**1. Introduction**

FINNOVER "Innovative strategies for the development of crossborder green supply chains" is an Interreg ALCOTRA Italy/France transfrontier cooperation project (2017–2020) whose main target is the "green" implementation of some agro-industrial chains [1]. Particularly, the project supports the creation of both innovative and eco-sustainable production chains of botanicals in order to valorize the biodiversity of the ALCOTRA territory. One of the main natural products studied in FINNOVER are bud derivatives, which represent a relatively new category of botanicals marketed, in the European Community, as plant food supplements according to the Directive 2002/46/EC of the European

Parliament [2,3]. Bud derivatives are obtained by cold maceration in solvents (i.e., ethanol and glycerol) of fresh meristematic tissues of trees and plants (i.e., buds and young sprouts) as reported in the European Pharmacopoeia VIII edition (2014) [2,4,5]. These botanicals are still poorly studied, although they are widely used in gemmotherapy, a branch of phytotherapy that exploits the properties of these plant extracts for medicinal purposes [6]. The peculiarity of meristematic tissues in this particular phenological stage concerns their fragile texture and the high content of compounds which constitute the bud phytocomplex. In fact, these substances, including mainly flavonoids, enzymes, vitamins, aminoacids, nucleic acids, and plant hormones, are often present only in trace in the corresponding adult tissues [5].

In this research, a sonochemical application with a green chemistry approach was presented. Particularly, pulsed ultrasound-assisted extraction (PUAE) was employed as alternative method to quickly produce new bud derivatives in comparison to the long traditional maceration in solvent (21 days) taking under control their total phenolic fraction and antiradical activity to monitor possible PUAE-induced degradations. *Ribes nigrum* buds (RNB) were used as case study due to their common use in herbal medicine for their potential health properties. The most important industrial products of *R. nigrum* are its berries, which contain very high amounts of bioactive compounds, particularly flavonoids, phenolic compounds, and anthocyanins [7–9]. However, *R. nigrum* bud derivatives also contain high amounts of polyphenols, representing more than 60% of the bud phytocomplex [7], and they are widely used for inflammatory, circulatory, respiratory, and cutaneous disorders [10].

Ultrasounds (UAE—ultrasound-assisted extraction) together with microwaves (MAE—microwaveassisted extraction), supercritical fluids (SCF), and pulsed electric fields (PEF), are emerging "green" extraction technologies [11]. According to the six principles of the green extraction introduced by Chemat and colleagues [12] and to the twelve principles of green chemistry set by the Environmental Protection Agency of USA [13], these eco-compatible extraction techniques, with respect to conventional methods, aim to reduce the environmental impact in terms of time and energy. Moreover, they reduce both the quantities of solvents employed, preferring alternative solvents (water or food-grade solvents), and the generation of waste, hazardous substances, and consequently pollution [11,14]. In particular, UAE is a relatively simple, cheap, and efficient alternative to conventional extraction techniques whose main benefits are faster kinetics and increased extraction efficiency [15,16]. In fact, UAE allows one to quickly extract, with high reproducibility both on small and large scale, a wide variety of bioactive compounds (i.e., aromas, pigments, antioxidants, and organic and mineral compounds) from several animal tissues, plants, or food matrices [15,17]. However, the effects of ultrasound on the extraction yield may be linked to the nature of the matrix. Therefore, the experimental conditions of UAE must be optimized for each matrix [18]. In a solid/liquid media, the ultrasound waves originate the cavitation phenomena, a succession of different phases of compression and rarefaction which generates cavitation bubbles in the liquid. The implosion of the cavitation bubbles on the surface of the solid material generate microjets, at very high temperature and pressure, which destroy the wall cells of the matrix, with the consequent recovery of the intracellular content in the extraction solvent. There are several mechanisms involved (i.e., fragmentation, erosion, sonoporation, detexturation, capillarity) which independently or in combination influence the final ultrasound extraction yield [17]. When UAE is used in a pulsed mode (PUAE), the ultrasound processor works intermittently during the entire extraction process (active time vs. inactive time). This extraction mode reduces the operating temperature, allowing the extraction of thermolabile compounds and decreasing the possibility to produce alterations (i.e., oxidation products) in the final extract [19].

Untargeted phytochemical fingerprints coupled with chemometrics [2,20], particularly the UV-Visible and fluorescence spectra of each extract as multivariate response variables, have been employed to quickly screen the best experimental conditions of PUAE investigated by the design of experiment (DoE) method [21]. Finally, the extract obtained by PUAE in the optimized conditions has been characterized using targeted phytochemical fingerprinting by HPLC [2,22,23] in order to identify and quantify the main polyphenolic compounds. The polyphenolic fraction has been selected as the marker of activity and degradation susceptibility in order to make a comparison with the corresponding *R. nigrum* glyceric macerate (RNGM), representing the commercial product.
