plants

Article
Eggplant Peels as a Valuable Source of Anthocyanins:
Extraction, Thermal Stability and Biological Activities
Nina-Nicoleta Condurache (Lazăr) 1,† , Constantin Croitoru 2,† , Elena Enachi 1 , Gabriela-Elena Bahrim 1 ,
Nicoleta Stănciuc 1 and Gabriela Râpeanu 1, *

1 Faculty of Food Science and Engineering, Dunarea de Jos University of Galati, 111 Domneasca Street,
E Building, 800201 Galati, Romania; [email protected] (N.-N.C.); [email protected] (E.E.);
[email protected] (G.-E.B.); [email protected] (N.S.)
2 Academy of Agricultural and Forestry Sciences, 61 Marasti Blvd, 011464 Bucharest, Romania;
[email protected]
* Correspondence: [email protected]; Tel.: +33-613-0177
† Both authors contributed equally to this work.

Abstract: This study aimed to use eggplant peels as a potential source of anthocyanins with biological
activities. Two different extraction methods were tested in order to obtain extracts with a high
anthocyanin content. The selected methods were the solid–liquid extraction (SLE) and ultrasound-
assisted extraction (UAE) methods. For each method, two concentrations of ethanol (EtOH) were
used, while varying the extraction time and temperature. Based on the results, the extracts obtained by
SLE using EtOH 96% after 30 min of extraction at 50 ◦ C showed the highest anthocyanin concentration.
The UAE allowed the best results with EtOH 96% after 30 min at 25 ◦ C. Both selected extracts showed





 similar chromatographic profiles, with delphinidin 3-O-rutinoside as the major anthocyanin, but in a
higher concentration in UAE. The extracts also presented inhibitory activity against lipoxygenase
Citation: Condurache (Lazăr), N.-N.;
(LOX), lipase, and α-amylase, thus suggesting a possible involvement in reducing the risk of various
Croitoru, C.; Enachi, E.; Bahrim, G.-E.;
Stănciuc, N.; Râpeanu, G. Eggplant
disorders. The first order kinetic model was used to predict the changes that can occur in the
Peels as a Valuable Source of anthocyanin content and antioxidant activity from the eggplant peel extract. The calculated kinetic
Anthocyanins: Extraction, Thermal and thermodynamic parameters confirm the irreversible degradation of phytochemicals.
Stability and Biological Activities.
Plants 2021, 10, 577. https://doi.org/ Keywords: eggplant peels; bioactive compounds; anthocyanins; extraction methods; thermal degra-
10.3390/plants10030577 dation; biological activity

Academic Editor: Enrico Doria

Received: 22 February 2021 1. Introduction

Accepted: 17 March 2021
Eggplant fruits (Solanum melongena L.) are widely consumed around the world due to
Published: 18 March 2021
their generous composition of nutraceuticals [1]. In recent years, many researchers concen-
trated their work on the biorecovery of valuable compounds from plant by-products. [2].
Publisher’s Note: MDPI stays neutral
The by-products that come from the processing of vegetables and fruits are rich in both
with regard to jurisdictional claims in
published maps and institutional affil-
primary and secondary metabolites. A special interest from the academic community and
iations.
industry is given to the secondary ones [1]. These compounds comprise terpenes, alkaloids,
and phenolic compounds [3]. Phenolic compounds have gained great attention due to
their properties, which can make them beneficial against a variety of disorders. Metabolic
syndrome (MS) is among them and refers to a series of cardio-metabolic disorders, such as
obesity, insulin resistance, dyslipidemia, and hypertension. Moreover, polyphenols can be
Copyright: © 2021 by the authors.
involved in reducing the risk of MS due to the high antioxidant activity they exert, which
Licensee MDPI, Basel, Switzerland.
can trigger other defense mechanisms [1]. However, their ability to cross the intestinal
This article is an open access article
distributed under the terms and
barrier and to enter into the blood system is low. To achieve any health effect, they must be
conditions of the Creative Commons
absorbed into the circulatory system and delivered to the appropriate location within the
Attribution (CC BY) license (https://
body [4].
creativecommons.org/licenses/by/ Similar to other vegetables and fruits, eggplants contain a wide range of phenolics.
4.0/). For example, the eggplant pulp is the richest source of phenolic acids from all vegetables

Plants 2021, 10, 577. https://doi.org/10.3390/plants10030577 https://www.mdpi.com/journal/plants

Plants 2021, 10, 577 2 of 16

that belong to the Solanaceae family. The eggplant’s leaves are a good source of kaempferol.
In the peels are found several types of compounds, although the most important are the
anthocyanins [1]. The purple color of the eggplant peels is given by the glycosides of
delphinidin [5,6].
Different in vitro and in vivo studies have shown the importance of anthocyanins.
Their antioxidant activity could provide antioxidative stress [7] and anti-inflammatory ef-
fects [8], protection against UV radiation playing a key role in skin aging, antidiabetic [9,10]
and antimicrobial activity [11], and anticancer and antitumor effects [12–15].
Anthocyanins are found in the outer layers of eggplants. The fact that they can bind
to sugars and proteins or that each plant matrix has a different composition makes their
extraction difficult [16]. The recovery of phenolic compounds from different raw materials
or agro-industrial by-products can be performed by traditional extraction methods. In
general, these methods are characterized by a low efficiency due to the prolonged heat
treatment [5]. This could cause the degradation of the sensitive compounds and also induce
energy, time, and solvent consumption, as explained by Barba et al. [17]. The so-called
assisted methods, such as ultrasounds, have been recently used extensively to preserve
the sensitive compounds. The main advantages provided by these techniques are high
extraction efficiency, high quality of the final product at a lower temperature, and less
energy/solvent consumption [17].
Despite all the benefits that anthocyanins can provide, they can degrade, easily turn-
ing into colorless or insoluble brown pigments. There are various factors affecting the
anthocyanins’ stability, including temperature. Heat treatment is an industrial process
applied to extend and preserve the shelf life of products and usually leads to significant
losses of the phenolic compounds [18].
In the literature, solid–liquid extraction (SLE) and ultrasound-assisted extraction
(UAE) methods have been used to extract the anthocyanins from many plant materials,
even eggplant peels. However, there are no reported data on the comparison between
the SLE and UAE of eggplant peel anthocyanins. In this regard, SLE and UAE methods
were comparatively used in our study to obtain a high-quality extract, in terms of high
anthocyanin content and biological activities. For each extraction method, different sol-
vent concentrations, times, and temperatures were used. The extracts were analyzed in
terms of total anthocyanin content (TAC) and antioxidant activity. Further, the selected
extracts with the highest anthocyanin content were analyzed by High-Performance Liquid
Chromatography (HPLC) to identify and characterize the major compounds. Additionally,
the extracts were analyzed for their inhibitory activity against lipase, α-amylase, and LOX,
enzymes associated with MS. A precise understanding of the kinetic and thermodynamic
parameters is necessary to predict the nutritional and functional changes during industrial
processing. Therefore, the extract with the highest TAC was thermally treated, and the
kinetic and thermodynamic parameters were calculated.

2. Results and Discussion

2.1. The SLE Method
SLE is among the most widely used and easiest methods that consists of direct ex-
traction of the plant material with a suitable solvent. This type of extraction involves the
phenolics mass transfer from the cell walls into the extraction solvent [16]. Temperature,
solvent concentration, or time could influence the extraction efficiency. To extract the
anthocyanins from the eggplant peels with the SLE method, two different EtOH concen-
trations (70% and 96%) acidified with 1N hydrochloric acid (HCl) were used. The EtOH
was chosen due to the recovery rate of the phenolic compounds, its nontoxicity, and its
suitability for polar compounds. Because every matrix has a distinct composition, and
phenolics have variable solubility characteristics, it is necessary to investigate the solvent
concentration efficiencies. Additionally, the mixture of solvent and water can decrease
solvent consumption [16]. In order to select the most suitable extraction parameters, two
temperatures (25 and 50 ◦ C) and different extraction times (30, 60, and 120 min) were tested.
Plants 2021, 10, 577 3 of 16

The extraction of anthocyanins is also affected by its duration. The extraction yield may
increase as time increases, or it may cause the degradation of anthocyanins mainly due
to oxidation. Additionally, high temperatures can improve the extraction efficiency by
increasing the solubility, diffusion coefficients, and mass transfer rate of the compounds.
However, high temperatures combined with a longer extraction time can lead to phenolic
degradation [16].
Table 1 presents the extracts’ phytochemical content obtained by the parameter vari-
ation. According to our results, it could be appreciated that certain parameters had a
significant effect on the anthocyanin extraction (p < 0.05). Although different studies have
claimed that pure EtOH has a lower extraction yield compared to aqueous EtOH (50% and
70%) [5], our results showed the opposite. The highest TAC was obtained for the extraction
with EtOH 96% at 50 ◦ C (Table 1a). This could be explained by the fact that the acid strength
combined with the solvent concentration and the temperature resulted in stronger cell
wall breaking. The other extracts also presented high TAC. The increased temperature was
positively associated with a higher TAC for both EtOH concentrations (p < 0.05). However,
it can be noticed that there is no significant influence of the extraction time (p > 0.05) on
TAC, regardless of the temperature and solvent concentrations. Hosseini et al. [19] tested
different EtOH concentrations and reported higher TAC by the conventional extraction
of anthocyanins from eggplant peels with water, EtOH, and HCl (50:48:2 ratio) than with
the concentrated EtOH. Boulekbache-Makhlouf et al. [20] reported lower eggplant peel
anthocyanin content after using EtOH 70% acidified with 0.2% formic acid.

Table 1. The total anthocyanin content (TAC) and antioxidant activity of the extracts obtained with solid–liquid extraction
(SLE) and ultrasound-assisted extraction (UAE) methods by varying the extraction parameters.

(a) TAC, mg D3G/g dw

EtOH 70% EtOH 96%
Samples t, min
25 ◦ C 50 ◦ C 25 ◦ C 50 ◦ C
30 0.84 ± 0.05 a*1 1.01 ± 0.08 a#1 0.93 ± 0.06 a*2 1.11 ± 0.08 a#1
SLE 60 0.84 ± 0.07 a*1 0.96 ± 0.09 a#1 0.92 ± 0.09 a*1 1.13 ± 0.10 a#2
120 0.87 ± 0.03 a*1 1.02 ± 0.09 a#1 1.01 ± 0.10 a*2 1.06 ± 0.06 a#1
15 0.91 ± 0.08 A*1 0.87 ± 0.05 A*1 0.98 ± 0.09 A*1 0.79 ± 0.07 A#2 •
UAE 30 0.90 ± 0.08 A*1 0.93 ± 0.09 A*1 1.04 ± 0.10 A*2 0.75 ± 0.07 A#2
45 0.94 ± 0.09 A*1 0.90 ± 0.08 A*1 0.97 ± 0.07 A*1 0.95 ± 0.09 B*1
(b) Antioxidant Activity, mM TE/g dw
EtOH 70% EtOH 96%
Samples t, min
25 ◦ C 50 ◦ C 25 ◦ C 50 ◦ C
30 25.34 ± 1.88 a*1 39.36 ± 2.87 a#1 31.89 ± 3.02 a*2 34.92 ± 3.41 a*2
SLE 60 28.80 ± 2.42 b*1 39.85 ± 3.19 a#1 30.96 ± 2.81 a*1 38.97 ± 1.75 b#1
120 27.67 ± 1.35 ab*1 38.17 ± 3.47 a# 29.75 ± 2.04 a*1 32.61 ± 2.5a*2
15 34.31 ± 0.42 A*1 32.10 ± 1.99 A*1 22.46 ± 1.01 A*2 32.64 ± 2.44 A#1
UAE 30 32.74 ± 0.38 B*1 33.54 ± 0.63 A#1 20.64 ± 1.93 A*2 32.86 ± 1.76 A#1•
45 32.59 ± 0.76 B*1 32.10 ± 1.99 A*1 21.69 ± 0.99 A*2 32.65 ± 1.34 A#1
The influence of extraction time was highlighted using lowercase and uppercase letters for SLE and UAE, respectively, in a column. The
influence of extraction temperature and solvent concentration was highlighted using symbols (* , # ) and digits, respectively, in lines. The
differences between the extraction methods were highlighted using symbols ( , •) in a column. Values that share a letter/symbol/digit
are not significantly different (p > 0.05). TAC = total anthocyanin content; EtOH = ethanol; t = time; D3G = delphinidin 3-O-glucoside;
TE = Trolox Equivalent; dw = dry weight.

Phenolic compounds from eggplants are known for their in vitro free radical scav-
enging activity. Table 1b presents the extracts’ antioxidant activities obtained after the
SLE parameters combination. The highest antioxidant activity corresponds to the extracts
obtained with EtOH 70% at 50 ◦ C. Probably, the polarity of EtOH and water combined with
the temperature facilitates the extraction of phenolics, thus providing a high antioxidant
activity to the extract. However, the other extracts also presented high antioxidant activities.
The extraction with EtOH 70% was significantly influenced by the temperature, with higher
results being obtained at a higher temperature (p < 0.05). In contrast, for the EtOH 96%, the
Plants 2021, 10, 577 4 of 16

temperature presented a significant influence only after 60 min of extraction (p < 0.05). At
25 ◦ C, EtOH 70% allowed significantly higher results after 60 min of extraction than 30 min
(p < 0.05). The same behavior was observed at 50 ◦ C with EtOH 96%. Our results are in
agreement with those reported by other authors. Hosseini et al. [19] obtained a higher
antioxidant activity for an eggplant peel extract after using EtOH, water, and HCl in a
50:48:2 ratio at room temperature rather than with pure EtOH.

2.2. The UAE Method

The UAE represents a faster alternative to conventional extraction methods. This
technique is based on the energy derived from sound waves with frequencies higher than
20 kHz [21]. Table 1a presents the phytochemicals extracted with two EtOH concentrations
at 25 and 50 ◦ C and different treatment times. The highest TAC was acquired by the
extraction at 25 ◦ C with EtOH 96%. The solvent concentration significantly influenced the
TAC after 30 min of extraction, EtOH 96% extracting higher contents than EtOH 70% for
both temperatures (p < 0.05). The extracts obtained with EtOH 96% at 50 ◦ C presented a
significantly lower TAC compared to those at 25 ◦ C (p < 0.05). The time did not significantly
affect the extraction (p > 0.05). The results exhibited that the exposure for 30 min to
ultrasounds is enough to extract a high content of anthocyanins. Horincar et al. [22] also
reported lower TAC values from an eggplant peel extract obtained with EtOH 70% acidified
with 1N HCl after 30 min at 40 ◦ C. Dumitrascu et al. [23] reported higher TAC for higher
EtOH concentrations at 50 ◦ C after 40 min of phenolic compound extraction from cornelian
cherry fruits.
As in the SLE case, using EtOH 70% led to extracts with a significantly higher antioxi-
dant activity at 25 ◦ C (p < 0.05). Thus, the highest antioxidant activity was obtained with
70% EtOH after 15 min of extraction at 25 ◦ C (Table 1b). At 50 ◦ C, the EtOH concentration
did not significantly influence the antioxidant activity (p > 0.05). The temperature increase
led to a significant increase in the antioxidant activity only for the extracts obtained with
EtOH 96%, regardless of the time, but also for the extract with EtOH 70% after 30 min of
extraction (p < 0.05). Time significantly influenced the antioxidant activity only for the
extract with EtOH 70% at 25 ◦ C (p < 0.05). Compared to our results, Horincar et al. [22]
obtained lower antioxidant activity by eggplant peels ultrasound extraction using EtOH
70% acidified with 1N HCl for 30 min at 40 ◦ C.
Both extraction methods led to the highest anthocyanin contents using EtOH 96%
(Table 1a). At 25 ◦ C, there were no significant differences between the TAC obtained with
both methods after 30 min (p > 0.05). However, at 50 ◦ C, SLE extracted significantly higher
TAC than UAE (p < 0.05) after 30 min. SLE extracts obtained with EtOH 70% at 50 ◦ C and
with 96% at 25 ◦ C presented higher antioxidant activities than those obtained by UAE
(Table 1b). On the contrary, UAE extraction with EtOH 70% at 25 ◦ C provided extracts with
significantly higher antioxidant activity than SLE (p < 0.05).
By comparison, at the same EtOH concentration, the use of UAE brings the advantage
of halving the temperature, which enables energy savings and reduces the risk of phenolic
degradation. Authors such as Caldas et al. [24] reported higher phenolic compound
recovery from grape skin in a shorter time with UAE than SLE.
Based on the TAC obtained, the extracts chosen for further experiments were the
following: the one obtained by SLE using EtOH 96% after 30 min of extraction at 50 ◦ C and
the one obtained by UAE using EtOH 96% after 30 min at 25 ◦ C.

2.3. HPLC Analysis of the Anthocyanins

In order to characterize the anthocyanin profile from the extracts obtained by the two
extraction methods, the analysis of the chromatographic profile was performed by HPLC
(Figure 1).
 literature. The identification wavelength was 520 nm. The chromatographic profile re-
Plants 2021, 10, 577 5 of 17
vealed the presence of five compounds: delphinidin 3-O-rutinoside-5-glucoside (Figure
2a), delphinidin 3-O-glucoside (Figure 2b), delphinidin 3-O-rutinoside (Figure 2c), cya-
nidin 3-O-rutinoside (Figure 2d), and petunidin 3-O-rutinoside (Figure 2e). Three of the
Plants 2021, 10, 577
2.3. HPLC Analysis ofwere
compounds the Anthocyanins
assessed based on the available standards, while the other two com- 5 of 16
Inpounds
order towere
characterize the anthocyanin
presumptively identifiedprofile
basedfrom thedata
on the extracts obtained
reported byscientific
in the the two litera-
extraction
turemethods,
[5,25]. the analysis of the chromatographic profile was performed by HPLC
(Figure 1). The anthocyanins from the eggplant peel extracts were identified based on the
retention time and by comparison with the available standards and existing data in the
literature. The identification wavelength was 520 nm. The chromatographic profile re-
vealed the presence of five compounds: delphinidin 3-O-rutinoside-5-glucoside (Figure
2a), delphinidin 3-O-glucoside (Figure 2b), delphinidin 3-O-rutinoside (Figure 2c), cya-
nidin 3-O-rutinoside (Figure 2d), and petunidin 3-O-rutinoside (Figure 2e). Three of the
compounds were assessed based on the available standards, while the other two com-
pounds were presumptively identified based on the data reported in the scientific litera-
ture [5,25].

(a) (b)
Figure
Figure1.1.Chromatographic
Chromatographic profile of of
profile eggplant peel
eggplant extracts
peel obtained
extracts by SLE
obtained withwith
by SLE EtOH 96%96%
EtOH for 30
formin at 50at
30 min (a)◦ C
°C50 and
(a)
UAE with EtOH 96% for 30 min at 25 °C (b):
◦ Peak 1—delphinidin 3-O-rutinoside-5-glucoside; Peak 2—delphinidin
and UAE with EtOH 96% for 30 min at 25 C (b): Peak 1—delphinidin 3-O-rutinoside-5-glucoside; Peak 2—delphinidin 3-O-
glucoside; Peak 3—delphinidin 3-O-rutinoside; Peak 4—cyanidin 3-O-rutinoside; Peak 5—petunidin 3-O-rutinoside.
3-O-glucoside; Peak 3—delphinidin 3-O-rutinoside; Peak 4—cyanidin 3-O-rutinoside; Peak 5—petunidin 3-O-rutinoside.
The anthocyanins from the eggplant peel extracts were identified based on the re-
tention time and by comparison with the available standards and existing data in the
literature. The identification wavelength was 520 nm. The chromatographic profile re-
vealed the presence of five compounds: delphinidin 3-O-rutinoside-5-glucoside (Figure 2a),
(a) delphinidin 3-O-glucoside (Figure 2b), delphinidin (b)3-O-rutinoside (Figure 2c), cyanidin
3-O-rutinoside (Figure 2d), and petunidin 3-O-rutinoside (Figure 2e). Three of the com-
Figure 1. Chromatographic profile of eggplant peel extracts obtained by SLE with EtOH 96% for 30 min at 50 °C (a) and
pounds were assessed based on the available standards, while the other two compounds
UAE with EtOH 96% for 30 min at 25 °C (b): Peak 1—delphinidin 3-O-rutinoside-5-glucoside; Peak 2—delphinidin 3-O-
were presumptively identified based on the data reported in the scientific literature [5,25].
glucoside; Peak 3—delphinidin 3-O-rutinoside; Peak 4—cyanidin 3-O-rutinoside; Peak 5—petunidin 3-O-rutinoside.

(a) (b)

(a) (b)

(c) (d)

(c) (d)

Figure 2. Cont.
Plants 2021, 10, 577 6 of 16
6 of 17

(e)
Figure 2. Chemical structure of Figure
the anthocyanin glycosides
2. Chemical structureidentified in the eggplant
of the anthocyanin peel extracts:
glycosides identified in the eggplant peel extracts:
delphinidin 3-O-rutinoside-5-glucoside (a); delphinidin 3-O-glucoside (b); delphinidin 3-O-rutino-
delphinidin 3-O-rutinoside-5-glucoside (a); delphinidin 3-O-glucoside (b); delphinidin 3-O-rutinoside
side (c); cyanidin 3-O-rutinoside (d), and petunidin 3-O-rutinoside (e). [26].
(c); cyanidin 3-O-rutinoside (d), and petunidin 3-O-rutinoside (e). [26].

The anthocyanins from the eggplant

The SLEpeel extracts
extract were1a)
(Figure identified
showedbased on the reten-
the following relative percentage concentra-
tion time and by comparisontionwithtothe
the concentration of total anthocyanins: in
available standards and existing data the litera- 3-O-rutinoside-5-glucoside
delphinidin
ture. The identification wavelength
(9.35%), was 520 nm.3-O-glucoside
delphinidin The chromatographic profile revealed
(9.72%), delphinidin 3-O-rutinoside (73.55%), cyanidin
the presence of five compounds: delphinidin 3-O-rutinoside-5-glucoside
3-O-rutinoside (1.97%), and petunidin 3-O-rutinoside(Figure 3a), del- The UAE extract showed
(0.42%).
phinidin 3-O-glucoside (Figure 3b), delphinidin 3-O-rutinoside (Figure 3c), cyanidin 3-O-
the followed concentrations of anthocyanins (Figure 1b): delphinidin 3-O-rutinoside-5-
rutinoside (Figure 3d), and petunidin
glucoside3-O-rutinoside (Figure
(7.30%), delphinidin 3e). Three of(12.18%),
3-O-glucoside the compounds
delphinidin 3-O-rutinoside (75.34%),
were assessed based on the cyanidin
available3-O-rutinoside
standards, while the other
(1.47%), two compounds
and petunidin were (0.54%). As such, the quan-
3-O-rutinoside
presumptively identified based on the
titative data reported
analysis followedinthe
the scientific
use literaturecalibration
of the standard [5,25]. curves for the three aforemen-
tioned compounds, such as delphinidin 3-O-glucoside, that presented a concentration of
4 mg/100 g dw for the SLE and 5 mg/100 g dw for the UAE; delphinidin 3-O-rutinoside
as the major anthocyanin presented a concentration of 157 mg/100 g dw for the SLE and
562 mg/100 g dw for the UAE, whereas cyanidin 3-O-rutinoside presented a content lower
than 1 mg/100 g dw for both of the analyzed samples. Our results are in agreement with
Ferarsa et al. [5] and Dranca and Oroian [25] who also identified five anthocyanins in
different eggplant peel extracts, with delphinidin 3-O-rutinoside as the major one.

2.4. In Vitro Enzymes Inhibition by Eggplant Peels Extract

LOX catalyzes the formation of hydroperoxides from polyunsaturated fatty acids,
which leads to several inflammation-related disorders, such as MS [27]. High levels of
pancreatic lipase in the human body are responsible for pancreatic disorders and obesity
(a) (b)
due to their function in catalyzing the hydrolysis of triglycerides into fatty acids. Various
researchers have demonstrated in vivo and in vitro that polyphenols can decrease and even
inhibit the enzymatic activity of lipase in the human body if they are absorbed in the intesti-
nal tract [28,29]. α-amylase is among the enzymes associated with diabetes that catalyze the
hydrolysis of carbohydrates into simple sugars. A therapeutic approach that can decrease
hyperglycemia is to stop the production and/or absorption of glucose by inhibiting the
responsible enzymes [30]. Phenolic compounds are positively associated with reducing the
risk of MS by inhibiting the responsible enzymes. Cyanidin 3-O-glucoside, delphinidin
3-O-glucoside, and petunidin 3-O-glucoside are known to inhibit cyclooxygenase enzyme
activities related to various disorders [31]. Metformin hydrochloride is the most commonly
used synthetic α-amylase inhibitor in the treatment of type 2 diabetes [32]. Orlistat is the
(c) only lipase inhibitor available on the market
(d) involved in the fight against obesity [33]. The
side effects of these drugs led to research for natural alternatives with lesser side effects.
These two drugs were used as positive controls in our study.
In our study, the in vitro effects of the selected eggplant peel extracts on the enzymes
associated with several disorders were assessed in three enzymes (LOX, lipase, and α-amylase)
and three extract concentrations. The inhibitory data are summarized in Tables 2 and 3.
Plants 2021, 10, 577 7 of 16

Table 2. The enzyme inhibition percentage of SLE and UAE selected extracts and positive controls in LOX, lipase, and
α-amylase enzymes at different concentrations.

LOX Lipase α-Amylase

Sample Concentration, mg/mL
Inhibition, % Inhibition, % Inhibition, %
1 50.87 ± 0.84 a 25.35 ± 0.35 a 58.58 ± 2.09 a
SLE 0.5 49.52 ± 0.35 d 23.31 ± 1.22 c 56.73 ± 1.36 bc
0.1 46.59 ± 1.81 g 22.29 ± 093 e 47.51 ± 1.22 d
1 53.64 ± 0.45 b 26.25 ± 1.04 a 58.02 ± 1.01 a
UAE 0.5 52.95 ± 0.75 e 25.34 ± 0.68 c 54.26 ± 1.57 c
0.1 49.13 ± 3.38 g 24.42 ± 1.72 e 48.50 ± 2.89 d
1 84.98 ± 3.28 c - -
Quercetin 0.5 79.37 ± 0.60 f - -
0.1 77.32 ± 1.20 h - -
1 - 34.70 ± 2.59 b -
Orlistat 0.5 - 32.19 ± 0.68 d -
0.1 - 30.82 ± 0.68 f -
1 - - 60.68 ± 0.83 a
Metformin
0.5 - - 58.91 ± 1.57 b
hydrochloride
0.1 - - 56.36 ± 1.01 e
Values from a column for a similar concentration that share a letter are not significantly different (p > 0.05). Measurements are expressed as
mean ± SD of triplicates. SLE—solid/liquid conventional extraction; UAE—ultrasound-assisted extraction; LOX—lipoxygenase.

Table 3. The enzyme inhibition results (IC50 values; µg/mL) of SLE and UAE selected extracts in LOX, lipase, and α-amylase
enzymes.

Sample IC50 (µg/mL)

LOX Lipase α-amylase
SLE 2.80 ± 0.68 a 3.31 ± 0.24 a 10.72 ± 1.06 a
UAE 2.83 ± 0.44 a 2.59 ± 0.24 a 7.27 ± 0.23 b
Quercetin 7.81 ± 0.66 b - -
Orlistat - 1.23 ± 0.09 b -
Metformin hydrochloride - - 4.31 ± 0.48 c
SLE 2.80 ± 0.68 a 3.31 ± 0.24 a 10.72 ± 1.06 a
Values from a column that share a letter are not significantly different (p > 0.05). Measurements are expressed as mean ± SD of triplicates.
SLE—solid/liquid conventional extract; UAE—ultrasound-assisted extract; LOX-lipoxygenase.

The extracts obtained by the two tested methods presented moderate enzyme inhibi-
tion activities. UAE extract showed significantly higher in vitro LOX inhibitory percentages
than SLE extract for all tested concentrations (p < 0.05). However, the IC50 values of both
extracts presented no significant differences (p > 0.05). When compared to the positive
control, both extracts presented significantly lower in vitro LOX inhibitory percentages
and IC50 values (p < 0.05). Both extracts showed similar in vitro lipase and α-amylase
inhibitory percentages, as shown in Tables 2 and 3. Compared with orlistat used as a
standard, all extracts were found to show significantly weak lipase inhibitory activity
(p < 0.05). However, when compared to metformin hydrochloride used as a standard for
α-amylase, there were significant differences only at 0.1 mg/mL concentration (p < 0.05).
Therefore, the eggplant peel extracts showed LOX, lipase, and α-amylase inhibition activity.
This suggests that by increasing the bioavailability of anthocyanins from the eggplant peels
in the human body, they might be involved in reducing glucose and lipids metabolism.
Our results are in agreement with Kwon, Apostolidis, and Apostolidis [34], who reported
moderate to high α-amylase inhibition capacity for different eggplant extracts. Mojica,
Berhow, and Gonzalez de Mejia [35] also reported that the anthocyanins from black beans
inhibit α-glucosidase and α-amylase.
Based on the HPLC chromatographic profiles obtained and biological activities, the
extract chosen for further experiments was the one obtained by UAE using EtOH 96% after
30 min at 25 ◦ C.
 Mojica, Berhow, and Gonzalez de Mejia [35] also reported that the anthocyanins from
black beans inhibit α-glucosidase and α-amylase.
Plants 2021, 10, 577 Based on the HPLC chromatographic profiles obtained and biological activities, 8the
of 16
extract chosen for further experiments was the one obtained by UAE using EtOH 96%
after 30 min at 25 °C.

2.5.
2.5. Thermal
Thermal Degradation
Degradation
Anthocyanins
Anthocyanins areare responsible
responsible forfor the
the purple
purple color
color ofofthe
theeggplant
eggplantpeels,
peels,which
whichare are
highly susceptible to degradation during processing, resulting in color changes.
highly susceptible to degradation during processing, resulting in color changes. To eval- To evaluate
thethe
uate degradation
degradationbehavior
behavior of the phenolics
of the phenolics from eggplant
from peel
eggplant extract,
peel a thermal
extract, treatment
a thermal treat-
was performed at different temperature–time combinations. The
ment was performed at different temperature–time combinations. The combinations combinations tested
caused changes in TAC and antioxidant activity (data not shown, p < 0.05)
tested caused changes in TAC and antioxidant activity (data not shown, p < 0.05) except except for the
antioxidant activity at 90 ◦ C (Figure 3a,b). The thermal processing techniques used in the
for the antioxidant activity at 90 °C (Figure 4a,b). The thermal processing techniques used
inindustry usually
the industry have have
usually an impact on theon
an impact structures of the of
the structures phenolic compounds,
the phenolic influencing
compounds, in-
their bioavailability and bioactivities [18].
fluencing their bioavailability and bioactivities [18].

2 230
80°C 80°C
TAC, mg D3G/g dw

Antioxidant activity,
1.5 90°C 220 90°C

mM TE/g dw
1 100°C 210 100°C
110°C 110°C
0.5 200
120°C 120°C
0 190
130°C 130°C
0 10 20 30 0 10 20 30
Time, min Time, min
(a) (b)
Figure
Figure4. 3.
Degradation
Degradationofoftotal
totalanthocyanin
anthocyanincontent—TAC
content—TAC(a) (a)and
andantioxidant
antioxidant activity
activity (b)
(b) in
in eggplant peel extract
eggplant peel extract treated
treatedat
atdifferent
differenttemperatures;
temperatures;D3G=
D3G=delphinidin
delphinidin3-O-glucoside;
3-O-glucoside;TE=
TE= Trolox
TroloxEquivalent;
Equivalent;dw—dry
dw—dryweight.
weight.

InIn
our
ourstudy
study atat
8080
°C,◦ C,
the TAC
the TACdecreased
decreasedbyby20% 20%after
after3030min
minwhenwhencompared
comparedwith with
the
theuntreated
untreated extract (Figure
extract (Figure 4a).
3a).AtAt130
130 ◦
°C,C, the TAC
the TACdecreased
decreasedbyby59% 59%after after3030min.
min.The The
antioxidant
antioxidant activity
activity was
wasless heat
less heat sensitive,
sensitive,with
with a reduction
a reductionofof2.8%
2.8%and and 12% 12% after
after3030min
min
ofof
treatment
treatment atat
8080and
and130
130 ◦ C,
°C, respectively
respectively (Figure
(Figure 4b). InIn
3b). the
thewhole
whole temperature
temperature and
and time
time
range
range studied,
studied, a similar trend
a similar trend was
was observed
observed forforTAC
TAC and
andantioxidant
antioxidant activity.
activity.OurOurresults
results
are
are inin agreement
agreement with
with different
different studies.
studies. AA sequential
sequential reduction
reduction inin
thethe phytochemicals
phytochemicals and
and
the antioxidant activity from a sour cherry extract was also reported
the antioxidant activity from a sour cherry extract was also reported by Oancea et al. [36] by Oancea et al. [36]
ininthe
thewhole
wholetemperature
temperaturerange rangetheytheystudied.
studied.InIn contrast,
contrast, Slavu
Slavu et etal.al.[37]
[37]reported
reportednono
significant
significant heat-induced
heat-induced changes
changes inin
thethe total
total anthocyanin
anthocyanin content
content from
from the thepurple
purplemazemaze
extract ◦ C.
extract inin the
the temperature
temperature range
range ofof8080
toto
110110°C.

2.5.1.
2.5.1. Kinetic
Kinetic Analysis
Analysis
Kineticmodels
Kinetic modelsare
are often
often used
usedtotoassess
assessthethe
influence of processing
influence on the
of processing oncritical quality
the critical
parameters
quality of products
parameters [38]. For
of products the For
[38]. eggplant peel extract,
the eggplant peel the kinetic
extract, theparameters at constant
kinetic parameters
pH = 2.18 and temperatures ranging from 80 to 130 ◦ C for 0 to 30 min were studied by spec-
at constant pH = 2.18 and temperatures ranging from 80 to 130 °C for 0 to 30 min were
trophotometric
studied analysis. The linear
by spectrophotometric relation
analysis. between
The linear the logarithm
relation between oftheTAC and antioxidant
logarithm of TAC
activity and time indicated that the degradation followed the first-order reaction kinetics. The
and antioxidant activity and time indicated that the degradation followed the first-order
kinetic parameters describing the heat-induced changes are the degradation rate (1/min) and
reaction kinetics. The kinetic parameters describing the heat-induced changes are the deg-
degradation energy of activation (Ea) and are presented in Table 4.
radation rate (1/min) and degradation energy of activation (Ea) and are presented in Table
The k values for TAC increased with increasing temperature, suggesting a high thermal
4.
sensibility at higher temperatures. In the antioxidant activity case, the k values were lower
when compared to the TAC, but also increasing with the temperature from 80 to 130 ◦ C.
Our results are in agreement with other authors who have also reported the first-order
kinetic model for the degradation of phenolics. Turturica et al. [39] reported an accelerated
degradation effect on the TAC from plum extracts determined by the temperature increase
in the 70–110 ◦ C range. Qiu et al. [40] noticed that k values increased with the temperature
in the 50–80 ◦ C range after the thermal treatment of purple potato extract. Similar to our
study, Oancea et al. [41] noticed that the k values for the antioxidant activity of elderberry
Plants 2021, 10, 577 9 of 16

extract were lower when compared with the TAC thermal degradation, but also increasing
with the temperature.

Table 4. Estimated kinetic parameters of bioactives in eggplant peel extract.

Compounds T, ◦ C K × 102 , min−1 R2 t1/2, min Ea, kJ/mol

80 0.74 ± 0.001 0.87 93.67 ±1.05
90 0.79 ± 0.004 0.90 87.74 ± 0.78
100 1.53 ± 0.002 0.97 45.30 ± 0.44
TAC 34.63 ± 3.59
110 1.59 ± 0.001 0.94 43.59 ± 4.84
120 2.42 ± 0.003 0.94 28.64 ± 0.33
130 2.91 ± 0.01 0.99 23.82 ± 0.71
80 0.10 ± 0.001 0.95 693.14 ± 4.87
90 0.08 ± 0.001 0.86 866.43 ± 4.76
Antioxidant 100 0.16 ± 0.02 0.86 433.21 ± 5.21
37.24 ± 3.24
activity 110 0.22 ± 0.001 0.89 315.06 ± 3.50
120 0.31 ± 0.04 0.92 223.59 ± 1.51
130 0.38 ± 0.001 0.91 182.40 ± 1.46
TAC—total anthocyanin content; k—the degradation rate constant; t1/2—the half-life of degradation; Ea—the activation energies;
R2 —determination coefficient; T—temperature.

The t1/2 values of TAC and antioxidant activity of eggplant peel extract decreased
with increasing the heating temperature (Table 4) and were determined from the k values.
Therefore, the higher the k value, the shorter the t1/2 value. The anthocyanins were the
most susceptible to thermal degradation. This may be due to the anthocyanin oxidation or
cleavage of covalent bonds during heat treatment. The antioxidant activity instead had
the highest half-life, regardless of the heating temperature. Our results for the t1/2 at
80 ◦ C are higher than those reported by Turturica et al. [39] at the same temperature for
a plum extract regarding the TAC and antioxidant activity. Slavu et al. [37] also reported
lower values of t1/2 than ours for the TAC after thermally treating a purple maze extract
at 130 ◦ C. On the contrary, Oancea et al. [36] obtained higher t1/2 values than ours for a
thermally treated sour cherry extract at 130 ◦ C, regarding the TAC and antioxidant activity.
All the authors reported a higher susceptibility of anthocyanins to thermal degradation
than other compounds.
The activation energy helps to characterize the temperature dependence of the pa-
rameters. This parameter allows a deeper understanding of the thermal degradation
kinetics used to describe the required energy to reach the active state of a reaction. The
degradation constants fitted to the Arrhenius equation, and the resulting Ea is shown in
Table 4. Higher Ea values suggest that a smaller temperature change is required to faster
degrade the compounds. Our results for the Ea of the TAC are similar to those reported by
Turturica et al. [39] for a plum extract, but lower than those reported by Oancea et al. [36]
for a sour cherry extract and by Slavu et al. [37] for a purple maze extract.

2.5.2. Thermodynamic Parameters

Table 5 provides the results for the activation enthalpy (∆H), the Gibbs free energy of
inactivation (∆G), and the activation entropy (∆S) for the degradation of the anthocyanins
and antioxidant activity from the eggplant peel extract at each temperature tested.
The activation enthalpy describes the endothermic state between the activated complex
and the reactant. It is a measure of the energy barrier that must be overcome by the reacting
molecules, related to the strength of the bonds that are broken and remade during the
transition state [42]. The ∆H values calculated at different temperatures (Table 5) were
similar. Considering that the ∆H values are positive, we can assume that the bioactive
degradation was an endothermic reaction.
Plants 2021, 10, 577 10 of 16

Table 5. Thermodynamic parameters obtained for phytochemical degradation in eggplant peel extract.

Compounds T, ◦ C ∆H, kJ/mol ∆G, kJ/mol ∆S, J·mol−1 ·K−1

80 31.69 ± 1.03 113.36 ± 9.56 −231.34 ± 11.05
90 31.61 ± 0.40 116.46 ± 11.61 −233.74 ± 18.80
100 31.53 ± 1.20 117.70 ± 10.98 −231.08 ± 15.21
TAC
110 31.44 ± 1.10 120.82 ± 9.03 −233.35 ± 11.55
120 31.36 ± 3.01 122.68 ± 9.43 −232.37 ± 15.13
130 31.28 ± 0.10 125.27 ± 11.26 −233.23 ± 17.01
80 34.30 ± 1.72 119.23 ± 12.68 −240.60 ± 14.67
90 34.22 ± 2.01 123.37 ± 15.49 −245.59 ± 14.76
100 34.13 ± 1.99 124.70 ± 10.71 −242.80 ± 15.82
Antioxidant activity
110 34.05 ± 1.53 127.12 ± 11.35 −242.98 ± 13.35
120 33.97 ± 1.67 129.40 ± 12.99 −242.82 ± 11.01
130 33.89 ± 0.91 132.09 ± 11.47 −243.69 ± 11.89
TAC—total anthocyanin content; ∆H—activation enthalpy; ∆G—Gibbs inactivation free energy; ∆S—activation entropy; T—temperature.

The Gibbs free energy of inactivation represents the difference between the activated
state and the reactants state and describes its equilibrium and spontaneity [42]. The ∆G
values calculated (Table 5) were positive for all temperatures tested, demonstrating that
the bioactive degradation reaction is not spontaneous.
The activation entropy describes the change in the disorder of a system’s molecules [42].
The ∆S values calculated for each temperature (Table 5) were negative. These values demon-
strate that the molecules from the transition state are more organized than at the beginning
of the reaction, and the thermal degradation process is irreversible. Thus, the formation of
the activated complex is associated with a decrease in entropy values.

3. Materials and Methods

3.1. Chemicals
EtOH from Titolchimica (Pontecchio Polesine, Italy), HCl, HPLC purity methanol
(MeOH), Folin-Ciocalteau reagent (FC), potassium chloride solution (KCl), sodium acetate
solution (CH3 COONa), 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent, lipoxidase from
Glycine max (soybean) type I-B, 50,000 units/mg protein, pancreatin lipase (111.5 units/mg
protein), α-amylase from porcine pancreas (type I-A, 700–1400 units/mg protein), sodium
phosphate buffer solution (PBS), linoleic acid (≥99%), p-nitrophenyl palmitate, Arabic
gum, Triton X-100, starch solution, dinitrosalicylic acid (DNS), quercetin ≥95% (HPLC),
orlistat ≥ 98%, and 1,1-Dimethylbiguanide hydrochloride (metformin hydrochloride) were
purchased from Sigma Aldrich (Steinheim am Albuch, Germany). The standards used for
the HPLC analysis, delphinidin 3-O-glucoside, delphinidin 3-O-rutinoside, and cyanidin
3-O-rutinoside, were purchased from Sigma Aldrich (Steinheim am Albuch, Germany) as
primary reference standards.

3.2. Sample Preparation

The fresh fruits of an autochthonous eggplant (Solanum melongena L.) variety of a dark
purple color with an elongated shape, named “Dragaica”, were purchased from a local
market in Galat, i, Romania, in 2019. The fruits were washed, and the peels were removed
with a knife in strips of uniform thickness. Subsequently, the peels were washed with
ultrapure water, dried with paper towels, and frozen. The drying process was carried
out with a CHRIST Alpha 1-4 LD plus equipment (Osterode am Harz, Germany), at
−42 ◦ C, under a pressure of 0.10 mBar, for 48 h until a 90% dw. The freeze-dried peels
were grounded with MC 12 equipment, produced by Stephan (Germany), and stored
in a plastic container with a lid in darkness at room temperature. The variability of the
environmental conditions, such as the relative humidity and temperature, was set at the
maximum possible. The dried material was analyzed in a maximum of 2 days.
Plants 2021, 10, 577 11 of 16

3.3. The SLE Method

SLE was performed as a conventional method to extract the polyphenols from the
eggplant peels [24]. The EtOH was used as a solvent in 70% and 96% concentrations. Based
on previous preliminary results (data not shown), the 1N HCl was chosen to acidify the
solvent in a 4:1 ratio of solvent to acid. The extractions took place under stirring at 25 and
50 ◦ C, for 30 to 120 min, on an orbital shaker (SI-300R Medline Scientific, Chalgrove, UK)
at 150 rpm. The solid-to-liquid ratio was set at 1:15 of plant material to solvent based on
previous preliminary results (data not shown). Afterward, the samples were centrifuged
using a Hettich Universal 320R, Germany, for 10 min at 14,000 rpm and 4 ◦ C, and the
supernatant was phytochemically analyzed.

3.4. The UAE Method

UAE was carried out as the Dranca and Oroian [25] method stated but with slight
modifications. The method used EtOH 70% and 96% acidified with 1N HCl (4:1 ratio) at
25 and 50 ◦ C, for 15 to 45 min, in a Smart sonic cleaner ultrasonic bath (MRC. Ltd., Holon,
Israel). Previously, the samples were centrifuged for 10 min at 14,000 rpm and 4 ◦ C, and
the supernatant was further analyzed.

3.5. Characterization of Extracts

The TACs were quantified using the pH-differential method [43], and the results were
expressed in milligrams of delphinidin 3-O-glucoside per gram of dry weight of peels (mg
D3G/g dw) ±SD. The experiment was performed in triplicate, using a 0.025 M KCl buffer
at pH = 1.0 and 0.4 M CH3 COONa buffer at pH = 4.5. The absorbances were measured
at 520 and 700 nm with a double-beam UV-VIS spectrophotometer with data analysis
software (JENWAY, UK). The TAC was assessed according to Equation (1).

TAC (mg D3G/g dw) = (A × MW × DF)/ε × L (1)

where, A = (A520 nm − A700 nm) pH 1.0 − (A520 nm − A700 nm) 4.5 (2)
MW—molecular weight of delphinidin 3-O-glucoside (465 g/mol); DF—dilution
factor; ε—the molar absorptivity of delphinidin 3-O-glucoside (29,000 L × mol−1 × cm−1 );
L—the optical path (1 cm).

3.6. In Vitro Antioxidant Activity

To measure the in vitro antioxidant activity, DPPH (2,2-diphenyl-1-picrylhydrazyl)
free radical was used, and the results were expressed as millimoles Trolox Equivalent
per gram of dry weight of the peels (mM TE/g dw) ±SD [39]. A volume of 100 µL
of the extract was mixed with 3.9 mL of DPPH stock solution. Afterward, the mixture
was kept in darkness at room temperature for 30 min. The absorbance was recorded at
λ = 515 nm against the blank with a double-beam UV-VIS spectrophotometer with data
analysis software (Jenway, Staffordshire, UK). The results were calculated based on the
Trolox calibration curve (n = 3), using Equation (3).

Antioxidant activity (mM TE/g dw) = (CE × V × d)/m (3)

CE—concentration in the calibration curve; V—stock volume; d—sample dilution;

m—sample mass taken into analysis.

3.7. HPLC Analysis of the Anthocyanins

The anthocyanin chromatographic separation and identification were achieved by the
method described by Condurache et al. [44]. A Thermo Finnigan Surveyor HPLC system
coupled with a DAD detector and controlled by the Xcalibur software (Finnigan Surveyor
LC, Thermo Scientific, USA) was used. In order to separate and identify the anthocyanins
from the eggplant peels, a Synergi 4u Fusion-RP 80A (150 × 4.6 mm, 4 µm) column was
Plants 2021, 10, 577 12 of 16

used at an oven temperature of 25 ◦ C, at 520 and 280 nm wavelengths. The samples were
pre-filtered using a C18 Sep-Pack cartridge, in order to remove any unwanted compounds
other than the targeted ones. Afterward, they were filtered through 0.22 µm syringe filters
(Bio Basic Canada Inc., Markham ON, Canada). The solvents used for the elution step
were 10% formic acid (A) and 100% methanol (B) under the following gradient conditions:
0–20 min, 9–35% (A); 20–30 min, 35% (A); 30–40 min, 35–50% (A); and 40–55 min, 50–90%
(A). The injection volume was 10 µL at a flow rate of 1.0 mL/min. The identification and
separation of the compounds were achieved based on their standard curves (delphinidin
3-O-glucoside, delphinidin 3-O-rutinoside, and cyanidin 3-O-rutinoside), retention times,
and the data reported in the literature by Ferarsa et al. [5] and Dranca and Oroian [25]. All
the used standard compounds presented a purity of ≥95.0% and were bought from Sigma
Aldrich (Germany).
Further, the extracts were concentrated until to dryness under reduced pressure at
40 ◦ C (AVC 2-18, Christ, UK) and stored at 4 ◦ C in Falcon tubes. The dried extracts were
used for the following analysis in a maximum of 2 days.

3.8. In Vitro Enzymes Inhibition Capacity of Eggplant Peel Extracts

3.8.1. In Vitro LOX Inhibition Assay
The bioassay for the LOX inhibition was performed according to Costamagna et al. [45],
with minor modifications. Briefly, 50 µL of 1, 0.5, and 0.1 mg/mL of extract diluted in
ultrapure water was added to 50 µL of LOX solution (1 mg/mL in 0.1 M PBS, pH = 9.0).
After 5 min of incubation at room temperature, 50 µL of 0.05 mM linoleic acid dissolved in
0.1 M PBS (pH = 9.0) was added, and the mixture was incubated for 20 min at 37 ◦ C. Further,
the samples were diluted with 2 mL of 0.1 M PBS at pH = 9.0, and the absorbances were
read at 234 nm in quartz cuvettes with a double-beam UV-VIS spectrophotometer with data
analysis software (Jenway, UK). Quercetin was used as a standard inhibitor. The inhibitory
effect of the extracts was expressed as the IC50 value and the percentage of enzyme
activity inhibition. The IC50 value (µg/mL) represents the extract concentration at which
50% of the enzyme activity is inhibited and was determined graphically by plotting the
logarithmic concentration versus the inhibition percentage. The enzyme activity inhibition
was calculated using the following equation:

% inhibition = (A0 − As )/A0 × 100 (4)

A0 is the absorbance of the control sample without extract; As is the absorbance of the
sample with the extract.

3.8.2. In Vitro Lipase Inhibition Assay

The lipase inhibitory effect of the eggplant peels extract was also estimated using
the method described by Costamagna et al. [45], with minor modifications. A volume of
50 µL of 1, 0.5, and 0.1 mg/mL extract was added to 50 µL of pancreatin lipase solution
(1 mg/mL in 0.1 M PBS, pH = 8.0). After 5 min of incubation at room temperature, 50 µL of
the substrate obtained from 0.01 M p-nitrophenyl palmitate, Arabic gum, and Triton x-100
was added, and the mixture was incubated for 20 min at 37 ◦ C. Further, the samples were
diluted with 1 mL of 0.1 M PBS at pH = 8.0, and the absorbances were read at 400 nm with a
double-beam UV-VIS spectrophotometer with data analysis software (Jenway, UK). Orlistat
was used as the standard inhibitor. The inhibitory effect of the extracts was expressed as
IC50 and as the percentage of enzyme activity inhibition, calculated using Equation (4).

3.8.3. In Vitro α-Amylase Inhibition Assay

In order to assess the α-amylase inhibitory effect of the eggplant peel extract, 100 µL
of 1, 0.5, and 0.1 mg/mL samples was added to 100 µL of α-amylase solution (1 mg/mL in
0.1 M PBS, pH 6.9). After 5 min of incubation at room temperature, 100 µL of soluble starch
1% solution dissolved in distilled water and previously boiled for 5 min was added to each
tube and incubated for another 20 min at 37 ◦ C. Finally, in each test tube, 200 µL of DNS
Plants 2021, 10, 577 13 of 16

reagent was added, followed by heating at 100 ◦ C for 5 min in a thermostatic water bath
(Digibath-2 BAD 4, Raypa Trade, Barcelona, Spain). Further, the samples were diluted with
2 mL of distilled water, and the absorbance was read at 540 nm with a double-beam UV-VIS
spectrophotometer with data analysis software (Jenway, UK). Metformin hydrochloride
was used as the standard inhibitor. The inhibitory effect of the extracts was expressed as
IC50 and as the percentage of enzyme activity inhibition, calculated using Equation (4) [45].

3.9. TAC and Antioxidant Activity Thermal Degradation

3.9.1. Heat Treatment
For the kinetic studies, glass tubes were filled with 2 mL of the extract solution
(10 mg/mL in ultrapure water, pH = 2.18) and heated in the 80 to 130 ◦ C temperature
range for 10 to 30 min, using a digital heating block (Stuart, UK). The parameters were
chosen considering the pasteurization and sterilization temperatures and times. After the
thermal treatment, the tubes were cooled in a mixture of ice and water to prevent further
degradation. Finally, the TAC and antioxidant activity of the thermally treated extract were
analyzed using the spectrophotometric methods described above.

3.9.2. Mathematical Models and Kinetic Analysis

The degradation kinetics of TAC and antioxidant activity was described by fitting the
experimental data to the first-order kinetic model (Equation (5)):

C/C0 = e − kt (5)

where C0 and C are the TAC or antioxidant activity before and after thermal treatment
(mg/g dw), respectively; t is the heating time (min); and k is the degradation rate constant
at T temperature.
Starting from Equation (5), the following kinetic parameters were calculated: the
degradation rate constant (k), the half-life of degradation (t1/2), and the activation energies
(Ea), as described by Slavu (Ursu) et al. [37].

3.9.3. Thermodynamic Parameters

The activation enthalpy (∆H), the Gibbs free energy of inactivation (∆G), and the
activation entropy (∆S) at each thermal treatment temperature were calculated as suggested
by Radu (Lupoae) et al. [46].

3.10. Statistical Analysis of Data

The statistical analysis of data was performed using the IBM SPSS 2020 Statistics
Software. The differences between the samples were assessed by the Tukey test with the
One-way ANOVA method for the data that followed the normal distribution and equal
variances conditions. For the other ones, the Kruskal–Wallis and Dunn’s nonparametric
tests were used. All the experiments were expressed as average values with a standard
deviation of triplicates.

4. Conclusions
Two different methods (SLE and UAE) were evaluated in terms of the best extraction
yield and high quality of extracts. Two different concentrations of EtOH were chosen, at
different extraction times and temperatures. Both extraction methods presented a high
anthocyanin content at the same EtOH concentration (96%) and time (30 min). The chro-
matographic profile revealed the presence of five compounds: delphinidin 3-O-rutinoside-5-
glucoside, delphinidin 3-O-glucoside, delphinidin 3-O-rutinoside, cyanidin 3-O-rutinoside,
and petunidin 3-O-rutinoside. The anthocyanin with the highest concentration from each
extract was delphinidin 3-O-rutinoside, with a concentration 3.5 times higher in UAE
extract than in SLE extract. Both extracts presented moderate in vitro inhibitory activity in
LOX, lipase, and α-amylase. This fact suggests that the phenolics from the eggplant peel
extracts might be involved in reducing the occurrence of various disorders when there
Plants 2021, 10, 577 14 of 16

is a high bioavailability in the human body. However, UAE inhibited the enzymes in a
significantly higher percentage than SLE (p < 0.05). Thereby, UAE is preferable to SLE due
to the lower temperature used, higher delphinidin 3-O-rutinoside content, and enzymes
inhibitory activity. The degradation data of eggplant peel anthocyanins and antioxidant
activity follow the first-order reaction kinetics, allowing an accurate prediction of the ther-
mal degradation kinetic parameters. The kinetic and thermodynamic parameters showed
that the temperature increase had to accelerate the degradation effect, demonstrating a
higher temperature dependence of anthocyanins and antioxidant activity. This information
is useful for optimizing the industrial processing conditions in order to minimize losses in
phenolic compounds. The eggplant peel extracts have an outstanding potential to be used
as paint ingredients, curing agents, and textile dyes, etc., though stability is among the
main challenges that must be overcome. To overcome the limitations of the use of eggplant
peel anthocyanins, alternatives, such as encapsulation, can allow loss minimization and
increase the compounds’ half-life. In this regard, more research will be further addressed.

Author Contributions: Conceptualization, N.-N.C. and C.C.; methodology, N.-N.C. and C.C.; soft-
ware, N.-N.C. and E.E.; validation, N.S. and G.R.; formal analysis, N.-N.C., C.C., and E.E.; investiga-
tion, N.-N.C. and C.C.; resources, G.-E.B., G.R., and C.C.; data curation, N.-N.C.; writing—original
draft preparation, N.-N.C.; writing—review and editing, G.R. and N.S.; visualization, G.-E.B.; super-
vision, N.S.; project administration, G.-E.B.; funding acquisition, G.-E.B. and G.R. All authors have
read and agreed to the published version of the manuscript.
Funding: This work was supported by project number PN-III-P1-1.2-PCCDI-2017-0569-PRO-SPER
(10PCCI), within the PNCDI program.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data that support the findings of this study are available from the
corresponding author, [G.R.], upon reasonable request.
Acknowledgments: This work was supported by the Integrated Center for Research, Expertise and
Technological Transfer in Food Industry (Bioaliment TehnIA), which is acknowledged for providing
technical support.
Conflicts of Interest: The authors declare no conflict of interest.

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