Journal of Agriculture and Food Research 10 (2022) 100460

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Journal of Agriculture and Food Research

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Phytochemicals and in-vitro antioxidant activity analysis of Aloe vera

by-products (skin) in different solvent extract
Md Munnaf Hossen a, Mohammad Lokman Hossain b, Kanika Mitra a, Billal Hossain b,
Ummey Hafsa Bithi a, Md Nazim Uddin a, *
a
Institute of Food Science and Technology, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhanmondi, Dhaka, 1205, Bangladesh
b
Department of Chemistry, Jagannath University, Dhaka, 1100, Dhaka, Bangladesh

A R T I C L E I N F O A B S T R A C T

Keywords: Aloe vera (A. vera), one of the most popular medicinal plants, is regularly used throughout the world to prevent or
Medicinal plant treat cancer, metabolic problems, cardiovascular diseases, and skin concerns. In this study, we investigated the
Phenolics antioxidant properties of A. vera skin extract, which are neglected during gel extraction. In the current work, we
Free radical
investigated the phytochemical and antioxidant profiles of an A. vera by-product using three different solvents
DPPH
and two different drying methods. The findings of the study showed that sample drying and solvent extraction
And ABTS
methods had a significant impact on the phytochemical content and in vitro antioxidant activity. The oven-dried
sample of the A. vera by-product contained more phytochemicals, antioxidants, and in vitro activity than the sun-
dried sample does. The study of solvent effects also indicated that an acidified methanolic solvent for A. vera by-
products was the most effective extraction medium for phytochemicals and antioxidants than methanolic or
aqueous ethanolic extract. Additionally, the comparison of solvent effects in the assessment of antioxidant ac­
tivity in various in vitro models was investigated, and findings revealed that the maximum antioxidant activity
and radical scavenging activity were found in oven-dried acidified methanolic (O-AM) extracts. The principal
component analysis (PCA) has shown that the biggest variation was 87.3% and O-AM has the highest contri­
bution to the dimension 1 (68.7%) in the PCA. These findings will spur attempts to describe the bioactivities of
A. vera by-products and increase their application in the cosmetics, food, and healthcare sectors.

1. Introduction
Aloe vera, a genus of the Liliaceae family, is a perennial succulent or
xerophyte with stems that are either absent or extremely short. It has
elongated, peaked leaves, and the tissue of these leaves stores a bunch of
water [1]. Aloe vera gel, also known as inner leaf, inner leaf fillet, or
fillet, is what makes up the majority of the plant. The upper green layer
is also known as the A. vera skin which is mostly considered as waste or
by-products during processing [2]. The circular agroeconomy concept
has gained popularity in recent years as a result of rising environmental
concerns. It is an eco-friendly way to stop waste from being produced
during production. According to recent studies, a wide range of
agro-wastes, including the skin of A. vera plants, are potentially
renewable sources of bioactive substances or biopolymers [3,4].
Since ancient times, A. vera has been attributed to therapeutic or
healing-promoting characteristics. Research has shown that it has anti­
oxidant, anti-inflammatory, antiviral, anti-microbial, and anti-cancer
properties [5,6]. Aloe plants have been shown to contain micro­
nutrients and phytonutrients that may have biological and toxicological
effects [1,6,7]. Recent study highlighted that rat liver damage caused by
pesticides might be significantly reduced by administering an aqueous
extract of A. vera leaves [8]. Additionally, study reported that the
harmful effects of cartap were shown to be significantly reduced by
pre-administration of an aqueous extract of A. vera leaves, which
shielded the levels of oxidative indicators (MDA and GSH) in Wistar rats
[9]. Aloe vera may have an ameliorative effect since it contains
numerous antioxidant molecules that can fight off the oxidative stress
brought on by cartap stress [10]. However, use of A. vera frequently
results in interactions with other medications, kidney problems, diar­
rhea, and electrolyte imbalance [11].
Importantly, most of the A. vera product e.g., gel is used in the cos­
metics, food, and pharmaceutical industries [12]. The outer skin, which
makes up more than 30% of the overall leaf weight and is typically
separated from the interior gel in A. vera processing factories, results in

* Corresponding author.Institute of Food Science and Technology, Bangladesh Council of Scientific and Industrial Research, Dhanmondi, Dhaka, 1205, Bangladesh.
E-mail address: nazimbio@yahoo.com (M.N. Uddin).

https://doi.org/10.1016/j.jafr.2022.100460
Received 25 September 2022; Received in revised form 3 November 2022; Accepted 22 November 2022
Available online 23 November 2022
2666-1543/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M.M. Hossen et al.
significant waste production. This agricultural waste typically has no
economic application and is disposed of, composted, or outright burned
[2]. Aloe vera skin has been reported to be a prospective source of
bioactive substances that might be employed in food, food packaging, or
biomedical applications, despite the fact that there is little knowledge
about it compared to the inner gel, which has been widely studied. The
successful extraction of these bioactive molecules from various plant
sources remains a difficult process despite the great commercial and
health-related values of natural antioxidants, as there are several com­
pounds with diverse physicochemical properties and solubility. Because
phenolic compounds come in a variety of free and conjugated forms,
their solubility, extraction yield, and antioxidant properties may be
affected by the varied structures and polarities of these compounds in
various solvents [13]. The polarity of the target solute is typically taken
into account while choosing the extraction solvents. For the extraction of
polyphenols, polar solvents including methanol, ethanol, and ethyl ac­
etate were chosen, whilst non-polar solvents like hexane were appro­
priate for less polar chemicals. The best extraction conditions vary
depending on the plant species and matrix [14], and there is currently no
advice for a particular extraction solvent system for the best recovery of
plant phenolic chemicals [15,16]. Most importantly, the levels of phy­
tochemicals can be affected by a variety of drying conditions since they
are heat, light, and oxygen sensitive [17,18]. Determining the ideal
drying conditions for each type of material is crucial. The drying of food
products, including plants, has traditionally been done using traditional
drying processes like sun, shade, and oven drying [17]. In this study, we
used A. vera by-product (skin) and characterized its phytochemicals and
antioxidants content by utilizing different drying and solvents extraction
methods. In addition to the manufacturing of A. vera gel, this study fo­
cuses on the usage of A. vera by-products as medicinal or therapeutic
components.
2. Materials and methods
2.1. Collection and sample preparation
A. vera was harvested in Bangladesh’s Natore district and cleaned in
pure water. After that, it was left to dry for 4 h in the shade at room
temperature. After that, the gel was removed, and the skin was collected
as a by-product. The skin was cut into little pieces and dried in the sun
for 12 h and oven (65 ◦ C for 12 h), then pulverized in a blender. In this
study, we used sun and oven drying methods rather than freeze drying
for by-product.
2.2. Preparation of the sample extract in different solvents
Three separate solvents (methanol, methanol + HCl, and ethanol +
water) were used for preparing the A. vera sample extract for phyto­
chemicals and antioxidant activity analysis. The samples from two
different drying methods used for solvent extractions. The solvent ex­
tractions were divided into oven-dry methanol extract (O-ME), oven-dry
acidic methanolic (O-AM), oven-dry aqueous ethanolic (O-AM), sun-dry
methanol extract (S-ME), sun-dry acidic methanolic (S-AM), and sun-dry
aqueous ethanolic (S-AE). In a flat-bottomed container, 100 gm dry
powders were dissolved in 500 ml three different solvents for three days
with intermittent shaking and stirring. Then it was filtered through filter
paper and the supernatant was collected, and the process was repeated
up to three times. The solvents were then evaporated at 60◦ Celsius in a
rotatory evaporator (RE 200 Sterling, UK). Water bath drying was used
to dry the concentrated extracts. The dried extracts were then used to
prepare the final sample and kept at 4 ◦ C until needed.
2.3. Determination of total phenolics content
With slight changes, the amount of total phenolic content was
determined using the established method outlined by Nazim and his
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Journal of Agriculture and Food Research 10 (2022) 100460
colleagues [19]. A mixture of 0.5 ml extract (1.0 mg/ml concentration)
and 0.5 ml Folin-Ciocalteau reagent (0.5 N) was combined and incu­
bated at room temperature for 5 min. Then 2.0 mL saturated sodium
carbonate was added, and the mixture was incubated at room temper­
ature for 30 min before the absorbance was measured at 765 nm using
spectrophotometer. As a standard solution, gallic acid was used. The
result was given in milligrams of gallic acid equivalent per gram of
extract.
2.4. Determination of total flavonoid content
Using rutin hydrate as a standard, the total flavonoid content was
measured using the aluminum chloride technique [20]. Pour 4.0 mL of
distil water into 1.0 mL of sample, then add 0.3 mL of 5% NaNO2 so­
lution and mix thoroughly. After 5 min of incubation, add 0.3 mL of 10%
AlCl3 solution and thoroughly mix. Allow the mixture to settle at room
temperature for 6 min. The mixture was then given a final volume of 10
mL by adding 2 mL of 1 M NaOH solution and double-distilled water.
After allowing the mixture to sit for 15 min, the absorbance was
measured using spectrophotometer at 510 nm. The result was given in
milligrams of rutin hydrate equivalent per gram of sample extract.
2.5. Analysis of total tannin content
Based on the Hagerman and Butler method [21], the tannin con­
centration was determined. A standard curve generated from tannic acid
(0–100 g/ml) solution. In terms of milligrams of tannic acid equivalent
per gram of sample (mg TAE/g), the final tannin concentration was
calculated.
2.6. Analysis of total alkaloid content
The total alkaloid content of A. vera samples was determined using
the 1- 10-phenanthroline method [22], which was slightly modified. 1
ml A. vera extract, 1 ml 0.025 M FeCl3 in 0.5 M HCl, and 1 ml 0.05 M 1,
10-phenanthroline in ethanol made up the reaction mixture. In a hot
water bath at a constant temperature of 70 ◦ C, the mixture was incu­
bated for 30 min. At 510 nm, the absorbance of the red color complex
was measured against a reagent blank. The alkaloid content was
assessed and determined using the Pipredine standard curve.
2.7. Determination of total antioxidant capacity
The total antioxidant was determined by following previously
described methods [19]. A mixture of 1 ml extract and 3 ml reagent
solution was mixed (0.6 M sulfuric acid, 28 mM sodium phosphate and 4
mM ammonium molybdate). After that, the tubes holding the reaction
solution were covered and incubated for 90 min at 95 ◦ C. The absor­
bance of the solution was measured at 695 nm against a blank after the
samples had cooled to room temperature. The blank was made up of 1
mL of methanol instead of extract. The antioxidant activity was
measured in milligrams of Gallic acid equivalents.
2.8. Determination of ferric reducing antioxidant power assay (FRAP)
With some modifications, the FRAP assay was conducted using the
previously described methodology [19]. Acetate buffer (pH 3.6; 1.6 g
sodium acetate and 8 ml acetic acid make up 500 ml), 10 mM TPTZ
solution in 40 mM HCL, and 20 mM iron (III) chloride solution were used
to prepare the FRAP reagent, respectively. Every day, the FRAP reagent
was freshly made and reheated in an oven to 37 ◦ C before use. In a test
tube, combine 0.5 ml of the extract sample with 0.5 ml of distilled water.
After that, add 4 ml of the FRAP reagent and well mix. Using water as a
blank, the absorbance at 593 nm was measured using a UV–visible
spectrophotometer. Using a similar process, standard curves for gallic
acid (0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, and 1 ml) were prepared.
M.M. Hossen et al.
2.9. Determination of reducing power (RP) of extract
The methodology outlined by Jayanthi et al. was used to determine
the reducing power [19]. Phosphate buffer (2.5 ml) and potassium
ferricyanide were combined with different amounts of the powder ex­
tracts in each solvent (2.5 ml). For 20 min, this mixture was maintained
in a water bath at 50 ◦ C. After cooling, 2.5 ml of 10% trichloroacetic acid
was added, and the mixture was centrifuged for 10 min at 3000 rpm. A
freshly made ferric chloride solution (0.5 ml) and distilled water were
combined into the upper layer of the solution (2.5 ml). At 700 nm, the
absorbance was measured by UV spectrophotometer. The control was
made in a similar manner without samples. As a benchmark, ascorbic
acid in a range of concentrations was used. The reaction mixture’s
increased absorbance is a sign of increased reducing power.
2.10. DPPH scavenging activity
The free radical scavenging activity was assessed using DPPH, which
was modified slightly from the previously described approach [19]. In a
test tube, add water to a final volume of 1.0 ml after adding 0.2, 0.4, 0.6,
0.8, and 1.0 ml of various extract. After that, add 3.0 ml of the DPPH
stock solution (0.004%) and well mix. The mixture was incubated for 10
min in a dark place. A control was prepared using methanol and DPPH
solution. Using methanol as a blank, absorbance was determined using
spectrophotometer at 517 nm. The percentage (%) of inhibition can be
calculated by:
Inhibition (%) = (A0 – A1 / A0) × 100
Where; A0 is the absorbance of the control and A1 is the absorbance of
test.
The IC50 value is the quantity of antioxidant required to eliminate
half of all free radicals in the body.
2.11. Free radical scavenging activity of ABTS
Free radical scavenging activity of ABTS was conducted according to
Jiri Sochor method with some modification [19]. 4.95 mmolL-1 potas­
sium peroxo disulphated (m = 0.01338 g/10 mL) are mixed and dis­
solved in distilled water with seven mmolL-1 ABTS (m = 0.03841 g/10
mL). The solution was diluted in 1:9 v/v ratio with distilled water.
Incubated the solution mixture at dark for 12 h and stored at 4 ◦ C
temperature for to up to 7 days. Fill different test tubes with 0.2 ml, 0.4
ml, 0.6 ml, 0.8 ml, and 1.0 ml of various extract and made volume up to
1.0 ml with water. 3.0 mL ABTS reagent mixture was then added,
properly mixed, and incubated at room temperature. After 5 min incu­
bation, the absorbance was measured at 670 nm. Control was prepared
with water and reagent. Gallic acid was used as a standard solution. The
percentage (%) of inhibition can be determined by following equation
and expressed as IC50,
Inhibition (%) = (A0 – A1 / A0) × 100
Where; A0 is the absorbance of the control and A1 is the absorbance of
test.
2.12. Statistical analysis
There were three replications of each experiment. To express the
data, the mean and standard deviation were utilized. One-way ANOVA
was calculated using the R program (haven package) for group com­
parison, and the Tukey’s Honest Significant Difference (HSD) test was
performed for pair-wise comparisons between various samples. We
performed principal component analysis (PCA) by using R packages
FactoMineR [23] and factoextra on the basis of ggplot2 [24]. A statis­
tically significant level of probability was defined as * is equal to p <
0.05, ** is equal to p < 0.01, and *** is equal to p < 0.001.
3
Journal of Agriculture and Food Research 10 (2022) 100460
3. Results
3.1. Determination of phytochemical contents of A. vera skin
The phytochemical content (phenols, flavonoids, tannins, and alka­
loids) of A. vera skin extract was determined by using three different
solvents (methanolic, acidic methanolic, and aqueous ethanolic) for the
extract, and Fig. 1 represents the phytochemical content of A. vera skin
extracts. Two drying methods and three different solvents were used for
sample preparation. The results demonstrated that the total phenolic
content of A. vera skin extract is significantly (p < 0.001) higher in oven-
dried samples compared to sun-dried samples (Fig. 1a). In sun-dried
samples, methanolic extract (S-ME) contains significantly (p < 0.01)
higher phenolics content (2.55 ± 0.01 mg/g) compared to other sun-
dried extracts against gallic acid standard (Fig. 1a). On the other
hand, the acidic extract of A. vera skin extract showed higher phenolic
content (3.52 ± 0.1 mg/g) among oven-dried and sun-dried samples. In
contrast, compared to three different extracts, the acidic solvent extract
is exhibited significantly (p < 0.01) higher phenolic content (average
2.9 mg/g) compared to other extracts (Fig. 1a).
Furthermore, the results also described that the flavonoid content of
A. vera skin extract remained constant in both sun-dried and oven-dried
samples (Fig. 1b). The data demonstrated that flavonoid content of skin
extract is significantly higher in aqueous ethanolic extract (0.75 mg/g)
compared to methanolic extract against the rutin hydrate standard.
Importantly, the analysis results reported that the flavonoid is absent in
the acidic methanolic extract (Fig. 1b).
Fig. 1c also represents the alkaloid content of A. vera skin extract.
The results showed that the alkaloid content of A. vera skin extract
significantly (ANOVA, p < 0.001) varies from solvent to solvent, even
though it depends on sample drying methods. The results showed that
the oven-dried sample had a higher average alkaloids content than the
sun-dried sample. The data revealed that the oven-dried acidic methanol
(O-AM) extract contains significantly (p < 0.001) higher alkaloid (296.6
± 0.1 mg/g) content compared to other extracts and is almost two times
higher than sun-dried samples of acidic extract. Likewise, results also
demonstrated that the sun-dried aqueous ethanolic (S-AE) extract con­
tains significantly (p < 0.05) lower alkaloid content than other extracts.
Furthermore, we also analyzed the effects of different drying
methods and solvents on the tannin content of A. vera by-product
extract. The analyzed results demonstrated that the average tannin
content is significantly (ANOVA, p < 0.001) higher in oven-dried sam­
ples than sun-dried samples (Fig. 1d). In oven-dried samples, the acidic
methanolic (O-AM) extract shows significantly (p < 0.05) higher tannin
content (1.92 ± 0.01 mg/g) compared to O-AE and O-ME. On the other
hand, in sun-dried samples, S-AE contained significantly (p < 0.05)
lower tannin content (1.13 ± 0.03 mg/g) than S-AM and S-ME. Alto­
gether, these results demonstrated that the A. vera by-products (skin)
contain a significant number of phytochemicals and the number of
phytochemicals is higher in oven-dried samples plus acidic methanol
and methanolic extract samples.
3.2. Determination of antioxidant content of A. vera by-product
The effects of drying methods on total antioxidant content were also
investigated, and the results are summarized in Fig. 2. The findings show
that there is substantial (ANOVA, p < 0.001) differences between the
antioxidant contents of various dried samples. According to Fig. 2a,
oven-dried methanol extract (O-ME) samples had a considerably (p <
0.05) higher antioxidant content (17.3 ± 0.05 mg/g) than other oven
dry and sun-dry samples. Oven-dried aqueous ethanol (O-AE) also
contains significantly (p < 0.05) higher antioxidant content (12.5 ±
0.06 mg/g) content compared to sun-dried aqueous ethanol (S-AE). On
the other hand, the oven-dried acidic methanol (O-AM) shows signifi­
cantly lower antioxidant content (0.40 ± 0.04 mg/g) than all the other
samples. The results also revealed that oven-dried samples contain
M.M. Hossen et al.
significantly (p < 0.001) more antioxidants than sun-dried samples
(Fig. 2a). In sun-dried samples, the methanol extract shows higher
antioxidant content (9.95 ± 0.02 mg/g) compared to other sun-dried
samples. These results suggest that oven dry plus methanol extract
samples show higher antioxidant content compared to other samples.
Furthermore, we also analyzed the antioxidant content of different
4
Journal of Agriculture and Food Research 10 (2022) 100460
Fig. 1. Phytochemicals contents of different solvent
extract of aloe vera by-product (skin). a) Total phe­
nolics content, b) Total flavonoids content, c) Total
alkaloids content, and d) Total tannin content. We
applied the ANOVA test for multiple groups and pair-
wise Tukey’s Honest Significant Difference (HSD) test
between two groups. Where, super script a, b, c, d, e,
and f represents the statistically significant (p < 0.05)
of each group. Similar letter indicates no significant
difference between groups. O-ME: Oven-dry meth­
anol extract, O-AM: Oven-dry acidic methanolic, O-
AE: Oven-dry aqueous ethanolic, S-ME: Sun-dry
methanol extract, S-AM: Sun-dry acidic methanolic,
and S-AE: Sun-dry aqueous ethanolic.
Fig. 2. Antioxidant contents of different solvent
extract of aloe vera skin. a) Total antioxidant content
and b) Total antioxidant content by FRAP assay. We
applied the ANOVA test for multiple groups and pair-
wise Tukey’s Honest Significant Difference (HSD) test
between two groups. Where, super script a, b, c, d, e,
and f represents the statistically significant (p < 0.05)
of each group. Similar letter indicates no significant
difference between groups. O-ME: Oven-dry meth­
anol extract, O-AM: Oven-dry acidic methanolic, O-
AE: Oven-dry aqueous ethanolic, S-ME: Sun-dry
methanol extract, S-AM: Sun-dry acidic methanolic,
and S-AE: Sun-dry aqueous ethanolic.
samples of A. vera skin extract by FRAP assay. The results found that the
antioxidant content determined by the FRAP assay significantly
(ANOVA, p < 0.001) varies among different solvents and drying
methods (Fig. 2b). Among the two drying methods, oven-dried samples
exhibited more antioxidants than sun-dried samples. In oven-dried
samples, the acidic methanol (O-AM) contains significantly (p <
M.M. Hossen et al.
0.001) higher antioxidants (12.1 ± 0.01 mg/g) than other samples. On
the other hand, sun-dried aqueous ethanol (S-AE) extract shows higher
antioxidant content among sun-dried samples, and S-AM contains lower
antioxidants. Combining these findings revealed that, for the oven-dry
extraction method, acidic methanolic solvents are preferable for anti­
oxidant extraction.
3.3. In vitro free radical scavenging power of A. vera by-product extract
Following analysis of the phytochemical and antioxidant content of
A. vera by-products, we investigated the extract’s capacity to scavenge
free radicals in vitro. Different in vitro free radical models, including
ABTS, DPPH, and reducing power assays, were used in this research. The
results of various drying techniques and the capacity of various A. vera
skin extracts to scavenge free radicals are summarized in Fig. 3, with the
results expressed as IC50 (the minimum concentration required for
reducing or inhibiting 50% of free radicals) equivalent with gallic acid
standard for ABTS and DPPH assays, and ascorbic acid for the reducing
power assay. The findings showed that the IC50 for the ABTS assay dif­
fers considerably (ANOVA, p < 0.001) between the two dried groups as
well as in various solvent extraction groups (Fig. 3a). A lower IC50 in­
dicates that the models have a greater potential for scavenging free
radicals. O-AM exhibits a markedly (p < 0.01) lower IC50 (0.203 ± 0.15
mg/ml) in the ABTS experiment compared to other extracts (Fig. 3a), as
well as higher levels of free radical inhibition (Fig. 4a). Contrarily, the S-
AM exhibits lower percentages of inhibition and a higher IC50 (1.09 ±
0.05 mg/ml) in the ABTS assay when compared to other samples.
Likewise, we demonstrated the ability of several extracts to remove
free radicals in the DPPH model. The findings showed that different
extracts’ IC50s varied considerably (ANOVA, p < 0.001) depending on
the samples’ extracts and drying methods. Compared to other drying
5
Journal of Agriculture and Food Research 10 (2022) 100460
methods, the oven-dried samples show a significantly (p < 0.05) lower
IC50 than sun-dried samples. Similarly, when compared to other skin
extracts, the O-AM has a lower IC50 (0.73 ± 0.1 mg/ml) and higher
percentages of free radical inhibition (Figs. 3b and 4b). On the other
hand, S-AM shows a higher IC50 (1.99 ± 0.9 mg/ml) and lower free
radical inhibitions compared to other sun and oven-dried samples. These
results represent that comparatively O-AM shows better free radical
scavenging capacity than other oven and sun-dried samples.
Furthermore, we again analyzed the reducing power capacity of
different extracts. Fig. 3c depicted the decreasing power capacity of
various samples, with the results represented by EC50. EC50 stands for
the extract’s half-maximum effective concentration for decreasing free
radicals. The lower EC50 represents the low concentration required to
reduce half of free radicals. Results illustrated that the EC50 of different
sun and oven-dried extract significantly (ANOVA, p < 0.001) varies. The
results revealed that O-AM shows significantly (p < 0.01) lower EC50
values (0.9 ± 0.03 mg/ml) among oven-dried samples as well as sun-
dried samples. In contrast, O-ME exhibited a higher EC50 (5.15 ±
0.08 mg/ml) compared to other extracts. On the other hand, in sun-dried
samples, S-ME shows a lower EC50 (2.95 ± 0.03 mg/ml) than S-AM and
S-AE. To summarize these results demonstrated that O-AM has shown
lower IC50 and EC50 in the ABTS, DPPH, and reducing power assays
compared to sun-dried and other extracts. These could indicate that the
antioxidant derived by oven-drying and acidic methanol (O-AM) extract
has a greater capacity to scavenge free radicals than antioxidants
extracted by sun-drying and other methods.
3.4. Principal component analysis (PCA)
A chemometric technique named PCA [15] is used to pinpoint the
variable elements that affect A. vera skin extract activity in the most
Fig. 3. In vitro antioxidant activity of different sol­
vent extract of aloe vera skin. a) Free radical scav­
enging capacity of different extract in ABTS assay, b)
Free radical scavenging capacity of different extract
in DPPH assay, and c) Free radical scavenging ca­
pacity of different extract in reducing power assay.
We applied the ANOVA test for multiple groups and
pair-wise Tukey’s Honest Significant Difference
(HSD) test between two groups. Where, super script a,
b, c, d, e, and f represents the statistically significant
(p < 0.05) of each group. Similar letter indicates no
significant difference between groups. O-ME: Oven-
dry methanol extract, O-AM: Oven-dry acidic meth­
anolic, O-AE: Oven-dry aqueous ethanolic, S-ME: Sun-
dry methanol extract, S-AM: Sun-dry acidic meth­
anolic, and S-AE: Sun-dry aqueous ethanolic.
M.M. Hossen et al. Journal of Agriculture and Food Research 10 (2022) 100460

Fig. 4. The percentages of inhibition of different extract in ABTS and DPPH assay. a) Percentages of Inhibition in ABTS assay, and b) Percentages of Inhibition in
DPPH assay. O-ME: Oven-dry methanol extract, O-AM: Oven-dry acidic methanolic, O-AE: Oven-dry aqueous ethanolic, S-ME: Sun-dry methanol extract, S-AM: Sun-
dry acidic methanolic, and S-AE: Sun-dry aqueous ethanolic.

Fig. 5. Principal components analysis of different variables and individuals. A) Scree plot identifying the total number of PCA, B) Biplot of Principal components
analysis, C) Contribution of variables to the dimension 1, D) Contribution of variables to the dimension 2, E) Contribution of individuals to the dimension 1 and 2.

6
M.M. Hossen et al.
significant ways. Fig. 5 shows a PCA analysis with the main experi­
mental variables (extraction solvent, phytochemicals, and antioxidant
levels) being assessed in A. vera skin extracts. Two main dimensions
were among the five PCA dimensions shown in Fig. 5A. The biggest
variation (87.3%) in the data set was given by the combination of the
first (dim1, 68.7%) and second (dim2, 18.6%) principal components
(Fig. 5B). Analysis of dim1 revealed that the primary components ABTS,
total phenolics, total alkaloids, and FRAP were provided by oven-dry
acidic methanolic (O-AM) extract (Fig. 5B and C). However, the re­
sults of the dim2 analysis showed that five extracts were responsible for
the principal components of the extract (Fig. 5B and D). More signifi­
cantly, the results of the individual extract contributions to the dim1 and
dim2 analyses were very consistent, demonstrating that O-AM has the
biggest contribution in principal components (Fig. 5E). These results
summarized that O-AM has highest contribution in principal compo­
nents and highest extraction capacity from A. vera skin.
4. Discussion
One of the most well-liked medicinal plants, A. vera is frequently
used to prevent or treat cancer, metabolic disorders, cardiovascular
ailments, and skin conditions worldwide [25,26]. Aloe vera gel is typi­
cally used for medical, cosmetic, and other purposes. Several studies
reported that A. vera gel contains phenolics, flavonoids, and antioxi­
dants [5,27,28], but no study reported the phytochemicals and antiox­
idant content in A. vera skin. In this work, we examined A. vera skin
extract’s antioxidant capabilities, which are skipped over during gel
extraction. In the present study, we used three solvents and two distinct
drying techniques to examine the phytochemical and antioxidant profile
of an A. vera by-product. The by-product contains phytochemicals and
antioxidants, and the amount varies depending on the drying and
extraction processes used, according to our results of analysis.
The phytochemical analysis results show that A. vera by-products
contained significant (p < 0.01) amounts of phytochemicals (Fig. 1).
The results found that the phytochemical content of A. vera by-product
was significantly higher in acidic extract and oven-dried samples
compared to other samples. Previous studies have illustrated that the
phytochemical content significantly depends on sample drying and
extraction methods [18,29]. In our study, results demonstrated that the
phenolics, flavonoids, alkaloids, and tannin content are significantly
higher in oven-dried samples compared to sun-dried samples. Study
reported that the phytochemical content was higher in oven-dried
samples [29]. Furthermore, when compared to hot air-dried, low tem­
perature, and pressure-dried samples, the vacuum-dried (110 ◦ C) sample
contained the highest total phenolics and flavonoids content equivalent
to gallic acid [18]. This evidence suggested that the phytochemical
content significantly depends on sample drying methods.
Furthermore, the study suggested that the phytochemicals and
antioxidant activity significantly depend on the different solvent ex­
tractions of a sample, and our results reported that acidic methanol
extraction showed higher phytochemicals and antioxidant activity.
Similarly, the flavonoids, alkaloids, and tannin content also significantly
vary from solvent to solvent extract [27]. Consistent with previous
findings, our results described that the phenolics, flavonoids, alkaloids,
and tannin contents are significantly (p < 0.05) different between
methanolic, acidic methanolic, and aqueous ethanolic extracts (Fig. 1a
and c). Study also described that the A. vera extract contained 17.52 mg
phenolics per gram of extract equivalent with gallic acid which is lower
than our study [30]. Similarly, study also revealed that A. vera extract
contained 5.3 mg/g flavonoids. The results showed that the phenolics,
alkaloids content in O-AM samples is significantly (p < 0.05) higher than
in other extracts. On the other hand, flavonoids are higher in O-AE and
S-AE, and tannin content is significantly higher in O-AM extract samples
compared to others (Fig. 1b and d). In the present study, we mostly
found higher levels of phytochemicals and antioxidants in acidic ex­
tracts, which is consistent with previous findings [31]. The study
7
Journal of Agriculture and Food Research 10 (2022) 100460
highlighted that acidic extract (HCL and ethanol) contained the highest
phenolics and antioxidant content [31]. In food or plant components,
phytochemicals or antioxidants exist in two forms (free and bound), and
bound forms cannot be extracted in a standard aqueous solution [32,
33]. Acid hydrolysis may break the hydrogen or covalent bonds in bound
phytochemicals and antioxidants and release more phytochemicals and
antioxidants. More importantly, a study reported that acid hydrolysis
significantly increased the bioavailability of phytochemicals in Fagonia
indica and exhibited anti-proliferation effects against breast cancer cells
in vitro [34].
Similar to methanol and acidic methanol extract, oven-dried samples
of A. vera skin extracts have a much-increased antioxidant content. The
oven-dried methanolic (O-ME) samples from among the six samples
exhibit higher antioxidant levels in comparison to other samples that are
equivalent to the gallic acid standard. According to the study, A. vera
leaf extract (gel) exhibits antioxidant activity in amounts of 1.1 mmol of
Trolox/g in comparison to the Trolox standard [35]. Similarly, re­
searchers revealed that A. vera skin extracted with chloroform-ethanol
had the highest antioxidant content (289.6 μg/g extract), which was
equivalent to the d’α-tocopherol standard [36]. According to another
study, the antioxidant content of A. vera gel extract greatly varies
depending on the extraction solvent and ranges from 195 to 471 μg/g
extract [36]. On the other hand, the oven-dried sample’s acidic extract
exhibits the highest level of antioxidant activity in the FRAP assay
(Fig. 2b). Intriguingly, a different A. vera study discovered that acidified
methanolic extract had the highest antioxidant activity in the FRAP
assay [37]. The study also revealed that A. vera skin and gel extract
exhibit antioxidant activity in the FRAP assay, with FRAP values of
185.98 μM and 26.51 μM, respectively [38]. Keep supporting these, a
study described that A. vera gels showed FRAP activity and the value was
0.38 mM equivalent of trolox/g fresh weight [39], and our study results
exhibited higher FRAP values than previous findings. The analysis’s
findings are in line with the amount of total phenolics and flavonoids,
both of which are highest in acidified methanolic solvent. According to
the findings, A. vera skin extract may be able to convert ferric ions into
ferrous ions.
A. vera skin extracts demonstrated their ability to scavenge free
radicals in DPPH, ABTS, and reducing power assays. We observed
remarkably consistent findings for oven-dried, acidified methanolic
samples. We used gallic acid and ascorbic acid as standards when
describing the in vitro free radical scavenging activity of A. vera extract
in terms of IC50. Gallic acid’s IC50 values (19.26 μg/ml for ABTS and
15.11 μg/ml for DPPH) were applied in the study’s respective assays.
Additionally, the ascorbic acid EC50 value (9.38 μg/ml) was used for the
reducing power assay. According to the findings, O-AM displayed the
lowest IC50 values for the ABTS (0.20 ± 1.3 mg/ml) and DPPH (0.73 ±
0.25 mg/ml) assays, the lowest EC50 values for the reducing power assay
(0.9 ± 0.03 mg/ml), and the strongest inhibitory capacities when
compared to other extracts. Extracts from A. vera demonstrated modest
ABTS, DPPH, and reducing activity when compared to standards,
although these results were in line with previous research. A study on
A. vera extract found that it had free radical scavenging capacity in an
ABTS assay, with an IC50 of 0.501 mg/ml [40]. The study also revealed
that A. vera gel can inhibit free radicals in a dose-dependent manner,
with an IC50 of 0.57 mg/ml in the DPPH experiment [41]. Nearly all of
our findings are supported by the IC50 value for inhibiting 50% free
radicals in the ABTS and DPPH experiments. The results also correlate
with the quantity of total phenolics, flavonoids, and antioxidants found
in the acidified methanol extract. Most notably, the results of our PCA
analysis were consistent with those of earlier research. O-AM has the
biggest contribution to the principal components’ variables according to
PCA analysis (Fig. 5), which is clearly supported by the phytochemicals
and antioxidant activity of O-AM extract.
Moreover, in this study, we investigated the skin, a by-product of
A. vera, utilizing three distinct solvents and two different drying tech­
niques. The findings showed that antioxidants and phytochemicals were
M.M. Hossen et al.
present in A. vera skin extract in significant amounts, although the
amount varied greatly depending on the procedures used for extraction
and processing. According to the results’ summaries, O-AM exhibits the
highest levels of phytochemicals and antioxidant activity in the FRAP,
DPPH, and ABTS assays. Although we have studied the effects of various
solvent extracts on A. vera by-products, further research needs to be
done on the mechanism of acidified methanolic extract. In additional
research, it is also necessary to analyze the precise phytochemicals and
antioxidant components found in A. vera by-products.
5. Conclusion
The present study demonstrated the phytochemicals and in vitro
antioxidant activity of poorly studied A. vera by-products (skin) in
different solvent extracts. The findings of the study showed that sample
drying and solvent extraction techniques had a significant impact on the
phytochemical content and in vitro antioxidant activity of A. vera by-
product extract. The phytochemicals, antioxidant content, and in vitro
activity of the A. vera by-product were the highest in the oven-dried
sample rather than the sun-dried sample. The investigation of solvent
effects also suggested that the most efficient extraction medium for
phytochemicals and antioxidants was an acidified methanolic solvent
for A. vera by-products. A comparison of solvent effects in the charac­
terization of antioxidant activity in different in vitro models was also
investigated and results showed that oven-dried acidified methanolic
(O-AM) extracts had the highest radical scavenging activity and ferric-
reducing antioxidant power activity. Additionally, the PCA analysis
confirmed that O-AM has the highest contribution compared to other
extracts. Despite our research on the effects of different solvent extracts
on A. vera by-products, more study is required to understand how
acidified methanolic extract works. This study will motivate efforts to
characterize the bioactivities of A. vera by-products and expand their use
in the healthcare, food, and cosmetics industries.
Conflicts of interest
The authors declare that there is no conflict of interest.
Funding
For conducting all of the experimental work for this study, we were
received a fund from the Bangladesh Council of Scientific and Industrial
Research (BCSIR), Dhaka, Bangladesh.
Author contribution
Md. Munnaf Hossen, and Mohammad Lokman Hossain equally
contributed to the whole study. Kanika Mitra, Mohammad Lokman
Hossain, and Md. Nazim Uddin conceived and supervised the study. Md.
Munnaf Hossen, Billal Hossain, and Ummey Hafsa Bithi design and
performed all experiments. Md. Munnaf Hossen, Mohammad Lokman
Hossain, Kanika Mitra, and Md. Nazim Uddin wrote, revised, and edited
the English language in this manuscript. Md. Munnaf Hossen and Md.
Nazim Uddin statistically analyzed the inputted data.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
8
Journal of Agriculture and Food Research 10 (2022) 100460
Acknowledgement
The authors appreciate the assistance of the entire laboratory tech­
nical staff. The Bangladesh Council of Scientific and Industrial Research
(BCSIR) provided financial assistance for this study, and the authors are
grateful for that.
References
[1] M.D. Boudreau, F.A. Beland, An evaluation of the biological and toxicological
properties of Aloe barbadensis (miller), Aloe vera, J. Environ. Sci. Health C
Environ. Carcinog. Ecotoxicol. Rev. 24 (1) (2006) 103–154.
[2] I. Solaberrieta, A. Jiménez, M.C. Garrigós, Valorization of aloe vera skin by-
products to obtain bioactive compounds by microwave-assisted extraction:
antioxidant activity and chemical composition, Antioxidants (Basel, Switzerland)
11 (6) (2022).
[3] M.L. Flores-López, A. Romaní, M.A. Cerqueira, R. Rodríguez-García, D. Jasso de
Rodríguez, A.A. Vicente, Compositional features and bioactive properties of whole
fraction from Aloe vera processing, Ind. Crop. Prod. 91 (2016) 179–185.
[4] L. Lucini, M. Pellizzoni, R. Pellegrino, G.P. Molinari, G. Colla, Phytochemical
constituents and in vitro radical scavenging activity of different Aloe species, Food
Chem. 170 (2015) 501–507.
[5] M. Hęś, K. Dziedzic, D. Górecka, A. Jędrusek-Golińska, E. Gujska, Aloe vera (L.)
webb.: natural sources of antioxidants - a review, Plant Foods Hum. Nutr. (Dordr.)
74 (3) (2019) 255–265.
[6] B. Salehi, S. Albayrak, H. Antolak, D. Kręgiel, E. Pawlikowska, M. Sharifi-Rad,
Y. Uprety, P.V. Tsouh Fokou, Z. Yousef, Z. Amiruddin Zakaria, E.M. Varoni,
F. Sharopov, N. Martins, M. Iriti, J. Sharifi-Rad, Aloe genus plants: from farm to
food applications and phytopharmacotherapy, Int. J. Mol. Sci. 19 (9) (2018).
[7] K.V. Gupta, S.N. Yarla, M. de Lourdes Pereira, J.N. Siddiqui, B. Sharma, Recent
advances in ethnopharmacological and toxicological properties of bioactive
compounds from Aloe barbadensis (Miller), Aloe vera, Curr. Bioact. Compd. 17 (5)
(2021) 3–24.
[8] V.K. Gupta, N.J. Siddiqi, A.K. Ojha, B. Sharma, Hepatoprotective effect of Aloe vera
against cartap- and malathion-induced toxicity in Wistar rats, J. Cell. Physiol. 234
(10) (2019) 18329–18343.
[9] V.K. Gupta, N.J. Siddiqui, B. Sharma, Ameliorative impact of aloe vera on cartap
mediated toxicity in the brain of wistar rats, Indian J. Clin. Biochem. : Indian J.
Clin. Biochem. 37 (1) (2022) 51–59.
[10] V.K. Gupta, A. Kumar, M.d.L. Pereira, N.J. Siddiqi, B. Sharma, Anti-inflammatory
and antioxidative potential of aloe vera on the cartap and Malathion mediated
toxicity in wistar rats, Int. J. Environ. Res. Publ. Health 17 (14) (2020) 5177.
[11] X. Guo, N. Mei, Aloe vera: a review of toxicity and adverse clinical effects,
J. Environ. Sci. Health C Environ. Carcinog. Ecotoxicol. Rev. 34 (2) (2016) 77–96.
[12] K. Eshun, Q. He, Aloe vera: a valuable ingredient for the food, pharmaceutical and
cosmetic industries–a review, Crit. Rev. Food Sci. Nutr. 44 (2) (2004) 91–96.
[13] S.F. Sulaiman, N.A.M. Yusoff, I.M. Eldeen, E.M. Seow, A.A.B. Sajak, Supriatno, K.
L. Ooi, Correlation between total phenolic and mineral contents with antioxidant
activity of eight Malaysian bananas (Musa sp.), J. Food Compos. Anal. 24 (1)
(2011) 1–10.
[14] K.L. Ho, C.G. Tan, P.H. Yong, C.W. Wang, S.H. Lim, U.R. Kuppusamy, C.T. Ngo,
F. Massawe, Z.X. Ng, Extraction of phytochemicals with health benefit from
Peperomia pellucida (L.) Kunth through liquid-liquid partitioning, Journal of
Applied Research on Medicinal and Aromatic Plants 30 (2022), 100392.
[15] Z.X. Ng, S.N. Samsuri, P.H. Yong, The antioxidant index and chemometric analysis
of tannin, flavonoid, and total phenolic extracted from medicinal plant foods with
the solvents of different polarities, J. Food Process. Preserv. 44 (9) (2020), e14680.
[16] Q.D. Do, A.E. Angkawijaya, P.L. Tran-Nguyen, L.H. Huynh, F.E. Soetaredjo,
S. Ismadji, Y.-H. Ju, Effect of extraction solvent on total phenol content, total
flavonoid content, and antioxidant activity of Limnophila aromatica, J. Food Drug
Anal. 22 (3) (2014) 296–302.
[17] P. Ghosh, N. Venkatachalapathy, Processing and drying of coffee–a review, Int. J.
Eng. Res. Technol. 3 (12) (2014) 784–794.
[18] T.M. Kieu Tran, T. Kirkman, M. Nguyen, Q. Van Vuong, Effects of drying on
physical properties, phenolic compounds and antioxidant capacity of Robusta wet
coffee pulp (Coffea canephora), Heliyon 6 (7) (2020), e04498.
[19] R.B. Robbani, M.M. Hossen, K. Mitra, M.Z. Haque, M.A. Zubair, S. Khan, M.
N. Uddin, Nutritional, Phytochemical, and in vitro antioxidant activity analysis of
different states of soy products, International Journal of Food Science (2022),
9817999, 2022.
[20] M. Uddin, M. Samad, M. Zubair, M. Haque, K. Mitra, T. Khan, M. Hossain, A. Syed,
A. Afroze, Potential bioactive phytochemicals, antioxidant properties and
anticancer pathways of Nymphaea nouchali, Asian Pac. J. Trop. Biomed. 10 (12)
(2020) 555–562.
[21] A.E. Hagerman, L.G. Butler, Protein precipitation method for the quantitative
determination of tannins, J. Agric. Food Chem. 26 (4) (1978) 809–812.
[22] D.K. Singh, B. Srivastava, A. Sahu, Spectrophotometric determination of Rauwolfia
alkaloids: estimation of reserpine in pharmaceuticals, Anal. Sci. : the international
journal of the Japan Society for Analytical Chemistry 20 (3) (2004) 571–573.
[23] S. Lê, J. Josse, F. Husson, FactoMineR: an R package for multivariate analysis,
J. Stat. Software 25 (1) (2008) 1–18.
[24] H. Wickham, Ggplot2: Elegant Graphics for Data Analysis, Springer, New York,
2009, p. 212, 2009.
M.M. Hossen et al.
[25] E. Teplicki, Q. Ma, D.E. Castillo, M. Zarei, A.P. Hustad, J. Chen, J. Li, The effects of
aloe vera on wound healing in cell proliferation, migration, and viability, Wounds :
a compendium of clinical research and practice 30 (9) (2018) 263–268.
[26] C. Liu, Y. Cui, F. Pi, Y. Cheng, Y. Guo, H. Qian, Extraction, purification, structural
characteristics, biological activities and pharmacological applications of
acemannan, a polysaccharide from aloe vera: a review, Molecules 24 (8) (2019).
[27] O.A. Wintola, A.J. Afolayan, Phytochemical constituents and antioxidant activities
of the whole leaf extract of Aloe ferox Mill, Phcog. Mag. 7 (28) (2011) 325–333.
[28] H. Tariq, M. Zia, H. Ihsan Ul, S.A. Muhammad, S.A. Khan, N. Fatima, A. Mannan, A.
M. Abbasi, M. Zhang, Antioxidant, antimicrobial, cytotoxic, and protein kinase
inhibition potential in aloe vera L, BioMed Res. Int. (2019), 6478187, 2019.
[29] Z. Aghaei, S.M. Jafari, D. Dehnad, Effect of different drying Methods on the
physicochemical properties and bioactive components of saffron powder, Plant
Foods Hum. Nutr. (Dordr.) 74 (2) (2019) 171–178.
[30] T. Debnath, M. Ghosh, Y.M. Lee, N.C.D. Nath, K.-G. Lee, B.O. Lim, Identification of
phenolic constituents and antioxidant activity of Aloe barbadensis flower extracts,
Food Agric. Immunol. 29 (1) (2018) 27–38.
[31] N. Pengkumsri, K. Kaewdoo, W. Leeprechanon, B. Sundaram Sivamaruthi,
Influence of extraction Methods on total phenolic content and antioxidant
properties of some of the commonly used plants in Thailand, Pakistan J. Biol. Sci. :
PJBS 22 (3) (2019) 117–126.
[32] I.M. Sani, S. Iqbal, K.W. Chan, M. Ismail, Effect of acid and base catalyzed
hydrolysis on the yield of phenolics and antioxidant activity of extracts from
germinated brown rice (GBR), Molecules 17 (6) (2012) 7584–7594.
Journal of Agriculture and Food Research 10 (2022) 100460
[33] S. Schefer, M. Oest, S. Rohn, Interactions between phenolic acids, proteins, and
carbohydrates-influence on dough and bread properties, Foods 10 (11) (2021).
[34] C.L. Miranda, Y. Kumbi, W. Wu, H.S. Lee, R.L. Reed, J.F. Stevens, Phytochemical
characterization and bioactivity toward breast cancer cells of unhydrolyzed and
acid-hydrolyzed extracts of Fagonia indica, Nat. Prod. Commun. 17 (7) (2022).
[35] V. Cesar, I. Jozić, L. Begović, T. Vuković, S. Mlinarić, H. Lepeduš, S. Borović Šunjić,
N. Žarković, Cell-type-specific modulation of hydrogen peroxide cytotoxicity and
4-hydroxynonenal binding to human cellular proteins in vitro by antioxidant aloe
vera extract, Antioxidants (Basel, Switzerland) 7 (10) (2018).
[36] S. Miladi, M. Damak, In vitro antioxidant activities of Aloe vera leaf skin extracts,
J Soc Chim Tunisie 10 (10) (2008) 101–109.
[37] M.N. Uddin, S.C. Roy, A.A. Mamun, K. Mitra, M.Z. Haque, M.L. Hossain,
Phytochemicals and In-Vitro Antioxidant Activities of Aloe Vera Gel.
[38] M. Moniruzzaman, B. Rokeya, S. Ahmed, A. Bhowmik, M.I. Khalil, S.H. Gan, In
vitro antioxidant effects of Aloe barbadensis Miller extracts and the potential role
of these extracts as antidiabetic and antilipidemic agents on streptozotocin-induced
type 2 diabetic model rats, Molecules 17 (11) (2012) 12851–12867.
[39] C. Quispe, M. Villalobos, J. Bórquez, M. Simirgiotis, Chemical composition and
antioxidant activity of aloe vera from the Pica Oasis (Tarapacá, Chile) by UHPLC-
Q/Orbitrap/MS/MS, J. Chem. (2018), 2018.
[40] O. Wintola, A. Afolayan, Phytochemical constituents and antioxidant activities of
the whole leaf extract of Aloe ferox Mill, Phcog. Mag. 7 (28) (2011) 325–333.
[41] A. Ray, S.D. Gupta, S. Ghosh, Evaluation of anti-oxidative activity and UV
absorption potential of the extracts of Aloe vera L. gel from different growth
periods of plants, Ind. Crop. Prod. 49 (2013) 712–719.