Thinned Nectarines, an Agro-Food Waste with Antidiabetic Potential: HPLC-HESI-MS/MS Phenolic Characterization and In Vitro Evaluation of Their Beneficial Activities

Due to the side effects of synthetic drugs, the interest in the beneficial role of natural products in the management of diabetic conditions is growing over time. In the context of agro-food waste products, a screening of different fruit thinning by-products identified thinned nectarines (TN) as the richest matrices of abscisic acid (ABA), a phytohormone with well-documented hypoglycemic potential. These waste-food matrices may represent not only precious sources of ABA but also other bioactive molecules with potential health benefits, such as polyphenols. Therefore, we aimed to perform a qualitative and quantitative characterization of a polyphenolic profile of a TN-based nutraceutical formulation through HPLC-HESI-MS/MS and HPLC-DAD-FLD analyses. Additionally, the in vitro antioxidant and antidiabetic potential of TN was investigated. HPLC analyses allowed us to identify forty-eight polyphenolic compounds, nineteen of which were quantified. Moreover, the results obtained through different in vitro assays showed the antioxidant and antidiabetic potential exerted by the tested nutraceutical formulation. In conclusion, the concomitant presence of different bioactive compounds in TN-based nutraceutical formulation, such as ABA and polyphenols, would reasonably support TN as an innovative nutraceutical formulation useful for the management of glucose homeostasis. Further in-depth animal-based studies and clinical trials are needed to deepen these aspects.


Introduction
Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by decreased β-cells insulin secretion and/or insulin resistance, resulting in chronic hyperglycemia (elevated blood glucose levels) [1]. This metabolic disease has reached epidemic proportions: the latest edition of the IDF Diabetes Atlas shows that 10.5% of adults aged 20-79 years are currently affected by diabetes, and this percentage is set to rise even further [1]. Due to the significant individual, social and economic impact of this pathology, the correct management of diabetes represents a primary need, especially in order to avoid microvascular and macrovascular complications related to this condition [2]. The management of T2DM involves a wide range of approaches, such as changes in lifestyle, including diet and physical activity, and pharmacological treatment to achieve metabolic control [3]. Although these approaches can substantially reduce diabetes-related morbidity and mortality, the conventional medications in diabetes treatment can cause unwanted side effects to patients, leading to incompliance and treatment failure [4]. As a result, natural agents from plants and plant products have been the alternative target source for new antioxidant and antidiabetic agents based on their traditional use [5]. Among bioactive molecules of plant origin, a specific interest was recently placed on the investigation of abscisic acid (ABA). In this regard, the literature from the last decades reported the role of ABA as an endogenous hormone produced in humans by pancreatic β-cells, adipocytes and myoblasts in response to glucose [6]. Specifically, the evidence concerning the AMPK-mediated signaling pathways of ABA in glycemic control and the non-overlapping metabolic effects with insulin highlight a leading role of ABA as the first hormonal response in the physiological regulation of plasma glucose levels in humans [7].
Additionally, the role of ABA as a terpenoid phytohormone majorly responsible for the regulation of plant growth and differentiation is widely described by the scientific literature, mainly due to the ability to inhibit germination and promote plant dormancy [8]. A progressive accumulation of this phytohormone is reported during fruit ripening, reaching its maximum concentration at a specific stage after the full bloom and then decreasing to its minimum level at the fruit's fully ripe/harvest stage [9]. In this scenario, immature fruits derived from the thinning stage would represent rich sources of this phytohormone. Of note, the interest of the nutraceutical industry in agro-food waste products is increasingly growing since they represent still rich sources of bioactive compounds that can be conveniently recovered for the formulation of food supplements useful for the management of diabetic conditions. In the context of fruit by-products, thinned fruits come from the excessive thinning of immature fruits. This process is carried out to improve fruit size and quality in crop management but can lead to a large number of unripe fruits being discarded every year [10]. Regarding their possible industrial applications, unripe fruits are widely used as sources of bioactive compounds to be exploited in food preservation and as functional additives [11]. Nevertheless, thinned fruits have long been underestimated as potential high value-added plant resources, and their physiochemical profile and bioactive capacity remain poorly studied.
Interestingly, a screening of different fruit thinning waste products (i.e., peaches, nectarines, apples, pears and plums) conducted by our research group allowed us to identify nectarines as the richest matrices in terms of ABA content [10]. Thus, a novel nutraceutical formulation based on TN polyphenolic extract was chosen as the ideal candidate to be tested for its hypoglycemic potential. The obtained results showed the ability of this formulation to positively influence postprandial glycemia in healthy human subjects in association with an insulin-sparing mechanism of action [10]. From this point of view, thinned fruits would represent not only precious sources of ABA but also other bioactive molecules with potential health benefits, such as polyphenols. Accordingly, growing scientific evidence describes the potential role of polyphenolic compounds in the treatment of diabetes, as it is able to act on the control of blood sugar on different levels [12,13]. In the light of these considerations, natural products containing bioactive molecules with antioxidant potential would be highly desirable for more efficient management of the diabetic condition. Therefore, in order to investigate high added-value components eventually contained in TN-based nutraceutical formulation, we aimed to perform a qualitative and quantitative characterization of TN polyphenolic profile through HPLC-HESI-MS/MS analysis. Additionally, the in vitro antioxidant and antidiabetic potential of a nutraceutical formulation based on TN was evaluated.

Sample Collection and Sample Preparation for HPLC Analyses
TN were collected in June 2019 at the orchards of "Giaccio Frutta" society (Vitulazio, Caserta, Italy, 41 • 10 N-14 • 13 E), at 20-25 days after full bloom, coinciding with the fruit thinning stage. The whole fruits were frozen at −80 • C, freeze-dried and ground to obtain a uniform powder that represented the production batch used for the analysis. For TN polyphenols extraction, 1 g of homogenized sample was suspended in 5 mL of 80% aqueous methanol containing 1% formic acid for 10 min, mixed on a vortex mixer for 1 min, sonicated (Branson Fisher Scientific 150E Sonic Dismembrator) for 10 min, and centrifuged for 10 min at 9000× g. The supernatant was decanted, and the pellet was re-extracted with 5 mL of the previous solution. Finally, the combined supernatants were filtered with a 0.22 µm nylon filter (CellTreat, Shirley, MA, USA) and stored at −20 • C until analysis [14].

Qualitative Polyphenols by HPLC-HESI-MS/MS
An HPLC DIONEX UltiMate 3000 (Thermo Fisher Scientific, San Jose, CA, USA) equipment, coupled with an autosampler, a binary solvent pump and an LTQ XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA), was used for the analysis. The chromatographic analysis was performed according to Maisto et al., with slight modifications [15]. Elution was performed on a Kinetex ® C18 column (250 mm × 4.6 mm, 5 µm; Phenomenex, Torrance, CA, USA). The mobile phases were water at 2% formic acid (A) and 0.5% formic acid in acetonitrile and water 50:50 (v/v). After 2 min hold at 10% solvent B, elution was performed according to the following conditions: from 10% (B) to 55% (B) in 50 min and to 95% (B) in 10 min, followed by 10 min of maintenance; then the column was equilibrated to the initial conditions for the remaining 10 min to recondition the column. The separation conditions were as follows: column temperature was set at 30 • C, inject volume was 10 µL, the flow rate was set at 1 mL/min. The source was a heated electrospray interface (HESI), operated in negative ionization with full scanning (FS) and data-dependent acquisition (DDA). Collision-induced fragmentation was made using argon, with a collision energy of 35.0 eV. The ion source was set using the following parameters: sheath gas flow rate: 30; auxiliary gas flow rate: 10; capillary temperature: 320 • C; source heated temperature: 150 • C; source voltage: 3 kV; source current: 3.20 µA; capillary voltage: −20 V; tube lens: −106.40 V.

Quantitative Polyphenols Analysis by HPLC-DAD-FLD
An HPLC Jasco Extrema LC-4000 system (Jasco Inc., Easton, MD, USA), coupled with an autosampler, a binary solvent pump, a diode-array detector (DAD) and a fluorescence detector (FLD). The chromatographic analysis was performed according to Maisto et al., as previously described [16]. Phenolic acids, hydroxycinnamic acids, flavanols and flavanones were monitored at 280 nm, while flavonols were monitored at 360 nm. Procyanidins were monitored by a fluorescence detector that was performed with an excitation wavelength of 272 nm and an emission wavelength of 312 nm. Peak identifications were based on retention times and standard addition to the samples. Compounds were quantified according to a calibration curve made with six different concentrations over a concentration range of 0.1-1000 ppm and triplicate injections at each level.

HPLC-DAD-FLD Method Validation
Method validation was performed according to the ICH validation guideline (ICH.Q2[R1], 1995), which included the evaluation of a set of parameters, such as linearity range, the Foods 2022, 11, 1010 4 of 18 limit of detection (LOD), the limit of quantification (LOQ), precision and accuracy [17]. Data relating to method validation are reported in Supplementary Tables S1-S3. As regards polyphenolic quantification, the construction of a six-point calibration curve using diluted standard solutions was performed. Eight polyphenols (gallic acid, catechin, epicatechin, chlorogenic acid, procyanidins B1 and B2, quercetin and rutin) were selected for method development and validation. In order to assess these parameters, calibration curves were prepared in triplicate. The LOD and LOQ were calculated using the following equations: LOD = 3.3 S a /b and LOQ = 10 S a /b, where Sa is the standard deviation of the intercept of the regression line and b is the slope of the calibration curve. The precision of the method was evaluated through the percentage coefficient of variation (CV%), while method accuracy was evaluated through bias. The intra-day precision and accuracy were assessed with three concentration levels in one day. The inter-day precision and accuracy were assessed with three concentration levels over three consecutive days.

Total Phenol Content (TPC)
The total phenol content (TPC) was determined through Folin-Ciocalteau's method, using gallic acid as standard (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 0.125 mL of sample (properly diluted with water to obtain an absorbance value within the linear range of the spectrophotometer) underwent an addition of: 0.5 mL of distilled water, 0.125 mL of Folin-Ciocalteau's (Sigma-Aldrich, St. Louis, MO, USA) reagent and 1.25 mL of an aqueous solution of Na 2 CO 3 7.5% (w/v%), bringing the final volume to 3 mL with water. After mixing, the samples were kept in the dark for 90 min. After the reaction period, the absorbance was measured at 760 nm using a V-730 UV-visible/NIR spectrophotometer operated by Spectra Manager ™ Suite (Jasco Inc., Easton, MD, USA). Each sample was analyzed in triplicate, and the concentration of total polyphenols was calculated in terms of gallic acid equivalents (GAE) [14]. The antioxidant activity of TN was measured with respect to the radical scavenging ability of the antioxidants present in the sample using the stable radical 2,2-diphenyl-1picrylhydrazyl (DPPH) (Sigma-Aldrich St. Louis, MO, USA). Briefly, 200 µL of the sample was added to 1000 µL of a methanol solution of DPPH (0.05 mM). The antioxidant effect was evaluated by following the decrease in UV absorption at 517 nm with a UV-visible spectrophotometer (Jasco Inc., Easton, MD, USA). The absorbance of DPPH radical without antioxidant, i.e., the control, was measured as blank. All determinations were in triplicate. Inhibition was calculated according to the formula: where A i is the absorbance of the sample at t = 0, A f is the absorbance after 9 min, and A c is the absorbance of the control at time zero. The 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox) was used as a standard antioxidant, and the results were expressed in µmol Trolox Equivalent (TE). Furthermore, the results were also reported as EC 50 , which is the amount of antioxidant necessary to decrease the initial DPPH • concentration by 50% [18].

TEAC (Trolox Equivalent Antioxidant Capacity) Assay
The method is based on the ability of antioxidant molecules to quench ABTS •+ radical (2,20-azinobis(3-ethylbenzotiazoline-6-sulfonate)), a blue-green chromophore with characteristic absorption at 734 nm. The assay was performed according to the method described by Babbar et al. (2011) [19], with slight modifications. ABTS solution was prepared by dissolving 2.5 mL of ABTS 7.0 mM solution and 44 µL of potassium persulfate 140 mM solution, which was left to react for at least 7 h, at 5 • C in the dark. Then, ethanol-water was added to the solution until an absorbance value of 0.700 (0.05) at 754 nm (Jasco Inc., Easton, MD, USA). The assay was performed by adding 1 mL of diluted ABTS working solution to 100 µL of the sample. The determination of sample absorbance was accomplished after 2.5 min of reaction. All determinations were in triplicate. Blank was performed with ethanol in each assay. Inhibition was calculated according to the formula: where A i is the absorbance of the sample at t = 0, A f is the absorbance after 6 min and A c is the absorbance of the control at time zero. Trolox was used as a standard antioxidant. The results were expressed both as µmol of TE and EC 50 , which is the amount of antioxidant necessary to decrease the initial ABTS •+ concentration by 50% [18].

Ferric Reducing/Antioxidant Power (FRAP) Assay
When a Fe 3+ -TPTZ complex is reduced to the Fe 2+ form by an antioxidant under acidic conditions, an intense blue color with absorption maximum develops at 593 nm [18]. Therefore, the antioxidant effect (reducing ability) of the TN sample was evaluated by monitoring the formation of a Fe 2+ -TPTZ complex with a spectrophotometer (Beckman, Los Angeles, CA, USA). The assay was performed according to Benzie and Strain (1996) and Surveswaran et al. (2007) [20,21], with some modifications. The FRAP assay reagent was prepared by adding 10 vol of 0.3 M acetate buffer, pH 3.6 (3.1 g sodium acetate and 16 mL glacial acetic acid), 1 vol of 10 mM TPTZ prepared in 40 mM HCl and 1 vol of 20 mM FeCl 3 . All solutions were used on the day of preparation. The mixture was pre-warmed at 37 • C. This reagent (2.85 mL) was mixed with 0.15 mL diluted test samples similar to those used for the ABTS and DPPH assays. The mixture was shaken and incubated at 37 • C for 4 min, and the absorbance was read at 593 nm (Jasco Inc., Easton, MD, USA). The blank is represented by the only reagent solution. Blank absorbance must be subtracted from the absorbances of the samples. All determinations were in triplicate. A standard curve was made with Trolox, and the results were expressed as µmol TE.
2.6. Antidiabetic Activity 2.6.1. α-Amylase Inhibitory Assay The α-Amylase inhibitory assay was performed using a modified procedure by Schisano et al. (2019) [22]. A total of 40 µL of TN extract at different concentrations was placed in a plastic tube, adding 160 µL of distilled water and 200 µL of α-amylase solution (4 U/mL). This solution was pre-incubated at 37 • C for 10 min. The starch solution (1% w/v) used as the substrate was prepared by boiling potato starch in 0.02 M sodium phosphate buffer (pH 6.9). An amount of 400 µL of the above-mentioned starch solution was added to the reaction mixture, then further incubated at 37 • C for an additional 20 min. The reaction was terminated by adding 200 µL of DNS solution (20 mL 96 mM 3,5-dinitrosalicylic acid, 12 g sodium potassium tartrate in 8 mL of 2 M NaOH and 12 mL deionized water). The tubes were then incubated in boiling water for 5. After cooling the tube, the reaction mixture was diluted with distilled water (2 mL). Finally, the sample's absorbance was read at 540 (Jasco Inc., Easton, MD, USA). A control and a blank were prepared using the same procedure, replacing extract and enzyme, respectively, with distilled water. Acarbose, a well-known antidiabetic medicine, was used as a positive control. Three sets of experiments were conducted for the test: standard, blank and control. The α-amylase inhibitory activity was calculated as follow: where A cb is the absorbance of the control blank, A c is the absorbance of the control, A sb is the absorbance of the sample blank and A s is the absorbance of the sample. The results were reported as IC 50 , which is the amount of sample necessary to decrease the initial α-amylase activity by 50%. IC 50 values were determined by plotting percent inhibition (Y-axis) versus log 10 extract concentration (X-axis) and calculated by logarithmic regression analysis from the mean inhibitory values [23].

Advanced Glycation End-Product (AGE) Inhibition
The inhibition of AGE formation by TN extract and the standard phenolic rutin was measured through the method described by Justino et al. (2019) [24], with slight modifications. An amount of 500 µL of progressive dilutions of the samples (0.075-70 mg/mL of final concentrations for TN and 0.05-2 mg/mL for rutin) was prepared in distilled water, then added to an assay mixture containing 500 µL of bovine serum albumin (50 mg/L), 250 µL fructose (1.25 mol/L) and 250 µL of glucose (25 mol/L). All the components of the reaction mixture were solubilized in phosphate buffer (200 mmol/L; pH 7.4), containing sodium azide (0.02% w/v). The solution was incubated at 37 • C for 7 days; thereafter, fluorescence was measured at an excitation wavelength of 355 nm and an emission of 460 nm (Perkin-Elmer LS 55, Waltham, MA, USA). Distilled water was used as a negative control, while the blank was carried, substituting fructose and glucose with phosphate buffer. The inhibitory activity was calculated as a percentage of glycation inhibition (GI) by using the following formula: where F s is fluorescence intensity in the presence of sample; F sb is fluorescence intensity in the absence of fructose and glucose; F c is fluorescence intensity in the absence of sample; and F cb is fluorescence intensity in the absence of sample, fructose and glucose. Finally, the results were reported as EC 50 .

Statistics
Unless otherwise stated, all the experimental results were expressed as the mean ± standard deviation (SD) of three determinations. Graphics and IC 50 values determination were performed using GraphPad Prism 8 software.

Qualitative Polyphenols Analysis by HPLC-HESI-MS/MS
TN polyphenolic extract was characterized by HPLC-HESI-MS/MS. Based on a comparison with the literature data, 48 compounds were putatively identified. A total of 27 phenolic acids, 10 flavans and 11 flavonolds are displayed in Table 1. The identity of 19 compounds (3, 6-9, 13, 14, 16, 20, 24, 28, 29, 32, 34, 36, 37, 38, 43, 45) was verified by comparison with analytical standards.  The absence of a prominent ion caused by CO loss, typical of the phenolic moiety, allowed distinguishing the alcoholic aliphatic group from the phenolic scaffold. Based on the literature data, compound 5 was tentatively identified as quinic acid [50]. The presence of two caffeoylquinic acids (compounds 6 and 13) was confirmed by the [M-H] − ion at m/z 353 and two fragment ions at m/z 191 and m/z 179, corresponding to quinic acid and caffeic acid ions, respectively. The identification of these compounds was supported by comparison with the authentic standards. In this way, compounds 6 and 13 were identified as neochlorogenic acid and chlorogenic acid, respectively [29].  (14). Therefore, these compounds were tentatively identified as three caffeoylshikimic acid isomers [41]. Compound  Furthermore, the fragmentations of the two compounds followed the fragmentation pattern of chlorogenic acid and neochlorogenic acid (compounds 6 and 13) and was in agreement with the literature data [48].

Flavans Identification
Flavans are a class of 2-phenylchroman compounds largely distributed in plants. The main flavan class is represented by the flavan-3-ols, which include monomeric derivatives, such as (+)-catechin and (−)-epicatechin and oligomeric and polymeric compounds named procyanidins. Procyanidins can be divided into A-type or B-type based on the linkage between monomers. While B-type linkage procyanidins dimers show an interflavan single carbon-carbon bond between monomers, A-type linkage ones are characterized by two linkages between flavan units, an interflavan single carbon-carbon bond and an ether bond [51]. The ether linkage connects the monomers by a six-membered ring, resulting in a difference of 2 Da units. Procyanidins give a characteristic fragmentation pattern, which includes three types of fragmentation mechanisms: the heterocyclic ring fission (HRF), the retro Diels-Alder fission (RDA) and the quinone methide cleavage (QM). The heterocyclic ring fission (HRF) is given by the loss of a phloroglucinol unit (−126 Da), preserving the interflavanic bond between the two monomers. The quinone methide cleavage (QM) represents the fragmentation of the interflavanic bond between two monomers. Therefore, the resulting ions indicate the number of monomers in the oligomeric compounds [31].  QM cleavage, respectively [31]. One procyanidin dimer A-type linkage (44) [30,31]. Flavanones show a different fragmentation pattern than flavan-3-ols. In fact, these compounds occur in the plants as aglycone or glycosides and exhibit a characteristic fragmentation pattern, with prominent fragment ions due to the loss of sugars and retro Diels-Alder fission (RDA) [45].

Flavonols Identification
Flavanols are a class of 3-hydroxy-2-phenylchromen-4-one polyphenols and exhibit a flavanone-like fragmentation pattern. The aglycon ion represents the main fragment peak due to the cleavage of sugars, but other fragments include the neutral losses of H 2 O, CO and CO 2 and retro Diels-Alder fission (RDA). A typical fragment ion is represented by the m/z 179 due to the RDA fragmentation [44]. However, the glycosylated flavonols displayed two prominent fragment ions due to the loss of glucidic units and the cross-ring cleavage of the sugar moiety with a neutral loss of C 4

Quantitative Polyphenols Analysis by HPLC-DAD-FLD
Chromatographic analysis for the quantification of TN polyphenolic composition was conducted as reported in Section 2.3. The HPLC-DAD-FLD analysis allowed the quantification of 19 different compounds, including flavanols, procyanidin compounds, phenolic acids and flavonol derivatives. The results are reported in Table 2. Values are expressed in µg/g ± standard deviation (SD) of three repetitions. * Procyanidins B1 and B3 peaks were partially overlapped and were quantified as a mixture of two compounds using the procyanidin B1 calibration curve.

Total Polyphenols and In Vitro Antioxidant Activity of Thinned Nectarine
In order to obtain an overview of the total polyphenolic content, Folin-Ciocalteau's assay was performed on hydroalcoholic TN extract. The TN sample exhibited a total phenol content of 17.01 ± 0.35 mg GAE/g of extract. Additionally, the antioxidant activity was evaluated by using DPPH, ABTS and FRAP assays. As reported in Table 3, results were expressed as µmol of TE per g of dried extract. In order to standardize the results from various studies, DPPH and ABTS assays were also reported as EC 50 , which is the amount of antioxidant necessary to decrease the concentration of the initial solution by 50% [18]. As displayed in Figure 1, TN extract exhibited an EC 50 of 1.57 mg/mL for DPPH assay and 1.58 mg/mL for ABTS assay.
In order to standardize the results from various studies, DPPH and ABTS assays were also reported as EC50, which is the amount of antioxidant necessary to decrease the concentration of the initial solution by 50% [18]. As displayed in Figure 1, TN extract exhibited an EC50 of 1.57 mg/mL for DPPH assay and 1.58 mg/mL for ABTS assay.

3.4.1.α-. Amylase Inhibitory Assay
The α-amylase inhibition activity of TN was tested by an in vitro assay. Figure 2 shows the percentage inhibition as IC50, which is the amount of compound necessary to inhibit the enzyme activity by 50% [23]. The results revealed that both acarbose and the TN inhibited α-amylase activity in a concentration-dependent manner. The IC50 values generated from the percentage inhibition revealed a result of 8.11 and 0.43 mg/mL for TN and acarbose, respectively.

Advanced Glycation End-Product (AGE) Inhibition Assay
The inhibitory activity of TN on AGEs formation was also investigated. The assay is based on the inhibition of specific fluorescence generated during the course of glycation and AGEs formation. Figure 3 shows the percentage inhibition as EC50, which is the concentration required to obtain a 50% effect. Under our experimental conditions, TN and rutin produced a concentration-dependent inhibition of AGE, with an EC50 of 11.0 and 0.1 mg/mL for TN and rutin, respectively ( Figure 3).

α-Amylase Inhibitory Assay
The α-amylase inhibition activity of TN was tested by an in vitro assay. Figure 2 shows the percentage inhibition as IC 50 , which is the amount of compound necessary to inhibit the enzyme activity by 50% [23]. The results revealed that both acarbose and the TN inhibited α-amylase activity in a concentration-dependent manner. The IC 50 values generated from the percentage inhibition revealed a result of 8.11 and 0.43 mg/mL for TN and acarbose, respectively. The α-amylase inhibition activity of TN was tested by an in vit shows the percentage inhibition as IC50, which is the amount of comp inhibit the enzyme activity by 50% [23]. The results revealed that both TN inhibited α-amylase activity in a concentration-dependent manne generated from the percentage inhibition revealed a result of 8.11 and 0 and acarbose, respectively.

Advanced Glycation End-Product (AGE) Inhibition Assay
The inhibitory activity of TN on AGEs formation was also investi based on the inhibition of specific fluorescence generated during the c and AGEs formation. Figure 3 shows the percentage inhibition as EC50 centration required to obtain a 50% effect. Under our experimental c rutin produced a concentration-dependent inhibition of AGE, with an mg/mL for TN and rutin, respectively ( Figure 3).

Advanced Glycation End-Product (AGE) Inhibition Assay
The inhibitory activity of TN on AGEs formation was also investigated. The assay is based on the inhibition of specific fluorescence generated during the course of glycation and AGEs formation. Figure 3 shows the percentage inhibition as EC 50 , which is the concentration required to obtain a 50% effect. Under our experimental conditions, TN and rutin produced a concentration-dependent inhibition of AGE, with an EC 50 of 11.0 and 0.1 mg/mL for TN and rutin, respectively ( Figure 3).

Discussion
Polyphenols, a family of plant secondary metabolites naturally occurring in fruits, are considered critical not only for fruit quality but also for human health benefits [52]. Scientific data showed that polyphenolic compounds contained in nectarine and peach could prevent cellular oxidative stress resulting from free radicals [53]. Interestingly, it was shown that thinned young fruits might exhibit significantly higher antioxidant capacity than those of their ripe counterparts. This was mainly due to the 5-10 times higher total polyphenol content found in thinned young fruits compared to ripe fruits [54]. In the present work, HPLC analyses allowed us to tentatively identify 48 polyphenolic compounds, 19 of which were quantified with analytical standards, confirming the high polyphenolic content of these waste-by products. The quantitative analysis of the polyphenolic profile of TN extract was in line with the available literature data. Specifically, Guo et al. displayed that chlorogenic acid and neochlorogenic acid represent the main polyphenols of unripe nectarines, with a content in the matrix of 100-500 μg/g and 270-1250 μg/g, respectively [54].
Data obtained in this work demonstrated a concentration-dependent inhibitory activity of TN extract on the α-amylase enzyme, with results expressed as IC50. It is wellknown that the IC50 of an inhibitor is very dependent on the assay conditions, such as enzyme concentration and origin, substrate type and concentration, reaction duration, temperature and pH [55]. This makes data comparison with the literature a difficult task. However, by utilizing acarbose as a benchmark, a comparison of the general inhibitory trend could be achieved. Recently, long-term excessive intake of starchy food has been reported to be one of the reasons for hyperglycemia that can even lead to type II diabetes disease [56]. In this regard, reducing the hydrolysis rate of starch through inhibiting digestive enzymes is one suggested way of relieving postprandial hyperglycemia [57]. Of note, the same test conducted with a standard of abscisic acid did not show any inhibitory capacity on the enzyme (data not shown), highlighting that the inhibitory activity of our sample would not be ascribable to the ABA content but to the contribution of other bioactive compounds. In this regard, the role of polyphenols in modulating starch digestion and glycemic levels was widely investigated [57]. As reported by the scientific literature, compounds such as quinic acid derivatives and mono and diglycosyl flavonols (e.g., neochlorogenic acid, chlorogenic acid and rutin) are closely correlated to the inhibition of αamylase [58]. Therefore, the high content of these polyphenols may justify TN inhibitory efficacy on this enzyme.
Moreover, concentration-dependent inhibition of AGEs formation was observed after testing TN extract through an opportune in vitro assay. AGEs are proteins or fats combined with blood sugars after exposure to a glycation process through the Maillard reaction [59]. These compounds result in being highly stable and resistant to enzymatic degradation, leading to their high accumulation in different tissues, modification in cells and

Discussion
Polyphenols, a family of plant secondary metabolites naturally occurring in fruits, are considered critical not only for fruit quality but also for human health benefits [52]. Scientific data showed that polyphenolic compounds contained in nectarine and peach could prevent cellular oxidative stress resulting from free radicals [53]. Interestingly, it was shown that thinned young fruits might exhibit significantly higher antioxidant capacity than those of their ripe counterparts. This was mainly due to the 5-10 times higher total polyphenol content found in thinned young fruits compared to ripe fruits [54]. In the present work, HPLC analyses allowed us to tentatively identify 48 polyphenolic compounds, 19 of which were quantified with analytical standards, confirming the high polyphenolic content of these waste-by products. The quantitative analysis of the polyphenolic profile of TN extract was in line with the available literature data. Specifically, Guo et al. displayed that chlorogenic acid and neochlorogenic acid represent the main polyphenols of unripe nectarines, with a content in the matrix of 100-500 µg/g and 270-1250 µg/g, respectively [54].
Data obtained in this work demonstrated a concentration-dependent inhibitory activity of TN extract on the α-amylase enzyme, with results expressed as IC 50 . It is well-known that the IC 50 of an inhibitor is very dependent on the assay conditions, such as enzyme concentration and origin, substrate type and concentration, reaction duration, temperature and pH [55]. This makes data comparison with the literature a difficult task. However, by utilizing acarbose as a benchmark, a comparison of the general inhibitory trend could be achieved. Recently, long-term excessive intake of starchy food has been reported to be one of the reasons for hyperglycemia that can even lead to type II diabetes disease [56]. In this regard, reducing the hydrolysis rate of starch through inhibiting digestive enzymes is one suggested way of relieving postprandial hyperglycemia [57]. Of note, the same test conducted with a standard of abscisic acid did not show any inhibitory capacity on the enzyme (data not shown), highlighting that the inhibitory activity of our sample would not be ascribable to the ABA content but to the contribution of other bioactive compounds. In this regard, the role of polyphenols in modulating starch digestion and glycemic levels was widely investigated [57]. As reported by the scientific literature, compounds such as quinic acid derivatives and mono and diglycosyl flavonols (e.g., neochlorogenic acid, chlorogenic acid and rutin) are closely correlated to the inhibition of α-amylase [58]. Therefore, the high content of these polyphenols may justify TN inhibitory efficacy on this enzyme.
Moreover, concentration-dependent inhibition of AGEs formation was observed after testing TN extract through an opportune in vitro assay. AGEs are proteins or fats combined with blood sugars after exposure to a glycation process through the Maillard reaction [59]. These compounds result in being highly stable and resistant to enzymatic degradation, leading to their high accumulation in different tissues, modification in cells and tissues, progressive deterioration of structural integrity and physiological function across multiple organs and increased risk of death [60]. Regarding diabetes mellitus-related hyperglycemic conditions, it is well known that excess intracellular glucose is converted to sorbitol by the polyol pathway, mainly in tissues and organs with an insulin-independent glucose uptake (e.g., retina, peripheral nerves, kidney, erythrocytes). This signaling pathway often results in a complex cascade of events that can culminate in tissue and vascular damage, significantly contributing to the onset of diabetes chronic complications [61]. Therefore, the inhibition of AGEs formation would represent a useful tool for the prevention of diabetes complications. In this regard, growing evidence highlighted that polyphenols are able to prevent AGEs production [62], reasonably supporting the herein observed beneficial activity exerted by TN-based formulation. Polyphenols' antiglycation properties are mainly due to the inhibition of early Maillard reaction products, especially reactive dicarbonyl as methylglyoxal (MGO) [63]. Phenolic acids and flavans (e.g., gallic acid, p-coumaric acid and epicatechin) can directly reduce the carbonyl groups by a redox reaction, inhibiting the formation of advanced Maillard products. Differently, flavonols (e.g., quercetin, rutin) react with the MGO dicarbonyl moiety, indirectly preventing the formation of glycation products [63,64]. Therefore, it is possible to hypothesize that TN extract may exert an antiglycation action with different mechanism of actions, due to the complexity of polyphenols fraction.
Considering the well-known link between diabetes and oxidative stress and considering the vegetal nature of our food matrix is highly likely to contain antioxidant polyphenols, the attention was focused on the investigation of in vitro antioxidant potential of TN. For these reasons, Folin-Ciocâlteu, DPPH, ABTS and FRAP assays were carried out on a polyphenolic extract of TN. The obtained results are in line with available studies conducted on immature nectarine, confirming the high antioxidant potential of these by-product matrices [25]. In this regard, a study conducted by Guo et al. reported that the antioxidant capacity of peaches and nectarines evaluated by DPPH, ABTS and FRAP assays were 1.3-11.2-fold higher in thinned young fruit compared to ripe fruit [54]. Notable, increasing evidence from in vitro and clinical studies suggests that oxidative stress plays a pivotal role in the pathogenesis of both types of diabetes mellitus. Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can indeed lead to damage to biological structures. These consequences of oxidative stress can promote the development of complications of diabetes mellitus [12]. Similarly, persistent hyperglycemia is recognized as one of the main causes of oxidative stress, supporting a direct cause and effect relationship between hyperglycemia and oxidative stress [13]. In the light of these considerations, the concomitant presence of bioactive molecules with antidiabetic and antioxidant potential in TN-based formulation would further support its supplementation for the management of diabetic pathology. Moreover, thinned unripe fruits revaluation fits properly with the concept of green economy and environmental sustainability, opening new paths for food by-products revaluation [11].

Conclusions
In conclusion, our data would reasonably support TN as an innovative and promising nutraceutical formulation with beneficial health effects, especially regarding the management of glucose homeostasis. According to previous data obtained from our research group, these beneficial actions may be related to the role of ABA occurred in TN, particularly relating to the insulin-sparing mechanism of action. Furthermore, the concomitant presence of other bioactive components in TN-based nutraceutical formulation, such as polyphenols, may contribute to the management of diabetes-related oxidative stress conditions. Overall, the herein obtained results could represent a starting point for further in-depth animal-based studies and clinical trials aimed at evaluating the antidiabetic effects of the TN-based nutraceutical formulation.

Data Availability Statement:
The data used to support the findings of this study are included in the article.