e.g., glucose, xylose, and galactose) radicals [25]. Finally, intestinal bacterial enzymes can metabolize the remaining aglycone fraction. In the intestinal and colonic epithelium, polyphenols can be involved in conjugation reactions with methyl, glucuronide, or sulfate groups, making the identification of the metabolites in the blood and tissue complex [26]. The glycosides of quercetin and the isoflavones (genistein and daidzein) are not recovered in plasma or urine [27–29]. Instead, anthocyanins glycosides are the most representative circulating forms [30,31]. Experimental studies showed that quercetin, without glycosides, is absorbed at the gastric level [32], anthocyanins in the stomach [33,34], and proanthocyanidins [33,35] and hydroxycinnamic acids are absorbed by the small intestine [36]. The remaining polyphenols are hydrolyzed in the colon by microflora enzymes into aglycones that are metabolized into benzoic acid derivatives [37,38].

The polyphenols' digestibility affects their biological properties [39]. Soluble polyphenols have more evident responses during gastrointestinal digestion since the cell wall does not protect them. Unfortunately, human enzymes cannot digest some cell wall materials.

The flavonoids linked to other macromolecules cannot exert their beneficial actions [37]. The heat and pressure application (processing parameters) can facilitate the disruption of the cell wall and their release improving their bioactivity [38].

The pH and number of OH groups in benzene rings can affect phenolic stability. Conjugated nonphenolic aromatic acids, such as trans-cinnamic acid, are stable at high pH. The aromatic acids with a single OH group (e.g., ferulic acid) are stable at high pH because they do not form quinone oxidation products. The aromatic acids, with two phenolic OH groups (e.g., caffeic acid) or three (e.g., gallic acid), are unstable at pH 7–11. The changes are ascribable to the two adjacent phenolic OH groups attached to the benzene ring. Flavonoid molecules (e.g., rutin) that have a wholly conjugated aromatic structure are influenced by pH because the spatial arrangement between the π-electron system and an OH group controls the extent of π-orbital overlap and susceptibilities to the chemical change. The flavonoids in which the first benzene ring is located in the meta-position (e.g., catechin, epicatechin) do not have planar structures. Therefore, the π-electrons of the two benzene rings cannot cooperate via conjugation and are less susceptible at high pH [40,41].

In plant-based food, polyphenols and cell wall polysaccharides co-exist, and their affinity may influence foods' physicochemical and nutritional properties during processing and digestion. The affinity of cell wall polysaccharides with polyphenols depends on their structures, concentrations, temperature, pH, ionic strength, and the presence of proteins [42].

The enzyme concentrations, solubility, pH, digestion time [43], and processing methods (e.g., washing, refrigerating, fermentation, grain milling, roasting, juicing, blanching, and thermal processing) impact polyphenols' bioaccessibility and absorption [9].

Cooking and freezing processes positively impact the polyphenols' bioaccessibility since they soften the cell wall. The cooking medium also influences their bioaccessibility [44,45].

Pasteurization affects the polyphenols' bioaccessibility in the function of the heat treatment intensity, steps involved in processing, and type of food, decreasing the adverse processing effects on small bioactive compounds and even increasing polyphenols content [46]. Pasteurization can enhance food polyphenols extraction since the temperature softens the cell wall [47].

Finally, the interactions between macronutrients, micronutrients, and other phytochemicals, in finished products may also impact polyphenols' stability [48].

#### **4. Polyphenols & Microbiota**

Gut microbiota can break the flavonoid C-ring in different positions, producing simple phenolics from the A and B rings. Most of these metabolites are acid or aldehyde phenolics with 1, 2, and (or) 3 hydroxyl and methyl ester radicals. Non-flavonoid phenolics (e.g., hydrolyzable tannins, stilbenes, lignans, and hydroxy-benzoic acid derivatives) are absorbed in the small intestine based on their chemical complexity. The gut bacteria can hydrolyze the ester bonds in tannins, dehydroxylate, and decarboxylate, the gallic acid [49], and reduce the resveratrol and its precursors [50].

The role of polyphenols and their metabolites on the gut microbiota is not elucidated. They probably have a prebiotic-like effect [51], since they can modulate the gut microbial profile. [52,53].

The polyphenols' prebiotic effect is associated mainly with the promotion of probiotics (e.g., Bifidobacteriaceae and Lactobacillaceae) or the inhibition of pathogenic bacteria (i.e., *E. coli*, *Clostridium perfringens*, and *Helicobacter pylori* [52]) resulting in reduced proinflammatory immune response and decreased risk of colon cancer, gastroenteritis, inflammatory bowel disease, and metabolic syndrome [54,55].

Some polyphenols prevent bacterial growth, binding the cell membranes in a concentration-dependent manner. Catechins can change the microbial (i.e., *Bordetella bronchiseptica*, *Klebsiella pneumonie*, *E. coli*, *Pseudomonas aeruginosa*, *Serratia marcescens*, *Bacillus subtilis Salmonella choleraesis*, and *Staphylococcus aureus*) cell membrane permeability by producing H2O2 [56]. In Gram-positive bacteria, polyphenolic compounds can delay the oligopeptides autoinducers that sense the bacterial quorum sensing. In Gram-negative bacteria, they can prevent the bacteria-acylated homoserine lactones autoinducers [57].

#### **5. Effects of the Food-Processing Techniques on Polyphenol Levels and Bioavailability**

The heat treatments (e.g., boiling, steaming, frying, stewing, baking, roasting, ovens, steam, and microwave) and the transformation food processing (e.g., roasting, toasting, drying, pasteurization, canning, and sterilization), can affect the polyphenols' bioavailability. The heat breaks cell walls, mobilizes the phenolic compounds, improves their availability, enhances their oxidation processes, and degrades them based on their thermostability. Domestic cooking and industrial thermal processes can cause losses in polyphenols, with significant variability depending on the nature of food matrices [58].

Boiling produces the most harmful polyphenol composition changes. Instead, steaming and frying can preserve them since the polar media (water) can extract higher levels of polyphenol than nonpolar media (oil) [59]. During boiling, heat decomposes the tissues, and the phenolics leak into the water [60]. Water volume can impact the polyphenol alteration during the heat process: small water volumes produce lower phenolic extraction than larger volumes [61]. Diverse boiling times produce different polyphenol profiles in foods, and a long time can cause more severe damage than a short one [62]. The type of heat treatment affects the polyphenol bioavailability. Steaming is the best heat method to preserve phenolic fractions since they are indirectly exposed to water [63]. The form in which phenolics are present also affects bioavailability [64].

Canning, a process employed to produce sterilized and microbiologically safe food products by applying heat treatment, can decrease phenolic compound levels [65] because they migrate into the surrounding medium [66].

Drying, the preservation process that aims to decrease the moisture content of food by using heat and mass transfer, can affect the phenolic levels in the function of the temperature regime. Freeze-drying is the most efficient method to preserve phenolic content, while hotair-drying is the least. The vast variety of chemical polyphenol classes also influences the variability in the effects caused by drying [67]. Oven-dried processes produce higher levels of bioaccessible phenolics than other drying processes [68–70]. Slow freezing enhances the bioavailability of the phenolic compounds since it forms ice crystals that favor the polyphenols extraction, oxidation, and degradation, during digestion [71].

Peeling fruits and vegetables determines the loss of high amounts of bioactive compounds since they are contained in the peel and external parts of the plants at higher levels than other parts [72]. Grinding, the technique that reduces the size of solid particles using mechanical forces, enhances the polyphenols extractions as a function of the particle size [73]. The ultrasound treatments pulsed electric field, high-pressure, and pulsed-light processing enhance polyphenols digestion, bioaccessibility, and bioavailability [74].

#### **6. Polyphenol Biological Activities**

Epidemiological studies have shown an inverse association between a polyphenolicrich diet and the risk of chronic human diseases. Polyphenolic compound-rich foods and beverages can have antioxidant, anti-inflammatory, anticancer, and anti-aging properties and reduce the risk of degenerative diseases such as cardiovascular, diabetes mellitus, and neuronal diseases.

#### *6.1. Antioxidant Activity*

Experimental evidence showed that polyphenols protect cell constituents against oxidative damage and degenerative diseases associated with oxidative stress [75]. The polyphenol-rich foods can improve plasma antioxidant capacity by scavenging radical species (e.g., ROS, RNS) or repressing radicals' formation by inhibiting the activities of the oxido-reductive enzymes' and/or chelating the metals that intercept free radical production. Their phenolic groups can accept an electron to form phenoxy radicals, interrupting chain oxidation reactions, and conjugated aromatic systems can delocalize an unpaired electron [76]. Polyphenols reduce the oxidation of lipids and other molecules by donating hydrogen to radicals (R). The resonance makes PO· (phenoxy radical) relatively stable (new chain reactions are not started) and acts as terminators of the propagation route when reacting with other free radicals (Figure 2).

**Figure 2.** Reactions between lipids and phenols.

The reduction activity of phenolic acids and their derivatives depends on the free hydroxyl groups in the molecule [77]. The hydroxycinnamic acids show better antioxidant activity than hydroxybenzoic acid equivalents due to the aryloxy-radical stabilizing effect of the –CH=CH–COOH linked to the phenyl ring by resonance [78]. The phenolic acids' antioxidant activity of free, esterified, glycosylated, and nonglycosylated phenolics is mainly ascribed to radical scavenging via the hydrogen atom donation mechanism [78,79].

The flavonoids' radical scavenging depends on the ortho-dihydroxy structure in the B ring, which allows higher stability to the radical form and participates in electron delocalization of the 2,3-double bond with a 4-oxo function in the C ring [77].

The chemical electron deficiency of anthocyanins is particularly reactive toward ROS/RNS.

The polyphenolic compounds with dihydroxy groups prevent metals-induced free radical formation by conjugating the transition metals that interact with hydrogen peroxide (H2O2) through the Fenton reaction to form hydroxyl radicals (·OH).

Phenolic compounds with catecholate and gallate groups can stop metal-induced oxygen radicals by improving metal ion autoxidation or forming an inactive complex with weaker interaction [79].

The metal ions can attack the flavonoids into positions 3 and 4 (B ring), 3 and 4, 3 and 5, 4-keto and 3-hydroxy, and 4-keto and 5-hydroxy (C ring) [80].

Moreover, polyphenols can improve cellular antioxidant activity by regulating Nrf2, which controls some detoxifying enzymes (SOD, GSH, GPx1, NADP(H) quinone oxidoreductase, HO-1, and GST) [77]. Finally, polyphenols can influence microRNAs [81].

MicroRNAs are a class of small, endogenous, noncoding RNAs. Some microRNAs (i.e., miR-21, miR-125, and miR-146) are involved in vascular inflammation and diseases [82–84]. Dietary polyphenols can influence the microRNAs' expression and biogenesis [30]. For example, curcuminoids can act as anti-atherosclerosis agents by upregulating miR-126 expression [85]. Resveratrol can act as a cardioprotective molecule by improving the mRNA activating SIRT1, and enhancing the SOD' levels [86–89].

Gallic acid can decelerate atherosclerosis progression by upregulating miR-145 and downregulating miR-21 expression [90].

Under certain conditions, the polyphenolic compounds can initiate an autoxidation process and perform as prooxidants. In these cases, the phenoxy radicals can interact with oxygen to make quinones and superoxide anions [91]. pH, high concentrations of transition metal ions, and oxygen molecules can induce the autooxidation of polyphenols [92]. Quercetin and gallic acid can have prooxidant activity; instead, the hydrolyzable tannins have little or no prooxidant activity [93].

#### *6.2. Anti-Inflammatory Activity*

Plant polyphenols can decrease the effect of the cytokine, affecting their receptors or reducing their secretion processes [94].

Phenolic compounds can suppress the binding of proinflammatory mediators, control eicosanoid synthesis, prevent stimulated resistant units, and impede the activity of COX-2 and NO synthase, acting on NF-κB [95]. Some phenolic acids, such as rosmarinic acid and isosalvianolic acid, can reduce the production of IL-6, TNF-α, and IL-1β at the gene and protein levels [30].

The catechols' enzymatic activity depends on the structure of the B ring and needs nucleophilic additions [96]. The procyanidins decrease the concentrations of NO, prostaglandin E2, and ROS [97].

The flavonoids (e.g., flavones) regulate IL-6 in the blood [98]. The flavonoids' antiinflammatory mechanism is related to the unsaturation in the C ring that affects the strength of binding interactions by resonance [99].

#### *6.3. Anticancer Activity*

Cancer development consists of initiation, promotion, progression, invasion, and metastasis [100]. Genetic mutations occur when DNA damage is not repaired, and a clone of mutated cells is reproduced during mitosis. Tumor promotion is a reversible and long-term process in which a selective clonal expansion of the cells forms a population of aggressively proliferating multi-cellular cells (premalignant tumor). Clonal expansion determines the development of the premalignant cells into tumors (tumor progression phase). Finally, some tumor cells may be cut off from the primary tumor mass, migrate toward blood vessels or lymphatic vessels and produce a second lesion (invasion and metastasis phases). The natural phenolic compounds can induce cell cycle arrest at G1, S, S-G2, and G2 phases by down-regulating cyclins and cyclins-dependent kinases or producing the expression of p21, p53, and p27 genes [80].

Polyphenols can act against tumor initiation and promotion, changing the redox status and affecting essential cellular functions (i.e., cell cycle, apoptosis, angiogenesis, inflammation, invasion, and metastasis) [101]. Oxidative damage can cause cancer since ROSs can damage the DNA and affect cell replication and signal transduction [102].

The flavonoid anticancer effects are related to their antioxidant and pro-oxidant activities [103,104]. The flavonol kaempferol can induce apoptosis and arrest in the S-phase of cancerous cells by modulating ROS levels [105]. When it acts as pro-oxidants it decreases NF-κB levels and produces cyclooxygenase-2 (COX) overexpression, inducing apoptosis, and cell-cycle arrest [106].

Some flavonoids and resveratrol can affect the procarcinogens' activation by impeding phase I metabolizing enzymes (e.g., cytochrome P450) [107–110]. They can help carcinogens' detoxification and removal inducing the phase II metabolizing enzymes (e.g., glutathione S-transferase, UDP-glucuronyl-transferase, and NAD(P)H quinine oxidoreductase) [111].

The polyphenols can produce apoptosis-inducing cell cycle arrest inhibiting the extracellular regulated kinase, c-Jun *N*-terminal kinase, and P38 mitogen-activated protein kinase pathway, transcription factors, NF-κB, activator protein-1 (AP1), protein kinase C (PKC), and growth factor-mediated pathways. The apigenin inhibits the growth of human thyroid carcinoma cells, probably by decreasing the phosphorylation of MAPK and by activating the protein kinases, and scavenging H2O2 [112].

The 3,4 dihydroxybenzoic acid stimulates apoptosis, in human gastric carcinoma cells, by ROS overproduction which can activate JNK/p38 MAPKs [113].

The polyphenolic compounds can negatively affect some factors involved in the inflammatory processes, such as the NF-κB, proinflammatory cytokines release, COX-2, lipoxygenases, inducible nitric oxide synthase, and MAPK-mediated pathway [80]. For example, Epigallocatechin gallate can block NFκ B activation in human epithelial cells and downregulate the expression of inducible nitric oxide synthase and nitric oxide production in macrophages [114]. Finally, the kaempferol can counteract malignant cell invasion and metastasis, down-regulating the matrix metalloproteases (MMP-2 and MMP-9), urokinaseplasminogen activator (uPA), and uPA receptor expression [115,116].

#### *6.4. Cardiovascular Protective Activity*

Cardiovascular (CVD) pathologies are the primary cause of morbidity and mortality (ischemic heart disease and stroke contribute 85%) [117]. Oxidative stress and inflammatory processes are considered promoters of endothelial dysfunction [118,119]. Polyphenols have antioxidant, anti-inflammatory abilities and can modulate lipid metabolism [120]. They (mainly quercetin and resveratrol) can decrease LDL oxidation [121], cholesterol synthesis, improve LDL receptor expression and activity [122,123], and the cholesterol transporters expression [124]. The anthocyanins and resveratrol can improve fecal cholesterol elimination [125,126] and decrease the triglyceride plasma level, decreasing the apolipoprotein B48 and apolipoprotein B100 production in the liver and intestine [127] or the lipoprotein lipase expression [128]. Flavonoids can also reduce blood pressure ameliorating flow-mediated dilation in humans (by improving the NO synthase activity) [129,130] and influencing the renin-angiotensin system [131,132]. Moreover, they can prevent platelet aggregation, decreasing the activity of cyclooxygenase 1, and thromboxane A2 that act as a vasoconstrictor and platelet aggregation's inducer, respectively [133]. Finally, the polyphenols' prebiotic-like activity can account for the amelioration of markers of CVD [124].

#### *6.5. Antidiabetic Activity*

There are two main types of diabetes (diabetes-1 and diabetes-2). Diabetes type-2 or diabetes mellitus is due to damage in glucose metabolism and advancing insulin resistance that leads to hyperglycemia. The leading causes of hyperglycemia are dietary carbohydrates' digestion and absorption, glycogen storage reduction, β-cell dysfunction, peripheral tissue insulin resistance, deficiency in insulin signaling pathways, and improved gluconeogenesis and production of hepatic glucose [134]. Polyphenols can decrease the intestinal absorption

of carbohydrates, control the enzymes that regulate glucose metabolism, and increase the β-cell functionality, insulin secretion, and the anti-inflammatory and antioxidant properties of these components (Figure 3).

The phenolic acids, flavonoids, and tannins can regulate the key enzymes responsible for the digestion of carbohydrates (α-glucosidase and α-amylase) [135]. The catechin, epicatechins, and chlorogenic, caffeic, ferulic, and tannic acids can decrease the glucose transporters Na+-dependent (SGLT1 and SGLT2) [136]. The coffee phenols, anthocyanin, and curcumin can regulate postprandial glycemia and decrease the progression of glucose intolerance by a simplified insulin response and improved secretion of glucagon-like polypeptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) [137]. Ferulic acid can decrease blood glucose by improving glucokinase activity and glycogen production in the liver [138]. Catechins and epicatechins can decrease hyperglycemia and hepatic glucose output, downregulating the expression of liver glucokinase, and upregulating the glucose-6-phosphatase and phosphoenolpyruvate carboxykinase [139].

**Figure 3.** The polyphenols' effects on glucose homeostasis and insulin resistance.

#### *6.6. Neurodegenerative Protection*

Neurodegenerative diseases are due to the deterioration of neurons' structure and/or function. Reactive oxygen and reactive nitrogen species can determine neuronal cell dysfunction and death. The phenolic compounds can interact with the amino acid residues of acetylcholinesterase's (AChE) active site, making hydrogen bonds and hydrophobic and π–π interactions. Multiple hydroxyl groups can improve the inhibition of AChE, increasing the binding capacity [30]. Resveratrol can protect against microglia-dependent β-amyloid toxicity by decreasing the nuclear factor κB [140]. Some polyphenols protect against Parkinson's disease by scavenging the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated radical formation [141] or decreasing free radicals' formation by chelating iron [142].

#### *6.7. Anti-Aging Action*

Aging determines detrimental changes in the cells and tissues. The cosmetic industry constantly strives in product development and reformulation to meet consumers' preferences. Today, nature-derived products are in demand on the market. Some botanical preparations that contain polyphenols (e.g., flavonoids, phenolic acids, and stilbenes) are employed in the composition of anti-aging products [143]. Free radicals and oxidative stress are the major contributors to aging damage. The phenolic hydroxyl groups on polyphenol molecules can scavenge ROS [144]. The polyphenolic compounds can regulate the production of oxidase enzymes (sodium oxide dismutase 1 in the cytosol, sodium oxide dismutase 2 in the mitochondria), and endogenous antioxidants [145–149] can improve the transcriptional factor Nrf2 DNA-binding activity and regulate protein expression [150–152]. Anti-aging formulations contain botanicals metabolites able to protect DNA, regulate the enzymes' action, decrease inflammation, and alter hormone imbalance [143].

The epigallocatechin-3-gallate in green tea decreases the UVB-induced hydrogen peroxide release from normal epidermal keratinocytes, MAPK phosphorylation, and inflammation by activating NFkB. The flavins in black tea decrease UVB-induced AP-1 induction, prevent UVB-induced phosphatidyl-inositol 3-kinase activation, decrease the amount of ROS in the skin, and offer photoprotection by reducing local and systemic immunosuppression UVB-induced [153]. Resveratrol is employed to reduce hydrogen peroxide, improve lipid peroxidation, and decrease the levels of COX-2 and ornithine decarboxylase. Moreover, it can decrease UVA-induced oxidative stress in human keratinocytes since it controls the Keap1-a protein that acts on Nrf2 [153]. Curcuminoids (found in Turmeric spice) can decrease inflammation by inhibiting the MAPK and NFkB signaling pathways and decreasing nitric oxide levels and COX2. Moreover, in keratinocytes and fibroblasts, curcuminoids can decrease UVB-induced TNF mRNA expression and matrix metalloproteinase-1 expression [154].

#### *6.8. Antiallergic Action*

Allergic diseases happen when an organism becomes sensitive to an innocuous allergen and releases many allergy-related intermediaries. Polyphenols limit the production of IgE, the release of allergic mediators, and allergy symptoms. Polyphenols can control hypersensitivity by regulating oxidation and interacting with inflammatory mediators [155]. Catechins can decrease Th2 cytokine production and T cell activation and proliferation. Caffeic, chlorogenic, and ferulic acids can irreversibly bind peanut allergens (Ara h1 and Ara h2), reducing their allergenicity [156]. Punicalagin, phloridzin, and rutin can improve the growth of probiotics such as *Lactobacillus* and *Bifidobacterium*, which positively impact food allergies [157].

#### *6.9. Antiosteoporotic Action*

Osteoporosis causes the loss of bone mineral density, decreased bone mass, and microstructural deterioration. Flavonoids and stilbenes can improve osteogenesis by controlling the bone morphogenetic protein, NF-κB, IGF, and MAPK, and can inhibit the osteoclastogenesis pathways through epigenetic regulations. They can activate SIRT-1 (histone deacetylase) and modify the NAD+/NADH ratio [158–160].

#### *6.10. Antimicrobial Action*

Some plant extracts rich in polyphenols can decrease the growth of fungi and bacteria (i.e., Listeria monocytogenes, *Salmonella* spp., and *Escherichia coli*) [161,162], minimize the exposure of humans to resistant bacteria [163], and can have a synergic action with other antimicrobials. These findings have suggested a potential use of polyphenol-rich extracts as food preservatives and in the pharmaceutical industry to improve efficacy and decrease antibiotic side effects, such as repressing antibiotic-resistant bacteria [164]. The polyphenol-rich extracts can be placed on the food surface by spraying, dipping, brushing, or mixing with other ingredients [165]. Unfortunately, in some cases, the interaction with

food components can cause a lack of antimicrobial efficacy. Therefore, it was thought to encapsulate them in carriers to increase their distribution in the food and reduce contact with food matrix molecules that reduce their effectiveness [166]. The mechanisms of antibacterial action are not yet entirely deciphered. However, it is known that many sites of action at the cellular level are involved. Polyphenols can modify the cell membrane permeability, destroy the cell wall integrity and change intracellular functions by binding some enzymes [167].

#### **7. Polyphenols Potentialities in the Nutraceutical Era**

Today, consumers include a high level of bioactive compounds in their standard diet, preferably derived from natural sources such as plants and fruits, in the hope of giving more life to the years by preventing disabling pathologies that decrease the skills that allow living life in all its manifestations [168]. Food and pharmaceutical companies develop nutraceutical foods and supplements that contain botanical extracts and metabolites alone or combined with other ingredients [169–171].

Numerous studies strongly suggested that including polyphenols or polyphenol-rich extracts in supplements or foods may protect the body tissues against oxidative stress and aging [172,173].

The primary issue in using plant extracts is that fungi, which can produce toxins, can contaminate the extracts (e.g., *Aspergillus* section Nigri that produces Ochratoxin A, a carcinogenic, teratogenic, nephrotoxic, neurotoxic, and immunotoxic toxin) [174]. The research should implement efforts to develop analytical controls that safeguard consumer safety.

#### **8. Polyphenols Extraction**

Extraction plays a pivotal role in the purification of polyphenols from foodstuffs. Extraction techniques can employ traditional or "conventional" such as percolation, maceration, and Soxhlet extraction, and modern methods, such as ultrasound or microwave, the latter is most extensively used (Figure 4). In both cases, extraction efficiency depends on various factors such as the nature of the solvent, solvent–solid ratio, temperature, and particle size. Polyphenol can be extracted from fresh, frozen, or dried plant samples. The extracts can be added to an organic solvent, such as methanol or ethanol, with low viscosity to accelerate mass transfer [175]. Before extraction, the pretreatment of the plant matrices (e.g., cleaning, washing, milling, grinding, drying, homogenizing) is crucial.

**Figure 4.** The polyphenols' traditional and modern extraction methods. Blue arrows: traditional extraction methods; red arrows: modern methods; in the orange circle: techniques used to improve polyphenol management.

Percolation extraction uses water as a solvent. It takes a long time to obtain the pure extract to be concentrated [176].

The maceration is a solid-liquid extraction method using different solvents depending on the target compounds' physical and chemical properties. It has low efficiency and extraction yield and employs a large volume of solvents compared to non-conventional techniques such as ultrasound-assisted extraction (UAE) [177]. A higher ratio of solid/solvent increases polyphenols recovery [178].

Decoction extracts plant materials by boiling. It is inefficient for heat and light-sensitive compounds [179]. The decoction of Citrus fruits produces by-products of Citrus peels with high concentration levels of polyphenol fraction [180].

Heat reflux extraction is a solid–liquid extraction method performed with repeated solvent evaporation and condensation at a constant temperature. It requires less extraction time and solvent than percolation or maceration and allows for a greater extraction yield [181]. Polyphenols from wastes of *Vitis vinifera* were extracted by Moldovan et al. by using heat reflux extraction [182].

The solid-phase extraction (SPE) method is considered quick and easy to extract polyphenols from vegetable oils. According to the experimental needs, different stationary phases were used (e.g., C8 cartridges, octadecyl C18, diol-bonded phase cartridges, aminophase cartridges, and octadecyl C18EC) [6]. The non-conventional techniques employ supercritical fluid, high-voltage electric discharge, and enzyme-assisted extraction. It is preferable to extract the bound polyphenols, also referred to as non-extractable polyphenols (NEP), from plant sources using one or more combinations of modern technology rather than conventional methods. A comparison between traditional and SFE extraction performed on black tea leftovers showed that SPE technique gives the best performance in extraction of phenolic compounds (SPE gives 521 mg GAE/g; traditional gives 283 mg GAE/g) [183].

The extraction with supercritical fluids such as CO2, propane, argon, and SF6 allows for an easy penetration inside plant materials and high solvents. Power-pulsed electric fields (PEF) perform a gentle extraction due to the electroporation of cell membranes. It has been applied to exotic fruits, grapes, and oil crop components [184]. Microwave-assisted extraction (MAE) is an eco-friendly technique with higher efficiency in the recovery of polyphenols from waste products if compared to that of extracts prepared by ultrasoundassisted extraction (UAE) and conventional methods such as maceration [185].

Fruit skins, stem barks, grain seed coats, brans, and pods, are generally considered agro-waste, but they are essential resources for NEP recovery [186–188]. Table 1 reports conventional and non-conventional techniques applied to extract polyphenols from organic waste.

**Table 1.** Extraction of free and bound polyphenols.

#### **9. Polyphenol Nano Delivery Systems**

The potential of antioxidative and repair pathways decreases with age, causing several adverse effects such as the risk of neurodegenerative diseases such as Parkinson's disease, memory loss, Alzheimer's disease, atherosclerosis, and cancers due to the accumulation of reactive oxygen species. Polyphenols, due to their oxidizing ability, can protect from the damaging effects of ROS. Therefore, they can be used as active compounds in several formulations preventing oxidative stress [197,198]. The loading of polyphenols into lipid nanocarriers (NCs) is an essential tool for increasing bioavailability, reducing degradation, and protecting antioxidant polyphenols' activity. The NCs are biodegradable and have no significant toxicity. The nanoemulsion, liposome, phytosome, solid lipid nanoparticles (SLNs), nanostructured lipid carrier (NLCs), and lipid-polymer hybrid nanoparticles (LPHNs) can encapsulate polyphenols to improve their biophysiological target [199]. Nanoparticles (NPs) have diameters as small as 1–100 nm. They can enhance polyphenols delivery and promote their absorption and bioavailability [200]. The transcellular pathway is a route for the NPs transportation (via endocytosis or macropinocytosis mechanisms) and subjected to the degradative microenvironment of the cellular lysosome environment in the acidic endosomal lumen [201]. Carbohydrate-based delivery systems, such as mono, oligo, and polysaccharide, are employed to encapsulate polyphenols due to their abundance and low cost; for example, curcumin was encapsulated in chitosan using an injection-gelation method to increase its bioavailability, antioxidant properties, and to improve stability and effects on tumor cells [202].

Protein-based systems have been employed to prepare nanoparticles for carrying polyphenols. The proteins act as "host" and polyphenols as "guest" molecules. The polyphenols bind specific regions on the protein surfaces through hydrogen and/or hydrophobic non-covalent bounds. The β-lactoglobulin nano delivery was used to increase the water solubility 3-fold at pH 7 of the epigallocathechin-3-gallate and naringenin [203]. Finally, polyphenols are used to design polyphenol-based nanomaterials for biomedical applications. For example, the polyphenol-grafted polymers are used as antidiabetic agents [204], the curcumin encapsulated in chitosan and polyglycolic acid (PGA) particles for wound healing [205], and the polyphenol-loaded electrospun nanofibers to improve the remineralization and regeneration of bone [206].

#### **10. Polyphenols in Active Packaging**

In recent years, packaging technology has evolved, including intelligent or smart packaging. Biodegradable, active, and bioactive packaging are new trends in food packaging research. Food contact materials (FCM) are engineered to protect foods, and improve their shelf-life [207]. Plastic packaging materials, such as polycarbonate, polyethylene, and polyethylene terephthalate, widely used in food packaging, are nonbiodegradable and disadvantageous to the environment and human health [208–210]. To date, natural and biodegradable biopolymer-based packaging films and edible coatings represent the alternative to plastic packaging materials [211]. Natural biopolymers, including proteins, polysaccharides, and lipids, have been used in packaging manufacturing [212]. Fruit industrial manufacture generates large amounts of waste that harm the environment and causes considerable treatment expense [213]. Nevertheless, these by-products are rich in bioactive compounds [214], some of which can be incorporated into biodegradable plastics for food packaging to protect the polymeric matrix against thermal [162,213], photo-induced degradation, and preserve the food freshness and quality [215]. The solvent-based impregnation of biodegradable polymers with extracts of *Cistus linnaeus* is used to improve the polymers' thermal stability [216]. Chemically-synthesized and biomass-derived biodegradable polymers have been used as matrices to protect food during transportation, storage, and sale. Polyphenols are employed in the so-called "leaching systems" that are active-releasing systems able to release actives by direct contact between food and packaging material. For this purpose, the propolis is mixed with biopolymers, plasticizers, and reinforcing agents to produce active packaging and edible coatings [213,216].

Biopolymers can be dissolved in solvents depending on their hydrophilicity. Hydrophilic biopolymers, such as gelatin, κ-carrageenan, alginate, and agar in water, chitosan, can be dissolved in acidic solutions [217], while organic solvents, chloroform, and ethyl acetate can be employed for hydrophobic biopolymer such as polylactic acid (PLA) [218]. For example, the ethanolic propolis extract, and propolis in powder, together with plasticizers such as glycerol and (PEG) polyethylene glycol, have been added to biopolymeric solutions [219]. Tea polyphenols (TP) are employed as an active component in biopolymer materials for active food packaging. Shao et al. have incorporated TP into pullulancarboxymethylcellulose sodium (Pul-CMC) solutions on electrospun nanofiber films [220].

#### **11. Conclusions**

Polyphenols are secondary plant metabolites that can benefit human health and preserve food. The use of polyphenols as supplements, antibiotic drugs, cosmetics, and natural food preservatives is a promising trend in the industry because of the growing interest in natural products and the multiple biological activities of these products.

The great demand for polyphenols and the small quantity produced by the plants have determined the need to use extraction techniques that allow exhaustive extraction even when it is necessary to recover them from non-traditional sources such as organic waste. For this purpose, unconventional techniques such as supercritical fluid, high-voltage electric discharge, and enzyme-assisted extraction must be optimized.

The polyphenols' low oral bioavailability and interactions with other molecules negatively impact the possible industrial application. Therefore, different nanocarriers have been developed to protect, improve bioavailability, ensure achievement to the active site, and improve their effectiveness. It is essential to underline that polyphenols are commonly present in plant-based foods such as fruits and vegetables.

Guidelines for their consumption and supplementation should be provided by regulatory bodies to make consumers safe and informed.

**Author Contributions:** Conceptualization, writing—original draft preparation I.D. and L.G. All authors have read and agreed to the published version of the manuscript.

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

**Data Availability Statement:** Not applicable.

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

#### **References**

### *Review* **Dihydrochalcones in Sweet Tea: Biosynthesis, Distribution and Neuroprotection Function**

**Yong-Kang Wang 1, Si-Yi Hu 1, Feng-Yi Xiao 1, Zhan-Bo Dong 2, Jian-Hui Ye 1, Xin-Qiang Zheng 1, Yue-Rong Liang <sup>1</sup> and Jian-Liang Lu 1,\***

	- **\*** Correspondence<EMAIL_ADDRESS>Tel./Fax: +86-571-88982704

**Abstract:** Sweet tea is a popular herbal drink in southwest China, and it is usually made from the shoots and tender leaves of *Lithocarpus litseifolius.* The sweet taste is mainly attributed to its high concentration of dihydrochalcones. The distribution and biosynthesis of dihydrochaldones in sweet tea, as well as neuroprotective effects in vitro and in vivo tests, are reviewed in this paper. Dihydrochalones are mainly composed of phloretin and its glycosides, namely, trilobatin and phloridzin, and enriched in tender leaves with significant geographical specificity. Biosynthesis of the dihydrochalones follows part of the phenylpropanoid and a branch of flavonoid metabolic pathways and is regulated by expression of the genes, including *phenylalanine ammonia-lyase, 4-coumarate: coenzyme A ligase, trans-cinnamic acid-4-hydroxylase* and *hydroxycinnamoyl-CoA double bond reductase*. The dihydrochalones have been proven to exert a significant neuroprotective effect through their regulation against Aβ deposition, tau protein hyperphosphorylation, oxidative stress, inflammation and apoptosis.

**Keywords:** *Lithocarpus litseifolius*; trilobatin; phloridzin; phloretin; metabolic pathway; bioactivity

#### **1. Introduction**

Tea is one of the most popular beverages worldwide. In addition to the traditional tea made from the leaves of *Camellia sinensis* (L.) O. Kunzte, herbal teas are regionally consumed and usually made from the leaves or other tissues of multifarious plants, such as sweet tea (*Lithocarpus litseifolius*), camomile (*Chrysanthemum lavandulifolium*), jasmine (*Jasminum sambac*), *Dracocephalum rupestre*, honeysuckle (*Lonicera japonica*) and *Litsea coreana* var. *lanuginosa*. The *Lithocarpus litseifolius* has usually been called sweet tea or sweet leaf tree by the local residents since the products made from its leaves taste quite sweet. In China, the plucked shoots and tender leaves are often manufactured according to protocols similar to green tea or black tea, the annual output of the sweet tea product is estimated to exceed 2000 t, and these products are commonly consumed by local people in some provinces along the Yangtze River, such as Sichuan, Chongqing, Hunan and Jiangxi. The sweet tea has attracted much more attention recently because of its unique taste and health benefits, which are attributed to dihydrochalcones (DHCs) such as trilobatin, phloridzin and phloretin. DHCs are a class of flavonoids characterized by a basic C6-C3-C6 backbone structure and the absence of a heterocyclic C ring. DHCs are considered to be the primary precursors and represent important intermediates in the synthesis of flavonoids [1,2]. DHCs, especially phloretin, phloridzin and trilobatin, have a variety of health effects, like antioxidant activity [3], anti-inflammation [4,5], antidiabetic activity [6,7], cardioprotection [8], intestinal protection [9], hepatoprotective effect [10], anticancer activity [11] and neuroprotection [12]. With an increase in the population aging, the incidence of neurodegenerative diseases has increased dramatically [13]. Neurodegenerative diseases mainly include Alzheimer's disease (AD), Parkinson's disease (PD), stroke and so on. In folk experience, drinking sweet tea can often be used to prevent and treat neurodegenerative diseases, which might be attributed to the effect of DHCs. Nowadays, DHCs have been

**Citation:** Wang, Y.-K.; Hu, S.-Y.; Xiao, F.-Y.; Dong, Z.-B.; Ye, J.-H.; Zheng, X.-Q.; Liang, Y.-R.; Lu, J.-L. Dihydrochalcones in Sweet Tea: Biosynthesis, Distribution and Neuroprotection Function. *Molecules* **2022**, *27*, 8794. https://doi.org/ 10.3390/molecules27248794

Academic Editor: Nour Eddine Es-Safi

Received: 19 November 2022 Accepted: 9 December 2022 Published: 12 December 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

well studied in *Malus pumila* Mill., being 25.6–113.7 mg/g DW in leaves [14] and 99.9 mg/g DW in immature fruits. These are natural sources for the DHCs' separation and utilization. However, the sweet tea can also be considered as an alternative source of the DHCs because of its high level of these compounds. This review introduces the physiological and biochemical characteristics of the sweet tea, summarizes the DHCs biosynthesis and the influencing factors, and finally elaborates on the neuroprotection of the sweet tea and its related mechanisms.

#### **2. Physio-Biochemical Characteristics of the Sweet Tea**

#### *2.1. Morphological Characteristics*

*Lithocarpus polystachyus* or *Lithocarpus litseifolius* have been used to refer to the sweet tea tree in published reports. According to WFO (www.worldfloraonline.org (accessed on 1 January 2020)), *Lithocarpus polystachyus* is an evergreen arbor in the genus *Lithocarpus* of the family *Fagaceae*. It was first reported as *Quercus polystachya* Wall. ex A. DC. in 1864, then renamed as *Pasania polystachya* (Wall. ex A. DC.) Oerst. in 1871 and *Synaedrys polystachya* (Wall. ex A. DC.) Koidz. in 1916. Later, it was finally designated as *Lithocarpus polystachyus* (Wall. ex A. DC.) Rehder in 1919. *Lithocarpus litseifolius* is also a species belonging to the genus *Lithocarpus* in the family *Fagaceae*. This species was first reported as *Quercus litseifolia* Hance in 1884, then renamed as *Pasania litseifolia* (Hance) Schottky in 1912 and *Synaedrys litseifolia* (Hance) Koidz. in 1916, and finally designated as *Lithocarpus litseifolius* (Hance) Chun in 1928. However, according to FOC (www.eFloras.org, (accessed on 2 March 2021)), these two species are synonyms and belong to the same variety of *Lithocarpus litseifolius*, namely, *Lithocarpus litseifolius* var. *Litseifolius,* as shown in Figure 1, because of their similar morphological characteristics and overlapping geographical distributions. This variety mainly distributes in south China, Laos, northeast Myanmar, northern Vietnam and India [15,16]. In addition to the var. *litseifolius*, another var. *pubescens* Huang has also been found and identified in dense forests of Tian'e County, Guangxi, China. Main morphological differences have been screened out from these two varieties, i.e., the branchlets and infructescences rachis, the cupule size and the fruit ripening time. Glabrous branchlets, sparsely pubescent infructescences rachis, 0.8–1.4 cm cupule diameter and fruit ripening in Jun–Oct has been observed in the former, while puberulent branchlets and infructescences rachis, relatively big cupule (1.2–1.5 cm in diameter) and early fruit ripening time (in April–May) have been witnessed in the latter. In this review, *Lithocarpus litseifolius* is used to refer to all the sweet tea, including the two varieties and the synonymous plants. The sweet tea is a common light-loving and drought-tolerant tree species in the mountain area and usually possesses big elliptic leaves around 8–18 cm in length and 3–8 cm in broadness, ~25 cm male inflorescences in a panicle and ~35 cm female inflorescences with two to six spikes. Its tender leaves or shoots can be picked two to three times a year, but leaf-picking is quite difficult because the tree can grow up to 20 m under natural conditions; therefore, pruning usually has been performed to control the height of the tree below 1.5 m for improving the picking efficiency. Up to now, products in the market are mainly harvested from wild resources since this plant has not been popularly cultivated. Although sweet tea can be propagated through cuttings and seeds similar to the *Camellia sinensis*, sexual propagation through seeds is mainly adopted, which will lead to production difficulties because of the mixed genetic background of the trees.

#### *2.2. Concentration of the DHCs and Influence Factors*

The sweet tea contains diverse secondary metabolites. Eighteen terpenoids have been identified from cupules [17,18] and 35 flavonoids, including flavones, flavonols, dihydroflavones, isoflavones and DHCs, are detected in stems [19]. Meanwhile, seven triterpenoids are isolated from the leaves and twigs [20], 268 volatiles are identified from young leaves [21] and 68 phenolic compounds have been qualified and quantified from leaves [22]. In this respect, the leaves are the main economic parts of the sweet tea. Among these metabolites, DHCs are the most abundant component in sweet tea. Research showed that the content and composition of the DHCs are influenced by many factors, such as geographical distribution of the trees, leaf maturity and harvest time (Table 1). Yang et al. reported that the abundance of the DHCs is significantly correlated with latitude negatively

as well as temperature and soil organic matter and nitrogen content positively [23]. With the decline of latitude, the growth and development of the sweet tea tree will be positively stimulated by an increase in the average annual temperature and rainfall; therefore, increased leaf area, broadened vein distance and accelerated growth rate of the trees have usually been observed in the low latitude area [24–28]. In the sweet tea, trilobatin, phloridzin and phloretin are the main DHCs. The content of these compounds varies with the tissues and organs of the sweet tea tree. With an increase in the maturity of leaves, the phloridzin level increases, and trilobatin decreases [26]; meanwhile, the level of phloretin is relatively low and changes little. Thus, a high level of trilobatin is usually observed in the tender leaves, with a content of about 14–28%, while accumulated phloridzin is witnessed in the mature and old leaves [25]. For the same plant, the content of the three DHCs in tender leaves fluctuates with the harvest time. The content of trilobatin peaks in April, and the phloridzin peaks in April and August [24]. This implies that the DHCs may possess important biological functions for the growth and development of the plant. When the phloridzin biosynthesis was blocked through transgenic operation, the genetically modified 'Royal Gala' apple showed a series of severe phenotypic changes, including stunted growth, reduced internode length, narrowed leaf, increased lateral branches and weakened adventitious roots [29]. Exogenous supplementation of phloridzin to the genetically modified apple tree would enhance axial leaf growth and partially restore the leaf to a 'normal' shape [30]. Moreover, phloridzin biosynthesis would promote photosynthetic carbon accumulation but limit nitrogen accumulation via the shoot-dependent nitrogen assimilation pathway in apples [31]. In general, high-level phloridzin in mature leaves may help to maintain the morphological and physiological functions of the leaves by regulating photosynthesis and resistance. However, the exact physiological effect has not been clearly revealed till now, and much more research needs to be carried out to elucidate the accumulation mechanism and physiological roles of the DHCs, especially in sweet tea trees. The processing method will remarkably affect the composition and content of the DHCs in the sweet tea. At present, the harvested leaves of the sweet tea have usually been made into "green tea" and "black tea" by adopting traditional tea processing technologies. Fixation, as a characteristic step of green tea processing, can denature the enzymes of fresh leaves and maximally maintain the color and composition of the raw materials through heating at high temperatures in a short time. Four fixation styles, including roller-fixation, microwave-fixation, steam-fixation and fry-fixation, were tested, and the results showed that retention of the different DHCs changed with the fixation styles. The content of the phloretin in products followed the order: fry-fixation > roller-fixation > microwave-fixation> steam-fixation. The level of the phloridzin was highest in products through microwave-fixation, a medium through steamfixation and roller-fixation, and lowest through fry-fixation. The highest trilobatin was observed in roller-fixation treated products, medium in microwave-fixation and fry-fixation treated products and lowest in steam-fixation treated products [32]. This indicated that the different DHCs might possess various thermal sensitivities and could transform among them during heat treatment. Fermentation is the key step of black tea processing. After fermentation, the contents of total polyphenols, trilobatin and phloridzin in the sweet tea were decreased by 26.36%,10.24% and 39.37%, respectively [33]. This suggested that the redox activity of meta hydroxyls is higher than that of para hydroxyls. In addition, the level of phloridzin extracted from the sweet tea leaves fermented with *Saussurea* bacteria is much higher than that of the naturally fermented and unfermented leaves [34]. Studies also showed that the drying method would impact the level of phenolics in sweet tea, and freeze-drying could retain the highest level of phenolics [35].

**Figure 1.** The change of scientific name of the sweet tea.

**Table 1.** Effect of the location, picking time and leaf maturity on the content of DHCs in the sweet tea.

#### **3. DHCs Biosynthesis and Its Regulation**

Trilobatin and phloridzin, being positional isomers of the glycosidically bound phloretin, are produced through a side branch of the phenylpropanoids and flavonoids pathways (Figure 2) [38] which is initiated from cleavage of phenylalanine catalyzed by phenylalanine ammonia-lyase (PAL). The first committed step of the DHCs biosynthesis is the conversion of p-dihydrocoumaryl-CoA from p-coumaryl-CoA, which can be catalyzed by a hydroxycinnamoyl-CoA double bond reductase (HCDBR) [39]. Then phloretin will be produced from the p-dihydrocoumaryl-CoA and

three units of malonyl-CoA through decarboxylative condensation and cyclization mediated by chalcone synthase (CHS) [40]. Finally, biosynthesis of phloridzin and trilobatin requires the action of UDP-glycosyltransferases (UGTs), also called phloretin-2 -O-glycosyltransferase (P2 GT) and phloretin-4 -O-glycosyltransferase (P4 GT), to attach a glucose moiety at either 2 or 4 positions of the phloretin A-ring [1,41]. UGTs catalyze glycosylation of the flavonoids in the plant, and members of the *UGT88Fs* subfamily encode P2 GT and P4 GT, which are responsible for the glycosylation of phloretin in apples [41–45]. *UGT88F1* was the firstly cloned gene encoding the P2 GT in apples [42]. The exogenously expressed P2 GT by *UGT88F1* can specifically glycosylate the phloretin in the presence of UDP-glucose, UDP-xylose and UDP-galactose, but not toward caffeic acid, chlorogenic acid, coumaric acid, cyanidin, 3,4-dihydroxyhydrocinnamic acid, 3-hydroxybenzoic acid, naringenin, 3,4-dihydroxybenzoic acid, catechin, epicatechin, quercetin and rutin. Reports showed that the enzymes encoded by *UGT71A15* and *UGT71K1* of apple can also convert phloretin into phloridzin in vitro [45], while *UGT75L17* of apple encodes P4 GT which catalyzes the 4 position glycosylation of phloretin to produce the trilobatin in the presence of UDP-glucose [41]. Overexpression of the *UGT75L17* in *Escherichia coli* can be used for efficiently producing trilobatin from phloretin [46]. *UGT71A16*, *UGT71K2* and *UGT88F2* isolated from pear, the relative homolog of *UGT71A15*, *UGT71K1* and *UGT88F1* in apple, can encode the enzymes to synthesize phloridzin from phloretin [45]. This indicated that the biosynthesis limitation of phloridzin and analogs in pear is due to unable formation of phloretin or its precursor(s) rather than a lack of glycosyltransferases. Studies also revealed that trilobatin can be produced through the hydrogenation of the naringin and then hydrolysis with α-L-rhamnosidase in aqueous medium [47]; however, this pathway might not exist under physiological conditions. In the sweet tea tree, *chalcone isomerase*(*CHI*)*, leucoanthocyanidin reductase* (*LAR*)*, flavone 3-hydroxylase* (*F3H*) and *4-coumarate: coenzyme A ligase* (*4CL*) have been cloned, and expression of the *LAR* and *4CL* is positively correlated with DHCs content significantly [48–51].

The biosynthesis of phloridzin is mainly influenced by light quality, light intensity and photoperiod. The phloridzin content in the sweet tea leaves decreases in the red or blue light treatment and increases in the green light treatment compared with natural light. The content increases with prolonging the illumination time from 8 h to 14 h and with increasing the intensity from 12.5 to 37.5 <sup>μ</sup>mol·m−2·s−<sup>1</sup> in the white light treatment. The change in the phloridzin level is consistent with the expression of *PAL* and *4CL* genes [15]. Thus, the effect of the light on the phloridzin accumulation is mainly regulated by the expression of the genes involved in the early steps of the phenylpropanoid pathway, which might be used to explain the fluctuation of the DHCs along with the change of the season and latitude. In addition, DHCs biosynthesis may also be modulated by hormone level and development status because the accumulation of phloridzin and trilobatin regularly changes with the increase of leaf maturity.

**Figure 2.** Biosynthesis pathway of the DHCs.

#### **4. Neuroprotective Effects of the DHCs**

AD, PD and stroke are the main neurological disorders. The pathogenesis of AD is related to amyloid β-protein (Aβ) deposition, formation of nerve fiber tangles (NFTs) through abnormal phosphorylation of tubulin-associated unit (Tau) protein, and synaptic dysfunction. PD syndrome is characterized by the degenerative death of dopaminergic neurons in the midbrain substantia nigra, decreased dopamine concentration in the striatum, and the formation of a Lewy body. The DHCs might exert their neuroprotective effects either directly by inhibiting or alleviating the neurological damage or indirectly by preventing the nervous tissue from oxidative stress, inflammation and apoptosis (Table 2).

**Table 2.** Neuroprotective effects of the DHCs.

#### *4.1. Prevention and Treatment of Neurological Diseases*

Under normal physiological conditions, amyloid precursor protein(APP) can be digested by α-secretase to produce soluble α-APP fragment (sAPPα) and 83-amino-acid membranebound C-terminal fragment (C83), and then the C83 is cleaved by γ-secretase to produce non-toxic fragments. However, when the APP is cleaved by β-secretase, soluble β-APP (sAPPβ) and 99-amino-acid C-terminal (C99) fragments will be generated, and then the C99 will be digested by γ-secretase to produce a variety of Aβ peptides containing 39–42 amino acid residues. The Aβ monomers can spontaneously aggregate and deposit into oligomers, fibrils and senile plaques, which then induce oxidative injury, microglial and astrocytic activity as well as alter kinase/phosphatase activity, eventually leading to neuronal death and AD [62]. Recent reports showed that trilobatin and phloretin can decrease Aβ deposition by downregulating the expression of *beta-site APP cleaving enzyme 1* (*BACE1*) and consequentially decreasing the β-secretase [53]. Oral administration with 20 mg/kg trilobatin could protect 3 ×FAD (familial Alzheimer's disease) mice from neurological damage by alleviating Aβ deposition, synaptic degeneration, astrocytosis and microgliosis activation, neuronal loss and cognitive deficits [53]. Phloretin could also suppress Aβ aggregation in the dentate gyrus and CA1 (Cornu Ammonis region 1) regions of the hippocampus in the Aβ-induced AD rats [57]. Furthermore, the Aβ1–42 impaired plasticity at the neuronal and presynaptic levels could be restored to normal value by phloretin treatment [56].

Tau is a main microtubule-associated protein of neurons and plays a critical role in microtubule assembly and stability maintenance. Abnormal hyperphosphorylation of tau neutralizes the basic inhibitory domains and enables tau–tau interaction, resulting in the formation of NFTs in nerve cells, consequently causing the disintegration of tubulins and collapse of the delivery system, eventually leading to the death of the nerve cell. At present, it is well known that glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A

(PP2A) are the two most important enzymes which can regulate the phosphorylation of tau protein. In mice, treatment of trilobatin can directly inhibit the hyperphosphorylation of tau at Ser396 and Ser202 sites and indirectly alleviate the phosphorylation of tau through suppressing the hyperphosphorylation of GSK-3β which acts as a Tau kinase [53], as shown in Figure 3.

**Figure 3.** Directly neuroprotective effects of the DHCs. APP, amyloid precursor protein; sAPPα, soluble α-APP fragment; C83, 83-amino-acid membrane-bound C-terminal fragment; AICD, APP intracellular domain; sAPPβ, soluble β-APP; C99, 99-amino-acid C-terminal; Aβ, amyloid β-protein; GSK 3β, glycogen synthase kinase 3β; NFT, Nerve Fiver Tangles. Arrow (→) indicates activation, and the blunt arrow (–**|**) indicates inhibition.

#### *4.2. Antioxidative Effect*

It is widely presumed that excessive radical oxygen species (ROS) would lead to a variety of neurological diseases. Trilobatin is able to maintain mitochondrial ROS homeostasis by inhibiting overproduction and promoting the elimination of ROS. Regulation of trilobatin against ROS might carry out mainly by influencing the oxidative stress signal transduction (Figure 4). Pretreatment with trilobatin can stimulate AMP-activated protein kinase (AMPK) phosphorylation which will consequently trigger activation of peroxisome proliferators activated receptor-γ coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα), and then the activated ERRα will bind to the ERRα response element (ERRE) of sirtuin 3 (*Sirt3*) promoter to activate the transcription of *Sirt3* which encodes a major mitochondria NAD+-dependent deacetylase, or the activated PGC-1α will accelerate the disassociation of complex Kelch-like ECH-associated protein 1 (Keap-1)/Nuclear respiratory factor 2 (Nrf2) to provoke translocation of the key transcript factor Nrf2 from the cytoplasm into nucleus which can activate the transcription of *Sirt3* through recognizing and binding to antioxidant response element (ARE) of the *Sirt3* promoter. The elevated Sirt3 will promote superoxide dismutase 2 (SOD2) deacetylation, which can boost ROS scavenging. It was confirmed that trilobatin protected HT22 cells against isoflurane-induced neurotoxicity mainly via activating the Nrf/ARE pathway [55], and phloretin

could also eliminate the oxidative stress of the cerebral ischemia/reperfusion rats mainly through activating the Nrf2 defense pathway [60]. The study also showed that trilobatin could reverse the cytotoxicity of HT22 cells induced by the Aβ25–35 treatment partially by inhibiting the oxidative injury mediated by mitogen-activated protein kinase p38 (p38)/Sirt3 pathway [52]. In addition, trilobatin can exert antioxidant capacity by improving the activities of NADHubiquinone oxidoreductase and ATPase and balancing the NAD+/NADH ratio [12]. However, the antioxidative effects of phloretin and phloridzin cannot be achieved by increasing the activity of enzymes such as SOD or catalase (CAT). However, they can directly scavenge the superoxide anions generated from both the electron transfer chain reactions of NADHubiquinone oxidoreductase /FMN/Fe-S clusters (complex I) and ubiquinone-cytochrome c oxidoreductase/cytochrome b/ Fe-S clusters (complex III) in human SH-SY5Y neuronal-like cells treated with inducer rotenone [63].

**Figure 4.** DHCs exert neuroprotection through their antioxidative effect. DHCs, dihydrochalcones; AMPK, AMP-activated protein kinase; PGC-1α, peroxisome proliferators activated receptor-γ coactivator-1α; Keap-1, Kelch-like ECH-associated protein 1; Nrf2, Nuclear respiratory factor 2; ARE, antioxidant response element; ERRα, estrogen-related receptor α; ERRE, ERRα response element; Sirt3, sirtuin 3; SOD2, superoxide dismutase 2; ac, acetylation; ROS, radical oxygen species. Arrow (→) indicates activation, and the blunt arrow (–**|**) indicates inhibition.

#### *4.3. Anti-Neuroinflammation and Anti-Apoptosis*

Neuroinflammation is critical damage resulting in neurological diseases. It has been proved that trilobatin could inhibit the inflammation in middle cerebral artery occlusion (MCAO)-induced cerebral I/R injury rats by suppressing the toll-like receptor 4(TLR4)/ myeloid differentiation factor 88(MyD88)/tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) signaling pathway, and down-regulating the phosphorylation level of NFκBp65 which will restrain the release of proinflammatory factors, including IL-1β, IL-6, TNF-α and inducible nitric oxide synthase (iNOS) [54], as shown in Figure 5. Pretreatment with phloretin could also inhibit the activation of the glial cell and suppress the inflammatory responses [58].

**Figure 5.** DHCs exert neuroprotective effects through their anti-inflammation and anti-apoptosis. TLR4, Toll-like receptor 4; TIRAP, toll/interleukin 1 receptor domain-containing adaptor protein; MYD88, myeloid differentiation factor 88; TRAF6, TNF receptor-associated factor 6; IκBα, inhibitor of NF-κB; NF-κB, nuclear factor κ-B; TNF-α, tumor necrosis factor α; TNFR, tumor necrosis factor receptor; BDNF, brain-derived neurotrophic factor; Trk B, tyrosine kinase receptor B; PI3K, phosphatidylinositol 3- kinase. Arrow (→) indicates activation, and the blunt arrow (–**|**) indicates inhibition.

The experiment showed phloridzin could significantly elevate the brain-derived neurotrophic factor (BDNF) level and reduce the acetylcholinesterase (AChE) activity in the hippocampus and cortex of the lipopolysaccharide (LPS)-treated mice [61]. BDNF can bind to tyrosine receptor kinase B (Trk B), which activates phosphatidylinositol 3-kinase (PI3K), and then spurs the anti-apoptosis and protein synthesis [64]. AChE can degrade acetylcholine and terminate the excitatory effect of neurotransmitters on the postsynaptic membrane, and reduced AChE levels can inhibit neuron damage and prevent AD formation. A large amount of AChE was expressed when cells were in the state of apoptosis. The increased AChE protein can not only inhibit cell growth but also enter the nucleus and participate in the formation of apoptotic bodies and promote apoptosis [65]. Another research confirmed that inhibition of AChE activity through pretreatment and treatment with phloretin could improve spatial memory formation during the Morris water maze (MWM) test in scopolamine-induced amnesia mice [59]. Tests also showed that trilobatin could prevent HT22 cells from the injury induced by the Aβ25–35 treatment via suppression of the caspase-3-dependent apoptosis pathway. Caspase-3, the most important terminal cleavage enzyme in the process of apoptosis, can be activated by TNF, cyt-c and other caspase families. The activated caspase-3 further enlarges the cascading effects and finally leads to cell death [66].

#### **5. Conclusions**

The shoots and leaves of *Lithocarpus litseifolius* are rich in DHCs, which taste sweet and possess a variety of physiological effects. Phloretin, trilobatin and phloridzin are the main components of the DHCs. The level of these compounds usually fluctuates along with leaf maturity and distribution location, but some contradictory results have been reported because many samples usually have been collected without uniform picking standards from natural growth trees instead of cultivated plants. The biosynthesis of the DHCs begins with the phenylpropanoid pathway and turns to a branch of the flavonoid pathway at the step from p-coumarinyl CoA to dihydro-4-coumaroyl CoA rather than directly to naringenin chalcone. It has been speculated that the accumulation of the DHCs might be regulated at the transcriptional level at the early steps of the pathway. The DHCs can prevent the neuron tissues from damage by directly inhibiting Aβ deposition and tau hyperphosphorylation as well as indirectly eliminating oxidative stress, inflammation and apoptosis. However, up to now, many issues are not clear, especially why the *Lithocarpus litseifolius* accumulates high levels of DHCs in their leaves, what is the exact biological function of these compounds for the trees and how these compounds are synthesized and metabolized precisely. Meanwhile, many more studies need to be approached for better utilizing the *Lithocarpus litseifolius*, such as collection and improvement of the germplasms, technology development of standardization cultivation, optimization of the processing system and new biological function extension and its mechanism elaboration of the DHCs.

**Author Contributions:** Data curation: Y.-K.W. and Z.-B.D.; visualization: Y.-K.W. and S.-Y.H.; validation: F.-Y.X. and J.-H.Y.; formal analysis: F.-Y.X. and Z.-B.D.; writing—original draft: Y.-K.W. and S.-Y.H.; supervision: X.-Q.Z. and Y.-R.L.; writing—review and editing: J.-H.Y. and J.-L.L.; funding acquisition: X.-Q.Z. and J.-L.L.; project administration: Y.-R.L.; conceptualization: J.-L.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding-Tea Plant (No. 2021C02067-6), the National Natural Science Foundation of China (No. 32272763) and China Agriculture Research System of MOF and MARA.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no competing financial interests.

#### **References**

### *Article* **Enhancement of the Functional Properties of Mead Aged with Oak (***Quercus***) Chips at Different Toasting Levels**

**Juciane Prois Fortes 1,2, Fernanda Wouters Franco 1,2, Julia Baranzelli 1,2, Gustavo Andrade Ugalde 3, Cristiano Augusto Ballus 1, Eliseu Rodrigues 4, Márcio Antônio Mazutti 5, Sabrina Somacal 1,2,\* and Claudia Kaehler Sautter 1,2**

**Abstract:** Consumers increasingly prefer and seek functional beverages, which, given their characteristics, provide important bioactive compounds that help prevent and treat chronic diseases. Mead is a traditional fermented alcoholic beverage made from honey solution. The aging process of mead with oak chips is innovative and bestows functional characteristics to this beverage. Thus, in this study, we sought to develop and characterize a novel functional beverage by combining the health benefits of honey with the traditional aging process of alcoholic beverages in wood. Phenolic compounds, flavonoids, and antioxidant capacity were analyzed in mead using oak chips at different toasting levels and aged for 360 days. LC-ESI-QTOF-MS/MS was used to analyze the chemical profile of different meads. Over time, the aging process with oak chips showed a higher total phenolic and flavonoid content and antioxidant capacity. Eighteen compounds belonging to the classes of organic acids, phenolic acids, flavonoids, and tannins were identified in meads after 360 days. Our findings revealed that the addition of oak chips during aging contributed to *p*-coumaric, ellagic, abscisic, and chlorogenic acids, and naringenin, vanillin, and tiliroside significantly impacted the functional quality of mead.

**Keywords:** polyphenols; honey; functional beverage; characterization; alcoholic beverage; fermented beverage; phenolic compounds; antioxidant; functional foods; beneficial effects

#### **1. Introduction**

The increased prevalence of noncommunicable diseases in recent years has made consumers increasingly aware of healthy and natural diets [1]. Consequently, the food and beverages industry faces new challenges in designing functional foods. Among the different types of functional foods, beverages are the most acceptable due to logistic facilities, their distribution, and the ease of incorporating bioactive compounds as functional ingredients [2]. Furthermore, functional beverages have gained more market shares over the last decade [3].

Recently, wine and beer, two of the most popular alcoholic beverages, have been identified as functional beverages, and the benefits of their moderate consumption have been widely supported by the scientific community [4]. The main source of the beneficial potential of consuming these beverages is phenolic compounds. After consuming phenoliccompound-rich foods, such as functional beverages, the colon is the leading site of microbial fermentation. Intestinal microbiota transforms phenolic compounds into phenolic acids or

**Citation:** Fortes, J.P.; Franco, F.W.; Baranzelli, J.; Ugalde, G.A.; Ballus, C.A.; Rodrigues, E.; Mazutti, M.A.; Somacal, S.; Sautter, C.K. Enhancement of the Functional Properties of Mead Aged with Oak (*Quercus*) Chips at Different Toasting Levels. *Molecules* **2023**, *28*, 56. https://doi.org/10.3390/ molecules28010056

Academic Editor: Nour Eddine Es-Safi

Received: 15 November 2022 Revised: 7 December 2022 Accepted: 14 December 2022 Published: 21 December 2022

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

lactone structures, which produce metabolites with biological and antioxidant activity, and evidence suggests that these metabolites have health benefits for humans [3,5,6].

Mead or honey wine is a beverage traditionally produced by diluting honey in water and yeast and may present some variations through the addition of fruit or fruit juice, herbs, or spices [7]. Fermentation and maturation/aging are the two most time-consuming processes in mead production, often lasting a few days to months [8]. The final composition of the mead will depend on the type of honey used, the ingredients added, and the fermentation and storage conditions [9]. Mead has been produced since ancient times, especially in Eastern European countries; however, it is currently not as popular as other alcoholic beverages, highlighting the need for further research on this beverage and its potential functionality [10].

Honey is widely known for its health-promoting biological characteristics such as its anti-inflammatory, antiviral, antifungal, and antitumor properties [11]. Among the bioactive descriptors in honey, phenolic compounds, organic acids, carbohydrates, amino acids, proteins, minerals, vitamins, and lipids stand out [11,12], which directly influence the chemical and sensory characteristics of the mead [8,9,12]. Nevertheless, the composition of honey is quite variable and relies on the floral source and seasonal and environmental conditions, in addition to the processing and storage techniques used [9,11].

The aging process of alcoholic beverages in wood provides various changes in the composition and concentration of compounds in beverages [13,14]. Such modifications may be noticeable by changes in the beverages' taste, color, and aroma. In addition to sensory changes, many phenolic compounds are acquired or elevated during maturation [13,15]. Martínez et al. (2008) tested different quenching methods on the chemical composition of American (*Quercus alba*) and French (*Quercus petraea*) oak, and their findings showed the evolution of ellagitannins, a low molecular weight phenolic compound, and volatile compounds regarding oak species and the tempering method [16]. The natural process in the open air was considered superior to the artificial and mixed drying methods, as it showed greater effectiveness in reducing excess ellagitannins. In addition, the evolution of the aromatic potential of the wood was more positive, reaching higher concentrations of compounds such as volatile phenols, phenolic aldehydes, furanic compounds, and *cis*and *trans*-methyl-γ-octalactones. The wood toasting process takes place after tempering, where the wood is subjected to temperatures in the range of 150–240 ◦C for a certain period, according to the desired toasting level. In this step, thermal degradation reactions occur, transforming nonvolatile precursors into active aromatic volatile compounds [17].

Due to technological advances in the maturation/aging area of alcoholic beverages, alternative systems using wooden barrels to carry out this process have emerged. Staves, chips, shavings, and other alternative sources of extraction of compounds from oak or other species of forest wood have become common for the maturation and aging process of alcoholic beverages, generating a new use of wood residues in the cooperage [15,18].

Thus, this study aimed to develop a functional mead with multifloral honey and submit it to the aging process with oak chips (*Quercus*), a wood commonly used for the maturation and aging of alcoholic beverages that can provide bioactive compounds. In addition, we sought to evaluate the phenolic compounds content and antioxidant capacity after fermentation and identify the phytochemical compounds in mead aged with oak chips at two toasting levels.

#### **2. Results and Discussion**

Functional beverages can be a valuable component of the human diet given their ability to provide essential hydration and important bioactive compounds for maintaining health and/or contributing to preventing and treating chronic diseases [1,2,5,6]. Mead is a beverage that has been gaining more and more notoriety over time, although there are few studies on it; hence, this study is unprecedented as it presents data from meads aged with oak chips, a process so far only used for other types of alcoholic beverages [13] and which can improve the functionality of this beverage. In this study, the antioxidant capacity, total phenolic and flavonoid content, and characterization of phytochemical compounds of meads subjected to a 360-day aging period with oak chips (*Quercus*) at different toasting levels were determined.

Honey is a source of numerous biologically active compounds, including phenolic and volatile compounds, peptides, proteins, amino acids, enzymes, and minerals, which can be transferred to the mead during production [12]. Among all the substances, our attention was given to the phenolic compounds, which contribute to the mead functional quality. This phytochemical group includes flavonoids, tannins, and phenolic acids, which are natural antioxidants that play significant roles in the human body due to their capability to inhibit free radicals, which may cause cell damage, leading to chronic diseases [2,3,19]. Beverages are considered good dietary sources of phenolic compounds; moreover, the phenolic compounds in beverages are highly bioaccessible because they pass directly into intestinal fluids [5], making this study highly innovative in carrying out the aging process of mead with oak chips and monitoring it over time.

At time zero, all elaborated meads had a similar content of phenolic compounds, which came from the honey used (Figure 1). In the base mead (BM), these values remained constant for up to 180 days and then decreased with advancing maturation time. Meads with chips addition showed higher total phenolic compound levels until the aging period of 180 days. This increase may be related to the extraction of phenolic compounds from oak into mead throughout the maturation process. Canas et al. (2019) observed higher total phenolic compounds in wine spirits aged for 180 days with oak staves [20]. This increase was due to the combination of the thermal degradation of lignin and increased wood permeability during thermal treatment. Mead is still poorly studied, although due to its characteristics similar to young white wines, aging time may have provided oxidative reactions and/or condensation between the mead compounds with some wood molecules, reducing the phenolic compounds after 270 days of aging.

**Figure 1.** Composition of total phenolics (mg GAE L−1) of meads aged with oak (*Quercus*) chips. Data are presented as means ± SEM (*n* = 5). Lowercase letters indicate significant differences over time within the same experimental group (*p* ≤ 0.05). Capital letters indicate significant differences between experimental groups within the same time (*p* ≤ 0.05). GAE: gallic acid equivalent; BM: base mead; MOWT: mead aged with oak chips without toasting; MOMT: mead aged with oak chips at medium toasting; MOHT: mead aged with oak chips at high toasting.

Furthermore, flavonoids gradually increased in meads with oak chips throughout the aging period, with a maximum content at 270 days (Figure 2). We observed that the concentration of this class of phenolic compounds varied from 10.6 mg to 20.5 mg CAE L<sup>−</sup>1. Regardless of the toasting degree, the oak chips increased the mead flavonoids concentrations, which may have contributed to increasing the antioxidant capacity throughout the aging process (Figure 3) since these compounds have high free radical scavenging potential, as described elsewhere [8,19].

**Figure 2.** Composition of total flavonoids (mg CAE L−1) of meads aged with oak (*Quercus*) chips. Data are presented as means ± SEM (*n* = 5). Lowercase letters indicate significant differences over time within the same experimental group (*p* ≤ 0.05). Capital letters indicate significant differences between experimental groups within the same time (*p* ≤ 0.05). CAE: catechin equivalent; BM: base mead; MOWT: mead aged with oak chips without toasting; MOMT: mead aged with oak chips at medium toasting; MOHT: mead aged with oak chips at high toasting.

**Figure 3.** Antioxidant capacity (mM TEAC L−1) of meads aged with oak (*Quercus*) chips. Data are presented as means ± SEM (*n* = 5). Lowercase letters indicate significant differences over time within the same experimental group (*p* ≤ 0.05). Capital letters indicate significant differences between experimental groups within the same time (*p* ≤ 0.05). TEAC: Trolox equivalent antioxidant capacity; BM: base mead; MOWT: mead aged with oak chips without toasting; MOMT: mead aged with oak chips at medium toasting; MOHT: mead aged with oak chips at high toasting.

The antioxidant capacity of meads depends on the raw material's chemical composition, the environmental factors that directly affect the honey production process, and the technologies used to process it [8,9]. Apart from technological processes such as fermentation and aging, these beverages' antioxidant properties and chemical composition are determined by the additives used in their manufacturing [8]. In this investigation, the antioxidant capacity was determined with a 2,2 -azino-bis (3-ethylbenzothiazoline) 6-sulfonic acid (ABTS) assay and varied from 1.31 to 5.06 mM of Trolox equivalent antioxidant capacity per liter (TEAC L−1), showing a significant increase after 270 days of aging in meads with the addition of the oak chips (Figure 3). In meads, the antioxidant capacity is related to the presence of phenolic compounds, and the diversity of these compounds is directly linked to the honey used [8]. Hence, meads that only use honey and water in their composition tend to have lower compound diversity.

Nevertheless, meads with the addition of fruit juices or herbal extracts have a wider range of these phytochemicals [12]. The profile of the antioxidant capacity is a concomitant event to the behavior of the flavonoid content over time (Figures 2 and 3), especially at 270 days. A similar behavior was observed in unripened meads and other woodaged beverages [10,21]. However, one cannot rule out the possibility that other bioactive compounds, in addition to flavonoids, contributed to the antioxidant capacity observed in the final aging period of meads with oak chips in this investigation. Phenolic compounds are found in plants as they come from their secondary metabolism. Bees, by collecting pollen, transfer many of these compounds to the honey; consequently, these phytochemicals will be present in the mead, even in smaller amounts [9,11]. The Brazilian flora is vast, generating honeys with various phenolic compounds, with a predominance of many phenolic acids such as 3,4-dihydroxybenzoic, salicylic, caffeic, chlorogenic, *p*-coumaric, ferulic, gallic, syringic acids, and flavonoids including isorhamnetin, kaempferol, luteolin, naringenin, pinobanksin, quercetin, and rutin [22].

In this study, the phenolic compounds in the meads at the end of 360 days of aging were identified through MS/MS mass spectrometry analysis by combining the chromatographic behavior and collision spectra with compounds already described in the literature. Eighteen compounds belonging to the classes of organic acids, phenolic acids, flavonoids, and tannins were tentatively identified and are listed in Table 1 and in the Supplementary Material (Figure S1).

Among the compounds identified are citric acid, protocatechuic acid, sinapyl alcohol, syringic acid, ethylvanillin, 1-(2-hydroxy-4,6-dimethoxyphenyl)-ethanone, sebacic acid, and quercetin in all samples, which are therefore compounds from the honey used in the beverage's elaboration. Adding oak chips during aging, regardless of the toasting, contributed to vanillin, *p*-coumaric acid, ellagic acid, abscisic acid, and naringenin. However, chlorogenic acid and tiliroside were only present in the meads aged with oak chips without toasting (MOWT) and meads aged with oak chips at high toasting (MOHT). Various studies have demonstrated that chlorogenic acids are partially bioavailable and potentially beneficial to human health [6]. The antioxidant and anti-inflammatory effects of coffee chlorogenic acids are responsible for, at least to a certain extent, the association between coffee consumption and the lower incidence of various degenerative and nondegenerative diseases, in addition to higher longevity [6]. 2,3-Dihydroxy-1-guaiacylpropanone was only found in the BM, and the 3-hydroxy-3-(3-hydroxyphenyl) propionic acid was found in the BM and mead aged with oak chips without toasting (MOWT).

*Molecules* **2023**, *28*, 56

**Table 1.**

Phytochemical

 compounds

 detected in mead produced with multifloral honey aged with oak (*Quercus*) chips.

During the aging process, the transformations of lignin, present in woods such as French oak, are among the most important factors that affect the quality of beverages aged in contact with this material. Lignin is a polymer that undergoes thermal degradation during the manufacturing of barrels or by hydrolysis and ethanollysis during the aging of wines and alcoholic beverages [30]. The lignin macromolecule has ramifications of coniferyl alcohols (guaiacyl compounds) and sinapyl (syringyl compounds). Coniferyl alcohol generates coniferylaldehydes, which are converted into vanillin, while sinapyl alcohol gives rise to sinapaldehyde, which is transformed into syringaldehyde and later oxidized to syringic acid. Other compounds, such as hydrolyzable tannins, present in French oak, are more soluble in hydroalcoholic solutions; its transformation into ellagic acid is very common [31], corroborating the identification of this compound only in meads with the addition of oak chips.

In the present investigation, meads aged with oak chips showed a higher phenolic compound content and diversity. It is known that these compounds may be related to physicochemical and sensory characteristics in foods [8,12]. Thus, the sensory evaluation of meads is important to know the impact of compounds on sensory aspects, including color, flavor, and astringency. The absence of a sensory evaluation of different meads is one of the limitations of this study. These analyses were scheduled to be carried out in 2020–2021, and the study had already been approved by the institutional ethics committee (CAAE no. 58889316.3.0000.5346). Unfortunately, given the restrictions imposed by the SARS-CoV-2 pandemic, it was impossible to carry out the analyses. Until now, the classic wood-aging method employed in numerous alcoholic beverages has not yet been tested in mead. Hence, despite the absence of a sensory evaluation, this study is pioneering and can contribute to essential elucidations in the field of functional beverages.