3.2.2. Targeted Phytochemical Fingerprint

Antioxidant compounds (in particular, polyphenols) may play a critical health-promoting role in humans for disease prevention due to their synergistic or additive biological effects (phytocomplex) that influence human health better than a single molecule or a group of few compounds [37]. In this study, 13 biologically active compounds (grouped into four polyphenolic classes) were selected as markers for fingerprint analysis because they have been described as important health-effective substances in humans [38]. The phytochemical fingerprints of RNGM and RN8 are reported in Table 3.

**Table 3.** Targeted phytochemical fingerprint by HPLC-DAD of the polyphenolic compounds in the most promising *R. nigrum* extract (RN8) obtained by PUAE compared to the corresponding glyceric macerate (RNGM).


Results are reported as mg/100 g of bud fresh weight (FW) and expressed as mean value ± standard deviation (SD) (*n* = 3). \* n.d. = not detectable.

Among the analyzed compounds, chlorogenic acid, ferulic acid, hyperoside, and isoquercitrin were not detected. Bioactive compounds were separated and identified via HPLC-DAD. Adding other markers with demonstrated biological activity may be a necessary step for a better identification of the chromatographic pattern in further fingerprint studies together with a mass spectrometry detection of unknown peaks.

In RNGM samples, catechins were the most important bioactive class (44.36%), with flavonols as the second most abundant (27.82%), followed by benzoic acids and cinnamic acids (19.93% and 7.89%, respectively), while in PUAE extracts RN8, the quantitative relationships between catechins and flavonols were reversed: 29.81% for catechins and 42.39% for flavonols, while cinnamic acids (6.20%) and benzoic acids (21.60%) showed percentages similar to the bud macerates (Figure 4).

**Figure 4.** Contribution of each polyphenolic class to the total phytocomplex in RN8 and RNGM analyzed samples.

Figure 5 reports the polyphenolic chromatographic profile of analyzed samples: RNGM and RN8 presented qualitatively and quantitatively similar phenolic patterns. The most important differences were only detected in four compounds: (i) quercetin (48.53 ± 0.49 mg/100 g of bud fresh weight, FW, for RNGM and 80.14 ± 1.08 mg/100 g FW for RN8); (ii) quercitrin (30.86 ± 0.85 mg/100 g FW for RNGM and 48.18 ± 0.94 mg/100 g FW for RN8); (iii) catechin (95.88 ± 0.26 mg/100 g FW for RNGM and 55.85 ± 2.78 mg/100 g FW for RN8); (iv) epicatechin (59.83 ± 0.37 mg/100 g FW for RNGM and 49.08 ± 0.48 mg/100 g FW for RN8). Caffeic acid showed levels slightly higher in RNGM, while ellagic acid presented levels slightly higher in RN8.

Our results highlight that the traditional glyceric macerate and the alternative PUAE extract show similar total polyphenolic levels (and a qualitatively similar chromatographic pattern), but some differences in specific bioactive compounds (in particular, flavonols and catechins) were also detected, due to the different extraction method [39]. For this reason, PUAE yielded an extract rich in biological active molecules with potentially high health-promoting activity, but maybe with a practical use which could be different from traditional bud preparations. In any case, this research is only a preliminary study and further phytochemical, clinical, toxicological, and pharmaceutical in vitro and in vivo tests should be carried out to confirm this preliminary hypothesis.

Cinnamic acids and flavonols (**A**).

Benzoic acids and catechins (**B**).

#### **4. Conclusions**

In this work, PUAE, as an alternative time-saving method to produce *R. nigrum* bud derivatives, was presented according to the green chemistry principles. The unconventional extraction conditions were optimized by DoE at the lab scale using untargeted fingerprints coupled to chemometrics, but this same quick strategy could be analogously applied to transfer this method to an industrial scale. The impact of the UAE with respect to traditional maceration was evaluated in terms of recovering of the total phenolic content, the antiradical scavenging activity and the profiles of the most important bioactive compounds. In particular, PUAE provided the extract named RN8 in a few minutes, compared to the 21 day-long maceration, whose total polyphenolic levels and antiradical scavenging are similar or even slightly increased with respect to RNGM. Furthermore, RN8 presents a qualitatively similar chromatographic pattern, even if some differences in flavonols and catechins were detected. Due to

these differences, an LC-MS study of RN8 is mandatory in the near future. Nevertheless, this is a promising preliminary result to provide alternative uses of *Ribes nigrum* bud derivatives using this unconventional time-saving extraction method.

**Supplementary Materials:** Supplementary materials are available online at http://www.mdpi.com/2304-8158/8/ 10/466/s1. Table S1: score matrix; Table S2: loading matrix; Table S3: eigenvalue matrix; Table S4: chromatographic conditions. Figure S1: percentage of explained variance plot.

**Author Contributions:** Conceived the experiments, R.B., P.Z., A.P.; Designed the experiments, F.T. and D.D.; Followed the experiments, R.B. and G.L.B.; Performed the experiments and analyzed the data, P.Z., F.T., D.D. and R.B.; Wrote the paper, F.T.

**Funding:** This research was funded by an European Union project called FINNOVER (n◦ 1198), http://www. interreg-finnover.com/.

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