**2.6 Identification and quantification of phenolic compounds by RP–HPLC–DAD**

The polyphenolic profile of WBPs extracts was assessed by Reverse Phase - High Performance Liquid Chromatography - Diode Array Detector (RP-HPLC-DAD), in an Agilent HPLC 1100 series equipped with a photodiode array detector and a mass detector in series (Agilent Technologies, Waldbronn, Germany), in accordance with the method previously described [13]. The equipment consisted of a photodiode array detector (model G1315B), an autosampler (model G1313A), a binary pump (model G1312A), and a degasser (model G1322A). The HPLC system was controlled by Xcalibur software (Agilent, version 08.03). A C18 column (250 x 4.6 mm, 5 μm particle size; ACE, Aberdeen, Scotland) was used, being the reverse phase HPLC

**7**

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

method based on a polar mobile phase with the mixture of solvent A: H2O/HCOOH (99.9:0.1, v/v), and solvent B: CH3CN/HCOOH (99.9:0.1, v/v). The following linear gradient scheme was used (t in min; %B): (0; 5%), (15; 15%), (30; 30%), (40; 50%), (45; 95%), (50; 95%) and (55; 5%). At this last time (55 min), return to 5% of B to stabilize and prepare the column for the next sample. The analysis was performed at 25°C, with a flow rate of 1.0 mL/min and a sample injection volume of 20 μL. All samples were injected in triplicate. For the quantification of the identified compounds, the respective standards were used at 280 nm. Concentrations

The results are presented as mean (n = 3) ± standard deviation (SD). The data obtained were subjected to variance analysis (ANOVA) and a multiple range test (Tukey's test) for a *p* value <0.05, using IBM SPSS statistics 21.0 software (SPSS

In the present work, the determination of the phenolic composition and the antioxidant capacity of wine lees, grape stems, and grape pomace extracts of two grape (*Vitis vinvifera* L.) varieties (Sousão and Tinta Barroca) were performed. The phenolic content of these samples collected from Douro region (Northern Portugal) was presented in **Table 1**. As it can be observed, in general, grape stems were the WPBs with the highest content of total phenols (168.75 mg GA g−1 DW, on average), *ortho*-diphenols (166.39 mg GA g−1 DW, on average), and flavonoids (152.31 mg CAT g−1 DW, on average), followed by grape pomace and wine lees. Concerning this last winery by-product, it can be stated that the samples from Sousão variety showed the lowest content of these three studied parameters, being significantly different from the other variety and WBPs (p < 0.05). In fact, wine lees and grape stems presented the highest values of phenolic content in Tinta Barroca samples which has not been observed for the grape pomace extracts, which can be explained

Romero et al. obtained similar values in wine lees of total phenols (38–254 mg CAT g−1) and flavonoids (16–146 mg CAT g−1) content from the Tempranillo variety, with these ranges caused by the extraction solvent employed by these authors [16]. However, Pérez-Serradilha et al. [17] obtained higher values in this WBP of total phenols content (364 mg g−1) than those obtained in this work after a microwaveassisted extraction optimized. Our research group have analyzed the phenolic content of grape stem extracts prepared with conventional extraction methods

The values ranged between 32 and 123 mg GA g−1 DW for total phenols, between

35 and 116 mg GA g−1 DW for *ortho*-diphenols, and from 34 to 106 mg CAT g−1 DW for flavonoids, depending on cultivar, geographical localization, crop season, among other factors [4, 18, 19]. The values of the present work were slightly higher than the values obtained in those studies, maybe due to the new efficient extraction method performed in this work (UAE). Grape pomace has been also analyzed by other authors concerning its phenolic content, obtaining values around 40 mg GA g−1 DW for total phenols and around 14 mg CAT g−1 DW for flavonoids which are

**3.1 Phenolic content of wine lees, grape pomace and grape stems**

by the different phenolic compounds present in these WBPs.

(hydro-methanolic solvents) [3, 4, 11, 18].

significantly lower than those obtained in this work.

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

**2.7 Statistical analysis**

Inc., Chicago, IL, USA).

**3. Results and discussion**

were expressed in mg g−1 of dry weight (mg g−1 DW).

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

method based on a polar mobile phase with the mixture of solvent A: H2O/HCOOH (99.9:0.1, v/v), and solvent B: CH3CN/HCOOH (99.9:0.1, v/v). The following linear gradient scheme was used (t in min; %B): (0; 5%), (15; 15%), (30; 30%), (40; 50%), (45; 95%), (50; 95%) and (55; 5%). At this last time (55 min), return to 5% of B to stabilize and prepare the column for the next sample. The analysis was performed at 25°C, with a flow rate of 1.0 mL/min and a sample injection volume of 20 μL. All samples were injected in triplicate. For the quantification of the identified compounds, the respective standards were used at 280 nm. Concentrations were expressed in mg g−1 of dry weight (mg g−1 DW).

### **2.7 Statistical analysis**

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

**seeds**

**2.4 Phenolic content**

expressed as mg GA g−1 DW.

**2.5 Antioxidant capacity assays**

**RP–HPLC–DAD**

per gram of dry weight (mg CAT g−1 DW).

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

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)

The content in total phenols, flavonoids, and *ortho*-diphenols was determined

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

For the assessment of flavonoid content, the aluminum complex method was performed, using catechin as standard. Results were expressed as mg of catechin

All the assays were performed using 96-well micro plates (Nunc, Roskilde, Denmark) and an Infinite M200 microplate reader (Tecan, Grödig, Austria). For all

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].

The polyphenolic profile of WBPs extracts was assessed by Reverse Phase - High Performance Liquid Chromatography - Diode Array Detector (RP-HPLC-DAD), in an Agilent HPLC 1100 series equipped with a photodiode array detector and a mass detector in series (Agilent Technologies, Waldbronn, Germany), in accordance with the method previously described [13]. The equipment consisted of a photodiode array detector (model G1315B), an autosampler (model G1313A), a binary pump (model G1312A), and a degasser (model G1322A). The HPLC system was controlled by Xcalibur software (Agilent, version 08.03). A C18 column (250 x 4.6 mm, 5 μm particle size; ACE, Aberdeen, Scotland) was used, being the reverse phase HPLC

analyses, three replicates (n = 3) of each sample were assessed.

**2.6 Identification and quantification of phenolic compounds by** 

according to spectrophotometric methodologies previously reported [13].

and filtered. Samples were stored at 4°C until analysis.

**6**

The results are presented as mean (n = 3) ± standard deviation (SD). The data obtained were subjected to variance analysis (ANOVA) and a multiple range test (Tukey's test) for a *p* value <0.05, using IBM SPSS statistics 21.0 software (SPSS Inc., Chicago, IL, USA).
