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Antioxidants 2022, 11, 1933. https://doi.org/10.3390/antiox11101933 www.mdpi.com/journal/antioxidants
Article
Optimization of Phlorizin Extraction from Annurca Apple Tree
Leaves Using Response Surface Methodology
Maria Maisto 1,†, Vincenzo Piccolo 1,†, Ettore Novellino 2, Elisabetta Schiano 1, Fortuna Iannuzzo 1,*,
Roberto Ciampaglia 1, Vincenzo Summa 1 and Gian Carlo Tenore 1
1 Department of Pharmacy, University of Naples Federico II, via Domenico Montesano 59, 80131 Naples, Italy
2 Faculty of Medicine, University Cattolica, Largo Agostino Gemelli, 00168 Rome, Italy
* Correspondence: fortuna.iannuzzo@unina.it
† These authors contributed equally to this work.
Abstract: Phlorizin is a plant-derived molecule with relevant anti-diabetic activity, making this com-
pound a potential functional component in nutraceutical formulations for the management of gly-
cemia. It is noteworthy that promising sources for the extraction of phlorizin include apple tree
leaves, a by-product of apple fruit production. The main aim of this study was to optimize the ex-
traction process of phlorizin from Annurca apple tree leaves (AALs) using response surface meth-
odology (RSM), and to determine the potential nutraceutical application of the obtained extract. The
results of the RSM analysis indicate a maximum phlorizin yield of 126.89 ± 7.579 (mg/g DW) ob-
tained under the following optimized conditions: MeOH/H2O, 80:20 + 1% HCOOH as the extraction
solvent; 37.7 °C as the extraction temperature; and 170 min as the time of extraction. The HPLC-
DAD-HESI-MS/MS analysis performed on the extract obtained under such conditions, named opti-
mized Annurca apple leaves extract (OAALE), led to the identification of twenty-three phenolic
molecules, with fifteen of them quantified. To explore the nutraceutical potential of OAALE, the in
vitro antioxidant activity was evaluated by DPPH, ABTS, and FRAP assays, resulting in 21.17 ± 2.30,
38.85 ± 0.69, and 34.14 ± 3.8 μmol Trolox equivalent/g of extract, respectively. Moreover, the IC50 of
0.330 mg/mL obtained from the advanced glycation end-product inhibition assay, further sup-
ported the antidiabetic potential of OAALE.
Keywords: waste product; antioxidant activity; response surface methodology; antidiabetic activ-
ity; phlorizin
1. Introduction
Phlorizin (phloretin-2-O-β-D-glucopyranoside) is the glucoside of phloretin, a mem-
ber of dihydrochalcones, which are a family of bicyclic flavonoids. It was first isolated by
a French scientist from apple tree bark in 1835 [1]. This molecule was largely studied for
its multiple health effects, such as its anti-inflammatory, antioxidant, anticancer, and an-
tibacterial activities [2]. Particularly, phlorizin plays an important role as a dietary poly-
phenol that is able to regulate glucose homeostasis by reducing intestinal glucose uptake
[2]. More specifically, in diabetic rats, phlorizin was shown to inhibit intestinal and renal
glucose uptake via sodium-dependent glucose transporters (SGLTs), resulting in a reduc-
tion in hyperglycemia without altering insulin secretion [3]. Other studies reported that
phlorizin was not only able to reduce glucose plasma levels, but it also improved lipid
metabolism [4], accelerated liver glycogen synthesis [5], decreased hepatic gluconeogen-
esis [6], and exerted hypoglycemic effects in type 2 diabetes mellitus mice [6].
The main natural sources of phlorizin are the plants of the Malus genus, although it
also reaches a valuable concentration in other plant species, such as Punica granatum
(pulp) [7], Polygonum cuspidatum (flower), Prunus persica (pulp) [8], Rosa canina (flesh) [9],
Vaccinium vitis-idaea (flesh) [10], and Vaccinium macrocarpon [11]. Specifically, phlorizin is
Citation: Maisto, M.; Piccolo, V.;
Novellino, E.; Schiano, E.; Iannuzzo,
F.; Ciampaglia, R.; Summa, V.;
Tenore, G.C. Optimization of
Phlorizin Extraction from Annurca
Apple Tree Leaves Using Response
Surface Methodology. Antioxidants
2022, 11, 1933. https://doi.org/
10.3390/antiox11101933
Academic Editors: Silvana Hrelia,
Cristina Angeloni and Maria
Cristina Barbalace
Received: 2 August 2022
Accepted: 20 September 2022
Published: 28 September 2022
Publisher’s Note: MDPI stays
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Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/license
s/by/4.0/).
Antioxidants 2022, 11, 1933 2 of 15
not equally distributed in all parts of the apple tree, however, it reaches its maximum
concentration in the non-edible parts of the plant, e.g., leaves [12], twigs [13], root bark,
seeds [14], and unripe fruits [15]. Phloretin, and its glucoside phlorizin, are found to be
the major phenolic compounds in apple leaves, reaching a concentration ranging from
5.4% to 14% of leaf dry weight (DW) [12] Moreover, the phlorizin content of leaves seems
to be less affected by some variables, such as apple cultivar or harvesting period, than its
aglycone, making phlorizin concentration stable over time and in the type of apple culti-
var analyzed.
Considering the conventional method of cultivation, during the pruning period in
summer, unripe fruits and leaves are harvested to improve the quality of fruit production,
resulting in a high amount of non-utilized apple leaves that would be classified as agro-
food waste materials [12]. As widely reported, the interest of the nutraceutical industry in
the reutilization of agro-food waste products is progressively increasing, since they rep-
resent still rich sources of biologically active compounds that can be conveniently used
for the formulation of food supplements [16]. In this context, apple leaves may be consid-
ered an excellent source of bioactive compounds, especially phlorizin, their major phe-
nolic component. Currently, the main apple-derived waste product reutilized by
nutraceutical industries is root bark. Interestingly, compared to root barks, apple leaves
have a similar dihydrochalcones content and are also produced in higher amounts and in
every cycle of cultivation. Therefore, these byproducts could be considered a more con-
venient alternative raw material for the formulation of nutraceutical products rich in
phlorizin. Due to the aforementioned biological activities ascribed to phlorizin, several
extraction methods have been developed to optimize the extraction yield of this molecule
from plant materials. In this regard, similarly to other polyphenolic compounds, the hy-
drochloric mixture is considered the more exhaustive solvent for its extraction [2].
Annurca apple is the only apple cultivar native to Southern Italy, listed as a Protected
Geographical Indication (PGI) product by the European Council (Commission Regulation
(EC) No. 417/2006)). Annurca polyphenolic fraction is largely studied for its beneficial ef-
fects on the control and management of cholesterol plasma levels in healthy and mildly
hypercholesterolemic subjects [17]. On the other hand, there has been a lack of studies
regarding the chemical characterization of Annurca apple leaves (AALs) in the scientific
literature. Moreover, compared to other apple cultivars, both local (native of the same
region of Annurca apple, i.e., Rosa di Serino, Limoncella) and commercial ones (Pink Lady
and Golden Delicious), Annurca apple showed the highest title in dihydrochalcones, and
this trend would also be reproduced in the leaves [18,19].
In light of these considerations, the main goal of the present study was to investigate
the potential of Annurca apple tree leaves (AALs) as a source of phenolic compounds,
especially phlorizin. Moreover, the response surface methodology (RSM) was used to
reach the maximum phlorizin extraction rate from AAL. After the determination of the
optimum extraction condition (OEC), the polyphenolic composition of the extract ob-
tained in OEC, named OAALE (Optimized Annurca Apple Leaves Extract), was investi-
gated and its in vitro antioxidant and antidiabetic activities were studied.
2. Materials and Methods
2.1. Reagents
All chemicals, reagents, and standards used were analytical or LC–MS grade rea-
gents. The water was treated in a Milli-Q water purification system (Millipore, Bedford,
MA, USA) before use. Catechin (purity ≥ 98% HPLC), procyanidin B1 (purity ≥ 90%
HPLC), procyanidin B2 (purity ≥ 90% HPLC), procyanidin B3 (purity ≥ 95% HPLC), pro-
cyanidin C1 (purity ≥ 90% HPLC), chlorogenic acid (purity ≥ 95% HPLC), caffeic acid (pu-
rity ≥ 98% HPLC), syringic acid (purity ≥ 98% HPLC), gallic acid (purity ≥ 98% HPLC)
rutin (purity ≥ 94% HPLC), p-coumaric acid (purity ≥ 98% HPLC), epicatechin (purity ≥
98% HPLC), ferulic acid (purity ≥ 99% HPLC), quercetin 3-O-glucoside (purity ≥ 98%
Antioxidants 2022, 11, 1933 3 of 15
HPLC), kaempferol 3-O-glucoside (purity ≥ 90% HPLC), quercetin (purity ≥ 98% HPLC),
and the reagents for in vitro studies were purchased from Sigma-Aldrich (Milan, Italy).
2.2. Sample Collection and Extraction Protocol
AAL were harvested in October 2021 from the orchards of “Giaccio Frutta” society
(Vitulazio, Caserta, Italy, 41°100 N–14°130 E). The AALs were frozen at −80 °C, lyophi-
lized, and ground to obtain a homogeneous powder that constituted the production batch
used for the experiments. As reported in Table 1, for the optimization of phlorizin extrac-
tion protocol from AALs, different extraction times (30, 60, 120, 240 min), solvent compo-
sitions (80% aqueous methanol solution containing 0.1, 1, or 5% formic acid), incubation
temperatures (30, 35, and 45 °C), with and without a sonication stage of 30 mi n, were
opportunely combined. According to the general extraction protocol applied, 250 mg of
AALs were treated with 2 mL of extraction solvent, as previously optimized by Othman
et al. [12] the mixture obtained was left in incubation at selected temperatures and times
on an orbital shaker. At the end of the extraction time, where expected, 30 min of soni-
cation (continuous operative mode, 150 W Power, 40 kHz Frequency; Branson Fisher Sci-
entific 150E Sonic Dismembrator) was performed. After that, the samples were centri-
fuged for 5 min at 12,000× g. The supernatants were filtered with a 0.22 µm nylon filter
(Cell Treat, Shirley, MA, USA) and stored at −20 °C until analysis. All extractions were
performed in triplicate.
Table 1. Independent variables and their values used for the model set.
Independent Variable
Factor Levels
Incubation time (min)
30
60
120
240
% Acid in the extraction solvent
0.1
1
5
Temperature (°C)
45
35
25
Sonication
Yes
No
Total runs
69
2.3. HPLC Analyses of Samples
2.3.1. Qualitative Polyphenolic Composition by HPLC-DAD-HESI-MS/MS
An HPLC DIONEX UltiMate 3000 (Thermo Fisher Scientific, San Jose, CA, USA)
equipment, coupled with an autosampler, a binary solvent pump, a diode-array detector
(DAD), and an LTQ XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA),
were used for the analysis. The chromatographic analysis was performed according to
Maisto et al., with slight modifications [20]. The separation conditions were as follows:
column temperature was set at 35 °C, the injection volume was 5 µL, and the flow rate
was set at 1 mL/min. The selected column was the Kinetex® C18 column (250 mm × 4.6
mm, 5 µm; Phenomenex, Torrance, CA, USA). The mobile phases were water at 0.1% for-
mic acid (A) and acetonitrile at 0.1% formic acid (B). Elution was performed according to
the following conditions: 0–3 min hold at 5% solvent B, from 5% (B) to 40% (B) in 20 min
and 95% (B) in 10 min, followed by 5 min of maintenance; for the remaining 10 min, the
column was equilibrated to the initial conditions. Regarding the mass parameters, the
source was a heated electrospray interface (HESI), operated in negative ionization with
full scanning (FS) and data-dependent acquisition (DDA). Phenolic acids, hy-
droxycinnamic acids, flavanols, and flavanones were monitored at 280 nm, while flavo-
nols were monitored at 360 nm. 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: 3.5 kV; source current: 100 µA; capillary volt-
age: 31 V; and tube lens: 90 V.
Antioxidants 2022, 11, 1933 4 of 15
2.3.2. Quantitative Polyphenols Analysis by HPLC-DAD-FLD
The quantitative analysis of OECE was performed with the HPLC Jasco Extrema LC-
4000 system (Jasco Inc., Easton, MD, USA), equipped with an autosampler, a binary sol-
vent pump, a diode-array detector (DAD), and a fluorescence detector (FLD). The chro-
matographic analysis was performed according to our previously developed method [20].
Procyanidins were detected by a fluorescence detector that was set with an excitation
wavelength of 272 nm and an emission wavelength of 312 nm, while the phenolic acids,
hydroxycinnamic acids, flavanols, and flavanones were acquired at 280 nm, and flavonols
at 360 nm. The analyses were performed at a flow rate of 1 mL/min, with solvent A (2%
acetic acid) and solvent B (0.5% acetic acid in acetonitrile and water 50:50, v/v) using
Kinetex® C18 column (250 mm × 4.6 mm, 5 µm; Phenomenex, Torrance, CA, USA): 0–5
min of 10% (B), from 10% (B) to 55% (B) in 50 min and 95% (B) in 10 min, followed by 5
min of maintenance. Peak identifications were based on a comparison of retention times
with analytical standards and standard addition to the samples. The quantitative analyses
were performed using the calibration curve calculated with six different concentrations in
a concentration range of 0.1–1000 ppm and triplicate injections at each concentration level.
2.4. Total Phenolic Content Determination
The total phenol content (TPC) was performed by Folin–Ciocalteau’s assay, using
gallic acid as the reference standard (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 0.1 mL
of samples (appropriately diluted with water to achieve a measured absorbance value in-
cluded the linear range of the spectrophotometer) were added in sequence: 0.5 mL of Fo-
lin–Ciocalteau’s (Sigma-Aldrich, St. Louis, MO, USA) reagent and 0.2 L of an aqueous
solution of Na2CO3 7% (w/v%), bringing the final volume to 10 mL with water. Then, the
samples were mixed and left in incubation in the dark for 90 min. After the reaction time,
the absorbance was acquired at 760 nm (Jasco Inc., Easton, MD, USA). All the samples
were analyzed in triplicate and the concentration of total polyphenols was calculated in
gallic acid equivalents (GAEs).
2.5. Antioxidant Activity
2.5.1. DPPH• Radical Scavenging Assay
The radical scavenging ability of the antioxidants in the sample was evaluated using
the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) with a maximum absorbance at
517 nm. The analysis was performed by mixing 100 µL of each sample (opportunely di-
luted in extraction mixture) with 1000 µL of a methanol solution of DPPH (153 mmol/L).
The mixture obtained was left in incubation in the darkness for 9 min of reaction time. The
decrease in absorbance was evaluated using a UV–visible spectrophotometer (Beckman,
Los Angeles, CA, USA). All determinations were performed in triplicate. DPPH• inhibi-
tion was calculated according to the formula: [(Ai − Af)/Ac] × 100, where Ai is the absorb-
ance of the sample at t = 0, Af is the absorbance of the sample after the reaction time and
Ac was the absorbance of the control (1000 µL of a methanol solution of DPPH+100 µL of
methanol). The obtained results are expressed in µmol of Trolox (6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid) equivalent (TE). Moreover, the results were also re-
ported as EC50, which is the amount of antioxidant compound necessary to inhibit the
initial DPPH• concentration by 50% [21].
Antioxidants 2022, 11, 1933 5 of 15
2.5.2. Ferric Reducing/Antioxidant Power (FRAP) Assay
When a Fe3+-TPTZ complex is reduced to the Fe2+ ion by an antioxidant under acidic
conditions, a blue color develops, with maximum absorbance at 593 nm [18]. Thereby, the
antioxidant effect (reducing ability) of the sample was evaluated by monitoring the for-
mation of a Fe2+–TPTZ complex with a spectrophotometer (Jasco Inc., Easton, MD, USA).
The test was performed as reported by Benzie and Strain (1996) [22], with slight modifi-
cations. The Frap working solution was prepared by mixing 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 20mM FeCl3. All the components of the working solutions
were freshly prepared and used on the same day of preparation. Before performing the
assay, all the solutions were brought to 37 °C. The amount of 2.85 mL of working solution
was mixed with 0.15 mL diluted samples and incubated at 37 °C for 4 min. After the incu-
bation time, the absorbance was acquired at 593 nm (Jasco Inc., Easton, MD, USA). The
blank was represented by the only working solution. For the calculation of antioxidant
activity, the blank absorbance value was subtracted from the absorbances of the samples.
All analyses were performed in triplicate. A standard curve was plotted with Trolox, and
the results are expressed as µmol TE.
2.5.3. ABTS• Radical Scavenging Assay
The assay relied on the capability of antioxidant molecules to react ABTS•+ radical
(2,20-azinobis(3-ethylbenzotiazoline-6-sulfonate)), a chromophore with specific absorp-
tion at 734 nm. The test was performed according to the experimental protocol previously
performed by Babbar et al. (2011) [23]with some modifications.
ABTS solution was prepared by mixing 2.5 mL of ABTS 7.0 mM ethanol solution and
44 µL of potassium persulfate 140 mM solution, which was left to incubate for at least 7 h,
at 5 °C in darkness. After this time, to prepare the working solution, the obtained mixture
was diluted with the ethanol–water solution until an absorbance value of 0.700 ± 0.05 was
acquired at 754 nm (Jasco Inc., Easton, MD, USA). The assay was performed by mixing
1000 µL ABTS working solution with 100 µL of the sample opportunely diluted in the
extraction solvent. The mixture was incubated for 2.5 min in the dark. After this time, the
sample absorbances were read at 734 nm, with a visible discoloration of the sample with
high antiradical activity. The control was prepared by replacing the sample with the same
volume of ethanol. The radical inhibition was calculated according to the formula: [(Ai −
Af)/Ac] × 100, (2), where Ai is the absorbance of the sample at t = 0, Af is the absorbance
after 2.5 min, and Ac is the absorbance of the control at time zero. Trolox was used as a
standard antioxidant. The results are expressed both as µmol of TE and EC50, which is the
amount of antioxidant necessary to decrease the initial ABTS•+ concentration by 50% [21].
2.6. Advanced Glycation End-Product (AGE) Inhibition Assay
The inhibition of AGE generation by OAALE extract and the standard phenolic rutin
was performed according to the method reported by Schiano et al. [15] with slight modi-
fications. The amount of 500 µL of serial dilutions for each sample (0.075–70 mg/mL of
final concentrations for OAALE and 0.05–2 mg/mL for rutin) prepared in distilled water
were added to a working solution composed of 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 elements of
this reaction mixture were dissolved in phosphate buffer (200 mmol/L; pH 7.4) containing
sodium azide (0.02% w/v). The mixture was incubated at 37 °C for 7 days. After this incu-
bation time, the fluorescence was acquired 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 out by replacing the fructose and glu-
cose with phosphate buffer. The inhibitory activity was expressed as a percentage of gly-
cation inhibition (GI), using the following formula: GI (%) = [(Fs − Fsb)/(Fc − Fcb)] × 100, (4)
where Fs is the fluorescence intensity in the presence of the sample; Fsb is the fluorescence
Antioxidants 2022, 11, 1933 6 of 15
intensity in the absence of fructose and glucose; Fc is the fluorescence intensity in the ab-
sence of sample; and Fcb is the fluorescence intensity in the absence of sample, fructose,
and glucose. Finally, the results are reported as EC50.
2.7. Statistics
Unless otherwise stated, all experimental results are expressed as the mean ± stand-
ard deviation (SD) of three repetitions. Graphics and IC50 values determination were cal-
culated using GraphPad Prism 8 software. The RSM optimization was performed with
Minitab software version 21.1.0.
3. Results and Discussion
3.1. Optimisation of Phloridzin Extraction Using RSM Model
The choice to optimize the phlorizin extraction conditions in MeOH 80% was due to
the capacity of this solvent to reach the maximum extraction rate not only of phlorizin,
but also of total polyphenols [2,20,2]. The temperature was kept constantly below 40 °C to
avoid the temperature-dependent decomposition of polyphenols during the extraction
process. Generally, the stability of polyphenols at high-temperature values depends on
the class of polyphenols considered and, obviously, on their chemical structure. Specifi-
cally, it was well accepted that the polyphenols concentration was significantly stable (p <
0.05) during the extraction process at a temperature lower than 40 °C [24]. The experi-
mental data show that the phlorizin concentration ranged from 70.80 mg/g (p < 0.001) (60
min, 25 °C, 1% HCOOH with 30 min of sonication) to 141.59 mg/g (120 min, at 35 °C, +1%
HCOOH, without sonication). Initially, four independent commonly modified factors, i.e.,
extraction time (30, 60, 120, and 240 min), temperature (45, 35, and 25 °C), and % acid in
the extraction solvent (5, 1, and 0.1% of formic acid), combined with or without a single
cycle of sonication (30 min), were selected for the optimization of phlorizin yield in the
hydroalcoholic solvent. Considering the independent factors analyzed, according to pre-
liminary ANOVA analysis, only the extraction temperature (A) and extraction time (B),
without sonication assistance, were significantly correlated with the phlorizin extraction
rate, as explained in the Pareto-chart graphic with α = 0.05 (Figure 1).
Figure 1. (a) Pareto chart of the total parameters analyzed (significative and not significative); and
(b) Pareto chart of significative parameters only, i.e., temperature (A) and extraction time (B).
Based on the statistical results of model fitting, the best model to optimize the
phlorizin output would be by reducing the statistical analysis to two-factor interaction
(2FI, i.e., A and B) (Figure 1). The multiple regression analysis of phlorizin values showed
that the model was significant (p < 0.0001), did not present a lack of fit (p = 0.182), and a
percentage predictivity of the model was of 73.56% (R-sq 77.58%, R-sq(adj) 75.88%; R-
sq(pre) 73.41%). Second-order quadratic polynomial models were found to be adequate
to describe the effect of the two independent and significative factors on the phlorizin
output, as described by Equation (1), in terms of uncoded units.
Antioxidants 2022, 11, 1933 7 of 15
Phlorizin Concentration = −57.9 + 8.46 A + 0.3209 B – 0.1254 A*A – 0.001609 B*B + 0.00597 A*A
(1)
where factors A and B are the temperature and extraction time, respectively. According
to the process model (Equation (1)), factors A and B affected the phlorizin yield in different
ways. Specifically, temperature (A) was reported in the polynomial Equation 5 times vs.
three times of the extraction time (B), highlighting that the extraction temperature played
a predominant role in influencing the phlorizin yield. Moreover, as described by Equation
(1), the increase in factor B may lead to a decrease in phlorizin yield. It was well accepted
that the extended extraction time can damage the extracted phlorizin and degrade extract
quality [25]. The dominant role of temperature in influencing the polyphenols extraction
rate was largely described [25]. A high extraction temperature indeed decreases the vis-
cosity of the extraction medium, which helps the solvent penetrate the plant matrix, re-
sulting in faster kinetics [26]. Moreover, the increment in solvent temperature may de-
crease the surface tension and, consequently, enhances the wetting of the plant particles,
leading to a higher extraction yield [27] contrastingly, as confirmed by Equation (1), the
temperature value must be kept within some limits, beyond which it determines the deg-
radation of polyphenols. The same effect was also shown by the 3D response surface (Fig-
ure 2a). The predictive model studied indicates that the theoretical condition to achieve
the maximum phlorizin extraction consisted of the hydroalcoholic extraction
(MeOH/H2O, 80:20 + 1% HCOOH), conducted at 37.7 °C for 170 min, as reported in Figure
2. These variables were combined to set up another new extraction from LAA to verify
and confirm the theoretical phlorizin concentration (129.29 mg/g) described by the multi-
ple response prediction analysis.
Figure 2. (a) Surface plot of phlorizin concentration cv time and temperature; and (b) multiple re-
sponse prediction analysis.
Therefore, the experimental phlorizin concentration obtained in the extract, by the
application of these optimized conditions, was 126.89 ± 7.579, with an EA of 101.89%. Be-
cause of the low absolute error values achieved by the comparison between observed and
predicted values, the proposed model may be used to predict the experimental value.
3.2. Quantitative Polyphenols Analysis by HPLC-DAD-FLD
Chromatographic analysis for the quantification of OAALE polyphenolic composi-
tion was performed as previously described in Section 2.3. The HPLC-DAD-FLD analysis
resulted in the identification and quantification of 15 different selected phenolic com-
pounds, counting flavanols, procyanidins, phenolic acids, and flavonols. The obtained re-
sults are reported in Table 2. As expected, phlorizin and phloretin were some of the most
abundant and representative polyphenols contained in OAALE. Beyond dihydrochal-
cones, the second most representative class of polyphenols In OAALE were flavanols.
Quercetin-3-O-glucoside and Kaempferol-3-O-glucoside reached a valuable concentra-
tion in OAALE of 3.27 and 20.09 mg/g, respectively. Similarly, Othman et al. reported a
Antioxidants 2022, 11, 1933 8 of 15
relevant flavanol content in the apple leaf extract. Moreover, among the flavanols detected
by the same researchers, quercetin-3-O-rhamnoside was the most abundant polyphenolic
compound in extracts obtained from the leaves of different apple cultivars. Chlorogenic
acid was the major phenolic acid detected in OCE, followed by caffeic acid, 0.209 and
0.0785 mg/g of dry weight, respectively. Additionally, other studies related to apple leaf
extracts also reported chlorogenic acid as the most abundant phenolic acid [28]. As re-
gards the dimeric procyanidin content, a higher amount was observed for procyanidin B2
(0.454 mg/g), followed by procyanidin B1 and B3. Our results are in line with other evi-
dence about the procyanidin B2 as the most abundant procyanidin compound in apple
leaf extract [29].
Table 2. Quantitative analysis of OAALE determined by HPLC-DAD-FLD analysis.
Compound
Mean Value ± SD (mg/g)
Chlorogenic acid
0.2090 ± 0.0040
Caffeic acid
0.0785 ± 0.0013
p-Cumaric acid
0.0081 ± 0.0001
Procyanidin B1+B3
0.1634 ± 0.0003
Procyanidin B2
0.4540 ± 0.0080
Epicatechin
0.2000 ± 0.0037
Rutin
0.3510 ± 0.0010
Quercetin-3-O-glucoside
3.2740 ± 0.0010
Kaempferol-3-O-rhamnoside
0.1680 ± 0.0070
Kaempferol-3-O-glucoside
20.0970 ± 0.3820
Apigenin-7-O-glucoside
0.0081 ± 0.0001
Phloridzin
126.8900 ± 7.5790
Quercetin
0.0152 ± 0.0001
Phloretin
0.8650 ± 0.0070
Values are expressed in mg/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.
3.3. Qualitative Polyphenols Analysis by HPLC-HESI-MS/MS
OCE polyphenolic composition was characterized by HPLC-HESI-MS/MS, as re-
ported in Section 2.3. Based on a comparison with the literature data, 23 compounds were
putatively identified (Table 3). Compound 1 showed a [M-H]- ion at m/z 197. The base
peak ion at m/z 182 [M-H-CH3]− and the fragment ions of its tandem mass spectrum at m/z
179 [M-H-H2O]−, m/z 153 [M-H-CO2]− and m/z 138 [M-H-CO2-CH3]−, suggested the pres-
ence of a carboxylic acid, a methoxy and a phenolic group. According to the mass frag-
mentation pattern, compound 1 was identified as syringic acid [30]. Compounds 2 and 4
displayed a [M-H]− ion at m/z 163 and a base peak ion at m/z 119 [M-H-CO2]−. The fragment
ions at m/z 145 [M-H-H2O]− and at m/z 135 [M-H-CO]− indicated the presence of the hy-
droxycinnamic acid scaffold and a phenol group. In agreement with the literature data,
compounds 2 and 4 were annotated as p-coumaric acid isomers [31]. Compound 3 showed
a [M-H]− ion at m/z 353 and was putatively identified as a caffeoylquinic acid. The base
peak ion at m/z 191 [M-H-CA]− and the fragment ion at m/z 179 [M-H-QA]− were due to
the loss of the caffeic acid and the quinic acid group, respectively. By comparison with an
authentic analytical standard, compound 3 was identified as chlorogenic acid [32]. Caffeic
acid (5) displayed a [M-H]− ion at m/z 179. The base peak ion at m/z 135 [M-H-CO2]− and
the fragment ions at m/z 161 [M-H-H2O]− and m/z 107 [M-H-CO-CO2]− highlighted the
linkage of a carboxylic acid and a phenolic acid. One procyanidin dimer B-type linkage
(6) showed a [M-H]− ion at m/z 577 and a base peak ion at m/z 425 [M-H-C8H8O3]−, due to
the RDA fission. The fragment ions at m/z 451 [M-H-C6H6O3]−, at m/z 289 [M-H-C15H12O6]−
and at m/z 287 [M-H-C15H14O6]− were produced by the HRF and the QM cleavage,
Antioxidants 2022, 11, 1933 9 of 15
respectively. By comparison with the authentic analytical standard, compound 6 was
identified as procyanidin B2 [31]. Compound 7 displayed a [M-H]− ion at m/z 289 and was
putatively identified as epicatechin. The base peak ion at m/z 245 [M-H-C2H4O]− and the
fragment ion at m/z 137 [M-H-C8H8O3]−, due to the RDA fragmentation, were in agreement
with the literature data [33]. Two 4-O-coumaroylquinic acid isomers (compounds 8 and
9) were tentatively identified. They showed a [M-H]− ion at m/z 337 and three fragment
ions at m/z 191 [QA-H]−, at m/z 173 [QA-H-H2O]− and at m/z 163 [M-H-QA]−, due to the
fragmentation of the quinic acid moiety. However, the base peak ion at m/z 173 [QA-H-
H2O]− indicated the linkage between quinic acid and coumaric acid moieties with the 4-
OH group. Therefore, compounds 8 and 9 were putatively identified as 4-O-couma-
roylquinic acid isomers [34]. Four quercetin O-hexoside isomers (compounds 10, 14, 17,
and 20) were putatively detected. They showed an [M-H]- ion at m/z 463 and a base peak
ion at m/z 301 [M-H-Hex]− due to the fragmentation of the hexoside group. The fragments
at m/z 445 [M-H-H2O]− and at m/z 179 [M-H-Hex-C7H6O2]−, due to the RDA fragmentation,
confirmed the presence of the flavonol scaffold and agreed with literature data [35]. Two
quercetin O-rutinoside isomers (compounds 11 and 13) were tentatively identified and
displayed a [M-H]− ion at m/z 609. The base peak ion at m/z 301 [M-H-Glu-Rha]−, due to
the loss of the disaccharide group, and the fragment ions at m/z 463 [M-H-Rha]− and at
m/z 179 [M-H-Glu-Rha-C7H6O2]−, which derived from the RDA fragmentation, were con-
sistent with the literature data [35]. However, compound 13 was identified as rutin by
comparison with the authentic analytical standard. Compound 12 displayed a [M-H]− ion
at m/z 433. The base peak ion at m/z 271 [M-H-Hex]− and the fragment ions at m/z 313 [M-
H-C4H8O4]− and at m/z 151 [M-H-Hex-C8H8O]−, due to the RDA fragmentation, allowed
the identification of the flavanone scaffold. Therefore, compound 12 was annotated as
naringenin O-hexoside [35]. Compound 15 showed a [M-H]− ion at 431. The base peak ion
at m/z 269 [M-H-Hex]− and the fragment ion at m/z 311 [M-H-C4H8O4]− are derived from
the cleavage of the hexoside group and the RDA fragmentation, respectively. Based on
the tandem mass spectrum and by comparison with an analytical standard, compound 15
was identified as apigenin 7-O-glucoside. Compound 16 displayed a [M-H]− ion at m/z 593
and was annotated as kaempferol O-rutinoside. The base peak ion at m/z 285 [M-H-Pent-
Hex]− and the fragment ions at m/z 327 [M-H-Pent-C4H8O4]− and m/z 257 [M-H-Pent-Hex-
CO]− confirmed the linkage of the disaccharide rutinose and the aglycone kaempferol [36].
Compound 18 showed a [M-H]− ion at m/z 433. The base peak ion at m/z 301 [M-H-Pent]-
and the fragment ion at m/z 179 [M-H-Pent-C7H6O2]− allowed us to identify the presence
of the pentoside group and the flavanol scaffold. Based on the tandem mass spectrum,
compound 18 was annotated as quercetin O-pentoside. Quercetin O-rhamnoside (19) dis-
played an [M-H]- ion at m/z 447 and a base peak ion at m/z 301 [M-H-Rha]− for the loss of
the rhamnoside unit. The fragment ions at m/z 429 [M-H-H2O]−, m/z 179 [M-H-Rha-
C7H6O2]− and m/z 151 [M-H-Rha-C8H6O3]− confirmed the presence of the flavanol scaffold
and are consistent with the literature data. Compound 21 displayed a [M-H]− ion at m/z
435. The base peak ion at m/z 273 [M-H-Hex]− and the prominent fragment ion at m/z 167
[M-H-Hex-C7H6O]− indicated the presence of the chalcone scaffold and the linkage of the
hexoside group. Based on these data and by comparison with an analytical standard, com-
pound 21 was identified as phloridzin. Kaempferol 3-O-rhamnoside (22) showed a [M-
H]− ion at m/z 431. Its tandem mass spectrum displayed a base peak ion at m/z 285 [M-H-
Rha]− and two fragment ions at m/z 327 [M-H-C4H8O3]− and m/z 179 [M-H-Rha-C7H6O]−,
due to the loss of the sugar moiety and RDA fragmentation [36]. The identity of compound
22 was confirmed by comparison with the analytical standard. Compound 23 displayed a
[M-H]− ion at m/z 273. Its tandem mass spectrum is characterized by a base peak ion at m/z
167 [M-H-C7H6O]− and a fragment ion at m/z 125 [M-H-C9H8O2]−, which is linked to the
presence of the chalcone moiety. Based on these data and by comparison with an analyti-
cal standard, compound 23 was identified as phloretin [36].
Antioxidants 2022, 11, 1933 10 of 15
Table 3. Polyphenolic composition of OAALE extracts determined by HPLC-HESI–MS/MS analysis.
No.
Compound
Rt (min)
UV–Vis (nm)
m/z
Diagnostic Fragment
Ref.
1
Syringic acid
9.56
210, 260
197
182 [M-H-CH3]−, 179 [M-H-H2O]−,
153 [M-H-CO2]−, 138 [M-H-CO2-CH3]−
[30]
2
Coumaric acid isomer 1
11.17
215, 310
163
145 [M-H-H2O]−, 135 [M-H-CO]−, 119 [M-H-CO2]−
[31]
3
Chlorogenic acid
11.52
215, 295, 325
353
191 [M-H-CA]−, 179 [M-H-QA]−,
173 [M-H-CA-H2O]−, 161 [M-H-QA-H2O]−
[32]
4
Coumaric acid isomer 2
11.71
210, 305
163
145 [M-H-H2O]−, 135 [M-H-CO]−,
119 [M-H-CO2]−, 101 [M-H-CO2-H2O]−
[31]
5
Caffeic acid
11.95
205, 280
179
161 [M-H-H2O]−, 151 [M-H-CO]−,
135 [M-H-CO2]−, 107 [M-H-CO-CO2]−
[32]
6
Procyanidin B2
12.35
210, 295
577
451 [M-H-C6H6O3]−, 425 [M-H-C8H8O3]−, 289 [M-H-
C15H12O6]−, 287 [M-H-C15H14O6]−
[31]
7
4-O-Coumaroylquinic acid isomer 1
12.90
215, 310
337
319 [M-H-H2O]−, 191 [QA-H]−,
173 [QA-H-H2O]−, 163 [M-H-QA]−
[33]
8
4-O-Coumaroylquinic acid isomer 2
13.15
215, 310
337
319 [M-H-H2O]−, 191 [QA-H]−,
173 [QA-H-H2O]−, 163 [M-H-QA]−
[34]
9
Quercetin O-hexoside isomer 1
14.81
255, 355
463
445 [M-H-H2O]−, 301 [M-H-Hex]−,
179 [M-H-Hex-C7H6O2]−, 161 [M-H-Hex-C7H8O3]−
[34]
10
Quercetin O-rutinoside isomer 1
14.94
205, 280, 310
609
591 [M-H-H2O]−, 463 [M-H-Rha]−,
301 [M-H-Glu-Rha]−, 179 [M-H-Glu-Rha-C7H6O2]−
[35]
11
Naringenin O-hexoside
15.19
215, 280, 310
433
415 [M-H-H2O]−, 313 [M-H-C4H8O4]−,
271 [M-H-Hex]−, 151 [M-H-Hex-C8H8O]−
[35]
12
Rutin
15.21
210, 280, 320
609
591 [M-H-H2O]−, 463 [M-H-Rha]−,
301 [M-H-Glu-Rha]−, 179 [M-H-Glu-Rha-C7H6O2]−
[35]
13
Quercetin O-hexoside isomer 2
15.58
255, 355
463
445 [M-H-H2O]−, 343 [M-H-C4H8O4]−,
301 [M-H-Hex]−, 179 [M-H-Hex-C7H6O2]−
[35]
14
Apigenin O-hexoside
15.93
215, 280, 320
431
413 [M-H-H2O]−, 353 [?],
311 [M-H-C4H8O4]−, 269 [M-H-Hex]−
[35]
15
Kaempferol O-rutinoside
16.07
255, 350
593
575 [M-H-H2O]−, 327 [M-H-Pent-C4H8O4]−, 285 [M-H-
Pent-Hex]−, 257 [M-H-Pent-Hex-CO]-
[36]
16
Quercetin O-hexoside isomer 3
16.12
255, 350
463
445 [M-H-H2O]−, 343 [M-H-C4H8O4]−,
301 [M-H-Hex]−, 179 [M-H-Hex-C7H6O2]−
[36]
17
Quercetin O-pentoside
16.74
265, 320
433
415 [M-H-H2O]−, 301 [M-H-Pent]−,
179 [M-H-Pent-C7H6O2]−, 151 [M-H-Pent-C8H6O3]−
[37]
18
Quercetin O-rhamnoside
16.89
255, 345
447
429 [M-H-H2O]−, 301 [M-H-Rha]−,
179 [M-H-Rha-C7H6O2]−, 151 [M-H-Rha-C8H6O3]−
[37]
19
Quercetin O-hexoside isomer 4
17.02
280, 320
463
445 [M-H-H2O]−, 343 [M-H-C4H8O4]−,
301 [M-H-Hex]−, 179 [M-H-Hex-C7H6O2]−
[37]
20
Phloridzin
17.57
220, 285
435
417 [M-H-H2O]−, 273 [M-H-Hex]−, 167 [M-H-C13H16O6]−
[38]
21
Kaempferol 3-O-rhamnoside
18.16
215, 265, 315
431
403 [M-H-CO]−, 327 [M-H-C4H8O3]−, 285 [M-H-Rha]−, 179
[M-H-Rha-C7H6O]−
[36]
22
Quercetin O-rutinoside isomer 2
19.01
220, 280, 320
609
591 [M-H-H2O]−, 463 [M-H-Rha]−, 343 [M-H-Rha-
C4H8O4]−, 301 [M-H-Glu-Rha]−
[36]
23
Phloretin
22.28
220, 285
273
255 [M-H-H2O]−, 167 [M-H-C7H6O]−, 125 [M-H-C9H8O2]−
[36]
3.4. Total Polyphenols and In Vitro Antiradical Activity of OAALE
The antiradical potential of apple leaves, as vegetal matrices [39], considering their
well-accepted relation with diabetes and oxidative stress [40], prompted us to evaluate
the total phenolic content (TPC) and the in vitro antiradical activity of OAALE. Thus, to
obtain a general overview of its total polyphenolic content, Folin–Ciocalteau’s test was
performed on OAALE, resulting in 23.70 ± 1.23 mg GAE/g of Annurca apple leaves
(AALs). As expected, the TPC of AALs was higher than the TPC of Annurca apple f ruit
(AAF), which was 1.94 mg/g of DW of whole fruit (peel and pulp) [41]. The calculation of
antiradical activity was measured by the application of DPPH, ABTS, and FRAP assays
on OAALE, as described in Section 2. Results are reported in Table 4.
Antioxidants 2022, 11, 1933 11 of 15
Table 4. Antiradical activity of AAL extract evaluated by DPPH, ABTS, and FRAP assays.
Antiradical Activity (µmol TE/g AAL DW ± SD)
DPPH Assay
ABTS Assay
FRAP Assay
21.17 ± 2.30
38.82 ± 0.69
34.14 ± 3.83
The results are expressed as µmol TE per gram of AAL. Abbreviations: AALs, Annurca apple
leaves; DPPH, 2,2diphenyl-1-picrylhydrazyl; ABTS, 2,20-azino-bis (3-ethylbenzothiazoline-6-sul-
fonic acid); FRAP, ferric reducing antioxidant power; TE, Trolox equivalent, DW, dry weight. Val-
ues are mean ± standard deviation (SD) of three repetitions.
Regarding the antiradical activity, OAALE has shown a higher relevant activity com-
pared to AAF (antiradical activity, respectively, of 0.048 for ABTS, 0.01559 for DPPH, and
0.0266 µmol TE/g DW for FRAP) [41]. Moreover, in order to standardize the results of the
various activities studied, the results of DPPH and ABTS assays were also calculated as
EC50, which is the quantity of antioxidants necessary to decrease the concentration of the
initial solution by 50% [21]. Figure 3 reported that the OAALE extract exhibited an EC50
of 0.828 mg/mL for the DPPH assay and 0.542 mg/mL for the ABTS assay. Therefore, these
results would support the relevant potential application of OAALE as a source of anti-
radical agents, with the indubitable benefit of re-evaluating food waste. It is noteworthy
that increasing evidence from in vitro and clinical trials indicates that oxidative stress may
play a relevant role in the pathogenesis of diabetes. High levels of free radicals, and the
concomitant decrease in antioxidant defense mechanisms, may lead to the injury of bio-
logical structures, which is recognized as the main pathological origin for the generation
and development of diabetes-related complications [40].
Figure 3. Antiradical activity of OAALE expressed as (a) EC50 of DPPH assay and (b) EC50 of ABTS
assay. Values represent the mean ± standard deviation of triplicate reading.
3.5. In Vitro Antidiabetic Activity
Increasing evidence has identified the formation of advanced glycation end-products
(AGEs) as a major pathogenic risk agent related to hyperglycemia and diabetes-related
complications. It is also well known that the continuous AGEs accumulation in tissues
and organs is directly linked to the development of chronic diabetic-related complications,
such as retinopathy, nephropathy, neuropathy, and macrovascular disease [15,42]. AGEs
are proteins or fats combined with blood sugars after exposure to a glycation process
through the Maillard reaction [43]. These compounds are extremely and negatively stable
and resistant to enzymatic activities, resulting in their relevant accumulation in different
tissues, which may cause a remarkable morphological change in cell tissue, with a contin-
uous deterioration of tissue structure and the alteration of their physiological function
[15,42]. Therefore, the concentration-dependent inhibition of AGEs formation after the
treatment with OAALE was evaluated, with the results reported in Figure 4. The calcu-
lated IC50 value was 0.330 mg/mL. In this regard, phlorizin and phloretin may be
Antioxidants 2022, 11, 1933 12 of 15
considered the main actors of OAALE potential valuable biological activity. In support of
this hypothesis, these two molecules represent the main polyphenolic components of
OAALE and, as reported in other studies, both of them demonstrated the inhibition of
AGEs formation in a concentration-dependent manner, at a concentration range of 0.01–
1.0 mM [44].
In this context, although dihydrochalcones were the most abundant molecules in
OAALE, we also showed a valuable concentration of other classes of polyphenols (as re-
ported in Sections 3.2 and 3.3), which may contribute to the inhibition of AGEs formation.
Specifically, polyphenol antiglycation properties are due to their capacity to stop the for-
mation of a principal precursor of the Maillard reaction, the methylglyoxal (MGO) [45].
While phenolic acids and flavanols (e.g., gallic acid, p-coumaric acid, and epicatechin)
described a direct inhibition mechanism by a reduction in the carbonyl groups of MGO,
an indirect reaction with an MGO dicarbonyl moiety was reported [45,46]. Based on such
a consideration, the IC50 of 0.330 mg/mL would be attributed to the synergic action of di-
hydrochalcones and other polyphenols contained in OAALE.
Figure 4. Inhibition of advanced glycation end-product formation (%) by OAALE. Values represent
the mean ± standard deviation of triplicate readings.
4. Conclusions
The previously described results indicate that AAL could be considered an excellent
by-product source of bioactive compounds, especially phlorizin. Notably, the optimiza-
tion of the extraction protocol conducted using the RSM methodology allowed us to eval-
uate the maximum extractable phlorizin amount contained in AAL (126.89 mg/g). The
extract obtained under optimized conditions (OAALE) was also chemically characterized
and its in vitro potential biological activity was tested. The promising results about the
antioxidant activity and the inhibition of AGEs formation may suggest that AALs are a
powerful functional ingredient, useful for the formulation of nutraceutical products for
the management of diabetes disease. Further investigations about the beneficial potential
exerted by the formulation in a diabetes model are required to assess the effective appli-
cation in the management of this pathological condition. In addition, future perspectives
include the possibility of performing a toxicological analysis aiming to exclude the possi-
ble residues of the means used to treat apple trees.
Author Contributions: Coceptualization, M.M., V.P., G.C.T. and E.N.; methodology M.M., V.P., F.I.
and E.S.; Softwere V.P.; validation, M.M., V.P., E.S. and F.I.; formal analysis, M.M. and V.P.; inves-
tigation, M.M., V.P. and R.C., resources, G.C.T. and V.S.; data curation, M.M., V.P. and E.S., writing,
M.M., V.P., E.S. and G.C.T.; visualization, E.N. and V.S.; supervision, G.C.T., E.N. and V.S.; project
administration G.C.T., E.N. and V.S., funding acquisition G.C.T. All authors have read and agreed
to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Antioxidants 2022, 11, 1933 13 of 15
Informed Consent Statement: Not applicable.
Data Availability Statement: The data used to support the findings of this study are included in
this article.
Acknowledgments: The assistance of the staff is gratefully appreciated.
Conflicts of Interest: The authors declare no conflict of interest.
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