antioxidants

Article
Valorization of Aloe vera Skin By-Products to Obtain Bioactive
Compounds by Microwave-Assisted Extraction: Antioxidant
Activity and Chemical Composition
Ignacio Solaberrieta , Alfonso Jiménez and María Carmen Garrigós *

Department of Analytical Chemistry, Nutrition & Food Sciences, University of Alicante, San Vicente del Raspeig,
ES-03690 Alicante, Spain; solaberrieta@ua.es (I.S.); alfjimenez@ua.es (A.J.)
* Correspondence: mc.garrigos@ua.es; Tel.: +34-965-903-529

Abstract: Aloe vera skin (AVS) is a major by-product of Aloe processing plants all over the world.
In this study, response surface methodology was used to optimize microwave-assisted extraction
(MAE) of bioactive compounds from AVS. The influence of extraction parameters, such as ethanol
concentration (%Et), extraction temperature (T), time (t) and solvent volume (V), on extraction yield
(Y), total phenolic content (TPC), antioxidant activity (DPPH and FRAP methods) and aloin content,
was studied. Optimum extraction conditions were determined as 80% ethanol, 80 ◦ C, 36.6 min
and 50 mL and optimized extracts showed interesting contents of polyphenols and antioxidant
performance. The phenolic profile was determined by HPLC-DAD/MS and some major phenolic
compounds, such as aloin A, aloin B, aloesin, aloe-emodin, aloeresin D, orientin, cinnamic acid and
chlorogenic acid, were quantified while eight other compounds were tentatively identified. Moreover,
structural and thermal properties were studied by FTIR and TGA analyses, respectively. The obtained
Citation: Solaberrieta, I.; Jiménez, A.; results suggested the potential of AVS as a promising source of bioactive compounds, thus increasing
Garrigós, M.C. Valorization of Aloe the added value of this agricultural waste.
vera Skin By-Products to Obtain
Bioactive Compounds by Microwave- Keywords: Aloe vera; waste valorization; microwave-assisted extraction (MAE); antioxidant activity;
Assisted Extraction: Antioxidant phenolic profile; Box-Behnken design (BBD)
Activity and Chemical Composition.
Antioxidants 2022, 11, 1058. https://
doi.org/10.3390/antiox11061058

Academic Editors: Silvana Hrelia, 1. Introduction

Cristina Angeloni and Maria In recent decades, growing environmental concerns combined with the imminent
Cristina Barbalace depletion of fossil fuels have encouraged the concept of the circular economy as an envi-
Received: 8 May 2022
ronmentally friendly approach to prevent waste generation in productive processes, as
Accepted: 25 May 2022
well as to develop natural-based products in order to reduce dependence on fuel-based
Published: 26 May 2022
materials [1]. The use of biopolymers or active biomolecules obtained from agricultural
by-products or wastes has gained major importance in many countries around the world
Publisher’s Note: MDPI stays neutral
due to its environmental and economic advantages [2]. Recent studies have reported a
with regard to jurisdictional claims in
wide variety of agrowastes as renewable sources of bioactive compounds or biopolymers
published maps and institutional affil-
that are currently underexploited, such as Aloe vera skin [3,4], cocoa bean shells [5], tomato
iations.
seeds [6] and carob pod bark [7], among many others.
Aloe vera has been associated with curative or healing-promoting properties since an-
cient times. Several studies have demonstrated its anticancer, antiviral, anti-inflammatory,
Copyright: © 2022 by the authors.
antimicrobial and antioxidant activities [8,9]. Pharmaceutical, food and cosmetic industries
Licensee MDPI, Basel, Switzerland. have used Aloe vera in innumerable product formulations which have been continuously
This article is an open access article growing in recent years [10,11]. The most valuable part of the plant is the inner leaf gel,
distributed under the terms and which is used for product manufacturing. In Aloe vera processing plants, the inner gel
conditions of the Creative Commons is generally separated from the external skin, which accounts for over 30% of the total
Attribution (CC BY) license (https:// leaf weight, generating large amounts of waste. In most cases, this agrowaste lacks com-
creativecommons.org/licenses/by/ mercial use and is usually discarded, used as compost or directly incinerated [12]. Even
4.0/). though there is little information about Aloe vera skin compared to the inner gel, which

Antioxidants 2022, 11, 1058. https://doi.org/10.3390/antiox11061058 https://www.mdpi.com/journal/antioxidants

Antioxidants 2022, 11, 1058 2 of 25

has been extensively studied, it has been reported that Aloe vera skin could be a promising
source of bioactive compounds which might be used in food, food packaging or biomedical
applications [4,13,14].
There is a general consensus that biological activities of medicinal plants, including
Aloe vera, which present very complex phytochemical profiles and many constituents,
should be ascribed to the synergistic action of several compounds rather than to a single
molecule. More than 75 active compounds have been identified in Aloe vera, with some
of them, such as polysaccharides, related to wound healing, anti-inflammatory and an-
tidiabetic activities [8,15]. However, the specific roles of other components have not been
completely elucidated yet and some of them are suspected to be responsible for undesirable
effects when consumed. This is the case with aloin, an anthraquinone-C-glycoside which is
present, especially, in the latex of Aloe vera leaves and naturally occurs as a mixture of di-
astereoisomers, aloin A and aloin B [16]. Even though some studies have highlighted some
protective benefits of anthraquinones, such as hepatoprotective, anticancer, antimicrobial
and antioxidant activities [17–19], it has been reported to have a strong laxative effect, and
it might cause other undesirable effects [20,21]. The maximum content of aloin in products
intended for oral consumption is limited by specific regulations such as in North America,
where the International Science Aloe Council has set a limit of 10 ppm of aloin A and B [20].
On the other hand, the European Council Directive 88/388/EEC established an aloin limit
of 0.1 ppm in flavorings for beverages [22].
The extraction of bioactive molecules from plants could be carried out by using a vari-
ety of methods [23]. In recent years, several non-conventional and more environmentally
friendly extraction techniques, which usually lead to higher overall extraction yields with
less time and solvent consumption, have been developed [24,25]. Among them, microwave-
assisted extraction (MAE) has gained importance due to its multiple advantages compared
to conventional extraction techniques [26,27]. The synergistic combination of heat and
mass transfer phenomena, in which both gradients work in the same direction, combined
with volumetric heat dissipation inside the irradiated medium, are the most distinctive
characteristics of MAE [28]. Moreover, it has been reported that this heating mechanism
generates internal pressure into the plant materials, contributing to the rupture of cell walls
and promoting solvent penetration into the vegetal matrix. Therefore, active compounds
could be easily solubilized and extracted [28].
Most plant extracts comprise complex mixtures of different phytochemical constituents
with diverse chemical structures, and their analysis remains challenging. Currently, several
literature reviews on the identification and quantification of active compounds extracted
from plants are available [29–31]. Even though some analytical techniques, such as gas
chromatography and capillary electrophoresis, have been employed, high performance
liquid chromatography (HPLC) combined with mass spectrometry (MS) has been by far
the most frequently used technique for the identification and quantification of secondary
metabolites extracted from plants [32–35]. Successful phenolic profiling strongly relies on
proper optimization of chromatographic conditions, which are crucial for the selection of
adequate mobile phases and gradient elution programs.
MAE has been used to extract bioactive compounds from several agrowastes [36].
However, to the best of our knowledge, no studies have been carried out for the MAE
optimization of bioactive compounds with antioxidant activity from Aloe vera skin, adding
value to this underexploited renewable resource. In this context, this work aimed at
optimizing a new extraction procedure for bioactive compounds from Aloe vera skin in
order to increase the added value of this agricultural waste. The optimal MAE conditions
for maximizing the recovery of active phenolic compounds and the antioxidant activity of
the extracts were determined. The influence of four extraction variables (irradiation time,
extraction temperature, ethanol concentration in the extraction solvent and volume) was
studied and the MAE process was optimized using a Box-Benhken design (BBD). Total
phenolic content (TPC), antioxidant activity by DPPH and FRAP methods and aloin content
in Aloe vera skin extracts were evaluated as the response variables. Moreover, phenolic
Antioxidants 2022, 11, 1058 3 of 25

profile, thermal and structural properties of the optimized Aloe vera skin extract were also
determined by HPLC-DAD/MS, thermogravimetric analysis (TGA) and Fourier transform
infrared spectroscopy (FTIR), respectively. Finally, morphological changes on the AVS
structure due to the MAE process were studied by scanning electron microscopy (SEM).

2. Materials and Methods

2.1. Reagents and Raw Materials
Sodium carbonate, Folin-Ciocalteu reagent (2 N), 2,2-diphenyl-1-picrylhydrazyl (DPPH),
sodium acetate, glacial acetic acid, hydrochloric acid, 2,4,6-tripyridyl-s-triazine (TPTZ),
ferric chloride hexahydrate, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox), ethanol (absolute grade), methanol, acetonitrile (HPLC grade), formic acid
and cinnamic acid were purchased from Sigma Aldrich (Madrid, Spain). Aloin A, aloin B,
orientin, aloeresin D, aloesin and aloe-emodin were acquired from Cymit Química (Barcelona,
Spain). Chlorogenic acid and all other phenolic standards used in this study were purchased
from Extrasynthese (Genay, France). Ultrapure water from a Millipore Milli-Q system was
used (18.2 MΩ·cm at 25 ◦ C).
Fresh Aloe vera leaves, from three-year old plants, were kindly supplied by Las Coronas
(Carnota, Seville, Spain), and they were thoroughly washed with distilled water to remove
the adhering soil particles and other contaminants. Then, leaves were weighted and
measured for their length, thickness and width (Table 1). Subsequently, the base, tip and
spikes were removed, and the epidermis was carefully separated from the inner gel using a
sharp knife. The yield of Aloe vera waste was determined and expressed as a fraction of
the total leaf weight. The resulting Aloe vera skin was intensively washed with distilled
water and the residual gel was removed. Then, it was cut into small pieces and frozen at
−80 ◦ C. Afterwards, the skin was freeze-dried with a Telstar Lyoquest—55 PLUS (Terrassa,
Barcelona, Spain) and ground using a ZM 200 high-speed rotatory mill (Restch, Hann,
Germany). Particles passing through a 1.0 mm sieve were used to ensure the homogeneity
of the sample. Finally, the freeze-dried Aloe vera skin powder (AVS) was vacuum packed
and stored in the darkness until further analysis.

Table 1. Leaf dimensions, Aloe vera waste yield and chemical characterization of Aloe vera skin (AVS).
Results are expressed as mean ± SD.

Leaf Dimensions 1
Length (cm) 64.9 ± 3.7
Width at base (cm) 12.8 ± 0.8
Thickness (cm) 2.8 ± 0.3
Waste yield 1,2
Weight (g) 780 ± 90
Skin waste (%) 15.1 ± 2.1
Chemical characterization of AVS 3
Moisture (g 100 g FW−1 ) 84.9 ± 0.8
Ash (g 100 g DW−1 ) 15.5 ± 0.1
Protein (g 100 g DW−1 ) 6.5 ± 0.2
Lipids (g 100 g DW−1 ) 2.4 ± 0.1
1 n = 100; 2 Expressed as a fraction of the total leaf weight; 3 n = 3. FW: fresh weight; DW: dry weight.

2.2. AVS Characterization

AVS characterization was carried out by following AOAC official methods [37] and
reported works for the analysis of similar plant materials [3,38]. Total content of lipids was
gravimetrically determined after 8 h of Soxhlet extraction using petroleum ether. The crude
protein content was calculated using the Kjeldahl method by multiplying the nitrogen
value by 6.25. Total ash was determined by incineration in a muffle at 550 ◦ C for 4 h. The
Antioxidants 2022, 11, 1058 4 of 25

moisture content was determined after skin separation by freeze-drying. All analyses were
carried out in triplicate.

2.3. Microwave-Assisted Extraction (MAE)

MAE was performed using a Milestone flexiWAVETM microwave oven (Milestone srl,
Bergamo, Italy) in the open vessel configuration, and the solvent was heated and refluxed
through the sample. Ethanol:water mixtures were used as reported efficient solvents for the
extraction of phenolic compounds from plant materials [7,39,40], with ethanol showing low
toxicity [41]. According to preliminary tests, the AVS amount was fixed at 1.5 g. The initial
heating rate was optimized and held constant among the different extraction experiments.
During the extraction process, samples were stirred at 400 rpm and different combinations
of solvent composition (%Et), extraction temperature (T), extraction time (t) and volume
(V) were used according to Table 2. After extraction, the obtained Aloe vera skin extracts
(AVE) were cooled to room temperature and centrifuged at 5300 rpm for 10 min. The
solid residue was washed twice with the extraction solvent and then discarded. Then, the
supernatant was pooled with the washing solvent and stored overnight at −20 ◦ C in order
to remove possible interferences by precipitation. After that, the precipitates were removed
by centrifugation at 5300 rpm and 4 ◦ C for 10 min. The supernatant was collected, and the
ethanol was subsequently evaporated under reduced pressure. Afterwards, the extract was
frozen at −80 ◦ C, freeze-dried until completely dry and finally stored in vacuum-sealed
packs at −20 ◦ C in darkness until further analysis. AVE solutions were freshly prepared,
before analysis, at 500 mg kg−1 in ethanol:water (40%, v/v).

Table 2. Box-Behnken experimental design matrix and response values obtained from Aloe vera skin
extracts using microwave-assisted extraction (MAE).

Experimental Domain Response Variables

Yield TPC DPPH FRAP Aloin
Et T t V
Run (gAVE (mgGAE (mgTE (mgTE (mg
(%, v/v) (◦ C) (min) (mL)
100 gAVS−1 ) gAVE−1 ) gAVE−1 ) gAVE−1 ) gAVE−1 )
1 60 80 22.5 80 24.2 104.9 ± 1.8 59.6 ± 5.1 110.7 ± 3.2 46.1 ± 0.2
2 40 60 5.0 65 24.3 86.5 ± 0.9 51.4 ± 6.1 95.2 ± 3.7 39.2 ± 0.2
3 60 80 22.5 50 18.8 102.8 ± 1.4 65.0 ± 6.3 120.2 ± 4.2 50.3 ± 0.1
4 80 60 22.5 50 18.4 122.4 ± 1.2 68.4 ± 5.3 119.3 ± 3.2 53.0 ± 0.3
5 40 60 22.5 50 22.9 91.1 ± 0.4 53.5 ± 6.2 101.7 ± 1.7 42.0 ± 0.1
6 60 60 40.0 50 22.5 102.4 ± 0.5 58.7 ± 4.9 109.9 ± 3.8 42.7 ± 0.2
7 40 80 22.5 65 25.4 91.0 ± 0.9 61.6 ± 5.2 93.0 ± 2.0 39.0 ± 0.2
8 60 40 22.5 50 21.1 101.3 ± 0.5 54.9 ± 5.9 116.2 ± 3.5 48.2 ± 0.3
9 80 60 22.5 80 18.2 121.3 ± 1.7 61.6 ± 4.5 132.1 ± 0.7 54.2 ± 0.3
10 80 60 40.0 65 18.7 114.9 ± 1.6 59.3 ± 5.0 130.7 ± 3.6 56.6 ± 0.5
11 60 60 22.5 65 24.2 105.4 ± 1.1 58.3 ± 4.2 110.5 ± 5.7 46.9 ± 0.4
12 60 40 40.0 65 23.2 103.6 ± 1.0 54.4 ± 6.5 108.7 ± 4.2 44.5 ± 0.3
13 60 60 22.5 65 23.3 102.2 ± 1.1 57.8 ± 2.1 113.8 ± 3.0 46.1 ± 0.0
14 60 60 5.0 80 23.9 98.5 ± 1.1 55.2 ± 5.5 113.6 ± 3.9 45.4 ± 0.2
15 80 40 22.5 65 18.8 121.4 ± 1.1 68.1 ± 5.1 134.1 ± 2.8 57.5 ± 0.2
16 60 60 22.5 65 23.6 104.0 ± 1.3 55.2 ± 4.9 113.3 ± 4.3 47.1 ± 0.4
17 80 60 5.0 65 20.3 117.9 ± 1.2 64.3 ± 4.3 116.3 ± 5.1 52.9 ± 0.1
18 60 80 40.0 65 22.7 103.6 ± 1.4 62.5 ± 4.4 118.8 ± 1.9 47.1 ± 0.1
19 80 80 22.5 65 19.4 125.8 ± 1.3 73.4 ± 4.6 131.5 ± 6.0 53.3 ± 0.3
20 40 60 40.0 65 25.1 89.3 ± 0.4 49.9 ± 5.4 90.5 ± 2.4 36.5 ± 0.1
21 40 60 22.5 80 26.3 87.4 ± 0.7 50.5 ± 5.6 99.6 ± 2.5 41.0 ± 0.1
22 60 60 5.0 50 21.0 116.3 ± 2.2 62.1 ± 2.9 110.4 ± 1.8 44.8 ± 0.4
23 60 60 22.5 65 24.7 106.4 ± 0.9 58.8 ± 6.1 108.2 ± 1.0 43.5 ± 0.2
24 60 40 22.5 80 23.4 102.7 ± 2.4 53.5 ± 6.2 114.6 ± 5.4 47.6 ± 0.5
25 60 60 22.5 65 24.9 108.7 ± 2.3 59.1 ± 4.7 109.6 ± 9.5 43.0 ± 0.3
26 60 40 5.0 65 22.1 109.4 ± 1.2 61.2 ± 7.0 109.3 ± 0.2 45.6 ± 0.3
27 40 40 22.5 65 24.8 88.8 ± 0.3 53.9 ± 6.2 97.7 ± 3.8 38.4 ± 0.3
Antioxidants 2022, 11, 1058 5 of 25

Table 2. Cont.

Experimental Domain Response Variables

Yield TPC DPPH FRAP Aloin
Et T t V
Run (gAVE (mgGAE (mgTE (mgTE (mg
(%, v/v) (◦ C) (min) (mL)
100 gAVS−1 ) gAVE−1 ) gAVE−1 ) gAVE−1 ) gAVE−1 )
28 60 60 40.0 80 23.8 99.1 ± 0.6 54.1 ± 4.2 119.7 ± 14.1 44.3 ± 0.1
29 60 80 5.0 65 22.8 96.8 ± 1.2 53.4 ± 5.8 106.3 ± 3.5 44.4 ± 0.4
Et: ethanol concentration; T: extraction temperature; t: extraction time; V: solvent volume. AVS: Aloe vera skin;
AVE: Aloe vera extract; GAE: gallic acid equivalents; TE: trolox equivalents.

Box-Behnken Experimental Design (BBD)

MAE optimization of bioactive compounds from AVS was performed by using a BBD
with 29 runs and 5 central points in order to maximize extraction yield, total phenolic
content (TPC) and antioxidant activity (DPPH and FRAP), as well as to minimize aloin
content. The effect of four independent variables (ethanol concentration (%Et: 40–80%, v/v),
temperature (T: 40–80 ◦ C), time (t: 5–40 min) and solvent volume (V: 50–80 mL)) were
investigated at three levels. All experimental runs were performed randomly to minimize
the effect of unexpected variability in the response variables. The range of the studied
independent variables was selected on the basis of preliminary experiments (data not
shown), experimental limitations, constructive characteristics of the used equipment and
previously reported information in the literature.
Response surface methodology (RSM) was used, and the following second-order
polynomial model was applied for regression analysis of the experimental data:

Y = β0 + ∑ βi Xi + ∑ βi X2i + ∑ ∑ βij Xi Xj (1)

where Y represents the predicted response variable; Xi and Xj represent the independent
variables; β0 is a constant coefficient and βi , βii , βij are the regression coefficients of the
linear, quadratic and interaction effect terms, respectively. The lack of fit test and coeffi-
cient of determination (R2 ) were used to determine the adequacy of the model to predict
experimental data. Statistical significance of the model parameters was determined at 5%
probability level (α = 0.05), and graphic analysis of the principal effects and interactions of
independent variables on the studied responses was used.

2.4. AVE Characterization

2.4.1. Extraction Yield
The extraction yield was gravimetrically determined by using the following equation:
mAVE
Yield (%) = 100 (2)
mAVS

where mAVE is the weight of the extract obtained after freeze-drying and mAVS is the weight
of the dried starting material.

2.4.2. Total Phenolic Content (TPC)

The total phenolic content of AVE was determined using the Folin-Ciocalteu assay
according to Lucini et al. [4] with slight modifications. Aliquots (0.5 mL) of each extract
were mixed with 2.5 mL of the Folin-Ciocalteu reagent, previously diluted in distilled water
(1:10, v/v), and added with 2.0 mL of 7.5 wt% aqueous sodium carbonate. Then, the mixture
was vortexed, and the absorbance was recorded at 765 nm after 30 min of incubation at
45 ◦ C in the dark using a Biomate 3 UV-vis spectrophotometer (Thermospectronic, Mobile,
AL, USA). Gallic acid in EtOH:H2 O (40%, v/v) was used as a reference standard for
quantification (5–80 mg kg−1 , R2 = 0.9995). The results were expressed as milligrams of
gallic acid equivalents (GAE) per gram of AVE. Each extract was analyzed in triplicate.
Antioxidants 2022, 11, 1058 6 of 25

2.4.3. Antioxidant Activity

2.4.3.1. DPPH Radical Scavenging Assay
The DPPH scavenging activity of AVE was determined, in triplicate, as described
in previous studies [42–44] with some modifications. Briefly, 0.3 mL of AVE were mixed
with 2.2 mL of a freshly prepared DPPH solution (10−4 mol L−1 in ethanol). The mixture
was vortexed and incubated at room temperature in the dark for 90 min. Preliminary
studies demonstrated that this amount of time was required to accomplish the reaction and
reach the steady state. Then, absorbance was measured at 517 nm against a pure ethanol
blank. Trolox in EtOH:H2 O (40%, v/v) was used as the reference standard for quantification
(5–70 mg kg−1 , R2 = 0.9994). Results were expressed as milligrams of trolox equivalents
(TE) per gram of AVE.

2.4.3.2. FRAP Assay

The ability of AVE to reduce a ferric-tripyridyltriazine complex to its ferrous form
was assessed according to Benzie and Strain [45]. FRAP reagent was prepared by mixing
0.3 mol L−1 acetate buffer (pH = 3.6), 10 mmol L−1 TPTZ made up in 40 mmol L−1 HCl
and 20 mmol L−1 FeCl3 at a 10:1:1 ratio. Then, 0.1 mL of AVE were mixed with 3 mL of
the freshly prepared FRAP reagent pre-warmed at 37 ◦ C. The mixture was vortexed, and
the absorbance was measured at 593 nm after 30 min of incubation at 37 ◦ C in the dark-
ness. Trolox in EtOH:H2 O (40%, v/v) was used as the reference standard (5–100 mg kg−1 )
(R2 = 0.9996). The results were expressed as milligrams of trolox equivalents (TE) per gram
of AVE. Each extract was analyzed in triplicate.

2.4.4. Aloin Content Determination

The total aloin content (A and B stereoisomers) in AVE was determined as described
by Brown et al. [46] with slight modifications. An Agilent series 1260 Infinity Quaternary
HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a diode array
detector was used. Chromatographic separation was achieved with a Teknokroma Brisa
LC2 C18 column (150 × 4.6 mm I.D. × 5 µm film thickness). Then, 15 µL of samples were
injected, and a gradient of eluent A (water) and eluent B (acetonitrile) was used. The flow
rate was 1.0 mL min−1 and gradient elution conditions applied were: 17% B for 8 min
followed by a linear gradient to 100% B in 20 min (held 1 min), followed by a linear gradient
to 17% B in 5 min. Standard solutions were freshly prepared for quantification of aloin A
and B (0.005–100 mg kg−1 , R2 = 0.9998). All samples were filtered through 0.45 µm pore
size nylon membrane filters prior to HPLC analysis at 357 nm. Triplicate runs were carried
out for each sample. Linearity, limit of detection (LOD), limit of quantification (LOQ) and
precision were determined to validate the analytical method.

2.5. Characterization of AVE Obtained under Optimum Conditions

2.5.1. Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR)
The FTIR spectrum of AVE was recorded by using an infrared spectrophotometer
JASCO FTIR 4700 IRT-5200 (Easton, MD, USA) in the 4000–500 cm−1 range with a spectral
resolution of 4 cm−1 and 32 scans. The attenuated total reflectance (ATR) mode was used
with a Golden Gate accessory equipped with diamond crystal, and tests were performed
in triplicate.

2.5.2. Thermogravimetric Analysis (TGA)

The TGA of AVE was carried out using Mettler Toledo TGA/SDTA 851e equipment
(Schwarzenbach, Switzerland). Approximately 5 mg of the sample were heated from room
temperature to 700 ◦ C at 10 ◦ C min−1 under nitrogen atmosphere (50 mL min−1 ) using
alumina pans. The analysis was performed in triplicate.
Antioxidants 2022, 11, 1058 7 of 25

2.5.3. AVE Phenolic Profile

2.5.3.1. HPLC-MS Qualitative Analysis
The identification of phenolic compounds present in AVE was performed by high-
performance liquid chromatography coupled to mass spectrometry (HPLC-MS) according
to Lee et al. [47] and Quispe et al. [48], with some modifications. An Agilent 1100 HPLC
system equipped with a quaternary solvent delivery system coupled to an LC/MSD ion
trap mass spectrometer with electrospray ionization (ESI) source (Agilent Technologies,
Palo Alto, CA, USA) was used. Chromatographic separation was performed on a HALO
C18 column (100 mm × 4.6 mm I.D. × 2.7 µm) coupled to a HALO C18 guard column
90 Å (4.6 × 5 mm I.D. × 2.7 µm) operating at 25 ◦ C. The mobile phase was composed of
two solvents added with 0.1% formic acid (A: water, B: acetonitrile). The gradient elution
program was as follows: linear gradient from 10% B to 40% B in 20 min followed by a linear
gradient to 98% B in 5 min (held 5 min). The flow rate was 0.3 mL min−1 and the injection
volume 6 µL. MS spectra were recorded in the range 50–900 m/z in negative ionization
mode. The electrospray chamber was set at 3.5 kV with a drying gas temperature of 350 ◦ C.
The N2 pressure and flow rate of the nebulizer were 50 psi and 10 L min−1 , respectively.
AVE and standard solutions of phenolic compounds were freshly prepared in methanol
and filtered through a 0.22 µm nylon membrane prior to injection. Extracted ion chro-
matograms and mass spectra experimental data were used for identification of polyphe-
nols in AVE through comparison with the standards. Other phenolic compounds also
present in AVE were tentatively identified based on the information of ion fragments and
reported literature.

2.5.3.2. HPLC-DAD Quantitative Analysis

Quantitative analysis of previously identified compounds by HPLC-MS was carried
out by HPLC-DAD. An Agilent series 1260 Infinity Quaternary LC HPLC system (Agilent
Technologies, Palo Alto, CA, USA) equipped with a diode array detector was used. Op-
erating conditions detailed in Section 2.5.3.1 were applied, and simultaneous monitoring
was set at 254 nm (aloesin and aloe-emodin), 270 nm (cinnamic acid), 324 nm (chlorogenic
acid) and 357 nm (orientin, aloin B and aloin A) for quantification. The optimal detection
wavelength of each compound was set by previously verifying the obtained UV spectra.
Quantitative analysis was performed using the external calibration method based on the
preparation of calibration curves at ten concentration levels for the aforementioned stan-
dard compounds in methanol. All analyses were carried out in triplicate. Linearity, limit
of detection (LOD), limit of quantification (LOQ) and precision (RSD) were determined to
validate the analytical method.

2.6. Scanning Electron Microscopy (SEM)

SEM was employed to evaluate the damage in the vegetal material produced by the
MAE process. Raw and extracted AVS freeze-dried samples were mounted on aluminum
stubs and then coated with a gold layer under vacuum using an SCD 004 Balzers sputter
coater (Bal Tec., AG, Furstentum, Liechtenstein). SEM analysis was carried out using a
JEOL JSM 840 scanning electron microscope (Peabody, MA, USA) at an accelerating voltage
of 15 kV and 2500× magnification level.

2.7. Statistical Analysis

Statgraphics Centurion XVI (Statistical Graphics, Rockville, MD, USA) was used
to generate and analyze the BBD results. Graphic analysis of the principal effects and
interactions between variables was used to interpret the results. The coefficients of quadratic
polynomial models were determined by data regression analysis and the adequacy of the
fitted models was determined by evaluating the lack of fit, coefficient of determination (R2 ),
and F test obtained from the analysis of variance (ANOVA). The statistical significance of
the model parameters was determined at a 95% probability level (α = 0.05).
Antioxidants 2022, 11, 1058 8 of 25

3. Results and Discussion

3.1. AVS Characterization
The dimensions of Aloe vera leaves used in this work (Table 1) were very similar to the
morphological characteristics of plants studied by Zapata et al. [49]. Even though other
authors reported different dimensions for three- or four-year old Aloe vera plants [3,38,50],
it is well known that many factors, such as environmental and soil conditions, water
availability and plant variety, could significantly influence plant growth, thereby leading
to different leaves in terms of size and phytochemical constituents [51]. In this case,
after tissue separation of Aloe vera leaves, the yields of skin and other components of the
residue, such as the tip, base and spikes, accounted for up to 15.1 ± 2.1% and 17.5 ± 4.1%,
respectively. These results are in close agreement with those found by Femenia et al. [38]
and Flores-López et al. [3], who reported very similar yields for the epidermis fraction
in Aloe vera plants. Several drying techniques, including freeze drying, air drying and
oven drying at different temperature and time programs, were tested in order to find the
most suitable drying process to avoid leaf browning, which might be directly related to
oxidative processes and detrimental effects on bioactive substance content in plants. Similar
to Ng et al. [52], freeze drying was selected as the most adequate drying process and no
apparent oxidation of AVS was observed. On the other hand, it is well known that a smaller
particle size will enhance solute–solvent interaction during the extraction process; but
too many fine particles might make subsequent separation steps difficult. In this work,
particles passing through a 1.0 mm sieve were used.
The physicochemical characterization results of AVS are shown in Table 1. Moisture,
ash, protein and fat contents of AVS resulted in close agreement with experimental data
reported in other studies [3,12,38]. Proteins and lipids represented a minor fraction of
AVS, accounting for 6.5 ± 0.2 wt% and 2.4 ± 0.1 wt%, expressed on a dry matter basis,
respectively. On the other hand, the ash content was relatively high, and it accounted
for 15.5 ± 0.1%. It has been reported that calcium, potassium, sodium and magnesium
are the main mineral components present in AVS ash. It has also been suggested that
the presence of minerals in Aloe vera is essential for the proper functioning of enzymatic
systems and for improving resistance against microorganisms and water stresses. The
mineral content found in AVS was much more concentrated compared to other plant tissues,
such as gel or flowers [3]. Apart from these components, it has also been reported that AVS
is an interesting source of polysaccharides [53] and lignocellulosic matter [12]. Moreover,
Lucini et al. [4] and Quispe et al. [48] reported that AVS is rich in phenolic compounds and
its extracts exhibit antioxidant activity to a higher degree than other parts of the plant. In
this sense, AVS represents a low-cost, underused biomass resource with high potential to
be valorized, finding a wide range of applications in cosmetic, food or medicinal products.

3.2. MAE Optimization

3.2.1. Model Fitting and Analysis
The optimization of MAE conditions for the extraction of polyphenols from a wide vari-
ety of plant leaves, such as Myrtus communis L. [54], Achillea millefolium [55], strawberry [56],
Vitis vinifera L. [57] and artichoke [58], has been reported in recent years by using response
surface methodology (RSM). Even though these vegetal matrices might have results similar
to Aloe vera leaf skin, optimized MAE conditions could not be generalized to all plant mate-
rials due to the diverse nature of the existing bioactive phytochemicals and the different
levels of interaction between microwave irradiation and different plants and vegetable
parts. Moreover, it is well known that the MAE of bioactive compounds from plant mate-
rials could be affected by a wide number of experimental factors; in consequence, MAE
optimization should be performed in each study. In this work, the influence of ethanol
concentration (%Et), extraction temperature (T), extraction time (t) and solvent volume
(V) on the MAE of bioactive compounds from AVS was studied by using a Box-Behnken
experimental design with 29 independent runs, including 5 central points, which were
performed randomly. The experimental conditions and data obtained in terms of extraction
Antioxidants 2022, 11, 1058 9 of 25

yield, TPC, antioxidant activity (DPPH and FRAP assays) and total aloin content are shown
in Table 2.
Multiple regression analysis was applied to the experimental data, and all the studied
responses were fitted to second-order mathematical models as a function of the independent
factors. The resulting models are presented in Equations (3)–(7).

Extraction yield = −23.762 + 0.512 A + 0.122 B + 0.326 C + 0.824 D − 0.004 A2 + 0.000 AB − 0.002 AC
(3)
−0.003 A D − 0.002 B2 − 0.001 BC + 0.002 BD − 0.001 C2 − 0.001 CD − 0.005

TPC = 98.296 + 0.533 A − 0.245 B − 0.952 C − 0.295 D + 0.001 A2 + 0.001 AB − 0.004 AC

(4)
+ 0.002 AD − 0.001 B2 + 0.009 BC + 0.001 BD − 0.006 C2 + 0.014 CD − 0.002 D2
DPPH = 52.525 + 0.123 A − 0.575 B − 0.439 C + 0.504 D + 0.004 A2 − 0.002 AB − 0.0025 AC
(5)
− 0.003 AD + 0.006 B2 + 0.011 BC − 0.003 BD − 0.006 C2 + 0.002 CD − 0.002 D2
FRAP = 193.053 − 0.317 A − 0.490 B − 1.384 C − 2.129 D − 0.001 A2 + 0.001 AB + 0.014 AC
(6)
+ 0.012 AD + 0.005 B2 + 0.009 BC − 0.007 BD − 0.006 C2 + 0.006 CD + 0.013 D2
aloin = 56.258 − 0.066 A − 0.037 B − 0.340 C − 0.455 D + 0.003 A2 − 0.003 AB + 0.005 AC
(7)
+ 0.002 AD + 0.003 B2 + 0.003 BC − 0.003 BD − 0.004 C2 + 0.001 CD + 0.004 D2
where A, B, C and D represent ethanol concentration, extraction temperature, extraction
time and solvent volume, respectively.
In order to study the effect of the experimental factors in the response variables as
well as to evaluate the adequacy of the fitted models, analysis of variance (ANOVA) was
carried out and results are summarized in Table 3. The coefficient of determination (R2 )
values and the p-values obtained for lack of fit test were used as a measurement of the
degree of fitness of the proposed models to the experimental data. As shown in Table 3,
non-significance of the lack of fit test (p > 0.05) and acceptable R2 values were obtained
in all cases, accounting for 0.9498, 0.9308, 0.8691, 0.9329 and 0.9354 for yield, TPC, DPPH,
FRAP and aloin content, respectively. These values indicate the percentage of variability
being explained by the models for each response (87 to 95%), highlighting a high level of
correlation between the experimental data and predicted values. Moreover, adjusted R2
values did not differ greatly from R2 ones, and in all cases, the distribution of residuals was
randomly scattered around zero. These results confirmed the reliability of the models to be
used in subsequent optimization stages.

Table 3. ANOVA results for response surface quadratic models of AVS extraction.

Source Sum of Squares Df Mean Square F-Value p-Value

Yield
A 102.08 1 102.08 215.82 0.0001 *
B 0.00 1 0.00 0.00 0.9685
C 0.21 1 0.21 0.45 0.5386
D 19.00 1 19.00 40.17 0.0032 *
AA 13.00 1 13.00 27.49 0.0063 *
AB 0.00 1 0.00 0.00 1.0000
AC 1.44 1 1.44 3.04 0.1560
AD 3.24 1 3.24 6.85 0.0590
BB 5.29 1 5.29 11.19 0.0287 *
BC 0.36 1 0.36 0.76 0.4322
BD 2.40 1 2.40 5.08 0.0873
CC 1.26 1 1.26 2.67 0.1779
CD 0.64 1 0.64 1.35 0.3094
DD 9.01 1 9.01 19.04 0.0120 *
Lack-of-fit 6.02 10 0.60 1.27 0.4399
Pure error 1.89 4 0.47
Antioxidants 2022, 11, 1058 10 of 25

Table 3. Cont.

Source Sum of Squares Df Mean Square F-Value p-Value

Yield
Total (corr.) 157.55 28
R2 0.9498
Adj R2 0.8995
TPC
A 2995.68 1 2995.68 497.79 0.0000 *
B 0.44 1 0.44 0.07 0.8001
C 13.02 1 13.02 2.16 0.2153
D 41.81 1 41.81 6.95 0.0578
AA 1.15 1 1.15 0.19 0.6842
AB 1.21 1 1.21 0.20 0.6771
AC 8.41 1 8.41 1.40 0.3026
AD 1.69 1 1.69 0.28 0.6242
BB 0.55 1 0.55 0.09 0.7777
BC 39.69 1 39.69 6.60 0.0621
BD 0.12 1 0.12 0.02 0.8934
CC 26.36 1 26.36 4.38 0.1045
CD 52.56 1 52.56 8.73 0.0417 *
DD 1.81 1 1.81 0.30 0.6125
Lack-of-fit 212.62 10 21.26 3.53 0.1176
Pure error 24.07 4 6.02
Total (corr.) 3422.55 28
R2 0.9308
Adj R2 0.8617
DPPH
A 460.04 1 460.04 189.86 0.0002 *
B 72.52 1 72.52 29.93 0.0054 *
C 6.31 1 6.31 2.60 0.1819
D 65.80 1 65.80 27.16 0.0065 *
AA 20.94 1 20.94 8.64 0.0424 *
AB 1.44 1 1.44 0.59 0.4838
AC 3.06 1 3.06 1.26 0.3238
AD 3.61 1 3.61 1.49 0.2893
BB 40.43 1 40.43 16.69 0.0150 *
BC 63.20 1 63.20 26.08 0.0069 *
BD 4.00 1 4.00 1.65 0.2682
CC 22.88 1 22.88 9.44 0.0372 *
CD 1.32 1 1.32 0.55 0.5010
DD 1.99 1 1.99 0.82 0.4165
Lack-of-fit 108.49 10 10.85 4.48 0.0808
Pure error 9.69 4 2.42
Total (corr.) 902.51 28
R2 0.8691
Adj R2 0.7381
FRAP
A 2892.31 1 2892.31 499.79 0.0000 *
B 0.00 1 0.00 0.00 0.9910
C 61.65 1 61.65 10.65 0.0310 *
D 13.23 1 13.23 2.29 0.2051
AA 0.71 1 0.71 0.12 0.7432
AB 1.10 1 1.10 0.19 0.6850
AC 91.20 1 91.20 15.76 0.0165 *
AD 55.50 1 55.50 9.59 0.0363 *
BB 28.42 1 28.42 4.91 0.0910
BC 42.90 1 42.90 7.41 0.0528
BD 15.60 1 15.60 2.70 0.1759
CC 22.66 1 22.66 3.92 0.1189
Antioxidants 2022, 11, 1058 11 of 25

Table 3. Cont.

Source Sum of Squares Df Mean Square F-Value p-Value

FRAP
CD 10.89 1 10.89 1.88 0.2420
DD 56.67 1 56.67 9.79 0.0352 *
Lack-of-fit 214.98 10 21.50 3.71 0.1088
Pure error 23.15 4 5.79
Total (corr.) 3548.47 28
R2 0.9329
Adj R2 0.8658
aloin
A 696.16 1 696.16 186.04 0.0002 *
B 0.21 1 0.21 0.06 0.8230
C 0.03 1 0.03 0.01 0.9330
D 0.48 1 0.48 0.13 0.7383
AA 11.79 1 11.79 3.15 0.1505
AB 5.76 1 5.76 1.54 0.2825
AC 10.24 1 10.24 2.74 0.1734
AD 1.21 1 1.21 0.32 0.6000
BB 8.55 1 8.55 2.29 0.2051
BC 3.61 1 3.61 0.96 0.3816
BD 3.24 1 3.24 0.87 0.4048
CC 7.87 1 7.87 2.10 0.2205
CD 0.25 1 0.25 0.07 0.8088
DD 4.67 1 4.67 1.25 0.3266
Lack-of-fit 37.29 10 3.73 1.00 0.5501
Pure error 14.97 4 3.74
Total (corr.) 809.15 28
R2 0.9354
Adj R2 0.8708
A: ethanol concentration; B: extraction temperature; C: extraction time; D: solvent volume. * significant effect at
p < 0.05.

3.2.2. Effect of Extraction Variables on the Extraction Yield

The extraction yield ranged from 18.2 to 26.3 g AVE 100 g AVS−1 under the 29 ex-
periments performed (Table 2). According to Table 3, ethanol concentration and solvent
volume had significant effects (p < 0.05) on extraction yield, with the former being the most
significant factor by far. Moreover, three quadratic effects (A2 , B2 and D2 ) also showed
significant effects (p < 0.05). Particularly, ethanol concentration and all mentioned quadratic
effects had negative effects, while solvent volume showed a positive effect. Consequently,
significantly higher extraction yields were obtained at a low ethanol concentration level.
Solvent constitution is directly related to the type and quantity of compounds extracted
from plant materials. In this case, a low ethanol concentration might favor the co-extraction
of other untargeted plant components apart from polyphenols. It has been reported that
increasing water content in the extraction solvent resulted in higher polarity, which might
enhance the extraction of other polar compounds from AVS such as polysaccharides [59].
Furthermore, water addition can be used to improve solvent penetration into vegetal sam-
ple matrices as well as to enhance heating efficiency, usually leading to higher extraction
yields [26]. On the other hand, extraction yield was significantly affected (p < 0.05) by the
solvent volume. A positive effect was observed, so the higher the solvent volume, the
higher the obtained extraction yield. A low amount of solvent usually promotes mass
transfer barriers, which limit the movement of compounds out of the vegetal matrix since
active compounds are concentrated in certain regions near the vegetal surface [26]. On the
contrary, larger extraction volumes can cause more efficient swelling of plant materials,
enhancing the extraction procedure due to an easier rupture of the cell walls. However,
in the case of MAE, an excessive amount of solvent may cause poor microwave heating
 Antioxidants 2022, 11, 1058 12 of 25

Antioxidants 2022, 11, x FOR PEER REVIEW 13 of 27

efficiency as the majority of the irradiation would be absorbed by the solvent instead of the
plant material and additional power might be needed to obtain higher yields.

3.2.3. Effect of Extraction Variables on Total Phenolic and Aloin Contents

TPC and aloin content obtained under the 29 experiments carried out in the BBD
varied from 86.5 to 125.8 mg GAE g AVE−1 and from 36.5 to 57.5 mg g AVE−1 , respectively
(Table 2). ANOVA analysis (Table 3) showed that total phenolic content was significantly
(p < 0.05) influenced by the ethanol concentration and the interaction between extraction
time and solvent volume, both with positive effects, while the aloin content was only signif-
icantly affected (p < 0.05) by the ethanol concentration, also with a positive effect. Several
authors reported that high ethanol concentrations in the extraction solvent were adequate
for obtaining extracts with high TPC or antioxidant activities from aloe [4,13,43,44,60] and
other plant materials [40,55,61]. The solubility of phenolic compounds is strongly related to
their chemical nature and polarity. By increasing ethanol content in the extraction solvent,
the solubility of certain polyphenols resulted in enhanced and, consequently, higher TPC
and antioxidant activities, as expected. The significant interaction effect (p < 0.05) between
extraction time and solvent volume on the TPC response is shown in Figure 1a. It was
observed that the combination of short extraction times and low solvent volumes resulted
in higher TPC values. Similar results were found by other authors who reported that the
increase in the exposure time of the sample to microwave irradiation might promote the
extraction of other components, apart from polyphenols, from plant materials, causing a
relative decrease in TPC values on extracts [55,62]. Furthermore, microwave irradiation
time may lead to thermal degradation and oxidation of sensitive compounds such as
some polyphenols.

(a)

(b)
Figure 1. Cont.
Antioxidants
Antioxidants2022,
2022,11,
11,x1058
FOR PEER REVIEW 1314of of
25 27

(c)

(d)
Figure
Figure 1.
1. Response
Responsesurface
surfaceplots
plotsofofsignificant
significantinteractions
interactionsbetween
betweenindependent
independentvariables
variableson:
on:(a)
TPC (volume
(a) TPC vs time);
(volume (b) DPPH
vs. time); (b) DPPH(temperature
(temperaturevs time); (c) FRAP
vs. time); (ethanol
(c) FRAP concentration
(ethanol vs time)
concentration vs.
and (d)
time) FRAP
and (ethanol
(d) FRAP concentration
(ethanol vs solvent
concentration volume).
vs. solvent In all cases,
volume). the other
In all cases, the factors were fixed
other factors were at
their
fixedcentral
at theirvalues.
central values.

3.2.4. Effect
3.2.4. Effect of Extraction
Extraction Variables
Variableson onAntioxidant
AntioxidantActivity
Activity
The antioxidant activity,
The activity,expressed
expressedas asDPPH
DPPHradical
radicalscavenging
scavengingactivity,
activity,varied
variedfrom
from
49.9 to
to 73.4
73.4 mg −1 in the 29 MAE experiments carried out in this study (Table 2).
49.9 mg TE TE gg AVE
AVE−1 in the 29 MAE experiments carried out in this study (Table 2).
DPPH radical
DPPH radicalscavenging
scavengingactivity
activity was
was significantly
significantlyaffected (p < (p
affected 0.05) by three
< 0.05) linearlinear
by three effectsef-
(ethanol concentration, extraction temperature and solvent volume),
fects (ethanol concentration, extraction temperature and solvent volume), three quadratic three quadratic effects
(B2 , C2 and
effects (B2, A
2 and one interaction effect (BC). The linear effects of the ethanol concentration
C2) and A2) and one interaction effect (BC). The linear effects of the ethanol
and extraction and
concentration temperature
extraction were positive, while
temperature were thepositive,
solvent volume
while showed
the solvent a negative
volume
effect. Furthermore, all quadratic effects were positive except for extraction time. Overall,
showed a negative effect. Furthermore, all quadratic effects were positive except for ex-
the DPPH radical scavenging activity increased with increasing ethanol concentration and
traction time. Overall, the DPPH radical scavenging activity increased with increasing
extraction temperature, while decreasing solvent volume. Moreover, the interaction effect
ethanol concentration and extraction temperature, while decreasing solvent volume.
between the extraction temperature and time showed a significant (p < 0.05) positive effect
Moreover, the interaction
on DPPH antioxidant effectAs
activity. between
shown in theFigure
extraction
1b, antemperature and time showed
increase in extraction time at a
significant
low extraction temperature values led to a decrease in DPPH antioxidant activity of the1b,
(p < 0.05) positive effect on DPPH antioxidant activity. As shown in Figure
an increase
extract, in extraction
whereas increasingtime at lowtime
extraction extraction temperature
at high extraction values ledled
temperatures to to
a enhanced
decrease in
DPPH
DPPH antioxidant
values. Overall, activity
higher of antioxidant
the extract, activity
whereaswas increasing
obtainedextraction
◦
at 80 C and time at high
40 min. It isex-
traction
well knowntemperatures led to enhanced
that polyphenols could exert DPPH values.activity
antioxidant Overall, higher
and manyantioxidant activity
other interesting
was obtained
properties [63].at In
80 this
°C andcase,40similarly
min. It istowellTPC,known
a highthat polyphenols
ethanol could favored
concentration exert antioxi-
the
dant activity and many other interesting properties [63]. In this
extraction of certain components which might act as proton-donor substrates, scavenging case, similarly to TPC, a
high ethanolwhich
the radicals concentration favored
are involved the extraction
in DPPH reactionsofandcertain components
contributing which
to the might act
antioxidant
as proton-donor
activity of AVE. On substrates,
the otherscavenging the radicals
hand, high extraction which are involved
temperatures caused a in DPPH
drop reactions
in viscosity
and contributing
and surface tension, enhancing
to the antioxidant the capacity
activity ofofAVE.
the extraction
On the othersolvent
hand, to penetrate the
high extraction
temperatures caused a drop in viscosity and surface tension, enhancing the capacity ofInthe
vegetal matrix, resulting in an increase in the solubility of the target compounds [26].
extraction solvent to penetrate the vegetal matrix, resulting in an increase in the solubility
of the target compounds [26]. In addition, cell walls might be damaged by the action of
Antioxidants 2022, 11, 1058 14 of 25

addition, cell walls might be damaged by the action of high temperatures during the
extraction process, which could favor the release of active compounds in the extraction
solvent [26,28]. However, it has been reported that some phenolic compounds might suffer
thermal degradation when exposed to high temperatures [64], and optimal temperatures
for MAE usually do not exceed 100 ◦ C.
Concerning the antioxidant activity estimated by the FRAP method, the obtained
values ranged from 90.5 to 134.1 mg TE g AVE−1 under the 29 experiments performed in
the BBD (Table 2). FRAP values were significantly influenced (p < 0.05) by the linear effect
of the solvent concentration and extraction time, quadratic effect of solvent volume and
interaction effects of AC and AD. Overall, the significant effects (p < 0.05) were positive,
resulting in enhanced FRAP antioxidant activity values by increasing ethanol concentration
in the extraction solvent, extraction time and solvent volume. Moreover, the response
surface plots of the interaction effects between the ethanol concentration and extraction
time and solvent volume are shown in Figure 1c,d, respectively. A slight decrease in FRAP
values was observed with increasing extraction time at low ethanol concentrations in the
extraction solvent. However, FRAP antioxidant activity was enhanced with increasing
extraction time at high ethanol concentrations. Similar behavior was observed for the
interaction effect between the ethanol concentration and solvent volume. Altogether,
higher FRAP values were obtained at high ethanol concentration levels and long extraction
times as well as high solvent volumes.
As already detailed for TPC and DPPH values, a high ethanol concentration also
favors the extraction of some components, which contributes to enhancing FRAP antiox-
idant activity. Similar to the extraction temperature, higher extraction times contribute
to cell wall degradation and liberation of active compounds into the extraction solvent,
increasing the antioxidant activity of the extracts. On this point, it is worth mentioning
that different methods used to determine the antioxidant activity of AVE might evaluate
different fractions of antioxidant compounds, which might be partially overlapped. On
the one hand, the FRAP method evaluates the content of electron-donating species with a
certain redox potential, whereas the DPPH method assesses the free radical scavenging
capacity of a sample [43]. Moreover, the steric accessibility of DPPH radicals is a major
aspect of the reaction, as this method is much more sensitive to small molecules, and many
large antioxidant compounds might hardly participate in this reaction [65]. This effect
was already highlighted by Kim et al. [43], who extracted polyphenols and antioxidant
compounds from Aloe vera gel, and they observed that the antioxidant activity of aloe
extracts evaluated by the DPPH method may be underestimated to a certain degree.

3.2.5. Optimal Extraction Conditions

Table 4 shows the experimental conditions which optimized each response variable
individually and the optimum predicted values by the studied models. Great variability
was found among the different sets of experimental factors which maximized each response
variable, as expected from the analysis of linear, quadratic and interaction effects of the ex-
traction variables. Consequently, it was not possible to clearly determine the best extraction
conditions for AVS only considering these results. For this reason, a simultaneous opti-
mization procedure using the desirability function was carried out to find the experimental
conditions which simultaneously satisfy all the requirements for each response. First, the
optimization was performed in order to simultaneously maximize extraction yield, TPC,
DPPH and FRAP and minimize aloin content. However, due to the dissimilar extraction
conditions summarized in Table 4, a quite low desirability value was obtained (0.5802)
and further approaches were considered. According to ANOVA analysis (Table 3), the
ethanol concentration was by far the most significant (p < 0.05) factor affecting all responses.
Moreover, it exhibited a strong negative effect on the extraction yield with the optimum
at 40%, whereas its effect was positive in all the other responses with an optimum at 80%.
These results indicated an inverse correlation between extraction yield and the rest of the
responses, suggesting that MAE conditions leading to high extraction yields could not be
Antioxidants 2022, 11, 1058 15 of 25

selective for polyphenol extraction. A similar scenario was described by Kim et al. [43]
and Milutinovic et al. [66], who optimized the MAE of antioxidants from Aloe vera gel and
Equisetum arvense waste, respectively. In this sense, considering that extraction yield could
be interfered with by the co-extraction of other untargeted compounds and that TPC and
antioxidant activity responses are usually directly correlated with phenolic compound
concentrations, the extraction yield from the multi-response optimization was excluded.

Table 4. Single response optimized extraction conditions and predicted values.

Response Et (%) T (◦ C) t (min) V (mL) Predicted Value

Yield 40.0 67.7 26.7 80.0 26.8 g AVE 100 g AVS−1
TPC 80.0 40.0 5.0 56.0 127.4 mg GAE g AVE−1
DPPH 80.0 80.0 40.0 52.7 73.4 mg TE gAVE−1
FRAP 80.0 54.4 39.9 80.0 140.5 mg TE gAVE−1
aloinMAX 80.0 40.0 29.4 80.0 59.0 mg gAVE−1
aloinMIN 40.0 40.3 40.0 61.8 35.4 mg gAVE−1
Et: ethanol concentration; T: extraction temperature; t: extraction time; V: solvent volume.

In addition, it has been reported that aloin contributes to some extent to the antiox-
idant capacity exhibited by extracts from different aloe species [4,47,67]. Consequently,
maximization of antioxidant activity and total phenolic content while simultaneously mini-
mizing aloin content might lead to unavoidably low desirability values (0.5951). Therefore,
multiresponse optimization was finally performed, maximizing TPC, DPPH, FRAP and
aloin content and obtaining a desirability value of 0.8777, with optimal extraction condi-
tions of 80% ethanol, 80 ◦ C, 36.6 min and 50.0 mL. Subsequent purification or selective
extraction steps could be implemented in the case of limitations regarding aloin content.

3.2.6. Verification Test under Optimum Extraction Conditions

The predicted values determined at a 95% level of probability for response variables
obtained after multiresponse optimization in terms of extraction yield, TPC, DPPH, FRAP
and aloin content were 16.0 ± 2.2 g AVE 100 g AVS−1 , 118.4 ± 11.9 mg GAE g AVE−1 ,
74.2 ± 8.4 mg TE g AVE−1 , 134.8 ± 12.0 mg TE g AVE−1 and 56.6 ± 5.6 mg aloin g AVE−1 ,
respectively. Verification experiments under the obtained optimal conditions were carried
out, in triplicate, and the obtained experimental responses regarding extraction yield, TPC,
DPPH, FRAP and aloin were 17.3 ± 0.1 g AVE 100 g AVS−1 , 116.4 ± 4.5 mg GAE g AVE−1 ,
69.0 ± 1.9 mg TE g AVE− 1 , 131.9 ± 6.5 mg TE g AVE−1 and 55.6 ± 0.2 mg aloin g AVE−1 ,
respectively. Experimental results not significantly differing (p > 0.05) from predicted values
were obtained in all cases. Moreover, reproducibility of the whole AVE extraction and
characterization process was demonstrated, obtaining relative standard deviations ranging
from 0.36 to 4.92% for all analyzed response variables. Additionally, it was observed that
TPC, FRAP and aloin content in optimized AVE were almost higher than every individual
run of the design, while DPPH was surpassed by only one experiment (Table 2). Regarding
extraction yield, a value near 17% was obtained, as expected, due to the high ethanol
concentration used in the extraction solvent. In conclusion, the obtained quadratic models
were reliable for optimizing the extraction of bioactive compounds from Aloe vera skin in
the studied experimental domain, resulting in a high degree of correlation between the
experimental data and predicted values, indicating that the developed models could be
used to predict the studied responses.
Compared to conventional extraction techniques, MAE’s major advantages are its high
reproducibility and the noticeable reduction in extraction time and solvent consumption [26].
For instance, Sultana et al. [60] studied the effect of the extraction solvent on the yield
of antioxidant compounds from different plant materials using conventional extraction
techniques. They found that reflux extraction of Aloe barbadensis leaves with 200 mL of 80%
ethanol accounted for 18.1 ± 0.7 g 100 g DW. However, a much longer extraction time of 6 h
Antioxidants 2022, 11, 1058 16 of 25

was required. In another work, Quispe et al. [48] reported an extraction yield of 16.2 g 100 g
of AVS for a phenolic-enriched extract obtained after maceration with methanol for 48 h.

3.3. Characterization of AVE Obtained at Optimal Extraction Conditions

3.3.1. FTIR Analysis
It has been reported that Aloe vera plants contain a wide variety of bioactive com-
pounds, including phenolic acids and derivatives, flavonoids, chromones, anthraquinones,
polysaccharides and fatty acids. The FTIR–ATR spectrum of AVE obtained under op-
timal extraction conditions is shown in Figure 2a. The broad peak observed around
3275 cm−1 was attributed to the stretching vibration of different –OH groups of phenolic
compounds such as flavonoids, anthraquinones and chromones [68,69]. The bands at 2925
and 1379 cm−1 might be assigned to C-H stretching and bending vibration of aliphatic
hydrocarbons, respectively [68,70,71]. The absorption band at 1714 cm−1 was associated
with C=O stretching, indicating the presence of carbonyl functional groups [70,72]. Peaks
observed at 1222 and 877 cm−1 might be related to the stretching of C-O-C bonds of acetyl
groups of esters and phenols [68,72] and out of plane deformation of C-H bonds in aro-
matic rings [72], respectively. In addition, according to other authors who characterized
polyphenol-enriched extracts from other plant materials, such as Ilex paraguarensis [71],
Rosmarinus officinalis [73], Garcinia mangostana [74] and grape seeds [75], the observed peaks
at 1601, 1284 and 1036 cm−1 could be attributed to C=C ring stretching vibration [71,73,75],
Antioxidants 2022, 11, x FOR PEER REVIEW
ester C-O stretching [75] or the presence of methoxy groups [74], and C-N stretching 17 of 26

vibration of aliphatic amines [71], respectively. As a result of the aforementioned peak

assignations, it was concluded that AVE contained a variety of phenolic compounds.

(a) (b)
Figure
Figure2.2.FTIR
FTIRspectrum
spectrum(a)
(a)and
andTGA
TGAand
andDTGA
DTGA thermograms
thermograms (b)
(b) of
of AVE
AVE obtained under optimal
obtained under optimal
MAE
MAEconditions.
conditions.

3.3.2.
3.3.2.Thermogravimetric
ThermogravimetricAnalysisAnalysis (TGA)
(TGA)
The
Thethermal
thermalstability
stabilityof
ofAVE
AVE obtained
obtained under
under optimized MAE conditions was studied
by
bythermogravimetric
thermogravimetricanalysis.
analysis. Figure
Figure2b 2bshows
showsthe the TGA
TGA and
and DTGA
DTGA of of AVE obtained
under
underan an inert
inert nitrogen
nitrogen atmosphere.
atmosphere. The The thermal
thermal degradation
degradation process
process occurred over a
wide
widerange
rangeofoftemperatures,
temperatures,showing
showing multiple
multipleoverlapped
overlapped decomposition
decomposition steps which
steps are
which
in
areaccordance
in accordancewithwith
the complex composition
the complex compositionof AVE. Similar
of AVE. behavior
Similar waswas
behavior reported for
reported
for thermal
the the thermal degradation
degradation process
process of other
of other natural
natural extracts,
extracts, such such as those
as those from from
cocoacocoa
shell
shell [5],
bean bean [5], grape
grape seedand
seed [75] [75] yerba
and yerba
matemate
[71].[71]. According
According to Figure
to Figure 2b, 2b,
thethe initial
initial stepstep
of
of weight ◦ C, and it could be associated with water and volatile
weight lossloss started
started below
below 100 100
°C, and it could be associated with water and volatile phe-
phenolic
nolic compound
compound desorption.
desorption. The The subsequent
subsequent degradation
degradation stages
stages with
with peaks
peaks shown
shown at
at 215.0 ± 0.4, 301.6 ± 2.0 and 442.1 ± 1.3 ◦ C were relatively overlapped, and they were
215.0 ± 0.4, 301.6 ± 2.0 and 442.1 ± 1.3 °C were relatively overlapped, and they were at-
tributed to the thermal degradation of groups of biomolecules with different structural
features and characteristics present in AVE. The second degradation step occurred from
125 to 272 °C with an associated mass loss of 27.3%, and it could be related to the degra-
dation of some low to mid molecular weight components of AVE, such as the compounds
 Antioxidants 2022, 11, 1058 17 of 25

attributed to the thermal degradation of groups of biomolecules with different structural

features and characteristics present in AVE. The second degradation step occurred from 125
to 272 ◦ C with an associated mass loss of 27.3%, and it could be related to the degradation
of some low to mid molecular weight components of AVE, such as the compounds which
will be further discussed in Section 3.3.3. The third and fourth thermal degradation stages
extended from 272 to 400 ◦ C and from 400 to 800 ◦ C with mass losses of 17.3 and 14.7%,
respectively. These degradation processes may be ascribed to compounds with higher
molecular weights and more complex structures such as cellulose and lignin derivatives that
might be present in Aloe vera extracts [70]. Finally, nearly 34.1 ± 0.1% of the initial weight
remained at 800 ◦ C as char residue, which could be related to non-pyrolyzable compounds.

3.3.3. Determination of Phenolic Profile by HPLC

Optimization of chromatographic conditions (flow rate, mobile phase composition
and gradient step) was performed on AVE in order to obtain an adequate peak separation
and resolution. Contrary to some authors who reported that a methanol–water mobile
phase added with formic or acetic acid was suitable for phenolic profiling of different aloe
species [76–78], in this study it was found that peak shape and separation efficiency of phe-
nolic compounds in AVE were significantly improved by using an acetonitrile–water mix-
ture, which was selected as the mobile phase in accordance with previous works [47,48,79].
Antioxidants 2022, 11, x FOR PEER REVIEW 18 of 26
Aloesin, chlorogenic acid, orientin, aloeresin D, aloin B, aloin A, cinnamic acid and aloe-
emodin, whose chemical structures are shown in Figure 3, were identified in AVE ob-
tained under optimal MAE conditions by HPLC-ESI-MS/MS by comparing their retention
retention
times andtimes
massand mass
spectra spectra
with thosewith those of commercial
of commercial standards.standards. These
These results areresults are in
in general
general
agreement with previous works reporting the presence of these compounds in Aloe Vera in
agreement with previous works reporting the presence of these compounds
Aloe Vera[47,48,76].
extracts extracts [47,48,76].
It has beenItstated
has been
that stated that thehealth-promoting
the beneficial beneficial health-promoting
properties foundprop-
erties found
in Aloe in Aloe
leaves mightleaves mighttobethe
be related related
highlytocomplex
the highly complex phytochemical
phytochemical compositioncompo-
and,
sition and, in to
in particular, particular, to characteristic
characteristic phenolic which
phenolic compounds compounds which
could act could
against actradical
free against
free
and radical
oxidativeand oxidative
processes processes
[80]. However, [80]. However,
a general a general
consensus consensus
on the on the
role of some role of
of these
phenolic
some compounds
of these phenolichascompounds
not been achieved
has notyet, andachieved
been some studies
yet, have highlighted
and some studiestheir
have
potential adverse
highlighted effects [20].
their potential adverse effects [20].

(1) (2) (3) (4)

(5) (6) (7) (8)

Figure
Figure 3.
3. Chemical
Chemicalstructures
structuresofofeight
eightidentified
identifiedcompounds
compoundsby byHPLC-MS
HPLC-MSininAVE.
AVE.(1)
(1)aloesin;
aloesin;(2)
chlorogenic acid; (3) orientin; (4) aloeresin D; (5) aloin B; (6) aloin A; (7) cinnamic acid; (8) aloe
(2) chlorogenic acid; (3) orientin; (4) aloeresin D; (5) aloin B; (6) aloin A; (7) cinnamic acid;
emodin.
(8) aloe emodin.

Even
Even though some authors
though some authorshave
havereported
reportedthatthatAloe
Aloecontains
containsmany
manycommon
common phenolic
phenolic
acid derivatives(gallic
acid derivatives (gallic acid,
acid, protocatechuic
protocatechuic acid, acid, syringic
syringic acid, acid),
acid, gentisic gentisic acid), hy-
hydroxycin-
droxycinnamic acid derivatives
namic acid derivatives (p-coumaric(p-coumaric acid,caffeic
acid, ferulic acid, ferulic acid,
acid, caffeic
sinapic acid,
acid) andsinapic acid)
flavonoids
and flavonoids (naringenin, catechin, epicatechin, kaempferol, rutin, luteolin,
(naringenin, catechin, epicatechin, kaempferol, rutin, luteolin, myricetin) [4,42,81], only amyricetin)
[4,42,81], only
fraction of theatargeted
fraction standards
of the targeted standardsin
was identified was
AVE.identified in could
This fact AVE. This fact could
be related to
be related to the influence of some environmental conditions, such as water and sunlight
availability, soil characteristics (pH, conductivity, total N), geographical origin, plant va-
riety and age, harvest season, position of the leaves in the plant and post-harvest treat-
ment, among many other factors, which combined with the effect of the extraction tech-
Antioxidants 2022, 11, 1058 18 of 25

the influence of some environmental conditions, such as water and sunlight availability,
soil characteristics (pH, conductivity, total N), geographical origin, plant variety and age,
harvest season, position of the leaves in the plant and post-harvest treatment, among many
other factors, which combined with the effect of the extraction technique could significantly
affect secondary metabolite composition in plants [51,79,82]. In addition, it was noticed
that m/z signals corresponding to several aforementioned compounds were recorded at
different retention times compared to the standards, suggesting that isomeric compounds
of target polyphenols might be present in AVE.
The phenolic compounds identified in AVE were quantified by HPLC-DAD (Figure 4a).
Calibration curves of aloesin, chlorogenic acid, orientin, aloeresin D, aloin B, aloin A, cin-
namic acid and aloe-emodin were obtained, showing acceptable levels of linearity with
determination coefficients (R2 ) ranging from 0.9960 to 0.9999 for all the analyzed standards
at ten concentration levels (Table 5). The LOD and LOQ values that were obtained ranged
from 0.018 to 0.383 mg kg−1 and from 0.061 to 1.275 mg kg−1 , respectively, indicating the
feasibility of the proposed method for the quantification of the identified polyphenols. Fi-
nally, precision in terms of repeatability was evaluated by analyzing standard solutions, in
triplicate, for all concentration levels within the same day, with relative standard deviations
ranging from 0.9 to 2.6%, showing good repeatability. Diastereomeric anthraquinone deriva-
tives Aloin A and B were the main components present in AVE, accounting for 702 ± 2 and
308.1 ± 0.6 mg 100 g AVS−1 , respectively. Another major component found in AVE was
the chromone aloesin with a concentration of 292.6 ± 0.5 mg 100 g AVS−1 . Other quantified
phenolic compounds ranged from 3.6 ± 0.1 to 80.0 ± 0.2 mg 100 g AVS−1 . Quantification
results and analytical figures of merit for HPLC-DAD analysis are summarized in Table 5.

Table 5. Phenolic compounds identified in AVE by HPLC-MS, analytical figures of merit and
quantification results by HPLC-DAD.

(m/z) tR Calibration Range Linearity LOD LOQ RSD 2 AVE

Peak 1 Compound
[M-H]- (min) (mg kg−1 ) (R2 ) (mg kg−1 ) (mg kg−1 ) (%) (mg 100 gAVS−1 )
1 aloesin 393 9.3 0.06–61.80 0.9998 0.164 0.546 2.6 292.6 ± 0.5
2 chlorogenic acid 353 10.5 0.05–99.70 0.9960 0.213 0.711 1.3 80.0 ± 0.2
3 orientin 447 14.4 0.01–11.20 0.9991 0.061 0.203 2.3 46.5 ± 0.1
4 aloeresin D 555 19.1 0.05–9.82 0.9971 0.383 1.275 2.6 39.7 ± 1.1
5 aloin B 417 19.2 0.10–100.60 0.9998 0.087 0.292 1.1 308.1 ± 0.6
6 aloin A 417 20.0 0.20–202.10 0.9999 0.278 0.926 0.9 702.0 ± 2.0
7 cinnamic acid 147 25.2 0.004–3.700 0.9992 0.029 0.095 1.4 13.6 ± 0.5
8 aloe emodin 269 29.4 0.001–0.900 0.9961 0.018 0.061 1.8 3.6 ± 0.1
1peak assignation corresponding to Figure 4. 2 within-day precision (n = 3 at all concentration levels used in the
calibration range).

Phenolic compounds present in Aloe vera plant have been extensively studied in
recent decades due to their well-known beneficial and health promoting properties. How-
ever, the vast majority of the research was focused on the inner gel of the plant and
very few reports on Aloe vera skin phenolic profiles and contents are currently available.
Añibarro-Ortega et al. [13] reported that chromones and anthrones accounted for up to
44.9% and 43.8% of the phenolic compounds found in Aloe vera rind extract, respectively,
with the concentration of aloesin, aloin B and aloin A being 34.4 ± 0.7, 4.3 ± 0.3 and
9.9 ± 0.4 mg g AVE−1 , respectively. The relative concentration reported for aloin A and B
diastereomers is in close agreement with the results shown in Table 5, and aloin A was
2.3 times more concentrated than aloin B, although smaller quantities of both aloins were
reported. On the other hand, López et al. [42] found a lower chlorogenic acid concentration
in AVS, accounting for only 7.8 ± 0.2 mg 100 g AVS−1 . Lucini et al. [4] suggested that
hydroxycinnamic acids, anthrones and chromones might have a direct and relevant role in
the antioxidant potential of Aloe vera extracts.
 Quantification results and analytical figures of merit for HPLC-DAD analysis are summa-
rized in Table 5.
Antioxidants 2022, 11, 1058 19 of 25

(a)

(b)
Figure4.4. (a) HPLC-DAD
Figure HPLC-DAD chromatogram
chromatogram showing
showing the the phenolic
phenolic profile
profile of
ofAVE
AVEobtained
obtainedunder
underopti-
mum MAE
optimum MAEconditions; (b)(b)
conditions; HPLC-MS total
HPLC-MS totalion
ionchromatogram
chromatogram(TIC)
(TIC)of
of AVE. See Table
AVE. See Table5;5;Table
Table66 for
peak
for assignations.
peak assignations.

Table 5. Phenolic
Other studiescompounds identified in AVE
reported quantitative by HPLC-MS,
contents of aloesin,analytical
aloeresinfigures of merit
D, aloin andB,
A, aloin quan-
tification results
aloe-emodin, by HPLC-DAD.
orientin, cinnamic acid and chlorogenic acid [76,78,83–86]. However, different
plant species, Aloe plant tissues (i.e., whole leaf, gel, exudate) and processing and extraction
(m/z) tR Calibration Range Linearity LOD LOQ RSD 2 AVE
Peak 1 Compound techniques were used, and comparison of quantification results might not be straightfor-
[M-H] - (min) (mg kg −1) (R 2) (mg kg −1) (mg kg−1) (%)
ward. For instance, Lai et al. [83] reported 4.26 ± 0.14 and 25.81 ± 0.21 mg 100 g FW (mg 100 gAVS
−1 −1)
1 aloesin 393
of whole9.3 0.06–61.80
Aloe barbadensis 0.9998 acid
leaf for chlorogenic 0.164
and total0.546 2.6
aloin, respectively. 292.6 ± 0.5
Moreover,
2 chlorogenic acid Cardarelli
353 10.5
et al. [86]0.05–99.70
analyzed exudates 0.9960 0.213 Aloe
from six-year-old 0.711 1.3plants and
barbadendis 80.0found
± 0.2
3 orientin 447± 0.21,
2.81 14.40.042 ± 0.007, 0.016 ± 0.9991
0.01–11.20 0.001, 1.96 ± 3.27 ± 0.20
0.40 and 0.203
0.061 2.3100−1 g46.5of aloin,
± 0.1
4 aloeresin D aloe-emodin,
555 19.1 aloenin, aloesin
0.05–9.82 and aloeresin
0.9971 A, respectively.
0.383 Similarly,
1.275 Wu
2.6 et al. [76] found
39.7 ± 1.1
5 aloin B that
417 the 19.2
main component
0.10–100.60 Aloe barbadensis
present in0.9998 0.087 exudate was aloin
0.292 A, accounting
1.1 308.1 ± for
0.6
178 to 219 mg g−1 . Moreover, aloesin and aloe-emodin contents ranged from 0.90 to 19.3
6 aloin A 417 20.0 0.20–202.10 0.9999 0.278 0.926 0.9 702.0 ± 2.0
and from 0.96 to 2.27 mg g−1 , respectively. On the other hand, Aldayel et al. [84] reported
7 cinnamic acid 147 25.2 0.004–3.700 −1 0.9992 0.029 0.095 1.4 13.6 ± 0.5
43.3 ± 2.8 and 21.4 ± 2.0 mg g of Aloe vera gel extract for aloin B and A, respectively.
8 aloe emodin 269
In 29.4 a wide
conclusion, 0.001–0.900 0.9961 results
range of quantitative 0.018
could be0.061
found in 1.8 3.6 ±
the literature 0.1
using
1peak assignation
different approachescorresponding to Figure
for the analysis 4. vera
of Aloe within-day
2 precision (n = 3 at all concentration lev-
plant components.
els used in the calibration range).
Apart from the eight already quantified phenolic compounds, other peaks were also
detected in the negative ion mode as [M-H],- and they were tentatively identified based
on their accurate mass fragments, elution sequence and careful analysis of the literature
(Table 6). A typical total ion chromatogram (TIC) of AVE obtained by HPLC-MS is shown
Antioxidants 2022, 11, 1058 20 of 25

in Figure 4b. The MS spectrum found for peak a showed the most abundant fragment at
m/z 455. Añibarro-Ortega et al. [13] reported similar findings, although the structure of
this compound has not yet been elucidated. Peak b eluted at 13.0 min, showing fragments
at m/z 337 and 609, which were tentatively identified as cis or trans-5-p-coumaroylquinic
acid and luteolin-6,8-C-diglucoside, respectively, by the same authors. The mass spectrum
for peak c exhibited a main fragment at m/z 447, revealing a group of orientin isomers
which, according to the literature, might be identified as 8-O-methyl-7-hydroxyaloin [47,84]
or luteolin-6-C-glucoside [13]. Peaks d and e showed a main fragment at m/z 433. It
has been reported that Aloe vera extracts contain aloin-derived diastereoisomers such as
5, 7 or 10-hydroxyaloin, which were proposed for the tentative identification of these
peaks [13,47,48,76,85]. Similarly, peaks f, g, h and j were tentatively identified as dihy-
droisocoumarin glucoside [84], 60 -malonylnataloin B [47,48], 60 -malonylnataloin A [47,48]
and 5,30 -dihydroxy-6,7,40 -trimethoxyflavone [48], respectively. Finally, peak i showed a
main fragment at m/z 585. A compound with 585 g mol−1 of molecular weight was also
found by Añibarro-Ortega et al. [13], although its molecular structure was not determined.

Table 6. Tentative identification of unknown phenolic compounds in AVE by HPLC-MS.

tR 2 (m/z) Elemental
Peak 1 (min) [M-H]- Composition Tentative Identification Ref.

a 10.9 455 - Unknown [13]

- cis or trans
337 C16 H17 O8 [13]
b 13 5-p-Coumaroylquinic acid
609 C27 H29 O16 - luteolin-6,8-C-diglucoside [13]
447 C22 H23 O10 - 8-O-methyl-7-hydroxyaloin [47,84]
c 13.8
447 C22 H23 O10 - luteolin-6-C-glucoside [13]
433 C21 H21 O10 - 7-hydroxyaloin B [47]
d 15.2 433 C21 H21 O10 - 10-hydroxyaloin B [13,48,76,85]
433 C21 H21 O10 - 5-hydroxyaloin B [13]
433 C21 H21 O10 - 7-hydroxyaloin A [47]
e 15.9 433 C21 H21 O10 - 10-hydroxyaloin A [13,48,76,85]
433 C21 H21 O10 - 5-hydroxyaloin A [13]
Dihydroisocoumarin
f 17.7 505 C24 H25 O12 - [84]
glucoside
g 20.2 459 C23 H23 O10 - 60 -malonylnataloin B [47,48]
h 20.8 459 C23 H23 O10 - 60 -malonylnataloin A [47,48]
i 24.2 585 - Unknown [13]
5,30 -Dihydroxy-6,7,40 -
j 24.7 343 C18 H15 O7 - [48]
trimethoxyflavone
1Notation for peak identification refers to Figure 4. 2 Retention times from HPLC-MS according to extracted ion
chromatograms (EICs).

3.4. Scanning Electron Microscopy (SEM)

SEM was used to investigate the impact of the MAE process on the AVS structure.
Figure 5a,b show SEM micrographs obtained for raw AVS after drying and grinding steps
as well as for AVS residue obtained after MAE under optimal conditions, respectively. A
significant change in the surface morphology of the AVS particles was observed. Before
extraction, particles exhibited a relatively smooth and unaltered surface. However, after
MAE, AVS showed increased porosity and evidenced surface deterioration. These results
are in accordance with other authors reporting similar effects of microwave irradiation on
diverse vegetal materials such as cocoa bean shell [5], Pinus radiata bark [87] and brown
seaweeds [88]. In all cases, it was suggested that the damage to the vegetal structure can
facilitate solvent penetration, enhancing diffusion and extraction of bioactive compounds,
which helps to increase TPC and antioxidant activity values.
 extraction, particles exhibited a relatively smooth and unaltered surface. However, after
MAE, AVS showed increased porosity and evidenced surface deterioration. These results
are in accordance with other authors reporting similar effects of microwave irradiation on
diverse vegetal materials such as cocoa bean shell [5], Pinus radiata bark [87] and brown
seaweeds [88]. In all cases, it was suggested that the damage to the vegetal structure can
Antioxidants 2022, 11, 1058 21 of 25
facilitate solvent penetration, enhancing diffusion and extraction of bioactive compounds,
which helps to increase TPC and antioxidant activity values.

(a) (b)

Figure 5. SEM micrographs of raw AVS (a) and AVS after MAE under optimal conditions (b).

4. Conclusions
A new MAE methodology was developed for the extraction of bioactive compounds
from Aloe vera skin wastes as a green and fast strategy for the valorization of these
agrowastes. The combined effects of MAE experimental parameters such as ethanol com-
position, temperature, time and solvent volume on extraction yield, TPC, DPPH, FRAP and
aloin content of the extracts were studied and optimized by using a BBD. Second-order poly-
nomial regression models with high reliability were obtained and MAE conditions which
simultaneously optimize all responses were 80% ethanol, 80 ◦ C, 36.6 min and 50.0 mL.
Under these extraction conditions, the obtained responses regarding extraction yield, TPC,
DPPH, FRAP and aloin were 17.3 ± 0.1 g AVE 100 g AVS−1 , 116.4 ± 4.5 mg GAE g AVE−1 ,
69.0 ± 1.9 mg TE g AVE−1 , 131.9 ± 6.5 mg TE g AVE−1 and 55.6 ± 0.2 mg aloin g AVE−1 ,
respectively. Structural (FTIR) and thermal (TGA) characterization results were in accor-
dance with AVE composition, while significant differences in surface morphology were
evidenced by SEM in AVS before and after MAE. Moreover, eight major phenolic com-
pounds (aloesin, chlorogenic acid, orientin, aloeresin D, aloin B, aloin A, cinnamic acid
and aloe-emodin) were identified and quantified by HPLC-DAD/MS, while eight other
compounds were also tentatively identified. Diastereomeric anthraquinone derivatives
Aloin A and B were the main components present in AVE, followed by the chromone
aloesin. According to the obtained results, the proposed method could be a promising
procedure for obtaining antioxidant extracts rich in polyphenols with potential industrial
applications in the food, biomedical or cosmeceutical industries, as well as contributing to
the circular economy and reducing food waste and environmental impact issues.

Author Contributions: Conceptualization, A.J. and M.C.G.; methodology, A.J. and M.C.G.; valida-
tion, A.J. and M.C.G.; formal analysis, I.S.; investigation, I.S.; data curation, I.S.; writing—original
draft preparation, I.S.; writing—review and editing, A.J. and M.C.G.; supervision, A.J. and M.C.G.
All authors have read and agreed to the published version of the manuscript.
Funding: Authors would like to acknowledge Consellería de Educación, Investigación, Cultura y
Deporte de la Generalitat Valenciana (GRISOLIAP/2016/081) and Spanish Ministry of Science and
Innovation (Refs. PID2020-116496RB-C21, PDC2021-121345-C21) for their financial support.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: Authors acknowledge LAS CORONAS for providing Aloe vera leaves.
Conflicts of Interest: The authors declare no conflict of interest.
Antioxidants 2022, 11, 1058 22 of 25

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