Phytochemical Profile and Antioxidant, Antiproliferative, and Antimicrobial Properties of Rubus idaeus Seed Powder

In the context of the contemporary research on sustainable development and circular economy, the quest for effective strategies aimed at revaluation of waste and by-products generated in industrial and agricultural production becomes important. In this work, an ethanolic extract from red raspberry (Rubus idaeus) seed waste (WRSP) was evaluated for its phytochemical composition and functional properties in term of antioxidative, antiproliferative, and antimicrobial activities. Chemical composition of the extract was determined by both HPLC-ESI-MS/MS and spectrophotometric methods. Phytochemical analysis revealed that flavan-3-ols and flavonols were the major phenolic compounds contained in WRSP. The extract demonstrated very high radical-scavenging (4.86 ± 0.06 µmol TE/DW) and antioxidant activity in a cell-based model (0.178 ± 0.03 mg DW/mL cell medium). The WRSP extract also exhibited antiproliferative activity against three different epithelial cancer cell lines (MCF-7, HepG2, and HeLa cells) in a dose-dependent manner. Finally, microbiological assays showed the absence of colonies of bacteria and microscopic fungi (yeasts and molds) and revealed that the WRSP extract has a large inhibition spectrum against spoilage and pathogenic bacteria, without inhibitory activity against pro-technological bacteria. In conclusion, the obtained results show that WRSP is a rich source of phytochemical compounds exerting interesting biological activities. For these reasons WRSP could find applications in the nutritional, nutraceutical, and pharmacological fields.


Introduction
Plant foods are major sources of bioactive compounds, and their consumption is linked to potential human health benefits. This awareness has led to a significant increase in global demand for fruits and vegetables. On the other hand, large amounts of waste are inexorably generated not only by plant food consumption but also by overproduction and improper storage practices [1]. For many years, this waste was used as animal feed or disposed of in landfills. Currently, due to an increased perception of the benefits of the circular economy, plant food waste and by-products are now being investigated for recycling in food and non-food applications [2,3]. In particular, agri-food waste and by-products are still rich in bioactive compounds [4][5][6] and can therefore be used as raw materials for cosmetic, nutraceutical, and pharmaceutical formulations.
The red raspberry, or European raspberry, is the fruit of Rubus idaeus, a member of the Rosaceae family. Although it is a species native to western Asia, it is widespread in

Plant Materials and Preparation of Extracts
The raw material used for this work was obtained from a local company involved in the extraction of vegetable oils from seeds. The waste of the seed extraction process consists of the seed powder deprived of the oil fraction after a cold-pressing process. After oil extraction, the R. idaeus exhausted seeds were grinded in order to obtain a fine powder. The processed plant material was then stored at room temperature (RT) until extraction process. For the preparation of the extract, three different aliquots of WRSP were extracted using 70:30 (v/v) ethanol:water in 1:20 (w/v) ratio [21]. The samples were vortexed for 5 min, sonicated at RT for 20 min, and shaken for 24 h at 4 • C in the dark. Subsequently, samples were centrifuged at 2000 rpm for 10 min at 4 • C. The extraction procedure was repeated twice, and the supernatants were filtered (Millex HV 0.45 µm, Millipore, Billerica, MA, USA) and collected together. Meanwhile, the residue was extracted again with 10 mL of the same extraction solvent and the supernatant was separately stowed (exhausted). The obtained WRSP ethanolic extract was stored at −20 • C until further chemical and biological analyses.

Phytochemical Characterization
An Agilent Technologies 1200 liquid chromatography (LC) coupled to a Diode Array Detector (DAD) and a 6330 Series Ion Trap Mass Spectrometer (MS) System (Agilent Technologies, Santa Clara, USA) was employed for the phytochemical profile of WRPS. The separation was carried out using the chromatographic gradient and conditions previously reported [22]. Analyses were performed in triplicate.

Redox Active Proprieties
Radical scavenging activity of the WRSP extract was assessed by ABTS assay, as previously reported [23]. Briefly, the green-stable cationic radical ABTS •+ was freshly prepared incubating ABTS salt (Sigma Aldrich, St. Louis, MO, USA) with K 2 S 2 O 8 (VWR International, Radnor, PA, USA) at room temperature overnight. WRSP extract at different concentrations has been added with the radical previously diluted in ethanol. The reduction of the radical ABTS •+ was monitored spectrophotometrically at 734 nm, and the decolouration percentage (D) of the radical solution was evaluated using Equation (1): where A to is the absorbance of the radical solution before the addition of the WRSP extract and A t180 is the absorbance after 180" of the WRSP extract addition. The result was the average of three separate experiments. Trolox was used as standard, and the antioxidant activity of each assay was expressed as mmol of Trolox Equivalent (TE) per 100 g of DW.

Cellular Antioxidant Activity
The cellular antioxidant activity (CAA) assay was accomplished as previously described by Wolfe at al. [25] with some minor changes [26]. Briefly, 96-well plates were seeded with HepG2 cells in complete culture medium at the density of 6.0 × 10 4 where SA is the integrated area of the sample wells and CA is the integrated area of the control wells. The antioxidant activity was expressed as CAA 50 , that is the WRSP extract concentration necessary for 50% of DCF formation inhibition and CAA 50 was calculated from a concentration-response (CAA) curve using linear regression analysis, and it was expressed as mg of DW per mL of cell medium. The result, expressed as mg/mL of cell medium, is the mean value of three separate experiments.

Antiproliferative Activity
Exponentially growing cells were used for the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The assay was executed as previously described [21]. Briefly, the cells were seeded into standard 96-well plates at a density depending on the doubling times of each cell line. After 24 h of incubation, the WRSP extract at appropriate concentrations (500-50 µg DW/mL cell culture medium) was added and cells were incubated for another 48 hrs. Ethanol concentration never exceeded 0.25% (v/v). Control cells were incubated with culture medium containing 0.25% ethanol (v/v). After the incubation time, 0.5 mg/mL MTT reagent (Sigma Aldrich, St. Louis, MO, USA) was added and discarded after a 3-h incubation at 37 • C. The blue formazan produced in living cells was dissolved by dimethyl sulfoxide (DMSO). The absorbance was measured in a microplate reader (UV-1900i, Shimadzu ® , Milan, Italy) at 570 nm, and the percentage of growth (PG) with respect to control cells (untreated cells) was calculated according to Equation (3): where OD test is the average of optical density after cell exposure to the extract for a chosen period of time; OD tzero is the average of optical density before the extract addition; and OD ctr is the average of optical density after the chosen period of time with no exposure of cells to treatment. The concentration needed to induce 50% growth inhibition (GI 50 ) for each cell line was determined from concentration-response (PG) curves using linear regression analysis, and it was expressed as mg of DW per mL of cell medium. The results, expressed as µg/mL of cell medium, is the mean value of three separate experiments.

Microbiological Characterization of WRSP
Ten grams of WRSP were transferred into a sterile bag (BagFilter ® 400, Interscience, Saint Nom, France) diluted with 90 mL of Ringer's solution (Sigma Aldrich, Milan, Italy), and homogenized by the stomacher apparatus BagMixer ® 400 (Interscience, Saint Nom, France) at the highest speed (blending power 4) for 2 min. Homogenized WRSP was serially diluted and then plated on agar media for the enumeration of the main microbial groups belonging to the pro-technological, spoilage, and pathogenic populations following the approach of Messina et al. (2019) [27]. Briefly, the different microorganisms were inoculated as follows: total mesophilic microorganisms (TMM) on plate count agar (PCA); mesophilic lactic acid bacteria (LAB) rods and cocci on de Man-Rogosa-Sharpe (MRS) and Medium 17 (M17) agar, respectively; enterococci on kanamycin aesculin azide (KAA) agar; Pseudomonads on Pseudomonas agar base (PAB); members of the Enterobacteriaceae family on violet red bile glucose agar (VRGBA); coagulase-positive staphylococci (CPS) on Baird Parker (BP) supplemented with rabbit plasma fibrinogen (RPF); Listeria monocytogenes on Listeria selective agar base with SR0140E supplement; Salmonella spp. and Escherichia coli on hektoen enteric agar (HEA); and yeasts and moulds on malt agar (MA) supplemented with chloramphenicol (0.1 g/L). All media and chemicals were purchased from Microbiol Diagnostici (Uta, Italy). Plate counts were performed in triplicate.

Determination of Antibacterial Activity of WRSP
The ethanolic extract of WRSP was tested against bacteria of food origin. In particular, Enterococcus mundtii, Fructilactobacillus sanfranciscensis, Latilactobacillus sakei, Levilactobacillus brevis, Lactococcus lactis, and Leuconostoc mesenteroides were chosen among pro-technological species, Brochothrix thermosphacta, Pseudomonas endophytica, Pseudomonas fluorescens, Pseudomonas lactis, and Pseudomonas poae among spoilage bacteria, while Acinetobacter guillouiae, Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus, and Staphylococcus epidermidis among bacteria responsible for human diseases. All spoilage and pathogenic bacteria were sub-cultured in Brain Heart Infusion (BHI) broth (Condalab, Madrid, Spain) incubated for 24 h at 37 • C, En. munditii, Lc. lactis and Ln. mesenteroides were reactivated in M17 (Microbiol Diagnostici) incubated for 24 h at 30 • C, Lt. sakei and Lv. brevis in MRS (Microbiol Diagnostici) incubated for 24 h at 30 • C, while F. sanfranciscensis in modified MRS [28]. The inhibitory activity of WRSP extract was tested by the well diffusion assay (WDA) [27,29] using sterile water as negative control, while streptomycin (10% w/v) was used as positive control. The inhibitory activity was evaluated after incubation at the optimal temperature for each strain and was considered positive only in case of a definite clear halo surrounding the wells. The strains sensitive to the extract were subjected to the minimum inhibitory concentration (MIC) determination by broth microdilution method in 96-well microplates [30]. Briefly, the extract was serially diluted in the optimal growth media for each strain (1:2) and their concentrations ranged between 100 and 3.125 mg/mL. After inoculation with approximately 10 6 CFU/mL of each sensitive strain, the bacterial growth was measured using a ScanReady Microplate photometer P-800 (Life Real Biotechnology Co., Ltd., Hangzhou, China). WDA and MIC tests were carried out in triplicate.

Statistical Analysis
All the statistical analyses were carried out using SPSS ver. 24 software (IBM, Armonk, New York, USA). The results were expressed as mean ± standard deviation (SD).

Antioxidant Proprieties
Oxidative species physiologically generated by aerobic metabolism are involved in different processes, such as proliferation, apoptosis, and gene expression. However, an unbalance among endogenous antioxidant defenses and reactive species production causes cellular oxidative damage associated with etiology and progression of several chronic diseases [38][39][40]. Dietary antioxidants intake contributes to preventing this unbalance. In particular, from the literature it is known that phytochemicals, such as polyphenols, are able to increase the endogenous antioxidant defenses thanks to both their redox-active properties and modulating effects on antioxidant gene expression [21]. On the other hand, the bioactivity of phytochemicals has frequently been referred to by their antioxidant properties. Indeed, by influencing cellular redox homeostasis, dietary phytochemicals can induce structural changes in redox-sensitive targets and, consequently, modulate their function [41].
Different in solution methods have been used to estimate redox active proprieties of pure compounds, plant extracts, or biological fluids. These assays generally use radical generating systems and evaluate the ability of antioxidant compounds to scavenge synthetic free radicals. One of the most popular is the ABTS assay. Radical scavenging activity of the WRSP extract was equal to 4.86 ± 0.06 mmol TE/100 g DW (Figure 1, Panel C). With respect to the redox scavenging activity of WRSP of other berries seeds powder, the ABTS value measured for WRSP is comparable to that estimated for black currant and cranberry, meanwhile is it 2.5 and 50 folds higher than that of elder and rose hip, respectively [16,17]. In order to study whether the redox scavenging ability of WRSP extract prevent oxidative damage in a biological system, the antioxidant activity of the extract was also evaluated in a lipid peroxidation cell-based model. CAA assay not only allowed the evaluation of the antioxidant potential against peroxyl radicals but also the ability of antioxidant substances to interact with or cross membranes and their stability to cellular metabolism [42][43][44]. The estimated CAA 50 was 0.178 ± 0.03 mg DW/mL cell medium (Figure 1, Panel A and B). This value is indicative of a relevant antioxidant activity. Moreover, this data turns out to be two orders of magnitude lower, thus indicating a greater activity, compared to that determined by Wolfe 2008 for raspberry extracts [25,42] (togliere 43).
in a lipid peroxidation cell-based model. CAA assay not only allowed the evaluation of the antioxidant potential against peroxyl radicals but also the ability of antioxidant substances to interact with or cross membranes and their stability to cellular metabolism [42][43][44]. The estimated CAA50 was 0.178  0.03 mg DW/mL cell medium (Figure 1, Panel A and  B). This value is indicative of a relevant antioxidant activity. Moreover, this data turns out to be two orders of magnitude lower, thus indicating a greater activity, compared to that determined by Wolfe 2008 for raspberry extracts [25,42] (togliere 43).

Antiproliferative Activity
The antiproliferative potential of the WRSP extract was assessed via MTT assay, using three epithelial human tumor cell lines: cervical cancer cells (HeLa), hepatocarcinoma cells (HepG2), and breast cancer cells (MCF-7). The concentration required for 50% inhibition of cell growth (GI 50 , Growth Inhibitory 50%) was calculated by linear regression, using a concentration/percentage growth (PG) curve ( Figure 2). Cells were exposed to concentra-tions of WRSP extract ranging from 10 to 1000 µg DW/mL of cell culture medium. The results displayed an antiproliferative activity concentration-dependent, with variability of effects in function of the cell line considered. In particular, WRSP extract showed a stronger effect on HepG2 cells (299.866 ± 11.617 µg/mL) (Figure 2, Panel A), while a progressively smaller effects was observed on MCF-7 (469.739 ± 35.646 µg/mL) (Figure 2, Panel B) and HeLa (702.760 ± 55.986) cells (Figure 2, Panel C). This difference could be a consequence of peculiar protein expression patterns in the three cell lines. On the other hand, peculiar phytochemicals in WRSP, acting on differently expressed targets in the cells, could contribute, presumably in synergistic actions, to the observed activity.

Antiproliferative Activity
The antiproliferative potential of the WRSP extract was assessed via MTT assay, using three epithelial human tumor cell lines: cervical cancer cells (HeLa), hepatocarcinoma cells (HepG2), and breast cancer cells (MCF-7). The concentration required for 50% inhibition of cell growth (GI50, Growth Inhibitory 50%) was calculated by linear regression, using a concentration/percentage growth (PG) curve ( Figure 2). Cells were exposed to concentrations of WRSP extract ranging from 10 to 1000 µ g DW/mL of cell culture medium. The results displayed an antiproliferative activity concentration-dependent, with variability of effects in function of the cell line considered. In particular, WRSP extract showed a stronger effect on HepG2 cells (299.866 ± 11.617 µ g/mL) (Figure 2, Panel A), while a progressively smaller effects was observed on MCF-7 (469.739 ± 35.646 µ g/mL) (Figure 2, Panel B) and HeLa (702.760 ± 55.986) cells (Figure 2, Panel C). This difference could be a consequence of peculiar protein expression patterns in the three cell lines. On the other hand, peculiar phytochemicals in WRSP, acting on differently expressed targets in the cells, could contribute, presumably in synergistic actions, to the observed activity. Quercetin and catechin are the main dietary polyphenols and, with dimer B-type PAC, represent the most abundant phytochemicals in the WRSP extract. In particular, the quantity of quercetin is equal to over 40% of the total weight of the identified polyphenols. Bioavailability of quercetin and its glycosylated forms has been previously demonstrated [45] and several studies attest the antiproliferative properties of these polyphenols. Moreover, induction of cell cycle arrest and apoptosis by quercetin in cancer cells has been documented [46]. On the other hand, epidemiological studies have reported that intake of quercetin-rich food reduces the risk of different types of cancer [47].
HPLC-DAD-MS/MS analysis revealed that WRSP is also a very rich source of PACs. Along with other health protective effects [33], anti-carcinogenic activity of proanthocyanidins from different sources have been extensively documented and the involved molecular targets identified. Although the very low bioavailability of high-molecular-weight PACs, the literature data showed that smaller oligomeric procyanidins, in particular dimers and trimers, are both stable in digestive conditions and absorbed in the gut. WRSP Quercetin and catechin are the main dietary polyphenols and, with dimer B-type PAC, represent the most abundant phytochemicals in the WRSP extract. In particular, the quantity of quercetin is equal to over 40% of the total weight of the identified polyphenols. Bioavailability of quercetin and its glycosylated forms has been previously demonstrated [45] and several studies attest the antiproliferative properties of these polyphenols. Moreover, induction of cell cycle arrest and apoptosis by quercetin in cancer cells has been documented [46]. On the other hand, epidemiological studies have reported that intake of quercetin-rich food reduces the risk of different types of cancer [47].
HPLC-DAD-MS/MS analysis revealed that WRSP is also a very rich source of PACs. Along with other health protective effects [33], anti-carcinogenic activity of proanthocyanidins from different sources have been extensively documented and the involved molecular targets identified. Although the very low bioavailability of high-molecular-weight PACs, the literature data showed that smaller oligomeric procyanidins, in particular dimers and trimers, are both stable in digestive conditions and absorbed in the gut. WRSP extract contains mainly low-molecular-weight PACs. In particular, the dimer B-Type represents over 60% of the total identified PACs in the extract [31,45,48].
It must be emphasized that the combined action of multiple polyphenols, as occurs in a complex matrix, may be higher than that of a single polyphenol. A greater cell growth inhibition activity has been demonstrated for various combinations of polyphenols when compared to the activity of the single polyphenol. This evidence may not only be the result of the ability of phytochemical mixtures to affect multiple pathways involved in cancer simultaneously but also of the possible ability of different phytochemicals to reciprocally increase their bioavailability [49]. Anticancer effects of combinations of epigallocatechin gallate (ECGC) and quercetin have been demonstrated in ovarian and prostate cancer [50] and co-adjuvant therapy efficacy of catechin and procyanidin B2 with docetaxel was demonstrated in MCF-7 cells [51].

Microbiological Characterization and Inhibitory Properties of WRSP
In order to investigate the microbiological safety of WRSP and its potential application as a functional ingredient or natural antimicrobial food additive, microbiological characterization of WRSP was performed along with the evaluation of the inhibitory properties of the WRSP extract against bacteria growth.
The microbiological counts of WRPS did not reveal the presence of bacteria or microscopic fungi (yeasts and molds) (data not shown), highlighting the hygienic suitability of this by-product for food applications. In fact, it is well known that the presence of microorganisms in a by-product besides impairing its stability, when the by-product is used as a food additive, can endanger both consumer safety and stability of the food to which it is added [52].
The search for new antimicrobial food additives is a topic that has been attracting increasing attention over the past few years, since foodborne resistant diseases are one of the most important public health problems associated with the risk of emergence of multidrug-resistant bacterial strains in the food chain [53]. As a result, there is a raising demand for natural plant-derived preservatives because they are perceived as more natural and safe [54][55][56]. To evaluate a potential use of WRSP as food preservative, we evaluated its ability to inhibit the growth of undesirable microorganisms generally associated with foods [57].
The antibacterial activity of WRSP extract against spoilage and pathogenic bacteria is shown in Table 3. A considerable inhibition activity has been observed against both Br. thermosphacta, which is commonly associated with meat spoilage [58], and all Pseudomonas strains, commonly involved in the physicochemical change and generation of off-flavours in a wide range of foods of both plant and animal origin [59,60]. Regarding pathogenic bacteria, the WRSP extract was highly effective against four out of the nine tested strains, recording a diameter of the inhibition area around the wells of 13.5 mm for the B. cereus and of about 18 mm for A. guillouiae, St. aureus, and St. epidermidis. These microorganisms are relevant pathogens commonly associated with food consumption [61,62]. The antimicrobial activity of WRSP was also characterized in terms of MIC (Table 3). Only a MIC of 100 mg/mL was reached for the strain B. cereus ICE70, while a MIC of 25 mg/mL was observed for all the other strains. These results suggest that this extract has great potential applications as natural food preservative.
Interestingly, the development of pro-technological bacteria was not inhibited by WRSP (Table 3), indicating it is harmless against the lactic acid bacteria (LAB) used in food fermentation processes [63]. These results are very relevant because, in consideration of the previously demonstrated antioxidant and antimicrobial properties of WRSP, they indicate the suitability of WRSP as a functional or natural antimicrobial ingredient also in fermented foods.
The obtained inhibition profiles, resulting in no effects on LAB growth, are rather unexpected considering that usually antibacterial agents show greater activity against Grampositive rather than Gram-negative bacteria. The mechanism underlying the observed activity needs further investigation. Here we can just assume that the strain-dependent resistance to WRSP components can justify the observed inhibition profiles. Results indicate the mean value of three independent assays. Abbreviations: MIC, minimum inhibitory concentration; En., Enterococcus; F., Fructilactobacillus Lt., Latilactobacillus; Lv., Levilactobacillus; Lc., Lactococcus; Ln., Leuconostoc; Br., Brochothrix; P., Pseudomonas; A., Acinetobacter; B., Bacillus; E., Escherichia; L., Listeria; S., Salmonella; St., Staphylococcus; n.d., not determinated. Symbols:-no inhibition found.

Conclusions
This work demonstrates that the waste generated by the cold pressing of red raspberry seeds can be an interesting plant raw material for the extraction of bioactive compounds with different biological properties.
In particular, WRSP is shown to be really rich in several phytochemicals, including quercetin, catechin, and low weight proanthocyanidins. Further studies to elucidate the bioavailability of phytochemicals in WRSP are needed. However, considering the concentration of polyphenols in WRSP, the intake of even small quantities (5-10 g) of this plant matrix in a nutraceutical formulation or as a functionalizing ingredient can provide significant quantities of bioactive molecules with potential protective effects on human health. Therefore, antioxidant, anticancer, and antimicrobial properties evaluated and discussed in this article suggest a potential use of this agro-food waste in both food and non-food applications.