*2.4. Alternative Method to Produce R. nigrum Bud Derivatives: Pulsed Ultrasound-Assisted Extraction (PUAE)*

PUAE was carried out directly by an Hielscher UP200St (Teltow, Germany) in pulsed mode, with an ultrasonic titanium probe (7 mm diameter) able to transfer, with high efficiency, the acoustic energy into the treated media [2,24,25]. Fresh RNB were finely ground by a Grindomix 200 M (Retsch, Haan, Germany), for 20 s at 5000 rpm, and then sieved by a 150 µm sieve. Twenty grams of a glycerol/ethanol 96% mixture 1:1 *w*/*w* were added to 1 g (dry weight) of ground RNB in a polypropylene 50 mL centrifuge tube. The samples were processed in a 200 W ultrasonic processor at a constant frequency of 26 kHz, with an amplitude level of 30%, optimized in a previous paper from the authors [26], keeping temperature under control always below 70 ◦C. The pulse duration and pulse interval refer to "on" and "off" times of the sonicator. The same mixture of glycerol/ethanol 96% (1:1 *w*/*w*) and the same solid–solvent ratio 1:20 between buds and solvent (considering the dry weight), as described in the Section 2.3, were employed. The duty cycle (pulse) and the extraction time (65% and 20 min, respectively) were optimized by applying DoE (Table 1). The obtained suspension was then filtered by Buchner (Whatman n. 44 paper) and the filtrate was centrifuged at 3000 rpm for 10 min. The obtained solutions were stored at 4 ◦C until the analysis time.


**Table 1.** Experimental matrix of the Faced Central Composite Design, the experimental plan (in brackets), and the obtained response variable (Y).

#### *2.5. Untargeted Fingerprints of The R. nigrum Phytocomplex*

#### 2.5.1. UV-Visible Spectroscopy

An Agilent UV-Vis spectrophotometer Cary 100 (Varian Co., Santa Clara, CA, USA) with 0.5 nm resolution, was employed to record all the UV-Vis spectra of the extracts and the corresponding glyceric macerates of *R. nigrum*. Before being analyzed, all the samples were properly diluted 1:20 in the same extraction mixture (glycerol/ethanol 96% 1:1 *w*/*w*). The total spectrum of each analyzed sample was collected in duplicate at room temperature (25 ± 1 ◦C), against a blank solution (i.e., the extraction mixture), using rectangular quartz cuvettes with 1 cm path length. For each sample, the resulting spectra were averaged and used as vector of variables to build the data matrix.

#### 2.5.2. Fluorescence Spectroscopy

The excitation–emission fluorescence spectra were recorded in duplicate at room temperature (25 ± 1 ◦C) by a Perkin-Elmer LS55B luminescence spectrometer (Waltham, MA, USA) using the traditional right angle fluorescence spectroscopic technique [27]. A standard cell holder and a 10 mm quartz SUPRASIL® cell with volume of 3.5 mL by PerkinElmer were used. The emission spectra were recorded in the range of 450–800 nm, exciting samples at a fixed wavelength (λ ex = 430 nm) [20]. Both the excitation and the emission monochromator slits were set to 10 nm, with high gain and 600 nm/min of speed. The same dilution of all the samples, at the ratio of 1:20 with the solvent, was evaluated. For each sample, the resulting emission spectra were averaged and used as vector of variables to build the data matrix combing them together with the previous described UV-Vis spectra.

#### *2.6. Experimental Design and Multivariate Data Analysis*

DoE was used to optimize the experimental conditions of PUAE from RNB. A Faced Central Composite Design (2k + 2 <sup>k</sup> + 1) was applied with the aim to estimate the constant, the linear terms, the interactions between variables, and the quadratic terms, according to the following model [21]:

$$\mathbf{Y} = \mathbf{b}\_0 + \mathbf{b}\_1 \mathbf{X}\_1 + \mathbf{b}\_2 \mathbf{X}\_2 + \mathbf{b}\_{12} \mathbf{X}\_1 \mathbf{X}\_2 + \mathbf{b}\_{11} \mathbf{X}\_1^2 + \mathbf{b}\_{22} \mathbf{X}\_2^2$$

The experimental plan, illustrated in Table 1, summarizes the conditions of the nine experiments performed (namely from RN1 to RN9). The minimum, intermediate, and maximum value of each variables are labeled as −1, 0, and +1, respectively. A data matrix M9,1402 consisting of nine rows (the nine samples/extracts obtained by DoE) and 1402 columns (the vector of 701 absorbance values at different wavelengths in the range of 230–500 nm of the UV-Vis spectra plus 701 fluorescence emissions in the range of 450–800 nm of the fluorescence spectra), was prepared and further analyzed by the PCA, a multivariate statistical technique of unsupervised pattern recognition. The scores on PC1 have been

used as a response variable of the experimental design (Y). In detail, the standard normal variate (SNV) transform, or row autoscaling, was previously performed on the spectral data in order to revise both the baseline shifts and the global intensity variations [28]. Subsequently, PCA was performed using the nonlinear iterative partial least squares (NIPALS) algorithm on the column-centered data [29]. After the PCA, the scores on PC1, explaining the 90.3% of the total variance, were extracted and used as response to elaborate DoE. DoE and multivariate data analysis were performed by CAT (Chemometric Agile Tool) a chemometric software based on R, developed by the Chemistry Group of the Italian Chemical Society [30]. The data matrix and the detailed PCA analysis is available as Supplementary Materials.

#### *2.7. Analytical Determinations*

The most promising extract obtained by PUAE and the corresponding glyceric macerate (RNGM), in order to make a comparison, were characterized to evaluate their total phenolic contents (TPC) and their radical scavenging activity (RSA). All the measurements were performed in duplicate and the results are expressed as mean ± standard deviation (SD). Statistical analysis was performed by the Excel Data Analysis Tool (Microsoft Corporation, Seattle, WA, USA).

#### 2.7.1. Determination of The Total Phenolic Compounds (TPC)

The Folin-Ciocalteu spectrophotometric method was applied to estimate the TPC of the *R. nigrum* bud preparations [31]. 0.2 mL of sample appropriately diluted, 1 mL of Folin-Ciocalteu reagent (diluted 1:10 with deionized water), 0.8 mL of aqueous sodium carbonate 7.5% *w*/*v* solution were added in a test tube and vortexed. After an incubation period of 30 min at room temperature in the dark, the absorbance was recorded at 760 nm by an Agilent 8453 UV-Vis spectrophotometer with 1 nm resolution. A calibration curve, using gallic acid as a standard, has been used to evaluate the polyphenolic concentration. The TPC was expressed as milligrams of gallic acid equivalent (GAE) pulled-out from 100 mL of bud extract (mg GAE/100 mL).

#### 2.7.2. Determination of Radical Scavenging Activity (RSA)

The DPPH• assay was applied to evaluate the RSA of the *R. nigrum* bud preparations [32]. Determinations were performed as described in a previous paper [24]. The absorbance at 515 nm was recorded by an Agilent 8453 UV-Vis spectrophotometer with 1 nm resolution. A multilevel calibration with ascorbic acid as standard was used to evaluate the RSA and to express the results as milligrams of ascorbic acid equivalent (AAE) in 100 mL of bud extract (mg AAE/100 mL).

#### *2.8. HPLC Analysis*

HPLC methods were used for phytochemical analysis both on *R. nigrum* bud preparations and PUAE extracts. Analysis were focused on flavonols, phenolic acids (benzoic and cinnamic acids), and catechins, as polyphenolic markers with a demonstrated health-promoting activity [33]. Bioactive compounds were identified and quantified by comparison and combination of their retention times and UV spectra with those of authentic standards. The calibration parameters for all the employed analytical standards were previously reported by the authors [22,34]. The total bioactive compound content (TBCC) was determined as sum of the selected and identified markers with health-promoting activities and positive antioxidant effects on human health-status according to "multimarker approach" [35]: phytochemicals were grouped into different bioactive classes in order to evaluate each class contribution to phytocomplex composition. All analyses were triplicated and the results expressed as mg/100 g of fresh weight (FW).

Samples were filtered with circular pre-injection filters (0.45 µm, polytetrafluoroethylene membrane) prior to HPLC-DAD analysis. Chromatographic analysis was carried out using an Agilent 1200 High-Performance Liquid Chromatograph coupled to an Agilent UV-Vis diode array detector (Agilent Technologies, Santa Clara, CA, USA), based on HPLC methods previously validated for fresh fruits, herbal medicines, and other food products [2,22,23]. Chromatographic conditions

were set in order to obtain a phytochemical information with a good resolution and a reasonable analysis time.

Bioactive molecule separation was achieved on a Kinetex C18 column (4.6 mm × 150 mm, 5 µm, Phenomenex, Torrance, CA, USA). Different mobile phases were used for bioactive compound characterization and several linear gradients in different slopes were optimized because some compounds were similar in structure with each other in the same chemical class: (1) a solution of 10 mM KH2PO4/H3PO<sup>4</sup> and acetonitrile with a flow rate of 1.5 mL·min−<sup>1</sup> (method A—analysis of cinnamic acids and flavonols); (2) a solution of methanol/water/formic acid (5:95:0.1 *v*/*v*/*v*) and a mix of methanol/formic acid (100:0.1 *v*/*v*) with a flow rate of 0.6 mL·min−<sup>1</sup> (method B—analysis of benzoic acids and catechins). Selected wavelengths were suitable to achieve more specific peaks as well as a smooth baseline after a full scan on the chromatogram from 190 to 400 nm; in particular, UV spectra were recorded at 330 nm (A) and 280 nm (B). Information on used chromatographic methods and selected markers are reported in the Supplementary material.

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

### *3.1. Optimization of The PUAE Experimental Conditions by DoE Using Untargeted Phytochemical Fingerprint*

The PUAE conditions have been optimized by a Faced Central Composite Design (CCD), whose results are shown in Table 1. The DoE response variable to be optimized was obtained by an untargeted spectroscopic method combined with chemometrics previously described by the authors [2,20]. Briefly each of the nine extracts, obtained according the experimental plan and spectroscopic analyzed, was described by a vector of 701 UV-Vis absorbances plus 701 fluorescence emissions, as a holistic nontargeted fingerprint 1402-dimensional of the corresponding extract. Since these multivariate vectors of UV-Vis absorptions and fluorescence emissions (701 + 701 variables) of each extract have been proven to be strictly correlated to the whole polyphenolic fraction of the extracts they were combined in a multivariate data matrix: The DoE response matrix. This matrix, composed of nine rows and 1402 columns (M9,1402: nine objects corresponding to the nine experiments and 1402 variables which represent the spectral absorptions/emissions), has been elaborated by principal component analysis (PCA), an unsupervised patter recognition technique, in order to extract the useful analytical information and to reduce its dimensionality.

The untargeted polyphenolic phytochemical fingerprints (UV-Vis absorptions and Fluorescence emissions) of each extract obtained by DoE were reported in Figure 1 and compared with the corresponding commercial product RNGM.

Figure 2 shows the score-plot on the first two principal components (PCs), whose explained variance are 90.3% and 6.8%, respectively.

The first PC (PC1) retains all the useful information of the 1402 original variables and thus the corresponding scores were used as the response variable of each experiment in the experimental matrix. The other PCA details are reported in the Supplementary Materials (i.e., score matrix, loading matrix, eigenvalues, explained variance plot). The following model of the CCD has been obtained by applying multiple linear regression to the experimental matrix:

$$\text{Y} = -2.6708 - 0.6814 \text{X}\_1 \text{\*}-1.2487 \text{X}\_2 \text{\*\*}\*-0.1010 \text{X}\_1 \text{X}\_2 + 2.6000 \text{X}\_1 \text{\*}{"} \text{\*}{"} 1.4061 \text{X}\_2 \text{\*}"$$

\* indicates the significance of the coefficients: \* = *p* < 0.05, \*\* = *p* < 0.01.

**Figure 1.** Untargeted spectroscopic fingerprints of the *R. nigrum* bud (RNB) phytocomplex: (**a**) The UV-Vis averaged spectra (250–600 nm) of the nine experiments selected by the DoE and the corresponding *R. nigrum* glyceric macerate (RNGM) tested at the same dilution (1:20 in the extraction solvent); (**b**) The 2D Fluorescence averaged spectral emissions (450–800 nm) of the nine experiments selected by the DoE and the corresponding RNGM tested at the same dilution (1:20 in the extraction solvent).

**Figure 2.** The score plot on the first two principle components (PCs) selected by principle component analysis (PCA) using the vector of UV-Vis (250–600 nm) spectra coupled to fluorescence (450–800 nm) absorptions of each extract (RN1–RN9) as a multivariate untargeted signal. The red + represents the central point of the plot and the red circle highlights the extract obtained in the best experimental conditions.

All the linear and quadratic terms are significant as highlighted in the plot of the coefficients (Figure 3).

− − −

**Figure 3.** The coefficients of the model of Y (PC1 scores) obtained by the Faced Central Composite Design (X<sup>1</sup> : duty cycle; X<sup>2</sup> : extraction time). \* = *p* < 0.05, \*\* = *p* < 0.01.

Particularly, the linear term X<sup>2</sup> (\*\* = *p* < 0.01) corresponding to the extraction time, the quadratic term X<sup>1</sup> 2 (\*\* = *p* < 0.01), the linear term X<sup>1</sup> (\* = *p* < 0.05) corresponding to the duty cycle, and the quadratic term X<sup>2</sup> 2 (\* = *p* < 0.05) are the statistically significant coefficients. They should be increased or, on the contrary, decreased to improve or to minimize the Y response variable respectively. Experiments whose scores on PC1 are negative (Figure 2) correspond to the highest absorptions/emissions of the phytocomplex as shown in Figure 1, thus they must be decreased. RN8 represents the best experimental conditions and this extract has been analytically characterized and compared with RNGM.

#### *3.2. Analytical Characterization of The Most Promising PUAE Extract (RN8) and the Corresponding RNGM*

3.2.1. Determination of the Total Phenolic Compounds (TPC) and the Radical Scavenging Activity (RSA)

RN8, representing the most promising *R. nigrum* extract obtained by PUAE, and the corresponding RNGM, were analytically characterized to evaluate their total phenolic contents (TPC) and their radical scavenging activity (RSA). As reported in Table 2, both the bud preparations presented quantitatively similar RSA values: 1158.58 ± 73.24 mg/100 mL of bud extract for RN8 and 1137.04 ± 38.49 mg/100 mL of bud extract for RNGM, respectively. Regarding TPC, RN8 showed a higher value with respect to RNGM (415.56 ± 5.52 mg/100 mL vs. 276.44 ± 3.85 mg/100 mL). However, the Folin-Ciocalteu assay is a nonspecific method to quantify phenols and polyphenols. In fact, this reagent does not measure only phenols, but can react with some reducing substances (i.e., ascorbic acid) [36]. For this reason, the phenol content could be overestimated and further investigations on the phytocomplex composition should be carried out. Nevertheless, the higher value of RN8 is promising and indicative of almost no oxidative alterations potentially induced by ultrasounds with respect to maceration.


**Table 2.** Total phenolic compounds (TPC) and radical scavenging activity (RSA) of the most promising *R. nigrum* extract obtained by pulsed ultrasound-assisted extraction (PUAE) (RN8) compared to the corresponding commercial product (RNGM).

Results are reported as mg/100 mL of bud extract and expressed as mean value ± standard deviation (SD) (*n* = 2). GAE: gallic acid equivalent; AAE: ascorbic acid equivalent.
