sustainability

Article
Valorization of Banana and Red Beetroot Peels:
Determination of Basic Macrocomponent
Composition, Application of Novel Extraction
Methodology and Assessment of Biological Activity
In Vitro
Danijela Šeremet 1 , Ksenija Durgo 1 , Stela Jokić 2 , Ana Hud̄ek 1 , Aleksandra Vojvodić Cebin 1 ,
Ana Mandura 1 , Jasna Jurasović 3 and Draženka Komes 1, *
1 Faculty of Food Technology and Biotechnology, University in Zagreb, Pierottijeva 6, 10 000 Zagreb, Croatia;
dseremet@[Link] (D.Š.); kdurgo@[Link] (K.D.); ahudek@[Link] (A.H.); avojvodic@[Link] (A.V.C.);
amandura@[Link] (A.M.)
2 Faculty of Food Technology, Josip Juraj Strossmayer University of Osijek, Franje Kuhača 20, 31 000 Osijek,
Croatia; [Link]@[Link]
3 Institute for Medical Research and Occupational Health, Ksaverska Cesta 2, 10 000, Zagreb, Croatia;
jurasovic@[Link]
* Correspondence: dkomes@[Link]; Tel.: + 385-14-605-183




Received: 10 May 2020; Accepted: 1 June 2020; Published: 3 June 2020


Abstract: The nutritional and bioactive content of banana and red beetroot peels was investigated.
The basic macrocomponent composition was determined using standard AOAC (Association of
Official Analytical Chemists) methods, while the recovery efficiency of bioactive compounds was
investigated using conventional and innovative extraction techniques (subcritical water extraction,
ultrasound- and microwave-assisted extraction). Extracts were analyzed for biological effects in vitro
on human hepatic, tongue and colon cancer cell lines. A macrocomponent analysis revealed a notable
amount of dietary fiber in banana and beetroot peels (39.0 and 33.6% dmb) and a relatively high
content of protein in beetroot peel (18.3% dmb). Regarding the micronutrients-minerals, banana
and beetroot peels were shown to be a very good source of potassium (75.06 and 41.86 mg g−1 dmb).
Both extracts of banana and beetroot peels obtained by conventional extraction - decoction (100 ◦ C,
20 min) exhibited the highest total phenolic content and antioxidant capacity. Additionally, in
banana peel, these extracts were the richest in dopamine content (12.63 mg g−1 dmb). Extraction
by infusion (80 ◦ C, 30 min) yielded a beetroot peel extract with the highest total betacyanin content
(9.80 mg g−1 dmb). Biological effects in vitro were dose- and time-dependent, as well as influenced
by the presence of polysaccharides.

Keywords: bioactives; banana peel; red beetroot peel; novel extraction; cytotoxicity

1. Introduction
For the period between 2009 and 2050, the world population is predicted to grow by a third,
which will also result in a substantial increase in food demand [1]. Therefore, the agro-industrial
sectors, besides providing food to the whole population, are searching for the new eco-friendly and
sustainable forms of food production, including agro-industrial waste reutilization. Agro-industrial
waste includes stalks, stems, leaves, roots, molasses, husks, peels, etc., that are known to contain
high-value ingredients, such as nutrients and bioactive compounds [2], pointing to wider possibilities
of application in the food industry.

Sustainability 2020, 12, 4539; doi:10.3390/su12114539 [Link]/journal/sustainability

Sustainability 2020, 12, 4539 2 of 21

Banana (Musaceae) is a tropical fruit available throughout the whole year and, after tomato, the
most consumed fruit in the world [3], with an annual production of 115 million tons in 2018 [4].
Since approximately 30% of banana fruit is comprised of inedible peel [5], it is obvious that, on a global
scale, a lot of peel waste is generated annually in fruit industries, as well as in households. Regarding
the chemical composition, carbohydrates and crude fibers make up most of the banana peel’s dry
matter, but a significant amount of proteins, potassium, essential amino acids and polyunsaturated
fatty acids can also be found [6–8]. Banana peel also represents a great source of phenolic compounds,
like gallocatechin, and catecholamines, especially dopamine, whose content was found to be much
higher in the peel compared to the banana pulp [9,10].
Red beetroot (Beta vulgaris) has been listed in the top 10 vegetables with the highest antioxidant
activity [11], largely due to the nitrogen red-violet colored pigments called betacyanins, and especially
betanin [12]. Since the application of betanin as a food colorant has been approved by EFSA and
FDA [13], and due to the rise in demand for natural food colorants, the global consumption of beetroot
extracts has increased [14]. The quantity of betanin and phenolic compounds in the beetroot is higher in
the peel than in the flesh and crown [15], indicating a possibility for the peel’s reutilization. Red beetroot
peel is also known for a high content of ferulic acid, which is characteristic of many betalain-bearing
species [16].
To accomplish the maximum recovery of target bioactive compounds from plant material, the key
step is to choose the most adequate extraction technique. To overcome the limitations of conventional
extraction techniques, that are often long-lasting, result in the degradation of thermolabile compounds
and require organic solvents, some of them harsh, innovative methods of extraction have been
introduced, such as enzyme-, ultrasound- and microwave-assisted extraction, pulsed electric field
extraction, pressurized liquid extraction, etc. [17]. Ultrasound-assisted extraction (UAE) has been
investigated for many years and has found application in the industry in the form of an ultrasonic
reactor for the extraction and preparation of tinctures from different herbs, that was registered as a
patent [18]. Although microwave-assisted extraction (MAE) and subcritical water extraction (SWE)
have been introduced later than UAE, the advantages of their are currently well known. MAE is
characterized by a special heating system that allows for homogeneous internal heating throughout
the whole volume of the material, resulting in an increase of pressure inside the plant cells followed
by their disruption and the release of the compounds of interest [19]. SWE is based on maintaining
the water in a liquid state at temperatures higher than its boiling point, using high pressure and
thus improving the physical and chemical properties of water as a solvent, by changing the dielectric
constant, viscosity, surface tension, etc. [20]. MAE has been applied for the extraction of bioactive
compounds from different types of agro-industrial wastes, such as longan peel [21], potato peel [22]
and black rice husk [23]. SWE has been used for cocoa shell [24], orange peel [25] and potato peel [26].
In this study, conventional (infusion, decoction and maceration) and innovative (subcritical water
extraction, ultrasound- and microwave-assisted extraction) techniques of extraction were applied in
order to assess their extraction efficiency regarding the bioactive constituents of banana and beetroot
peels, possibly contributing to their valorization. The present study is the first reporting the use of
innovative techniques of extraction on red beetroot peel. Additionally, the obtained extracts were
evaluated for their biological activity (cytotoxicity and antioxidative/prooxidative effects) in vitro
test systems (human hepatic, tongue and colon cancer cell lines), to estimate a safe intake level
corresponding to the potential usage of these extracts as functional food ingredients, since these cells
come into contact with bioactive compounds during consumption, digestion and metabolism.

2. Materials and Methods

2.1. Materials and Chemicals

Banana (var. Cavendish, produced by Costanza Organic Bananas, Ecuador) and red beetroot
(grown at a local agricultural farm) were purchased in a local store. Hydrochloric acid (37%),
Sustainability 2020, 12, 4539 3 of 21

bromcresol green and methyl red indicators, boric acid and Folin–Ciocalteu’s reagent were supplied
from Kemika (Zagreb, Croatia). Kjeldahl tablets were purchased from CarlRoth (Karsruhe,
Germany). An integrated total dietary fiber assay kit was purchased from Megazyme (Wicklow,
Ireland). Dopamine hydrochloride, (S)-6-Methoxy-2,5,7,8-tetramethylchromane-2-carboxylic acid
(Trolox), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS), gallic acid, neutral red and dichlorofluorescein diacetate (DCFDA) were
purchased from Sigma-Aldrich (St. Louis, USA). Standards of D-glucose, D-fructose and sucrose were
purchased from Fluka (Taufkirchen, Germany). Methanol was supplied from Panreac (Barcelona,
Spain) and ethanol (96%), glacial acetic acid (85%) and acetonitrile from Carlo Erba (Val de Reuil,
France). All chemicals used for experimental procedures were of analytical grade.

2.2. Methods

2.2.1. Preparation of Banana and Red Beetroot Peel Powder

The maturity stage of bananas was determined following the standard color chart of Tapre &
Jain [27], by which they exhibited a value of 6 (full yellow). Bananas were peeled, and the collected
whole peels were submerged in boiling water and blanched for 7 min. Afterward, the peels were dried
using paper towels, cut into small pieces and freeze-dried (Alpha 1-2 LD plus freeze-dryer, Martin
Christ, Germany). Red beetroots were peeled, and the peels were left to air-dry at room temperature
for 48 h. The dried banana and red beetroot peels were milled into powder and sieved through a screen
with pores of 450 µm, to obtain fractions to be used in the analyses.

2.2.2. Determination of Macrocomponent Composition

The dry matter was determined according to the AOAC 930.15 method [28], by drying the
sample until constant mass. The crude protein content was determined according to the AOAC 976.05
method [29], by the Kjeldahl protocol (6.25 was used as a conversion factor). The crude fat content
was determined according to the AOAC 920.39 method [30], using the Soxhlet apparatus, and the
crude mineral content following the AOAC 942.05 method [31]. The high molecular weight insoluble
and soluble fiber content was determined using the Integrated Total Dietary Fiber Assay kit according
to the AOAC 2011.25 method [32]. The analysis of fatty acid composition, using the EN ISO 5509
method [33] and an Agilent Gas Chromatography 6890 series equipped with an Agilent 5973 Inert
Mass Selective Detector (Agilent Technologies, Santa Clara, CA, USA), was carried out only for banana
peel, since it contained a significant lipid fraction. The HPLC analysis of soluble sugars (glucose,
fructose and sucrose) was conducted on an Agilent Series 1200 chromatographic system (Agilent
Technologies, Santa Clara, CA, USA) coupled with RI detector (Agilent Technologies, Santa Clara,
CA, USA) and by using the Phenomenex Luna NH2 column (Phenomenex, Santa Clara, CA, USA)
in HILIC mode, following the method described by Vojvodić et al. [34]. The content of micro- and
macroelements was analyzed using an inductively coupled plasma mass spectrometer (Agilent 7500cx,
Agilent Technologies, Tokyo, Japan).

2.2.3. Extraction

Conventional Extraction Techniques

Infusion (INF), decoction (DEC) and maceration (MAC) were performed using distilled water
as a solvent, with the same ratio sample/solvent (1:20, w/v). The parameters for infusion, decoction
and maceration were as follows: 80 ◦ C for 30 min, 100 ◦ C for 20 min and room temperature for
48 h, respectively. The extractions were followed by centrifugation (9500 rpm, 20 min) and filtration
(Whatman® filter papers 4). The extracts were stored at +4 ◦ C until further analyses.
Sustainability 2020, 12, 4539 4 of 21

Innovative Extraction Techniques

All techniques of extraction were performed using distilled water as a solvent, with the same ratio
sample/solvent (1:20, w/v). Ultrasound-assisted extraction (UAE) was performed in the ultrasound
bath (Elmasonic 2 120, Elma, Singen, Germany) with a nominal power of 200 W and a frequency of
37 kHz during 30 (U30) and 60 (U60) min. Subcritical water extraction (SWE) was performed in an
appropriate system, previously described by Jokić et al. [35]. SWE was performed during 5 min at a
temperature of 150 ◦ C with an extraction pressure of 30 bar. Microwave-assisted extraction (MAE) was
carried out in the Micro SYNTH platform (Milestone, Sorisole, Italy) at a temperature 50 ◦ C during
5 min. All extractions were followed by centrifugation (9500 rpm, 20 min) and filtration (Whatman®
filter papers 4). The extracts were stored at +4 ◦ C until further analyses.

2.2.4. Characterization of the Bioactive Content of the Obtained Extracts

Determination of Total Phenolic Content (TPC) and Antioxidant Capacity (AC)

TPC in prepared extracts was determined spectrophotometrically (Genesys 10S UV-VIS
Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA), according to the method of
Singleton & Rossi [36]. The standard calibration curve was constructed with solutions of gallic acid
(25–200 µg L−1 ). The results were expressed as mg gallic acid equivalents/g of the sample’s dry matter
(mg GAE g−1 dmb). The AC of the prepared extracts was determined using the DPPH and ABTS radical
cation decolorization assays by Brand-Williams et al. [37] and Re et al. [38], respectively. Solutions
of Trolox (25–200 µg L−1 ) where used for the construction of the standard calibration curve, and the
results were expressed as mg Trolox equivalent/g of the sample’s dry matter (mg Trolox g−1 dmb).
All measurements were performed in triplicate.

HPLC Determination of Dopamine in Banana Peel Extracts

The HPLC analysis was performed on an Agilent Series 1200 chromatographic system (Agilent
Technologies, Santa Clara, CA, USA) using a Zorbax Extend C18 (4.6 × 250 mm, 5 µm i.d.)
chromatographic column (Agilent Technologies, USA) and coupled with a Photodiode Array
Detector (Agilent Technologies, Santa Clara, CA, USA). The elution was performed in a gradient
with a three-component mobile phase consisting of (A) 100% acetonitrile, (B) 2% (v/v) formic acid
solution in methanol and (C) 2% (v/v) formic acid solution in water, according to the following
regimen: 0 min – 0% A, 3% B, 97% C; 5 min – 0% A, 3% B, 97% C; 10. min – 0% A, 5% B, 95% C;
30 min – 30% A, 30% B, 40% C; 35 min – 30% A, 30% B, 40% C; 45 min – 0% A, 3% B, 97% C;
60 min – 0% A, 3% B, 97% C. The flow was 0.7 mL min−1 , the injection volume 20 µL and the column
temperature 25 ◦ C. The chromatograms were recorded at 280 nm. Dopamine identification was
performed by comparing the retention time and characteristic absorption spectrum (190–400 nm) with
a commercially available standard. Quantification was enabled by establishing a dopamine calibration
curve (25–300 µg mL−1 ). The analysis was performed in duplicate. All samples were filtered through a
0.45 µm membrane filter (Nylon Membranes, Supelco, Bellefonte, PA, USA) prior to the analysis.

Determination of Total Betalain Content in Red Beetroot Peel Extracts

The total betalain content was determined spectrophotometrically (Genesys 10S UV-VIS
Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA), as described previously [39].
The extracts were briefly diluted with distilled water without pH adjustment, so that the measured
absorption values, at 538 nm for betacyanins and 476 nm for betaxanthins, were in the range
0.8–1.0. The total betacyanin content was expressed as mg equivalents of betanin/g of sample’s dry
matter (mg betanin g−1 dmb), and the total betaxanthin content as equivalents of vulgaxanthin I
(mg vulgaxanthin I g−1 dmb). The total betacyanin and betaxanthin contents were calculated using the
Equation (1):
BC = [(A × Df × Mw × 1000)/(e × l) (1)
Sustainability 2020, 12, 4539 5 of 21

where A is the measured absorbance at 538 or 476 nm, Df the dilution factor, Mw the molecular weight
of betanin or vulgaxanthin I, e the molar extinction coefficients of betanin or vulgaxanthin I and l the
pathlength of the cuvette. The measurements were performed in triplicate.

2.2.5. Biological Effect of Banana and Red Beetroot Peel Extract

Preparation of Extracts for Biological Effects In Vitro

The extracts of banana and red beetroot peels with the highest bioactive content were further
analyzed for biological effects in vitro. Additionally, extracts with removed polysaccharides were also
prepared and analyzed. Polysaccharides in extracts were removed by ethanol precipitation (4-fold
volume of 96% ethanol). The precipitation lasted for 1 h, and the precipitated polysaccharides were
removed by filtration (Whatman® filter papers 4). Both extracts, with and without polysaccharides,
were evaporated under vacuum (Buchi Rotavapor R124, Flawil, Switzerland) and freeze-dried (Christ,
Alpha 1-2 LD plus, Osterode, Germany) to obtain powdered extracts that were subjected to further
analysis in vitro. Based on the known polyphenol concentration in both extracts, working solutions were
prepared in a range of 0.014–1 mg mL−1 . The lowest concentrations (0.014 mg mL−1 and 0.2 mg mL−1 )
corresponded to the prescribed daily intake of polyphenols and were expressed per volume of body
weight (70 kg) and blood volume (5 L), respectively. The other concentrations, 1 mg mL−1 and
10 mg mL−1 , were 5x and 10x higher than the prescribed ones.

Cytotoxicity Assay
The cytotoxicity of banana and red beetroot peel extracts was determined by a neutral red (NR)
assay, as described previously [40]. Human hepatic (HepG2), tongue (CAL 27) and colon (Caco-2)
cancer cell lines were briefly seeded in a 96-well plate and grown to confluency. Afterward, the cells
were treated with different concentrations of banana and red beetroot peels extracts (final concentrations
to which the cells were exposed: 0.014, 0.1, 1 and 10 mg mL −1 ) for 0.5, 1 and 2 h. The time of incubation
was chosen according to the stability of polyphenols in banana and red beetroot peel extracts under
previously determined incubation conditions. After incubation, NR solution was added into each
well after 45 min. The intensity of absorbance was measured at 540 nm in a microtiter reader (Cecil
Instruments Ltd., Cambridge, UK). Each extract concentration was tested in quadruplicate. The cell
viability was calculated using the Equation (2):

% cell viability = Absorbancesample /Absorbancecontrol × 100% (2)

where Absorbancesample is the absorbance of cells treated with extracts, and Absorbancecontrol is the
absorbance of the growth medium with 0.1% DMSO.

Reactive Oxygen Species Determination

The reactive oxygen species (ROS) formation in the cells (human hepatic (HepG2), tongue (CAL
27) and colon (Caco-2) cancer cell lines) after the treatment with banana and red beetroot peel extract
was determined by the dichlorohydrofluorescein (DCF) assay using a microplate reader, as described
previously [41,42]. After seeding the cells and obtaining confluency, they were treated with different
concentrations of banana and red beetroot peel extracts (final concentrations to which the cells were
exposed to: 0.014, 0.2, 1 and 10 mg mL−1 ) for 0.5, 1 and 2 hours. Each extract concentration was tested
in quadruplicate. The fluorescence (Cecil Instruments Ltd., Cambridge, UK) of the cells was measured
with an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The measured
fluorescence was compared to the fluorescence of control, and the results were expressed a as percentage
of ROS induction (Equation (3)):

% of fluorescence of samples = Fluorescencesample /% cell survivalsample /Fluorescence control /% of cell survivalcontrol ) (3)
Sustainability 2020, 12, 4539 6 of 21

where Fluorescencesample is the fluorescence of cells treated with extracts, and Fluorescencecontrol
is the fluorescence of non-treated cells.

2.2.6. Statistical Analysis

The results were statistically analyzed using the SPSS Statistics 17.0 program (IBM, Armonk, NY,
USA). The analysis of variance (ANOVA) and post-hoc analysis (Tukey’s HSD test) were performed
with a significance level of α = 0.05%.

3. Results and Discussion

The aim of this study was to present a valorization possibility of banana peel and red beetroot
peel by means of their macro- and micronutrient composition, as well as the recovery of bioactive
compounds by employing different extraction strategies. The macrocomponent composition was
determined using standard AOAC methods. The total phenolic content and antioxidant capacity were
determined spectrophotometrically, as well as the total betalain content in the case of red beetroot
peel. The dopamine content in banana peel was determined using the HPLC-DAD methodology.
The extracts exhibiting the highest bioactive content were further analyzed in vitro.

3.1. Macrocomponent Composition and Content of Macro- and Microelemenents

The macrocomponent composition and the content of macro- and microelements of banana peel
and red beetroot peel are presented in Tables 1 and 2, respectively.

Table 1. Macrocomponent composition of banana peel and red beetroot peel.

Banana Peel Red Beetroot Peel

Dry matter (%) 88.1 ± 0.1 90.8 ± 0.0
Crude protein content (% dmb*) 9.2 ± 0.3 18.3 ± 0.2
Crude fat content (% dmb) 7.5 ± 0.1 0.6 ± 0.0
• Lauric acid C12:0 (% fa*) 28.9 ± 0.2 np
• Palmitic acid C16:0 (% fa) 28.6 ± 0.1 np
• Stearic acid C18:1 (% fa) 4.7 ± 0.0 np
• Linolenic acid C18:3n3 (% fa) 37.8 ± 0.2 np
Crude mineral content (% dmb) 14.0 ± 0.3 12.1 ± 0.1
Total dietary fibre (% dmb) 39.0 ± 2.7 33.6 ± 1.0
• Insoluble dietary fibre (% dmb) 38.1 ± 2.9 26.6 ± 0.5
• Soluble dietary fibre (% dmb) 0.9 ± 0.3 6.9 ± 0.5
Soluble sugar content (% dmb) 19.1 ± 0.1 12.5 ± 0.2
• Fructose (% dmb) 12.7 ± 0.2 nd
• Glucose (% dmb) n.d. nd
• Sucrose (% dmb) 6.4 ± 0.1 12.5 ± 0.2
dmb—dry matter basis of the sample; fa—fatty acids; np—not performed; nd—not detected.
Sustainability 2020, 12, 4539 7 of 21

Table 2. Macro- and microelement content in banana peel and red beetroot peel.

Banana Peel Red Beetroot Peel

Macroelements (mg kg−1 dmb*)
Na 38 ± 0 4380 ± 189
Mg 150 ± 14 6570 ± 356
K 75061 ± 563 41854 ± 1366
Ca 2698 ± 3 2851 ± 153
Fe 33 ± 4 159 ± 10
P 2900 ± 31 7811 ± 393
Microelements (µg kg−1 dmb)
V 17 ± 1 308 ± 21
Mn 63 ± 0 52 ± 3
Cr 172 ± 31 343 ± 21
Co 35 ± 0 200 ± 7
Ni 391 ± 19 1370 ± 76
Cu 6±0 19 ± 1
Zn 14 ± 0 49 ± 2
As 8±0 91 ± 6
Se 13 ± 0 26 ± 1
Mo 135 ± 3 580 ± 11
Cd 11 ± 7 421 ± 8
Sn 23 ± 1 17 ± 1
Sb 8±1 9±1
Hg 49 ± 2 36 ± 1
dmb—dry matter basis of the sample.

Insoluble dietary fibers made up most of the banana peel’s dry matter (38.1% dmb), followed
by soluble sugars (19.1% dmb), crude minerals (14.0% dmb), crude protein (9.2% dmb), crude fat
(7.5% dmb) and soluble dietary fiber content (0.9% dmb) (Table 1). Regarding soluble sugars, they
were evaluated as fructose, glucose and sucrose content, and fructose was the main sugar found
in banana peel, making up 67% (12.7% dmb) of the total soluble sugar content, while the rest was
sucrose (6.4% dmb). A high content of insoluble dietary fibers in agro-industrial wastes correlates
with an abundance in cellulose, hemicellulose and lignin [43,44]. Anhwange [6] also reported a high
content of crude fiber for banana peel (31.7%), while Emaga et al. [8] also reported an increase of
fiber content in banana peel during maturation. Further, the results on sugar content presented
in this study are consistent with the study of Tapre & Jain [27], who reported a sugar content of
18.48% in banana peel in maturity stage 7. Moreover, the lipid fraction we have determined is in
accordance to previously reported values: 7.0 and 7.8% dmb in peels of Pelipita and CRBP039 banana
varieties in maturity stage 7 [8]. Additionally, the collected lipid fraction in this study was further
analyzed for fatty acid composition, using the GC method. Linolenic acid, an omega-3 fatty acid, was
found to be the predominant fatty acid in the lipid fraction of banana peel, comprising 37.8% of all
presented fatty acids. Among other fatty acids, lauric, palmitic and stearic acids were also identified,
representing relative contents in total fatty acids of 28.9%, 28.6% and 4.7%, respectively. In the study
of Morais et al. [45], a different profile of fatty acids in freeze-dried banana peel was determined,
with palmitic acid being dominant (3.59 mg g−1 dmb), followed by linoleic (3.35 mg g−1 dmb) and
linolenic (2.42 mg g−1 dmb) acids. Regarding the macroelement composition (Table 2), banana peel
showed to be a rich source of potassium (75.06 mg g−1 dmb), as also reported by Anhwange [6],
who stated that banana peel consumption could contribute to the regulation of body fluids, the
maintenance of normal blood pressure and the mitigation of respiratory, kidney or heart problems
due to a significant amount of potassium. The content of other macroelements, including sodium,
magnesium, calcium, iron and phosphorus, was notably lower (Table 2). Among the microelements,
Sustainability 2020, 12, 4539 8 of 21

the highest contents were determined for nickel, chrome and molybdenum, as follows: 0.40, 0.17 and
0.14 µg g−1 dmb, respectively.
A relatively high content of insoluble dietary fiber was also noted in the beetroot peel’s dry
matter (26.6% dmb), and a higher content of soluble dietary fiber (6.9% dmb) than in banana peel.
The contents of crude protein, crude minerals and crude fat were 18.3% dmb, 12.1% dmb and 0.6% dmb,
respectively. Among soluble sugars, only sucrose was identified (12.5% dmb). A high content of
sucrose is characteristic of beetroot, since it is a root vegetable where energy is stored in the form of
sucrose [46]. The chemical composition of beetroot peel is not well covered in the available literature,
so it is not possible to make comparisons. Nevertheless, the determined protein content is close to
the one reported by Costa et al. [47] for beetroot waste containing peels, stalks and parings, in the
range of 12.64%–12.68% dmb. Further, potassium was the main macroelement (41.86 mg g−1 dmb)
found in beetroot peel, followed by a much lower quantity of phosphorus and magnesium (7.81 and
6.57 mg g−1 dmb, respectively), while among microelements, the quantity of nickel (1.37 µg g−1 dmb)
deviated considerably from the others (Table 2).

3.2. Bioactive Content of Obtained Extracts

3.2.1. Characterization of Banana Peel Extracts

The bioactive content—including TPC, antioxidant capacity and dopamine content—of the
obtained banana peel extracts is presented in Table 3.

Table 3. Bioactive content of differently prepared extracts of banana peel.

Antioxidant Capacity
Sample TPC Dopamine
DPPH ABTS
(mg GAE g−1 dmb) (mg g−1 dmb)
(mmol Trolox g−1 dmb) (mmol Trolox g−1 dmb)
INF 25.59 ± 0.38 a 0.156 ± 0.00 0.160 ± 0.00 10.29 ± 0.00 a
DEC 25.38 ± 0.53 a 0.163 ± 0.00 0.195 ± 0.00 12.63 ± 0.00
MAC 18.73 ± 0.48 bc 0.124 ± 0.00 0.125 ± 0.00 9.94 ± 0.02
U30 18.26 ± 0.59 bd 0.117 ± 0.00 0.123 ± 0.00 10.33 ± 0.01 a
U60 17.96 ± 0.13 cd 0.112 ± 0.00 0.132± 0.00 11.31 ± 0.02
SWE 16.06 ± 0.06 0.110 ± 0.01 0.097 ± 0.00 10.21 ± 0.03
MAE 3.46 ± 0.05 0.021 ± 0.00 0.020 ± 0.00 5.14 ± 0.01
dmb—dry matter basis of the sample; INF—infusion; DEC—decoction; MAC—maceration; U30 and
U60—ultrasound bath for 30 and 60 min, respectively; SWE—subcritical water extraction; MAE—microwave-assisted
extraction; Means in the same column denoted with the same superscript letters (a,b,c,d) are not significantly
different (p > 0.05).

The conventional extraction techniques INF and DEC resulted in the extracts with the highest
TPC (25.59 and 25.38 mg GAE g−1 dmb, respectively) and the highest antioxidant capacity, determined
by the DPPH (0.156 and 0.163 mmol Trolox g−1 dmb, respectively) and ABTS (0.160 and 0.195 mmol
Trolox g−1 dmb, respectively) assays, while the extract obtained by MAE was characterized by the
lowest TPC and antioxidant capacity (3.46 mg GAE g−1 dmb; 0.021 and 0.020 mmol Trolox g−1 dmb)
(Table 3). High correlations between TPC and DPPH (R2 = 0.99), as well as for ABTS (R2 = 0.95),
were observed. It is noteworthy to point out the similar values (p > 0.05) of TPC between MAC
(18.73 mg GAE g−1 dmb) and U30 (18.26 mg GAE g−1 dmb) extracts, implying the increased efficiency
of UAE, which has reached the effect of 48 h maceration in just 30 min. The mechanism of UAE has
been extensively reviewed by highlighting ultrasonically induced cavitation as the most influential
phenomenon in the extraction enhancement of UAE [48–50]. According to Vu et al. [51], optimal UAE
parameters using the ultrasonic bath for the recovery of phenolic compounds from Musa Cavendish
banana peel, with a 60% aqueous solution of acetone as solvent, are a temperature of 30 ◦ C, a time
period of 5 min and an ultrasonic power of 150 W. Under these conditions, the authors [51] obtained a
banana peel extract with a total phenolic content of 23.49 mg GAE g−1 dmb and an antioxidant capacity
of 47.09 and 48.38 mg Trolox g−1 dmb, measured with the DPPH and ABTS assays, respectively, which
Sustainability 2020, 12, 4539 9 of 21

is in good agreement with the present study. Among the other available literature, the TPC of banana
peel was reported to be 17.89 mg GAE g−1 dmb in an 80% aqueous solution of methanol [52], 5.85
and 6.85 mg GAE g−1 dmb for ripe and green Cavendish peel, respectively, and 0.91 and 1.6 mg
GAE g−1 dmb for ripe and green Dream banana peel, respectively, also measured in 80% methanolic
extracts [53]. Further, MAE, as already mentioned, resulted in the extract with the lowest content
of TPC and an antioxidant capacity which is inconsistent with studies reporting a high recovery of
bioactive compounds [21–23,54]. Kaderides et al. [54] reported the increased efficiency of UAE and
MAE for the extraction of phenolics and punicalagin from pomegranate peels under specific operating
conditions, as well as more intensive creases and ruptures on the surface of the sample caused by
MAE. Further, regarding the SWE in the present study, it has shown a moderate extraction efficiency,
resulting in a TPC value of 16.06 mg g−1 dmb and an antioxidant capacity of 0.110 and 0.097 mmol
Trolox g−1 dmb, determined by the DPPH and ABTS assays, respectively. Ishak et al. [55] studied the
SWE of phenolic compounds from Pisang Tanduk and Pisang Cavendish banana peel varying the
extraction temperature and time at 100 bar and obtained a highest recovery of 69.51 mg GAE g−1 dmb
(90 min) and 151.40 mg GAE g−1 dmb (120 min), respectively, at 200 ◦ C. The authors [55] studied
banana peels at maturity stage 3 (more green than yellow), which could explain the higher results in
comparison to the ones obtained here, since it has been reported that the phenolic content in banana
peel decreases with maturity [53].
The antioxidant capacity of the obtained banana peel extracts was mostly attributed to the presence
of dopamine, a strong water-soluble antioxidant from the group of catecholamines [10], since it was
the predominant bioactive compound detected by the HPLC. Additionally, high a correlation between
dopamine content and the DPPH and ABTS assays was observed (R2 = 0.80 and 0.83, respectively),
and the same was found in the case of TPC and antioxidant capacity. The DEC extract exhibited the
highest dopamine content (12.63 mg g−1 dmb) and the MAE extract the lowest (5.14 mg g−1 dmb). INF
(10.29 mg g−1 dmb) and U30 (10.33 mg g−1 dmb), both performed for 30 min, resulted in the extracts
with similar dopamine content (p > 0.05). Among the available literature reports, González-Montelongo
et al. [5] studied the effect of extraction time, temperature and different solvents on dopamine content
in banana peel extracts and reported the highest content (3.81 mg g−1 dmb) in methanolic extract
obtained at 55 ◦ C during 120 min for the Granda Naine cultivar and at 25 ◦ C during 12 min for the
Gruesa cultivar (3.42 mg g−1 dmb).

3.2.2. Characterization of Red Beetroot Peel Extracts

The bioactive content—including TPC, antioxidant capacity, total betacyanin and betaxanthin
content—of the red beetroot peel extracts is presented in Table 4.

Table 4. Bioactive content of differently prepared extracts of red beetroot peel.

Antioxidant Capacity Total Betalain Content
TPC Total Betacyanin
Sample DPPH ABTS Total Betaxanthin Content
(mg GAE g−1 dmb) Content
(mmol Troloxg−1 dmb) (mmol Trolox g−1 dmb) (mg vulgaxanthin I g−1 dmb)
(mg betanin g−1 dmb)
INF 45.03 ± 0.99 abcd 0.056 ± 0.00 0.164 ± 0.00 9.80 ± 0.14 8.41 ± 0.03 a
DEC 66.30 ± 0.41 0.098 ± 0.00 0.295 ± 0.00 6.15± 0.01 6.50 ± 0.06
MAC 44.75 ± 2.36 aef 0.066 ± 0.00 a 0.188 ± 0.00 0.85 ± 0.01 3.99 ± 0.02
U30 44.07 ± 0.22 beg 0.042 ± 0.00 b 0.140 ± 0.00 a 3.87 ± 0.03 a 8.61 ± 0.08 a
U60 47.99 ± 0.98 ch 0.048 ± 0.00 0.142 ± 0.00 a 3.84 ± 0.02 a 6.98 ± 0.06
SWE 45.77 ± 0.05 dfgh 0.064 ± 0.00 a 0.210 ± 0.00 0.03 ± 0.00 0.19 ± 0.04
MAE 39.72 ± 0.76 0.040 ± 0.00 b 0.132 ± 0.00 3.08 ± 0.02 1.74 ± 0.08

dmb—dry matter basis of the sample; INF—infusion; DEC–decoction; MAC—maceration; U30 and U60—ultrasound
bath for 30 and 60 min, respectively; SWE—subcritical water extraction; MAE—microwave-assisted extraction;
Means in the same column denoted with the same superscript letters (a,b,c,d,e,f,g,h) are not significantly different
(p > 0.05).

Among the obtained red beetroot peel extracts, TPC ranged between 39.72 mg GAE g−1 dmb,
measured in the extract obtained by MAE, and 66.30 mg GAE g−1 dmb in the DEC extract. A high
correlation was observed between TPC and antioxidant capacity in both the DPPH and ABTS assays
Sustainability 2020, 12, 4539 10 of 21

(R2 = 0.80), so the lowest antioxidant capacity was noted in the MAE extract (0.040 and 0.132 mmol
Trolox g−1 dmb) and the highest in the DEC extract (0.098 and 0.295 mmol Trolox g−1 dmb). Further,
the applied innovative techniques U30 (44.07 mg GAE g−1 dmb), U60 (47.99 mg GAE g−1 dmb) and
SWE (45.77 mg GAE g−1 dmb) exhibited an extraction efficiency, measured as TPC, similar (p > 0.05) to
that of the INF (45.03 mg GAE g−1 dmb) and MAC (44.75 mg GAE g−1 dmb) conventional techniques.
It is noteworthy to point out that U30, INF, U60 and MAC required 30, 60 min and 48 h, respectively, to
reach a similar recovery of phenolics (p > 0.05), while SWE reached the same recovery in only 5 min,
thus standing out in terms of time saving. Additionally, the extract obtained by SWE was characterized
by an antioxidant capacity 1.3–1.5 times higher than that of the U30 and U60 extracts, and 1.2–1.3
higher than that of INF and MAC—with the exception of the ABTS assay, where SWE and MAC
exhibited similar values (p > 0.05). The extraction of phenolics from beetroot peel is not extensively
studied. Among the few available studies, Kujala et al. [16] reported that the extraction method and
type of solvent have a noticeable effect on the extraction efficiency of phenolics from red beetroot peel.
The authors determined the highest TPC of 24.1 mg GAE g−1 dmb in methanolic extract.
The highest total betacyanin content (9.81 mg betanin g−1 dmb) was observed in the extract
obtained by the INF and the highest betaxanthin content in the U30 extract (8.61 mg vulgaxanthin
I g−1 dmb). It can be concluded that the temperature and time of extraction had a notable effect
on the betalain content. A temperature up to 80 ◦ C during 30 min, as applied in INF, enabled a
higher betalain content, while with the DEC parameters (100 ◦ C, 20 min), the betalain content in the
extract decreased 0.63 and 0.77 times for betacyanins and betaxanthins, respectively, in comparison to
INF. The results are consistent with the study by Wong and Siow [56], who reported the stability of
betacyanins in red-fleshed dragon fruit juice for a temperature range of 65 ◦ C–80 ◦ C during heating
times of 10, 20 and 30 min, and their further decrease while increasing the temperature up to 95 ◦ C.
Further, the results obtained in the present study correspond to the previously reported betacyanin
and betaxanthin content in red beetroot peel (12.79 mg betanin g−1 dmb and 4.46 mg vulgaxanthin
I g−1 dmb, respectively) when extracted with a mixture of water/methanol/formic acid [57]. Regarding
the applied innovative techniques, U30 resulted in the highest recovery of total betacyanins (3.87 mg
betanin g−1 dmb) and betaxanthins (8.61 mg vulgaxanthin I g−1 dmb). Laqui-Vilca et al. [58] studied
the UAE of betalains from colored quinoa hulls using sonotrode and reported an optimum extraction of
betacyanins (0.965 mg g−1 fresh weight of sample) at an amplitude of 70%, a 0.6 cycle and an extraction
time of 9.2 s, while the maximum recovery of betaxanthins (2.010 mg g−1 fresh weight of sample) was
achieved with an amplitude of 90%, a 0.7 cycle and an extraction time of 40 s. Although SWE showed
a good extraction efficiency of phenolics, similar to that od conventional and innovative techniques,
the contents of betalains in this extract were the lowest of all beetroot peel extracts (betacyanin 0.03 mg
betanin g−1 dmb and betaxanthin 0.19 mg vulgaxanthin I g−1 dmb). One of the explanations could be
the changed behavior of water as a solvent in its subcritical region, exhibiting properties similar to
those of organic solvents [20], and thus not effective for betalain extraction. In addition, the applied
high temperature (150 ◦ C) during SWE probably had a negative effect on the stability of betalains,
as they are known to be thermosensitive [56]. During heat processing, betanin, the predominant
betacyanin in red beetroot, may be degraded by isomerization, decarboxylation or cleavage followed
by the reduction of the red coloring, and eventually by the appearance of a light brown color [59].
As far as the application of MAE, it resulted in 3.08 mg g−1 dmb of betanin and 1.74 mg g−1 dmb of
vulgaxanthin I extracted from the red beetroot peels, which are 0.31 and 0.21 times lower, respectively,
than the values obtained for INF. The results obtained for MAE in the present study are inconsistent
with those of Cardoso-Ugarte [60], who reported a higher extraction yield of betalains from red beets
using MAE compared to the conventional aqueous ethanolic extraction. The authors reported optimal
MAE parameters of 400 W, 100% duty cycle and a duration of 90–120 s for betacyanins, and 140–150 s
for betaxanthins. A further increase of the extraction time resulted in a decrease of both betacyanins
and betaxanthins. This observation could explain the low extraction efficiency of MAE with respect to
betalains in the present study, since the extraction time was set to 5 min.
Sustainability 2020, 12, 4539 11 of 21

3.3. Biological Effect of Banana and Red Beetroot Peel Extracts

The extracts with the highest bioactive content were analyzed for biological effects in vitro. In the
case of banana peel, it was the DEC extract, and for the red beetroot peel, it was the INF extract.
Three different human cell lines were used to determine the cytotoxic and antioxidant/prooxidant
effects of banana and red beetroot peel extracts: human squamous cell carcinoma (CAL 27), human
colon adenocarcinoma (Caco-2) and human hepatocellular carcinoma cells (HepG2). At the time of
treatment with the extracts to test the cytotoxic and antioxidant/prooxidative effects, the cell lines
were subconfluent, and the treatment time was 0.5, 1 and 2 h. Results in the present study (data not
shown) showed that after 2 h of incubation, the stability of polyphenols in both extracts decreased
significantly. Consequently, an incubation longer than 2 h would not be representative of the effect
of the polyphenols from the extract. During the treatment with the tested extracts, conditions of
37 ◦ C and 5% CO2 were ensured. The extracts that were used for the treatment contained 0.014,
0.2, 1 and 10 mg mL1 of polyphenols and were prepared from freeze-dried extracts in which the
concentrations of polyphenols were previously determined. These concentrations were determined
given that the recommended dose of polyphenols for adults is 1 g per day. Therefore, a concentration
of 0.014 mg mL−1 represents the recommended daily dose of polyphenols for an adult body weight
of 70 kg. A concentration of 0.2 mg mL−1 is the concentration corresponding to the recommended
daily dose of polyphenol per 5 L of blood held by an adult. The remaining two concentrations (1 and
10 mg mL−1 ) are 5× and 50× higher than the daily recommended dose of polyphenols. Extracts with
and without polysaccharides were used to determine whether the polysaccharides in the extracts had
any effect on any of the properties tested.
The cytotoxic effect of the extracts was evaluated according to the standard "neutral red" (NR)
method [40]. The principle of the method is that a weak cationic dye enters the living cells by
non-ionic diffusion and binds to the lysosomal matrix. Color can only be bound within viable cells.
Accordingly, the amount of bound and then released color is proportional to the amount of cells
that have survived the treatment with a particular xenobiotic and have no damaged membrane.
The cytotoxic effect test results were expressed as a percentage of control survival, which was set
as 100% survival. The cytotoxicity results of banana and red beetroot peel extracts are presented in
Figures 1 and 2, respectively.
The cytotoxicity results of banana peel extract (Figure 1) indicate that there was no cytotoxic
effect on the cells of tongue epithelium (CAL 27). The survival rate of these cells did not differ with
respect to different concentrations or treatment times. Moreover, the polysaccharides in the extract
did not make a difference in the cytotoxic effect. In the case of colon epithelial cells (Caco-2), only the
concentration of 10 mg mL−1 of the original extract showed a slight proliferative effect in all three
treatment times (0.5, 1, and 2 h). Other concentrations showed neither cytotoxic nor proliferative
effects on cell survival relative to control. As regards liver cells (HepG2), banana peel extract with
polysaccharides showed no cytotoxic/proliferative effect at any of the tested concentrations or treatment
times. Only the polysaccharide-free banana peel extract at the concentration of the recommended daily
dose of polyphenols (0.014 mg mL−1 ) during a 2 h treatment showed a mild proliferative effect on liver
cells. It can be concluded that none of the cell lines tested showed any significant sensitivity to the
cytotoxic effects of banana peel extract. In contrast, certain concentrations showed a proliferative effect.
If we compare the results of other studies, Dahham et al. [61] found that banana peel extract obtained
by extraction with hexane had cytotoxic effects on the human colorectal cancer cell line HCT-116 and
human umbilical cord endothelial cells HUVEC (inhibition of 62.04 and 61.21%). On the other hand,
aqueous and ethanol extracts of banana peel showed low antiproliferative effects against HCT-116
and MCF-7, which is consistent with the present results of aqueous banana peel extract. The mild
antiproliferative effects of aqueous and ethanol extract may have been the result of a much longer cell
treatment (48 h), as opposed to our experiment (0.5, 1, and 2 h).
 method [40]. The principle of the method is that a weak cationic dye enters the living cells by non-
ionic diffusion and binds to the lysosomal matrix. Color can only be bound within viable cells.
Accordingly, the amount of bound and then released color is proportional to the amount of cells that
have survived the treatment with a particular xenobiotic and have no damaged membrane. The
cytotoxic effect test results were expressed as a percentage of control survival, which was set as 100%
Sustainability
survival. The12,cytotoxicity
2020, 4539 12 of121
results of banana and red beetroot peel extracts are presented in Figure
and Figure 2, respectively.
a) 140

120
% CAL 27 cell lines survival
100

80

60 0.5 h

1h
40
2h
20

0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Extract concentration (mg mL-1)

b) 160
140
% Caco-2 cell lines survival

120
100
80
60 0.5 h

40 1h
20 2h
0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Sustainability 2020, 12, x FOR PEER REVIEW 12 of 21
Extract concentration (mg mL-1)
c) 180
160 *d
% HepG2 cell lines survival

140 d
120
c a
100 c
80 0.5 h
60 1h
40 2h
20
0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS

Extract concentration (mg mL-1)

Figure 1. Cell survival (% of control) of (a) CAL 27 cells, (b) Caco-2 cells, (c) HepG2 cells determined
Figure 1. Cell survival (% of control) of (a) CAL 27 cells, (b) Caco-2 cells, (c) HepG2 cells determined
with the neutral red assay after 0.5, 1 and 2 h of exposure to different concentrations of banana peel
with the neutral red assay after 0.5, 1 and 2 h of exposure to different concentrations of banana peel
extract −1 with (+PS) and without polysaccharides (-PS); Statistically
extract(0.014,
(0.014,0.2,
0.2,1 1and
and10 10mgmgmLmL-1)) with (+PS) and without polysaccharides (-PS); Statistically
significant
significantdifference
difference < 0.05)
(p (p between
< 0.05) betweendifferent concentrations
different of the
concentrations extract
of the andand
extract the control: *-control;
the control: *-
a-0.014 mg mL −1 ; b-0.2 mg mL−1 ; c-1 mg mL−1 ; d-10 mg mL−1 .
control; a-0.014 mg mL ; b-0.2 mg mL ; c-1 mg mL ; d-10 mg mL .
-1 -1 -1 -1

The cytotoxicity results of banana peel extract (Figure 1) indicate that there was no cytotoxic
effect on the cells of tongue epithelium (CAL 27). The survival rate of these cells did not differ with
respect to different concentrations or treatment times. Moreover, the polysaccharides in the extract
did not make a difference in the cytotoxic effect. In the case of colon epithelial cells (Caco-2), only the
 treatment time was much longer (3 days), unlike in the present experiment (0.5, 1 and 2 h), so the
proven cytotoxic activity should be explained by this difference. In the work of Lee et al. [63], the
results showed that betanin obtained from beetroot had anticancer activity (49% inhibition of
proliferation), especially in HepG2 cells, at a relatively high concentration of betanin (200 µg mL-1)
after 48 h of treatment. Unfortunately, the authors that have treated cells for several days did not
Sustainability 2020, 12, 4539 13 of 21
provide neither information concerning the stability of the examined extracts nor the conditions
under which they achieved a stability of polyphenols for such a long time.
a) 180

% CAL 27 cell lines survival

160
140
120
100
80 0.5 h
60 1h
40 2h
20
0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Extract concentration (mg mL-1)

b) 160
% Caco-2 cell lines survival

140
120
100
80
0.5 h
60
1h
40
2h
20
0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Sustainability 2020, 12, x FOR PEER REVIEW 14 of 21
Extract concentration (mg mL-1)
c)
180
*b *
160
% HepG2 cell lines survival

140
d
120
100
80 0.5 h
60 1h
40 2h
20
0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Extract concentration (mg mL-1)

Figure 2. Cell survival (% of control) of (a) CAL 27 cells, (b) Caco-2 cells, (c) HepG2 cells determined
withFigure 2. Cell red
the neutral survival
assay(%after
of control)
0.5, 1 ofand(a)2CAL
h of27 cells, (b) Caco-2
exposure cells, (c)
to different HepG2 cells determined
concentrations of red beetroot
with the neutral red assay after 0.5, 1 and
−1 2 h of exposure to different concentrations
peel extract (0.014, 0.2, 1 and 10 mg mL ) with (+PS) and without polysaccharides (-PS), of red beetroot
Statistically
peel extract (0.014, 0.2, 1 and 10 mg mL-1) with (+PS) and without polysaccharides (-PS), Statistically
significant difference (p < 0.05) between different concentrations of the extract and the control: *-control;
significant difference (p < 0.05) between different concentrations of the extract and the control: *-
a-0.014 mg mL−1 ; b-0.2 mg mL−1 ; c-1 mg mL−1 ; d-10 mg mL−1 . -1
control; a-0.014 mg mL ; b-0.2 mg mL ; c-1 mg mL ; d-10 mg mL .
-1 -1 -1

The determination of the Reactive Oxygen Species (ROS) is based on the conversion of the non-
fluorescent compound 2', 7'-dichlorofluorescein diacetate (DCFH-DA) into the fluorescent derivative
of fluorescein. Specifically, non-ionic and non-polar DCFH-DA passes through the cell membrane
where, due to the action of cellular esterases, it is hydrolyzed to 2',7'-dichlorofluorescein (DCFH),
which is polar and thus remains in the cell and in contact with reactive oxygen groups (ROS) of
Sustainability 2020, 12, 4539 14 of 21

The beetroot peel extract showed a proliferative effect on the CAL 27 cell-line only for purified
extracts with precipitated polysaccharides after a 1 h treatment. For Caco-2 cells, the original beetroot
peel extract at a concentration of 0.2 mg mL−1 , after a 2 h treatment, showed a mild proliferative effect.
In the case of liver cells, the original beetroot peel extract at the highest concentration (10 mg mL−1 )
and after treatments for 0.5 h and 2 h exhibited a proliferative effect. None of the beetroot peel extracts
resulted in the cytotoxic effect. Kapadia et al. [62] found that the cytotoxic effect of beetroot pulp extract
was dose-dependent (0.29 - 290 µg mL−1 ) in all four stable lines used (human androgen-independent
prostate cancer cells (PC-3), human breast cancer cells (MCF) -7) normal skin (FC) and liver (HC) cell
lines) after 3 days of treatment. The concentrations used were consistent with those used in the present
experiment (daily recommended dose of polyphenols: 0.014–0.2 mg mL−1 ), but the treatment time
was much longer (3 days), unlike in the present experiment (0.5, 1 and 2 h), so the proven cytotoxic
activity should be explained by this difference. In the work of Lee et al. [63], the results showed that
betanin obtained from beetroot had anticancer activity (49% inhibition of proliferation), especially
in HepG2 cells, at a relatively high concentration of betanin (200 µg mL−1 ) after 48 h of treatment.
Unfortunately, the authors that have treated cells for several days did not provide neither information
concerning the stability of the examined extracts nor the conditions under which they achieved a
stability of polyphenols for such a long time.
The determination of the Reactive Oxygen Species (ROS) is based on the conversion of the
non-fluorescent compound 20 , 70 -dichlorofluorescein diacetate (DCFH-DA) into the fluorescent
derivative of fluorescein. Specifically, non-ionic and non-polar DCFH-DA passes through the cell
membrane where, due to the action of cellular esterases, it is hydrolyzed to 20 ,70 -dichlorofluorescein
(DCFH), which is polar and thus remains in the cell and in contact with reactive oxygen groups (ROS) of
fluorescent compound fluorescein. In this way, the amount of reactive oxygen groups in the cell can be
determined by the fluorescence intensity [41,42]. One of the results of excessive ROS levels is a change
in the structure and function of cellular proteins and lipids, leading to cellular dysfunction, including
impaired energy metabolism, altered cell signaling and cell cycle control, impaired cellular transport
mechanisms and overall dysfunctional biological activity, immunoactivation and inflammation.
Oxidative stress is manifested by elevated lipid peroxidation products, protein carbonylation and
decreased antioxidant status. The results of the antioxidant/prooxidative effect of banana and red
beetroot peel extracts are presented in Figures 3 and 4, respectively.
 be determined by the fluorescence intensity [41,42]. One of the results of excessive ROS levels is a
change in the structure and function of cellular proteins and lipids, leading to cellular dysfunction,
including impaired energy metabolism, altered cell signaling and cell cycle control, impaired cellular
transport mechanisms and overall dysfunctional biological activity, immunoactivation and
inflammation. Oxidative stress is manifested by elevated lipid peroxidation products, protein
Sustainability
carbonylation2020, 12,
and4539
decreased antioxidant status. The results of the antioxidant/prooxidative effect15ofof 21
banana and red beetroot peel extracts are presented in Figure 3 and Figure 4, respectively.
a) 160
*a, b, d
% induction of ROS (Cal 27 cells)
140 *a b
b c
120

100 c *a, d d c
b c
80
0.5 h
60
1h
40
2h
20

0
0.014 0.2 1 10 0.014 0.2 1 10
Sustainability 2020, 12, x FOR PEER REVIEW
´+ PS ´ - PS 15 of 21
Extract concentration (mg mL-1)
b) 140
% induction of ROS (Caco-2 cells)

120

100

80

60 0.5 h
1h
40
2h
20

0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Extract concentration (mg mL-1)

c) 140
a, b, c a, b
% induction of ROS (HepG2 cells)

120
d c, d c
c
100 *c *
*d *c, d *b *b *
*
80 * * *a, b
*b, d *
60 0.5 h

1h
40
2h
20

0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Extract concentration (mg mL-1)
Figure 3. Antioxidative/prooxidative effects of different concentrations of banana peel extract (0.014,
0.2,Figure mg mL−1 ) with (+PS) andeffects
1 and3.10Antioxidative/prooxidative of different
without concentrations
polysaccharides (-PS) of
onbanana
(a) CALpeel
27 extract (0.014,
cells (b) Caco-2
0.2,(c)
cells 1 and 10 mg
HepG2 mL-1during
cells ) with (+PS)
0.5, 1and
andwithout
2 h of polysaccharides (-PS) onsignificant
exposure, Statistically a) CAL 27 cells b) Caco-2
difference (p <
cells
0.05)
c) HepG2
between cells during
different 0.5, 1 andof
concentrations 2h ofextract
the exposure,
andStatistically significant
control: *-control; difference
a-0.014 mg mL (p−1
< ;0.05) mg mL−1 ;
b-0.2between
c-1different
mg mL−1 concentrations
; d-10 mg mLof −1 .the extract and control: *-control; a-0.014 mg mL-1; b-0.2 mg mL-1; c-1 mg
mL-1; d-10 mg mL-1.

The results for banana peel extract show a prooxidative effect of the original extract containing
polysaccharides at concentration 0.2 mg mL-1 for the CAL 27 cell-line after 2 h of treatment. The same
concentration of the purified extract with precipitated polysaccharides exhibited an antioxidative
effect for the same treatment duration. From these results, it can be concluded that the
Sustainability 2020, 12, 4539 16 of 21
Sustainability 2020, 12, x FOR PEER REVIEW 17 of 21

a) 180
* * * * * * *b, c, d
160
% induction of ROS (CAL 27 cells)
*
140
a a
120
a
100
80 0.5 h
60 1h
40 2h
20
0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Extract concentration (mg mL-1)

b) 140
% induction of ROS (Caco-2 cells)

120

100

80

60 0.5 h

1h
40
2h
20

0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Extract concentration (mg mL-1)

180
c) *b
b
160
% induction of ROS (HepG2 cells)

d d
140
120 c a
100
80 a, c 0.5 h
60 1h
40 2h
20
0
0.014 0.2 1 10 0.014 0.2 1 10

´+ PS ´ - PS
Extract concentration (mg mL-1)

Figure 4. Antioxidative/prooxidative effects of different concentrations of red beetroot peel extract

(0.014, 0.2, 1 and 10 mg mL−1 ) with (+PS) and without polysaccharides (-PS) on (a) CAL 27 cells,
(b) Caco-2 cells, (c) HepG2 cells during 0.5, 1 and 2 h of exposure; Statistically significant difference
(p < 0.05) between different concentrations of the extract and the control: *-control; a-0.014 mg mL−1 ;
b-0.2 mg mL−1 ; c-1 mg mL−1 ; d-10 mg mL−1 .
Sustainability 2020, 12, 4539 17 of 21

The results for banana peel extract show a prooxidative effect of the original extract containing
polysaccharides at concentration 0.2 mg mL−1 for the CAL 27 cell-line after 2 h of treatment. The same
concentration of the purified extract with precipitated polysaccharides exhibited an antioxidative effect
for the same treatment duration. From these results, it can be concluded that the pro/antioxidative
effect of the examined extracts strongly depends upon concentration and the environment to which
extract is exposed to. There is no clear dose-response effect that can help in the prediction of the
overall effect of banana peel extract. What is also true is that polysaccharides play a role in the overall
antioxidative effect. The prooxidative effects on the CAL 27 cell-line were also observed for 1 mg mL−1
of purified extract after a 0.5 h treatment. In the case of colon cells (Caco-2), the lowest concentration of
banana peel with polysaccharides (0.014 mg mL−1 ) showed a prooxidative effect after 1 h, while other
concentrations (0.2 - 10 mg mL−1 ) showed antioxidant effect only after 2 h of treatment. Moreover, in
the case of banana peel extract with polysaccharides, a positive dose-dependent effect was observed
during the longest treatment of the Caco-2 cell-line. As regards the purified banana peel extract effect on
Caco-2 cells, only the lowest and highest concentrations showed antioxidant effect after 2 h treatment.
Regarding liver cells (HepG2), the antioxidative effect was observed for lower concentrations (0.014
and 0.2 mg mL−1 ) after a longer treatment time (2 h), as well as for higher concentrations (1 and
10 mg mL−1 ) after a shorter treatment time (0.5 and 1 h). These results indicate the positive dependence
of the length of treatment and extract concentration on the antioxidant effect of the original banana
extract. The same effect could also be observed for purified extracts with precipitated polysaccharides.
It can be concluded that only the banana peel extract containing polysaccharides exhibited notable
antioxidant activity in the case of colon (Caco-2) and liver (HepG2) cells. There is a positive dose
dependence as well as the effect of exposure time. Ortiz et al. [64] did not observe clear effects of banana
peel extract on the ability to inhibit lipid peroxidation, which is the final result of an increased level of
free radicals in the cell. The results of Sathya [65] confirmed those of the present study and showed
that aqueous banana peel extract with polysaccharides had a significant percentage of inhibition of
lipid peroxidation, indicating that the extract effectively bound free radicals, so that further damage
to cellular macromolecules was prevented. The oxidation of unsaturated fatty acids in biological
membranes leads to the formation and propagation of lipid radicals, oxygen uptake, rearrangement of
double bonds in unsaturated lipids and the eventual destruction of membrane lipids by the production
of breakdown products. In addition, the results of the study by Baldi et al. [66] showed significant free
radical scavenging activity by an alcoholic extract of banana peel marked by a significant decrease in
lipid peroxidation level.
The results of the antioxidant/prooxidative effect of the red beetroot peel extract indicate that, in the
case of tongue epithelial cells (CAL 27), only the red beetroot peel extract containing polysaccharides in
all the tested concentration range (0.014 - 10 mg mL−1 ) exhibited prooxidative effect over a prolonged
period of treatment (1 and 2 h). In colon cells (Caco-2), neither prooxidative nor antioxidant effects
of beetroot peel extract with or without polysaccharide at all treatment times were demonstrated.
In liver cells (HepG2), prooxidative/antioxidative effect was not detected. It can be concluded that,
contrary to the effect of banana peel extract, the beetroot peel extract has no antioxidant effect on any
of the cell lines tested. In contrast, the original beetroot peel extract showed a statistically significant
pro-oxidative effect on cells of the tongue epithelium.

Author Contributions: Conceptualization, D.K.; methodology, D.Š., K.D., A.V.C. and A.H.; formal analysis, D.Š.,
A.V.C., A.H., K.D., S.J., J.J. and A.M.; writing—original draft preparation, D.K., D.Š. and K.D.; writing—review
and editing, A.V.C, A.M. and A.H., visualization, D.Š.; supervision, D.K. and K.D.; All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the Croatian Science Foundation (Project “Sustainable production of
biochemicals from waste lignocellulose containing feedstock”, OPB-SLS, 9717).
Conflicts of Interest: The authors declare no conflicts of interest.
Sustainability 2020, 12, 4539 18 of 21

References
1. FAO: How to Feed the World. 2050. Available online: [Link]
Issues_papers/HLEF2050_Global_Agriculture.pdf (accessed on 19 March 2020).
2. Sadh, P.K.; Duhan, S.; Duhan, J.S. Agro-industrial wastes and their utilization using solid state fermentation:
A review. Bioresour. Bioprocess. 2018, 5, 1–15. [CrossRef]
3. World Atlas: The Most Popular Fruit in The World. Available online: [Link]
[Link] (accessed on 19 March 2020).
4. Knoema: World–Bananas Production Quantity. Available online: [Link]
Agriculture/Crops-Production-Quantity-tonnes/Bananas-production (accessed on 24 March 2020).
5. González-Montelongo, R.; Lobo, M.G.; González, M. Antioxidant activity in banana peel extracts: Testing
extraction conditions and related bioactive compounds. Food Chem. 2010, 119, 1030–1039. [CrossRef]
6. Anhwange, B.A. Chemical Composition of Musa sapientum (Banana) Peels. J. Food Technol. 2008, 6, 263–266.
7. Abou-Arab, A.A.; Abu-Salem, F.M. Nutritional and Anti-Nutrtional Composition of Banana Peels as
Influenced by Microwave Drying Methods. Int. J. Nutr. Food. Sci. 2017, 11, 845–852.
8. Emaga, T.H.; Andrianaivo, R.H.; Wathelet, B.; Tchango, J.T.; Paquot, M. Effects of the stage of maturation
and varieties on the chemical composition of banana and plantain peels. Food Chem. 2007, 103, 590–600.
[CrossRef]
9. Someya, S.; Yoshiki, Y.; Okubo, K. Antioxidant compounds from bananas (Musa Cavendish). Food Chem.
2002, 79, 351–354. [CrossRef]
10. Kanazawa, K.; Sakakibara, H. High content of dopamine, a strong antioxidant, in cavendish banana. J. Agric.
Food Chem. 2000, 48, 844–848. [CrossRef]
11. Vinson, J.A.; Hao, Y.; Su, X.; Zubik, L. Phenol Antioxidant Quantity and Quality in Foods: Vegetables. J. Agric.
Food Chem. 1998, 46, 3630–3634. [CrossRef]
12. da Silva, D.V.T.; dos Santos Baião, D.; de Oliveira Silva, F.; Alves, G.; Perrone, D.; Del Aguila, E.M.;
Paschoalin, V.M.F. Betanin, a Natural Food Additive: Stability, Bioavailability, Antioxidant and Preservative
Ability Assessments. Molecules 2019, 24, 458. [CrossRef]
13. López, N.; Puértolas, E.; Condón, S.; Raso, J.; Alvarez, I. Enhancement of the extraction of betanine from red
beetroot by pulsed electric fields. J. Food Eng. 2009, 90, 60–66. [CrossRef]
14. Transparency Market Research: Beet Root Extract Market–Global Industry Analysis, Size, Share, Growth,
Trend and Forecast 2018–2026. Available online: [Link]
[Link] (accessed on 19 March 2020).
15. Kujala, T.S.; Loponen, J.M.; Klika, K.D.; Pihlaja, K. Phenolics and Betacyanins in Red Beetroot (Beta vulgaris)
Root: Distribution and Effect of Cold Storage on the Content of Total Phenolics and Three Individual
Compounds. J. Agric. Food Chem. 2000, 48, 5338–5342. [CrossRef] [PubMed]
16. Kujala, T.; Loponen, J.; Pihlaja, K. Betalains and Phenolics in Red Beetroot (Beta vulgaris) Peel Extracts:
Extraction and Characterisation. Zeitschrift für Naturforschung C 2001, 56, 343–348. [CrossRef] [PubMed]
17. Azmir, J.; Zaidul, I.S.M.; Rahman, M.M.; Sharif, K.M.; Mohamed, A.; Sahena, F.; Jahurul, M.H.A.; Ghafoor, K.;
Norulaini, N.A.N.; Omar, A.K.M. Techniques for extraction of bioactive compounds from plant materials: A
review. J. Food Eng. 2013, 117, 426–436. [CrossRef]
18. Vinatoru, M. An overview of the ultrasonically assisted extraction of bioactive principles from herbs.
Ultrason. Sonochem. 2001, 8, 303–313. [CrossRef]
19. Vinatoru, M.; Mason, T.J.; Calinescu, I. Ultrasonically Assisted Extraction (UAE) and Microwave Assisted
Extraction (MAE) of Functional Compounds from Plants Materials. Trac-Trend. Anal. Chem. 2017, 97, 159–178.
[CrossRef]
20. Zakaria, S.M.; Kamal, S.M. Subcritical Water Extraction of Bioactive Compounds from Plants and Algae:
Applications in Pharmaceutical and Food Ingredients. Food Eng. Rev. 2016, 8, 23–34. [CrossRef]
21. Pan, Y.; Wang, K.; Huang, S.; Wang, H.; Mu, X.; He, C.; Ji, X.; Zhang, J.; Huang, F. Antioxidant activity of
microwave-assisted extract of longan (Dimocarpus Longan Lour.) peel. Food Chem. 2008, 106, 1264–1270.
[CrossRef]
22. Singh, A.; Sabally, K.; Kubow, S.; Donnelly, D.J.; Gariepy, Y.; Orsat, V.; Raghavan, G.S.V. Microwave-Assisted
Extraction of Phenolic Antioxidants from Potato Peels. Molecules 2011, 16, 2218–2232. [CrossRef]
Sustainability 2020, 12, 4539 19 of 21

23. Jha, P.; Das, A.J.; Deka, S.C. Optimization of utrasound and microwave assisted extractions of polyphenols
from black rice (Oryza sativa cv. Poireton) husk. J. Food Sci. Techol. 2017, 54, 3847–3858. [CrossRef]
24. Jokić, S.; Gagić, T.; Knez, Ž.; Šubarić, D.; Škerget, M. Separation of active compounds from food by-product
(cocoa shell) using subcritical water extraction. Molecules 2018, 23, 1408. [CrossRef]
25. Lachos-Perez, D.; Baseggio, A.M.; Mayanga-Torres, P.C.; Maróstica Junior, M.R.; Rostagno, M.A.; Martínez, J.;
Forster-Carneiro, T. Subcritical water extraction of flavanones from defatted orange peel. J. Supercrit. Fluid.
2018, 138, 7–16. [CrossRef]
26. Singh, P.P.; Saldana, M.D.A. Subcritical water extraction of phenolic compounds from potato peel. Food Res. Int.
2011, 44, 2452–2458. [CrossRef]
27. Tapre, A.R.; Jain, R.K. Study of Advanced Maturity Stages of Banana. Int. J. Adv. Eng. Res. Stud. 2012, 1,
272–274.
28. Padmore, J.M. Animal feed - AOAC official method 930.15 - Moisture in animal feed. In Official Methods of
Analysis, 15th ed.; Helrich, K., Ed.; AOAC International: Arlington, VA, USA, 1990; Volume 1, pp. 69–70.
29. Padmore, J.M. Animal feed - AOAC official method 976.05 – Protein (crude) in animal feed, automated
Kjeldahl method. In Official Methods of Analysis, 15th ed.; Helrich, K., Ed.; AOAC International: Arlington,
VA, USA, 1990; Volume 1, p. 72.
30. Padmore, J.M. Animal feed-AOAC official method 920.39 – Fat (crude) or ether extract in animal feed.
In Official Methods of Analysis, 15th ed.; Helrich, K., Ed.; AOAC International: Arlington, VA, USA, 1990;
Volume 1, p. 79.
31. Padmore, J.M. Animal feed-AOAC official method 942.05–Ash of animal feed. In Official Methods of Analysis,
15th ed.; Helrich, K., Ed.; AOAC International: Arlington, VA, USA, 1990; Volume 1, p. 70.
32. McCleary, B.V.; DeVries, J.W.; Rader, J.I.; Cohen, G.; Prosky, L.; Mugford, D.C.; Okuma, K. Determination of
insoluble, soluble, and total dietary fibre (CODEX definition) by enzymatic-gravimetric method and liquid
chromatography: Collaborative study. J. AOAC Int. 2012, 95, 824–844. [CrossRef] [PubMed]
33. ISO 5509. Animal and Vegetable Fats and Oils–Preparation of Methyl Esters of Fatty Acids; International
Organization for Standardization: Geneva, Switzerland, 2000.
34. Vojvodić, A.; Komes, D.; Vovk, I.; Belščak-Cvitanović, A.; Bušić, A. Compositional evaluation of selected
agro-industrial wastes as valuable sources for the recovery of complex carbohydrates. Food Res. Int. 2016, 89,
565–573. [CrossRef]
35. Jokić, S.; Aladić, K.; Šubarić, D. Subcritical water extraction laboratory plant design and application.
Annu. Croat. Acad. Eng. 2018, 21, 247–258.
36. Singleton, V.L.; Rossi, J.A. Colorimetry of total phenolics with phosphotungstic acid reagents. Am. J. Enol.
Viticult. 1965, 16, 144–158.
37. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity.
Lebensm.-Wiss. Technol. 1995, 28, 25–30. [CrossRef]
38. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an
improved ABTS radical cation decolorisation assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [CrossRef]
39. Stintzing, F.; Schieber, A.; Carle, R. Evaluation of colour properties and chemical quality parameters of cactus
juice. Eur. Food Res. Technol. 2003, 216, 303–311. [CrossRef]
40. Babich, H.; Borenfreund, E. Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell
viability assay. Appl. Environ. Microbiol. 1991, 57, 2101–2103. [CrossRef] [PubMed]
41. Wang, H.; Joseph, J.A. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate
reader. Free Radic. Biol. Med. 1999, 27, 612–616. [CrossRef]
42. Hempel, S.L.; Buettner, G.R.; O’Malley, Y.Q.; Wessels, D.A.; Flaherty, D.M. Dihydrofluorescein diacetate is
superior for detecting intracellular oxidants: Comparison with 2’,7’-dichlorodihydrofluorescein diacetate,
5(and 6)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate, and dihydrorhodamine. Free Radic. Biol. Med.
1999, 27, 146–159. [CrossRef]
43. Abdolali, A.; Guo, W.S.; Ngo, H.H.; Chen, S.S.; Nguyen, N.C.; Tung, K.L. Typical lignocellulosic wastes and
by-products for biosorption process in water and wastewater treatment: A critical review. Bioresour. Technol.
2014, 160, 57–66. [CrossRef] [PubMed]
44. Okeke, B.C.; Obi, S.K.C. Lignocellulose and Sugar Composition of Some Agro-waste Materials. Bioresour.
Technol. 1994, 47, 283–284. [CrossRef]
Sustainability 2020, 12, 4539 20 of 21

45. Morais, D.R.; Rotta, E.M.; Sargi, S.C.; Bonafe, E.G.; Suzuki, R.M.; Souza, N.E.; Matsushita, M.; Visentainer, J.V.
Proximate Composition, Mineral Contents and Fatty Acid Composition of the Different Parts and Dried
Peels of Tropical Fruits Cultivated in Brazil. J. Braz. Chem. Soc. 2017, 28, 308–318. [CrossRef]
46. Vasconcellos, J.; Conte-Junior, C.; Silva, D.; Pierucci, A.P.; Paschoalin, V.; Alvares, T.S. Comparison of Total
Antioxidant Potential, and Total Phenolic, Nitrate, Sugar, and Organic Acid Contents in Beetroot Juice, Chips,
Powder, and Cooked Beetroot. Food Sci. Biotechnol. 2016, 25, 79–84. [CrossRef]
47. Costa, A.P.D.; Hermes, V.S.; Rios, A.O.; Flôres, S.H. Minimally processed beetroot waste as an alternative
souce to obtain functional ingredients. J. Food Sci. Technol. 2017, 54, 2050–2058. [CrossRef]
48. Shirsath, S.R.; Sonawane, S.H.; Gogate, P.R. Intensification of extraction of natural products using ultrasonic
irradiations—A review of current status. Chem. Eng. Process. 2012, 53, 10–23. [CrossRef]
49. Vilkhu, K.; Mawson, R.; Simons, L.; Bates, D. Applications and opportunities for ultrasound assisted
extraction in the food industry. Innov. Food Sci. Emerg. 2008, 9, 161–169. [CrossRef]
50. Chemat, F.; Rombaut, N.; Sicaire, A.G.; Meullemiestre, A.; Fabiano-Tixier, A.S.; Abert-Vian, M. Ultrasound
assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and
applications. A review. Ultrason. Sonochem. 2017, 34, 540–560. [CrossRef] [PubMed]
51. Vu, H.T.; Scarlett, C.J.; Vuong, Q.V. Optimization of ultrasound-assisted extraction conditions for recovery of
phenolic compounds and antioxidant capacity from banana (Musa cavendish) peel. J. Food Process. Pres. 2017,
41, 1–14. [CrossRef]
52. Aboul-Enein, A.M.; Salama, Z.A.; Gaafar, A.A.; Aly, H.F.; A bou-Elella, F.; Ahmed, H.A. Identification of
phenolic compounds from banana peel (Musa paradaisica L.) as antioxidant and antimicrobial agents. J. Chem.
Pharm. Res. 2016, 8, 46–55.
53. Fatemeh, S.R.; Saifullah, R.; Abbas, F.M.A.; Azhar, M.E. Total phenolics, flavonoids and antioxidant activity of
banana pulp and peel flours: Influence of variety and stage of ripeness. Int. Food Res. J. 2012, 19, 1041–1046.
54. Kaderides, K.; Papaoikonomou, L.; Serafim, M.; Goula, A.M. Microwave-assisted extraction of phenolics
from pomegranate peels: Optimization, kinetics, and comparison with ultrasounds extraction. Chem. Eng.
Process. 2019, 137, 1–11. [CrossRef]
55. Ishak, N.A.; Abdul Rashid, M.Y.; Zabidi, N.A. Local Banana Peels Extract by Using Subcritical Water
Extraction Technique. Ulum Islamiyyah 2019, 26, 19–30.
56. Wong, Y.M.; Siow, L.F. Effects on heat, pH, antioxidant, agitation and light on betacyanin stability using
red-fleshed dragon fruit (Hylocereus polyrhizus) juice and concentrate as models. J. Food Sci. Technol. 2015, 52,
3086–3092. [CrossRef]
57. Sawicki, T.; Baczek,
˛ N.; Wiczkowski, W. Betalain profile, content and antioxidant capacity of red beetroot
dependent on the genotype and root part. J. Funct. Food. 2016, 27, 249–261. [CrossRef]
58. Laqui-Vilca, C.; Aguilar-Tuesta, S.; Mamani-Navarro, W.; Montaño-Bustamante, J.; Condezo-Hoyos, L.
Ultrasound-assisted optimal extraction and thermal stability of betalains from colored quinoa (Chenopodium
quinoa Willd) hulls. Ind. Crop. Prod. 2018, 111, 606–614. [CrossRef]
59. Azeredo, H.M.C. Betalains: Properties, sources, applications, and stability–a review. Int. J. Food Sci. Technol.
2009, 44, 2365–2376. [CrossRef]
60. Cardoso-Ugarte, G.A.; Sosa-Morales, M.E.; Ballard, T.; Liceaga, A.; San Martín-González, M.F.
Microwave-assisted extraction of betalains from red beet (Beta vulgaris). LWT-Food Sci. Technol. 2014,
59, 276–282. [CrossRef]
61. Dahham, S.S.; Agha, M.T.; Tabana, Y.M.; Malik, A.; Abdul, S. Antioxidant Activities and Anticancer Screening
of Extracts from Banana Fruit (Musa sapientum). Acad. J. Cancer Res. 2015, 8, 28–34.
62. Kapadia, G.J.; Azuine, M.A.; Rao, G.S.; Arai, T.; Iida, A.; Tokuda, H. Cytotoxic Effect of the Red Beetroot
(Beta vulgaris L.) Extract Compared to Doxorubicin (Adriamycin) in the Human Prostate (PC-3) and Breast
(MCF-7) Cancer Cell Lines. Anticancer Agents Med. Chem. 2011, 11, 280–284. [CrossRef] [PubMed]
63. Lee, E.J.; An, D.; Nguyen, C.T.T.; Patil, B.S.; Kim, J.; Yoo, K.S. Betalain and Betaine Composition of Greenhouse-
or Field-Produced Beetroot (Beta vulgaris L.) and Inhibition of HepG2 Cell Proliferation. J. Agric. Food Chem.
2014, 62, 1324–1331. [CrossRef]
64. Ortiz, L.; Dorta, E.; Gloria Lobo, M.; González-Mendoza, L.A.; Díaz, C.; González, M. Use of Banana (Musa
acuminata Colla AAA) Peel Extract as an Antioxidant Source in Orage Juices. Plant Food Hum. Nutr. 2017, 72,
60–66. [CrossRef]
Sustainability 2020, 12, 4539 21 of 21

65. Sathya, M. Assaying the Antioxidant Activity of Banana Peel. Am. J. Biochem. Mol. Biol. 2014, 4, 122–129.
[CrossRef]
66. Baldi, A.; Pandit, M.; Ranka, P. Amelioration of in-vivo Antioxidant Activity by Banana Extracts. Int. J.
Pharm. Biol. Arch. 2012, 3, 157–161.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license ([Link]