**1. Introduction**

The main strategies for the valorization of food wastes are related to their biotechnological transformation into chemicals or even the recovery of important substances, such as polyphenols that typically appear in the winery by-products (WBPs). Currently, the implemented alternatives for reducing the environmental impact of agronomic residues involves the development of new feeds and their use as soils amendments. Actually, these are the primary alternatives considered. Given their low added-value there is a need to search for new valorization alternatives.

Based on these premises, the content of bioactive phytochemicals of agro-food materials in general has allowed envisaging their use as donors of these kind of molecules to obtain materials that could contribute to enhance medical/nursing treatments.

Given the relevance of the winemaking companies, particularly at Douro region, and the high amount of underexploited wastes produced, the development of innovative applications for these materials urges [1]. On these materials ongoing research (also relevant studies developed by the research group) has revealed the valuable quantitative profile of bioactive compounds in WBPs, namely a variety of (poly) phenols and stilbenes that could be responsible for remarkable biological activities, such as anti-inflammatory, antioxidant, and antibacterial, among others [2–5].

However, for envisaging new applications for these materials, it is important to be aware about the close dependency of the phytochemical composition and therefore the biological power on an array of factors, namely the geographical growing conditions [3], the cultivar studied [4] and, most important, the extraction methodology employed [6]. In fact, the extraction methodology no just condition the phytochemical compounds obtained from a given plant material, but also is associated to environmental constraints, as well as to economic and toxicological issues depending on the solvent used [7]. To overcome these limitations, special attention has been paid to the extraction methods for bioactive compounds [8]. So, the use of eco-friendly techniques blended with reusable and non-toxic solvents is gaining a wide acceptance, due to its contribution to minimizing costs, heath related risks, and environmental impacts. As a valuable alternative to the traditional extraction methods, UAE arises as exceptional option to extract (poly)phenols, revealing to be an environment-friendly technology that offers several advantages over the conventional and non-conventional ones, such as a lower cost, versatility, and easily scale-up [7]. This technique has been already employed in diverse plant matrices and the outcomes reported have revealed it as one of the best alternatives to extract phenolic compounds from winery wastes [7, 9].

Based on these compositional features, potential applications for these materials, and specifically for grape stems, have been described by the research team, such as the spirits production, leading to an industrial alternative to traditional distilled spirits [10]. Beyond this application, recently, a preliminary study developed by the team demonstrated the stem extracts capacity to inhibit the growth of foot wound ulcers multidrug resistance bacteria (*S. aureus* and *Enterobacter aerogenes*) through disc diffusion and minimum inhibitory concentration assays [5, 11]. Whereby, WBPs are valuable candidates as wound healing agents for instance. Additional studies of our group also revealed that the quantitative (poly)phenolic profile of grape stems remains almost constant during storage for months, leading to the possibility to access this by-product all year-round, due to the preservation of the phytochemical composition [2], and thus, the biological activity expected.

In this work, we intend to generate new knowledge on the potential ability of WBPs (wine lees, grape pomace, and grape stems) bioactive compounds to be further used in pharmaceutical and cosmetic industries, using a sustainable and green extraction way, namely ultrasound-assisted extraction (UAE), enhancing the regional and circular economy.

### **2. Material and methods**

#### **2.1 Chemicals**

Folin–Ciocalteu's reagent, 3,4,5-trihydroxybenzoic acid (gallic acid), acetic acid, both extra pure (>99%), and sodium hydroxide were purchased from Panreac

**5**

**Figure 1.**

*Geographical origin of WBPs from the* Região Demarcada do Douro *(Portugal).*

*Winery By-Products as Source of Bioactive Compounds for Pharmaceutical and Cosmetic…*

(Panreac Química S.L.U., Barcelona, Spain). Sodium nitrate, aluminum chloride, and sodium carbonate, all extra pure (>99%), and methanol were acquired from Merck (Merck, Darmstadt, Germany). Sodium molybdate (99.5%) was purchased from Chem-Lab (Chem-Lab N.V., Zedelgem, Belgium). The compounds 2,2-diphe-

phonic acid)diammonium salt (ABTS•+), and potassium phosphate were obtained from Sigma-Aldrich (Steinheim, Germany), as well as the standards compounds for the chromatographic separation. Additionally, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from Fluka Chemika (Neu-Ulm,

Ultrapure water was obtained using a Millipore water purification system.

The present work was carried on WBPs, namely, grape stems, grape pomace and wine lees of two varieties of *Vitis vinifera* L. (Sousão and Tinta Barroca), which are traditionally cultivated in the *Região Demarcada do Douro*, in northern Portugal. Plant material came from a farm located in Cima Corgo sub-region (Upper Corgo) (Sanfins do Douro - GPS: 41.1656, −7.2912, Average Altitude.: 730 m, vineyard altitude from 690 to 730 m), as demonstrated in **Figure 1**, where geology is essentially characterized by schist formations with occasional outcrops of granite in Mediterranean-like climatic conditions [12]. No irrigation was applied in the field trial of this investigation. WBPs were collected in 2020 growing season, at the wine company which possesses this vineyard. Once collected, samples were lyophilized, grounded to a fine powder, and stored, protected from light, at room temperature

Chromatography solvents were of HPLC grade according to the analysis

), 2,2-azino-bis(3-ethylbenzothiazoline-6-sul-

*DOI: http://dx.doi.org/10.5772/intechopen.97881*

nyl-1-picrylhidrazyl radical (DPPH•

Switzerland).

performed.

**2.2 Sampling**

until analysis.

*Winery By-Products as Source of Bioactive Compounds for Pharmaceutical and Cosmetic… DOI: http://dx.doi.org/10.5772/intechopen.97881*

(Panreac Química S.L.U., Barcelona, Spain). Sodium nitrate, aluminum chloride, and sodium carbonate, all extra pure (>99%), and methanol were acquired from Merck (Merck, Darmstadt, Germany). Sodium molybdate (99.5%) was purchased from Chem-Lab (Chem-Lab N.V., Zedelgem, Belgium). The compounds 2,2-diphenyl-1-picrylhidrazyl radical (DPPH• ), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt (ABTS•+), and potassium phosphate were obtained from Sigma-Aldrich (Steinheim, Germany), as well as the standards compounds for the chromatographic separation. Additionally, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from Fluka Chemika (Neu-Ulm, Switzerland).

Ultrapure water was obtained using a Millipore water purification system. Chromatography solvents were of HPLC grade according to the analysis performed.

#### **2.2 Sampling**

*Innovation in the Food Sector Through the Valorization of Food and Agro-Food By-Products*

to extract phenolic compounds from winery wastes [7, 9].

Based on these compositional features, potential applications for these materials, and specifically for grape stems, have been described by the research team, such as the spirits production, leading to an industrial alternative to traditional distilled spirits [10]. Beyond this application, recently, a preliminary study developed by the team demonstrated the stem extracts capacity to inhibit the growth of foot wound ulcers multidrug resistance bacteria (*S. aureus* and *Enterobacter aerogenes*) through disc diffusion and minimum inhibitory concentration assays [5, 11]. Whereby, WBPs are valuable candidates as wound healing agents for instance. Additional studies of our group also revealed that the quantitative (poly)phenolic profile of grape stems remains almost constant during storage for months, leading to the possibility to access this by-product all year-round, due to the preservation of the phytochemical composition [2], and thus, the biological activity expected.

In this work, we intend to generate new knowledge on the potential ability of WBPs (wine lees, grape pomace, and grape stems) bioactive compounds to be further used in pharmaceutical and cosmetic industries, using a sustainable and green extraction way, namely ultrasound-assisted extraction (UAE), enhancing the

Folin–Ciocalteu's reagent, 3,4,5-trihydroxybenzoic acid (gallic acid), acetic acid, both extra pure (>99%), and sodium hydroxide were purchased from Panreac

Based on these premises, the content of bioactive phytochemicals of agro-food materials in general has allowed envisaging their use as donors of these kind of molecules to obtain materials that could contribute to enhance medical/nursing treatments. Given the relevance of the winemaking companies, particularly at Douro region, and the high amount of underexploited wastes produced, the development of innovative applications for these materials urges [1]. On these materials ongoing research (also relevant studies developed by the research group) has revealed the valuable quantitative profile of bioactive compounds in WBPs, namely a variety of (poly) phenols and stilbenes that could be responsible for remarkable biological activities, such as anti-inflammatory, antioxidant, and antibacterial, among others [2–5]. However, for envisaging new applications for these materials, it is important to be aware about the close dependency of the phytochemical composition and therefore the biological power on an array of factors, namely the geographical growing conditions [3], the cultivar studied [4] and, most important, the extraction methodology employed [6]. In fact, the extraction methodology no just condition the phytochemical compounds obtained from a given plant material, but also is associated to environmental constraints, as well as to economic and toxicological issues depending on the solvent used [7]. To overcome these limitations, special attention has been paid to the extraction methods for bioactive compounds [8]. So, the use of eco-friendly techniques blended with reusable and non-toxic solvents is gaining a wide acceptance, due to its contribution to minimizing costs, heath related risks, and environmental impacts. As a valuable alternative to the traditional extraction methods, UAE arises as exceptional option to extract (poly)phenols, revealing to be an environment-friendly technology that offers several advantages over the conventional and non-conventional ones, such as a lower cost, versatility, and easily scale-up [7]. This technique has been already employed in diverse plant matrices and the outcomes reported have revealed it as one of the best alternatives

**4**

regional and circular economy.

**2. Material and methods**

**2.1 Chemicals**

The present work was carried on WBPs, namely, grape stems, grape pomace and wine lees of two varieties of *Vitis vinifera* L. (Sousão and Tinta Barroca), which are traditionally cultivated in the *Região Demarcada do Douro*, in northern Portugal. Plant material came from a farm located in Cima Corgo sub-region (Upper Corgo) (Sanfins do Douro - GPS: 41.1656, −7.2912, Average Altitude.: 730 m, vineyard altitude from 690 to 730 m), as demonstrated in **Figure 1**, where geology is essentially characterized by schist formations with occasional outcrops of granite in Mediterranean-like climatic conditions [12]. No irrigation was applied in the field trial of this investigation. WBPs were collected in 2020 growing season, at the wine company which possesses this vineyard. Once collected, samples were lyophilized, grounded to a fine powder, and stored, protected from light, at room temperature until analysis.

**Figure 1.** *Geographical origin of WBPs from the* Região Demarcada do Douro *(Portugal).*

## **2.3 Ultrasound-assisted extraction (UAE) of bioactive compounds from olive seeds**

For the phenolic compounds extraction, the protocol used was previously described by Lameirão et al. with some modifications [8]. The UAE was performed with an ultrasonic apparatus (VCX 500 Vibra-Cell™, Newtown, Connecticut, USA), using a 13 mm diameter tip with amplitude, temperature and time controller. The amplitude was employed at 50%. The powdered samples (2.5 g) were extracted with 50 mL of methanol:water (70:30, v/v) into the ultrasonic apparatus during 40 min and at 70°C. After ultrasonic extraction, the methanolic extracts were centrifuged (13,000 rpm, 4°C) for 15 min (Sigma Centrifuges 2–16 K, Germany) and filtered. Samples were stored at 4°C until analysis.

### **2.4 Phenolic content**

The content in total phenols, flavonoids, and *ortho*-diphenols was determined according to spectrophotometric methodologies previously reported [13].

Briefly, the content of total phenolics in olive seed extracts was evaluated by the Folin–Ciocalteu spectrophotometric method, using gallic acid as standard, being the results expressed as mg of gallic acid per gram of dry weight (mg GA g−1 DW).

The content of *ortho*-diphenols in olive seeds was determined by adding Na2MoO4 (50 g L−1) to the samples appropriately diluted, reading the absorbance at 375 nm. For the quantification, the gallic acid was used as standard. Results were expressed as mg GA g−1 DW.

For the assessment of flavonoid content, the aluminum complex method was performed, using catechin as standard. Results were expressed as mg of catechin per gram of dry weight (mg CAT g−1 DW).

All the assays were performed using 96-well micro plates (Nunc, Roskilde, Denmark) and an Infinite M200 microplate reader (Tecan, Grödig, Austria). For all analyses, three replicates (n = 3) of each sample were assessed.

#### **2.5 Antioxidant capacity assays**

The free radical scavenging capacity was determined by ABTS and DPPH spectrophotometric methods, according to the method described by [14]. FRAP methodology was also applied to measure ferric antioxidant power of WBPs extracts.

These assays were also performed using 96-well micro plates (Nunc, Roskilde, Denmark) and an Infinite M200 microplate reader (Tecan, Grödig, Austria), being the results expressed in mmol Trolox per gram of dried sample (mmol Trolox g−1 DW). All the analyses were made in triplicate (n = 3) for each sample [15].
