International Journal of

Molecular Sciences

Article
Comparative Analysis of Water Extracts from Roselle (Hibiscus
sabdariffa L.) Plants and Callus Cells: Constituents, Effects on
Human Skin Cells, and Transcriptome Profiles
Won Kyong Cho 1 , Soo-Yun Kim 2 , Sung Joo Jang 2 , Sak Lee 2 , Hye-In Kim 2 , Euihyun Kim 2 , Jeong Hun Lee 2 ,
Sung Soo Choi 3 and Sang Hyun Moh 2, *

1 College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 16419, Republic of Korea;
[email protected]
2 Plant Cell Research Institute of BIO-FD&C Co., Ltd., Incheon 21990, Republic of Korea;
[email protected] (S.-Y.K.); [email protected] (S.J.J.); [email protected] (S.L.);
[email protected] (H.-I.K.); [email protected] (E.K.); [email protected] (J.H.L.)
3 Daesang Holdings, Jung-gu, Seoul 04513, Republic of Korea; [email protected]
* Correspondence: [email protected]; Tel.: +82-32-811-2027

Abstract: Roselle (Hibiscus sabdariffa L.) is a plant that has traditionally been used in various food
and beverage products. Here, we investigated the potential of water extracts derived from Roselle
leaves and callus cells for cosmetic and pharmaceutical purposes. We generated calluses from
Roselle leaves and produced two different water extracts through heat extraction, which we named
Hibiscus sabdariffa plant extract (HSPE) and Hibiscus sabdariffa callus extract (HSCE). HPLC analysis
showed that the two extracts have different components, with nucleic acids and metabolites such
as phenylalanine and tryptophan being the most common components in both extracts. In vitro
assays demonstrated that HSCE has strong anti-melanogenic effects and functions for skin barrier
and antioxidant activity. Transcriptome profiling of human skin cells treated with HSPE and HSCE
Citation: Cho, W.K.; Kim, S.-Y.; Jang, showed significant differences, with HSPE having more effects on human skin cells. Up-regulated
S.J.; Lee, S.; Kim, H.-I.; Kim, E.; Lee, genes by HSPE function in angiogenesis, the oxidation-reduction process, and glycolysis, while
J.H.; Choi, S.S.; Moh, S.H. up-regulated genes by HSCE encode ribosome proteins and IFI6, functioning in the healing of
Comparative Analysis of Water radiation-injured skin cells. Therefore, we suggest that the two extracts from Roselle should be
Extracts from Roselle (Hibiscus applied differently for cosmetics and pharmaceutical purposes. Our findings demonstrate the
sabdariffa L.) Plants and Callus Cells: potential of Roselle extracts as a natural source for skincare products.
Constituents, Effects on Human Skin
Cells, and Transcriptome Profiles. Int.
Keywords: Roselle; water extracts; cosmetics; skin cells; transcriptome profiling
J. Mol. Sci. 2023, 24, 10853. https://
doi.org/10.3390/ijms241310853

Academic Editors: Konstantin Volcho

and Olga Luzina 1. Introduction
Received: 3 June 2023 Roselle (Hibiscus sabdariffa L.), also known as red sorrel, is a member of the genus
Revised: 25 June 2023 Hibiscus in the family Malvaceae [1]. It is native to Africa and is widely cultivated in tropical
Accepted: 27 June 2023 and subtropical regions, including India, Indonesia, and Malaysia [2]. The plant is best
Published: 29 June 2023 known for its distinctive red flower, which is composed of cup-shaped calyces. The thick
and fleshy calyces of the Roselle flower have been used worldwide as a raw material for a
variety of food and beverage products, including popular cold and hot drinks, jams, syrups,
and wines, for a considerable period of time [3]. Additionally, the calyces of the Roselle
Copyright: © 2023 by the authors. plant are frequently employed as natural food colorants [4].
Licensee MDPI, Basel, Switzerland.
Several previous studies have identified metabolites present in various tissues of the
This article is an open access article
Roselle plant [5]. For instance, the major organic acids found in the aqueous extract of
distributed under the terms and
Roselle flowers are citric, malic, and tartaric acids [6]. Interestingly, the concentration of
conditions of the Creative Commons
these organic acids increases during the development of the calyces but decreases as the
Attribution (CC BY) license (https://
calyces ripen. In a previous study, TLC and HPLC fingerprint analyses of methanol extracts
creativecommons.org/licenses/by/
4.0/).
from Roselle flowers revealed several flavonoids, including quercetin, luteolin, gossypetin,

Int. J. Mol. Sci. 2023, 24, 10853. https://doi.org/10.3390/ijms241310853 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 10853 2 of 21

and hibiscetin, as well as other known chemical components such as anthocyanins, includ-
ing delphinidin-3-glucosyl-xyloside (hibiscin) and cyanidin-3-glucosylxyloside, phenols
and phenolic acids like protocatechuic and pelargonidic acid, eugenol, plant sterols, and
ergosterol [7].
For a long time, Roselle flower extracts have been used in traditional medicine to treat
fever, high blood pressure, and liver diseases [5]. Numerous recent studies have revealed
the many pharmacological characteristics of Roselle flower extract. For instance, the leaves
and flowers of Roselle are rich in several anthocyanins that provide antioxidant activities
and have been used as a diuretic and sedative [8]. Moreover, anthocyanins derived from
Roselle demonstrated anticancer activity against leukemia in both rats and humans [9].
Additionally, extracts of Roselle flowers have shown antipyretic, antinociceptive, anti-
inflammatory activities, and anticholesterol effects [9].
Although many plants produce phytochemical constituents, such as secondary metabo-
lites, that are useful in pharmaceutics and cosmetics, obtaining high amounts of these
compounds directly from plants is often difficult due to the low amounts produced. To
obtain natural compounds in high quantities, plant cell culture technology is frequently
used to produce plant-derived metabolites [10]. This technology allows for the production
of secondary metabolites in vitro without the obstacles posed by environmental factors and
geographical regions.
The transcriptome refers to the collection of all RNA molecules transcribed from
the genome [11]. Thanks to the rapid development of high-throughput sequencing and
bioinformatic tools, it is now possible to examine the effects of plant extracts on specific
human cell lines [12]. Several previous studies have demonstrated the usefulness of RNA
sequencing in identifying human RNAs regulated by a specific plant extract [13–15].
In this study, we selected the leaves of Roselle because generating calluses from Roselle
flowers can be more challenging compared to using leaves. Callus formation depends on
various factors, including the tissue’s ability to undergo dedifferentiation and reprogram
into a totipotent state [16]. In many plant species, including Roselle, leaves generally have
a higher dedifferentiation capacity compared to other plant organs. Flower tissues, such
as petals or reproductive structures, have a specialized cellular composition and are more
differentiated than leaves. These specialized cells may be less responsive to in vitro culture
conditions and callus induction due to developmental changes. Therefore, we decided to
study leaves as they have a higher propensity for callus formation in this study.
In this study, we generated plant cells from the leaves of Roselles and propagated
them using a bioreactor. We prepared extracts from the plant cells and Roselles leaves using
a hot water extraction method and compared the constituents and transcriptomes between
the two water extracts.

2. Results
2.1. Generation and Propagation of Plant Cells from Roselle Leaves
Roselle seeds were sterilized using EtOH and NaOCl (Figure 1A) and germinated
on the growth medium (Figure 1B). The small pieces of leaves from the Roselle seedlings
were used for the induction of Roselle callus (Figure 1C,D). The Roselle callus was induced
and propagated on the Murashige and Skoog (MS) medium (Figure 1E,F). Mature Roselle
calluses was used for the suspension cell culture using a bioreactor.
Next, we prepared two different water extracts from Roselle leaves and plant cells
through heat extraction. The heat extraction method was chosen based on the intended
application of Roselle extract, which is being developed as a sterilized additive for inclusion
in cosmetics and non-prescription pharmaceuticals. The selection of this method aligns
with the manufacturing process of these products and consequently led to the utilization
of high-pressure sterilization extraction. For simplicity, we named two extracts: Hibiscus
sabdariffa plant extract (HSPE) and Hibiscus sabdariffa callus extract (HSCE).
Int. J. Mol.Int.
Sci.J. 2023, 24,2023,
Mol. Sci. x FOR PEER REVIEW
24, 10853 3 of 21 3 of 21

Figure 1. Generation
Figure 1. Generationof of
callus
callusfrom
fromRoselle
Roselle seedlings. (A)Sterilized
seedlings. (A) Sterilized Roselle
Roselle seeds
seeds to ensure
to ensure the the ab
sence of contaminants.
absence of contaminants.(B)(B)
Germination
Germination of theRoselle
of the Roselle seeds
seeds to obtain
to obtain healthy
healthy seedlings.
seedlings. (C) The (C) The
Roselle seedlings
Roselle cutcut
seedlings into
intosmall
smallpieces forfurther
pieces for furthertissue
tissue culture.
culture. (D) (D)
The The Roselle
Roselle leavesleaves
placedplaced
on on a
medium containing
a medium Murashige
containing Murashige and andSkoog
Skoog(MS)
(MS)nutrients.
nutrients. (E) Inductionofofcallus
(E) Induction callus formation
formation by by add
ing appropriate plantplant
adding appropriate hormones
hormones to tothe
themedium.
medium. (F)(F)The
The development
development of mature
of mature RoselleRoselle
calluses, calluses
which cancan
which be be
used
usedfor
forvarious
various applications.
applications.

2.2. Identification of Components in Water Extracts of Hibiscus sabdariffa Using

Next, we prepared
High-Performance two different water extracts from Roselle leaves and plant cells
Liquid Chromatography
throughWe heat extraction.
analyzed The heat present
the components extraction
in twomethod was chosen
water extracts, HSPE andbased on the
HSCE, intended
using
application
high-performance liquid chromatography (HPLC). Chromatograms of the two extracts inclu-
of Roselle extract, which is being developed as a sterilized additive for
sionwere
in cosmetics
obtained atandthreenon-prescription pharmaceuticals.
different wavelengths The
(210, 255, and 305 selection
nm) ofwhich
(Figure 2), this method
revealed the differences between HSPE (Figure 2A,C) and HSCE (Figure 2D,F).
aligns with the manufacturing process of these products and consequently led to the uti-Although
not all peaks from HSPE were identified, we were able to detect guanine, phenylalanine,
lization of high-pressure sterilization extraction. For simplicity, we named two extracts
and tryptophan from both HSPE and HSCE at 210 and 255 wavelengths (Figure 2A,B).
Hibiscus sabdariffa plant extract (HSPE) and Hibiscus sabdariffa callus extract (HSCE).
Conversely, adenine, uridine, adenosine, and guanosine, which are components of nucleic
acids, were only identified from HSCE (Figure 2D,E). Interestingly, the identified peaks
2.2. from
Identification of Components
HSCE appeared inthose
faster than Water Extracts
from HSPE of
in Hibiscus sabdariffa Using
the chromatograms, indicating that
High-Performance
the componentsLiquid Chromatography
of HSCE are more hydrophilic than those of HSPE.
WeAssessment
2.3. analyzedofthe components
the Cosmetical present
Effects of HSCEinthrough
two water
In Vitroextracts,
Studies HSPE and HSCE, using
high-performance
We evaluated liquid chromatography
the effect of HSCE on cell(HPLC). Chromatograms
cytotoxicity using the CCK-8of the After
assay. two extracts
were obtained
treatment at three
with threedifferent
different wavelengths
concentrations of (210,
HSCE255, and 305
on HaCaT nm)
cells, the(Figure 2), which
cell viabil-
revealed
ity wasthemeasured
differences between
compared HSPE
to the (Figure
control 2A,C)
treated withand HSCE
sterile water(Figure
(Figure2D,F).
3A). AsAlthough
not the concentration
all peaks from HSPEof HSCE
wereincreased from we
identified, 1% were
to 10%, the to
able celldetect
viability decreased
guanine, from
phenylalanine
85.56% to 76.16%.
and tryptophan from both HSPE and HSCE at 210 and 255 wavelengths (Figure 2A,B)
The melanin inhibitory activity was measured after treatment with different concen-
Conversely, adenine, uridine, adenosine, and guanosine, which are components of nucleic
trations of HSCE on B16F1 cells (mouse melanoma cell line) (Figure 3B). Compared to the
acids, werecontrol
positive only identified
treated withfrom
100 ppmHSCEkojic(Figure 2D,E).
acid (36.24% Interestingly,
of melanin theactivity),
inhibitory identified
all peaks
fromHSCE-treated
HSCE appeared samples faster than
showed those from lower
a significantly HSPEmelanin
in the chromatograms, indicating that
inhibitory activity ranging
the components of HSCE are more hydrophilic than those of HSPE.
from 5.81% to 11.48%, showing statistical difference with the positive control.
Filaggrin (FLG) is a structural protein required for the development and maintenance
of the skin barrier. Using the expression of FLG, we measured the skin barrier effect of HSCE
after treatment with three different HSCE concentrations on HaCaT cells (Figure 3C). The
relative expression of the FLG gene in the positive control sample treated with 1% glyceryl
Int. J. Mol. Sci. 2023, 24, 10853 4 of 21

glucoside was 3.71, while the expression of the FLG gene for 1%, 5%, and 10% of HSCE-
treated samples was 0.63, 2.06, and 5.57. Compared to the control treated with sterile
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 21
water, the positive control and 10% of HSCE-treated samples showed significant differences
supported by statistical results (p < 0.01 and p < 0.001).

Figure 2. The HPLC chromatograms of two different extracts obtained from Hibiscus sabdariffa (HS).
Figure 2. The HPLC chromatograms of two different extracts obtained from Hibiscus sabdariffa (HS).
The extracts include HSPE, which is the extract obtained from the plant, and HSCE, which is the
The extracts include HSPE, which is the extract obtained from the plant, and HSCE, which is the
extract obtained from the callus. The samples were analyzed with a water and acetonitrile mixture
extract obtained
containing from the callus.
0.1% trifluoracetic acidThe samples
as the mobilewere
phaseanalyzed with at
and detected a water and
210 nm, acetonitrile
255 nm, and 305mixture
nm
using a diode array detector. The chromatograms for (A) HSPE (@210 nm), (B) HSPE (@255 305
containing 0.1% trifluoracetic acid as the mobile phase and detected at 210 nm, 255 nm, and nm),nm
using
and a diode
(C) HSPE array
(@305detector. The chromatograms
nm) are shown forwhile
in the top row, (A) HSPE (@210 nm), (B) for
the chromatograms HSPE(D)(@255
HSCEnm), and
(@210
nm), (E) HSCE
(C) HSPE (@305(@255
nm) nm), and (F)inHSCE
are shown (@305
the top row,nm) arethe
while shown in the bottomfor
chromatograms row.
(D) HSCE (@210 nm),
(E) HSCE (@255 nm), and (F) HSCE (@305 nm) are shown in the bottom row.
2.3. Assessment of the Cosmetical Effects of HSCE through In Vitro Studies
The antioxidant effect of HSCE was measured based on the expression of SOD1 after
We evaluated
treatment the effect
with different HSCE of concentrations
HSCE on cell cytotoxicity
on HaCaT using the CCK-8
cells (Figure 3D). assay.
ComparedAfterto
treatment
the controlwith three
treated different
with sterile concentrations
water, the positiveof HSCE
control onshowed
HaCaTsignificant
cells, the cell viability
up-regulation
was measured compared to the control treated with sterile water
of SOD1 gene expression (1.13). Among the three different HSCE concentrations, (Figure 3A). As the con-
only
centration
10% of HSCE (1.47) showed a significant difference compared to the control, althoughto
of HSCE increased from 1% to 10%, the cell viability decreased from 85.56% the
76.16%.
expression of SOD1 was increased after treatment with 5% and 10% of HSCE.
The melanin inhibitory activity was measured after treatment with different concen-
trations of HSCE onAnalysis
2.4. Transcriptomic B16F1 cells (mouse
of HaCaT melanoma
Cells celltoline)
in Response (Figure
Extracts 3B).Derived
of HS Compared from to the
Plant
and Callus
positive control treated with 100 ppm kojic acid (36.24% of melanin inhibitory activity),
all HSCE-treated
To investigate samples showed a significantly
the transcriptional changes inlower
HaCaT melanin
cells ininhibitory
responseactivity rang-
to HS extracts,
ing from 5.81% RNA
we performed to 11.48%, showingWe
sequencing. statistical
collecteddifference
a total of with
ninethe positive
HaCaT control.from three
samples
Filaggrin
different (FLG) is a conditions:
experimental structural protein required
a control group fortreated
the development
with distilledand water,
maintenance
a group
of the skin
treated withbarrier.
HSPE (HSUsing the expression
extract of FLG,
from the plant), andwe measured
a group the with
treated skin HSCE
barrier(HSeffect of
extract
HSCE after treatment with three different HSCE concentrations on
from the callus). Each condition consisted of three biological replicates (Table 1). HaCaT cells (Figure
3C). The relative expression of the FLG gene in the positive control sample treated with
1% glyceryl glucoside was 3.71, while the expression of the FLG gene for 1%, 5%, and 10%
of HSCE-treated samples was 0.63, 2.06, and 5.57. Compared to the control treated with
sterile water, the positive control and 10% of HSCE-treated samples showed significant
differences supported by statistical results (p < 0.01 and p < 0.001).
The antioxidant effect of HSCE was measured based on the expression of SOD1 after
treatment with different HSCE concentrations on HaCaT cells (Figure 3D). Compared to
the control treated with sterile water, the positive control showed significant up-regula-
tion of SOD1 gene expression (1.13). Among the three different HSCE concentrations, only
10% of HSCE (1.47) showed a significant difference compared to the control, although the
expression of SOD1 was increased after treatment with 5% and 10% of HSCE.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW
Int. J. Mol. Sci. 2023, 24, 10853
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Figure 3. Evaluation of the effects of HSCE on cell cytotoxicity, melanin production, skin barrier, and
Figure 3. Evaluation of the effects of HSCE on cell cytotoxicity, melanin production, skin barrier,
antioxidant activity. (A) Cell viability assay of HaCaT cells treated with HSCE. Inhibition of melanin
and antioxidant activity. (A) Cell viability assay of HaCaT cells treated with HSCE. Inhibition of
production by HSCE on B16F1 cells. (B) Inhibition of melanin production by HSCE on B16F1 cells.
melanin production by HSCE on B16F1 cells. (B) Inhibition of melanin production by HSCE on
Controlcells.
B16F1 without any without
Control treatment; anypositive control
treatment; (P.C) treated
positive controlwith 100treated
(P.C) ppm kojic
withacid. (C) Effects
100 ppm of
kojic acid.
HSCE on skin barrier function in HaCaT cells. The control group (C) was treated
(C) Effects of HSCE on skin barrier function in HaCaT cells. The control group (C) was treated with with sterile water,
and thewater,
sterile positive
andcontrol (P.C) group
the positive was(P.C)
control treated withwas
group 1% glycerol
treated glucoside. (D) Antioxidant
with 1% glycerol glucoside. effect
(D)ofAn-
HSCE on HaCaT
tioxidant effect ofcells.
HSCE Theon control
HaCaT group (C)The
cells. was control
treated with
group sterile
(C) water, and thewith
was treated positive control
sterile (P.C)
water, and
group
the was treated
positive with
control 2 mM
(P.C) NAC was
group (N-acetylcy-L-cysteine).
treated with 2 mM The gene(N-acetylcy-L-cysteine).
NAC expression of FLG and SOD1 Thewasgene
expression
analyzed after of FLG and SOD1
treatment wasdifferent
with three analyzed after treatment
concentrations with (1%,
of HSCE three5%,
different
and 10%).concentrations
The symbols of
HSCE (1%,
*, **, and *** 5%, and 10%).
represent The significance
statistical symbols *, **,at pand *** prepresent
< 0.05, < 0.01, andstatistical
p < 0.001significance at p < 0.05, p <
levels, respectively.
0.01, and p < 0.001 levels, respectively.
We treated HaCaT cell samples with either distilled water (control) or extracts of
HS derived from the
2.4. Transcriptomic plant and
Analysis callus.Cells
of HaCaT Eachincondition
Response toincluded
Extractsthree
of HSdifferent
Derived biological
from Plant
replicates.
and Callus HS refers to Hibiscus sabdariffa.
Total RNA was extracted from each sample and used to prepare a library for RNA
To investigate
sequencing. the transcriptional
The library was sequencedchanges in HaCaT
in paired-end mode cells in response
using to HS
the NovaSeq extracts,
6000 sys-
we
tem.performed RNA sequencing.
The total number of sequenced We reads
collected a total
ranged from of10
nine HaCaTpairs
gigabase samples from three
(HSPE-R1) to
different
13 gigabaseexperimental conditions:
pairs (HSCE-R3), a control
while the group of
total number treated
reads with distilled
in each librarywater,
rangedafrom
group
treated with
99 million HSPE (HS
(HSPE-R1) toextract from(HSCE-R3)
131 million the plant), and a group
(Table 2). Thetreated
Q20 and with
Q30HSCE (HSscores
quality extract
from the callus).
indicated Each condition
high sequencing consisted of three biological replicates (Table 1).
read quality.

Table 1. Details of the nine HaCaT samples used for RNA sequencing.

Index Condition Treatment Library Name Replicate

1 Control Distilled water DW-R1 1
2 Control Distilled water DW-R2 2
3 Control Distilled water DW-R3 3
Int. J. Mol. Sci. 2023, 24, 10853 6 of 21

Table 1. Details of the nine HaCaT samples used for RNA sequencing.

Index Condition Treatment Library Name Replicate

1 Control Distilled water DW-R1 1
2 Control Distilled water DW-R2 2
3 Control Distilled water DW-R3 3
4 HSPE HS extract from plants HSPE-R1 1
5 HSPE HS extract from plants HSPE-R2 2
6 HSPE HS extract from plants HSPE-R3 3
7 HSCE HS extract from callus HSCE-R1 1
8 HSCE HS extract from callus HSCE-R2 2
9 HSCE HS extract from callus HSCE-R3 3

Table 2. Summary of raw RNA sequencing data obtained.

Library Name Total Read (bp) Total Reads GC(%) AT(%) Q20(%) Q30(%)
DW-R1 10,517,649,342 104,135,142 40.2 59.8 98.5 95.5
DW-R2 13,311,500,636 131,797,036 41.1 58.9 98.6 95.7
DW-R3 11,736,440,986 116,202,386 43 57 98.5 95.6
HSPE-R1 10,081,467,712 99,816,512 41.2 58.8 98.5 95.7
HSPE-R2 10,758,263,258 106,517,458 40.8 59.2 98.5 95.4
HSPE-R3 13,290,280,334 131,586,934 42.2 57.8 98.5 95.6
HSCE-R1 13,310,397,918 131,786,118 43.5 56.5 98.5 95.7
HSCE-R2 10,762,588,482 106,560,282 42.3 57.7 98.7 96.1
HSCE-R3 13,324,897,276 131,929,676 40.8 59.2 98.7 95.9
Abbreviations used: bp, base pairs; Q20 and Q30, quality scores of 20 and 30, respectively, for raw sequencing data.

2.5. Identification of Differentially Expressed RNAs in Response to HS Extract Treatment

We filtered out poor-quality reads and mapped the remaining clean reads from each
library onto the human genome using BWA with default parameters. The eXpress program
was used to calculate the number of mapped reads for each RNA (transcript) by analyzing
the aligned SAM files. We then used the DESeq2 program to analyze the differentially
expressed RNAs (DERs) by comparing the extract-treated condition to the control condition.
We identified DERs for HSPE, HSCE, and HS-All (comparison of all treated samples to
control samples) using an adjusted p-value less than 0.01 and a log10-converted fold change
greater than 2 as a cutoff. The volcano plots (Figure 4A–C) indicated that HSPE had a
large number of DERs compared to HSCE. However, when we compared all extract-treated
samples to the control samples, we did not identify any DERs (Figure 4C). We identified
several transcript variants encoded by the same gene, and for simplicity, we regarded
each transcript variant as a single DER. We identified 111 DERs from HSPE (Table S1),
which were further divided into 92 up-regulated RNAs and 19 down-regulated RNAs
(Figure 4D). In the case of HSCE, we identified five up-regulated and one down-regulated
RNA (Figure 4D).
Of the identified DERs, the majority were mRNAs, but we also found 10 noncod-
ing RNAs (ncRNAs), including WDR77, MT1L, CLCA4, FGF11, SDAD1P1, MIR210HG,
LOC107987250, LOC101930275, LOC101930275, and EGLN3-AS1. All of the ncRNAs, ex-
cept for WDR77, were up-regulated, with EGLN3-AS1, which encodes EGLN3 anti-sense
RNA 1, being the most highly up-regulated transcript. Interestingly, we also observed
that 39 DERs were unique transcripts encoded by a single gene, while 72 DERs were RNA
variants. For example, we identified six DERs encoded by the NDRG1 gene, which encodes
N-myc downstream regulated 1, and six DERs encoded by the SAMD4A gene, which
encodes the sterile alpha motif domain containing 4A. In total, we identified 12 down-
regulated genes and 51 up-regulated genes following HSPE treatment. The representative
highly up-regulated RNAs were BNIP3, RNASE7, MIR210HG, SLC6A8, PPP1R3C, and
SAMD4A, while the highly down-regulated RNAs were AKR1B10, NME1, and ALDH1A
(Table 3).
Int.Int.
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2023, FOR PEER REVIEW 7 of721of 21

Figure 4. Distribution of differentially expressed RNAs (DERs) and number of DERs in each condition.
Figure 4.plots
Volcano show theofdistribution
Distribution of adjusted
differentially expressedp-values
RNAs and foldand
(DERs) changes in HSPE
number (A), in
of DERs HSCE
each(B),
condi-
and HS-All (C) conditions. Blue and red dots indicate down- and up-regulated RNAs, respectively.
tion. Volcano plots show the distribution of adjusted p-values and fold changes in HSPE (A), HSCE
(D) and
(B), The number
HS-All (C)of identified DERs
conditions. Bluein and
HSPE and
red HSCE
dots conditions.
indicate down- and up-regulated RNAs, respec-
tively. (D) The number of identified DERs in HSPE and HSCE conditions.
Table 3. Ten representative genes differentially expressed by HSPE treatment.

Gene Name NCBI ID Of the identified DERs, the majority were mRNAs, Padj
Description but we also found log10 noncoding
2 FC
RNAs (ncRNAs), including WDR77, MT1L, CLCA4, FGF11, SDAD1P1, MIR210HG,
EGLN3-AS1 XR_943736.2 EGLN3 antisense RNA 1, ncRNA 9.76 × 10−38 2.832333775
BNIP3 LOC107987250,
NM_004052.3 LOC101930275, LOC101930275,
BCL2 interacting protein 3 and EGLN3-AS1.
2.64 × 10 − 114 All of2.533223896
the ncRNAs, ex-
RNASE7 cept
NM_032572.3 for WDR77, were up-regulated,
ribonuclease A family member 7 with EGLN3-AS1, which
1.85 × 10 − encodes
12 EGLN3
2.225154471anti-sense
MIR210HG RNA 1, being
NR_038262.1 the most
MIR210 highly
host gene, up-regulated
long non-coding RNA transcript.1.27 × 10 − 39
Interestingly, we also observed that
2.016904685
SAMD4A NM_015589.5
39 DERs were sterile alpha motif
unique domain containing
transcripts encoded by 4A a single × 10−while
1.77gene, 10
72 1.770832281
DERs were RNA
PPP1R3C NM_005398.6 −11
× 10the
variants. For phosphatase
example, we 1 regulatory
identifiedsubunit 3C encoded
six DERs 2.11 by NDRG11.840645949
gene, which en-
SLC6A8 NM_001142806.1 solute carrier family 6 member 8 2.60 × 10−22 1.771043453
codes N-myc downstream regulated 1, and six DERs encoded by the SAMD4A gene,
ALDH1A1 NM_000689.4 aldehyde dehydrogenase 1 family member A1 5.40 × 10−6 −1.302747218
NME1 which
NM_000269.2 encodes the sterile alpha motif
NME/NM23 nucleoside diphosphate kinase 1 domain containing 4A.
1.56 × 10 − 17In total,−1.355005295
we identified 12
AKR1B10 down-regulated
XM_011516416.1 genes
aldo-keto and 51
reductase up-regulated
family 1 member B10 genes following
1.04 × 10HSPE treatment.
− 7 −1.607186598The repre-
sentative highly up-regulated RNAs were BNIP3, RNASE7,
Abbreviations: padj, adjusted p-value; log2 FC, log2 converted fold change. MIR210HG, SLC6A8,
PPP1R3C, and SAMD4A, while the highly down-regulated RNAs were AKR1B10, NME1,
2.6. ALDH1A
and Functional Classification
(Table 3). of Differentially Expressed RNAs Associated with HSPE Treatment
Next, we examined the enriched functions of up- and down-regulated DERs associated
with HSPE
Table 3. Ten across seven different
representative databases expressed
genes differentially (Table S2 and Figure
by HSPE 5A). Based on the gene
treatment.
ontology (GO) term enrichment analysis, we identified 45 GO terms for up-regulated DERs
Gene Name NCBI IDand 3 GO terms for down-regulated
DescriptionDERs according to biological Padj log2FC
processes. According
EGLN3-AS1 XR_943736.2 EGLN3 antisense
to the cellular components, RNA 1,ofncRNA
the number enriched GO terms9.76
for ×up-regulated
10 −38 2.832333775
DERs
BNIP3 NM_004052.3
was lower than that BCL2 interacting protein
for down-regulated DERs.3Regarding the molecular
2.64 × 10−114 2.533223896
function, 48 and
RNASE7 41 enriched GO terms
NM_032572.3 were identified
ribonuclease for member
A family up- and down-regulated
7 DERs,
1.85 × 10respectively.
−12 The
2.225154471
MIR210HG enriched functions
NR_038262.1 MIR210 forhost
down-regulated DERs were higher
gene, long non-coding RNA than those
1.27for× up-regulated
10−39 DERs
2.016904685
across three databases such as Panther, Reactome, and WikiPathways, except for KEGG.
SAMD4A NM_015589.5 sterile alpha motif domain containing 4A 1.77 × 10 −10 1.770832281
PPP1R3C NM_005398.6 phosphatase 1 regulatory subunit 3C 2.11 × 10 −11 1.840645949
SLC6A8 NM_001142806.1 solute carrier family 6 member 8 2.60 × 10−22 1.771043453
 ated with HSPE across seven different databases (Table S2 and Figure 5A). Based on the
gene ontology (GO) term enrichment analysis, we identified 45 GO terms for up-regulated
DERs and 3 GO terms for down-regulated DERs according to biological processes. Ac-
cording to the cellular components, the number of enriched GO terms for up-regulated
DERs was lower than that for down-regulated DERs. Regarding the molecular function,
Int. J. Mol. Sci. 2023, 24, 10853 48 and 41 enriched GO terms were identified for up- and down-regulated DERs, respec- 8 of 21

tively. The enriched functions for down-regulated DERs were higher than those for up-
regulated DERs across three databases such as Panther, Reactome, and WikiPathways,
except comparing
When for KEGG. When comparing
the list thefunctions
of enriched list of enriched functions
for the for theonly
two groups, two two
groups, only
functions,
two functions, (GO:0005739)
mitochondrion mitochondrionand (GO:0005739)
fructose and and fructosemetabolism
mannose and mannose metabolism
(hsa00051), were
(hsa00051), identified
commonly were commonly identified
for both up- andfor both up- and down-regulated
down-regulated DERs (Figure 5B).DERs (Figure 5B).

Figure 5. Enriched functions of identified

identified DERs
DERs associated
associated with
with HSPE
HSPEtreatment.
treatment. (A)
(A)The
Thenumber
numberof
of enriched functions identified for up- and down-regulated DERs based on seven functional cate-
enriched functions identified for up- and down-regulated DERs based on seven functional categories.
gories. (B) A Venn diagram displaying the number of enriched functions identified for
(B) A Venn diagram displaying the number of enriched functions identified for up- and down- up- and
down-regulated DERs.
regulated DERs.
A total of 51 up-regulated
up-regulated genesgenes were
were assigned
assignedtotoknown
knownGO GOterms,
terms,asasshown
showninin
Figure 6A. Among
Among the the assigned
assigned functions,
functions, the
themetabolic
metabolicprocess
process(34 (34genes),
genes),biological
biological
regulation (27
(27 genes),
genes), andand response
responseto tostimulus
stimulus(25(25genes)
genes)werewerefrequently
frequentlyidentified
identified
ac-ac-
cording to
to the
thebiological
biologicalprocess.
process.Many
Manyup-regulated
up-regulatedgenes
geneswere targeted
were to the
targeted membrane
to the mem-
(19 genes),
brane nucleus
(19 genes), (16 genes),
nucleus and and
(16 genes), membrane-enclosed
membrane-enclosed lumenlumen(16(16
genes).
genes).Additionally,
Addition-
some up-regulated genes were associated with the microbody (one
ally, some up-regulated genes were associated with the microbody (one gene) and gene) and endosome
endo-
(one
some (one gene). The most abundant molecular functions for up-regulated genesprotein
gene). The most abundant molecular functions for up-regulated genes were were
binding (33 genes),
protein binding (33 ion binding
genes), (17 genes),
ion binding (17 and hydrolase
genes), activityactivity
and hydrolase (10 genes). Furthermore,
(10 genes). Fur-
molecular
thermore, functions
molecularsuch as the translation
functions such as theregulator activity,
translation lipid binding,
regulator activity, chromatin bind-
lipid binding,
ing, molecular
chromatin transducer
binding, activity,
molecular and electron
transducer transfer
activity, activity were
and electron exclusively
transfer identified
activity were ex-
in up-regulated
clusively genes.
identified in up-regulated genes.
A total of 12 genes were assigned to known GO terms as down-regulated genes by
HSPE treatment (Figure 6B). According to the biological process, the metabolic process
(10 genes) and biological regulation (nine genes) were frequently identified, whereas only
five genes were assigned to the response to stimulus. Many down-regulated genes were
targeted to three cellular components: cytosol (seven genes), nucleus (seven genes), and
membrane-enclosed lumen (seven genes). As compared to up-regulated genes, down-
regulated genes were highly identified in the cytoskeleton (five genes) and extracellular
space (four genes). According to the molecular functions, down-regulated genes were
mostly associated with protein binding (nine genes), nucleotide binding (seven genes), and
ion binding (six genes).
Next, we performed hierarchical clustering analysis of 111 genes in three different
conditions. The heatmap revealed that 111 genes could be categorized into two distinct
groups: Group I and Group II (Figure 7A). Group I consisted of 19 genes that showed
down-regulation in both HSPE and HS-All conditions, but their expression levels re-
mained unchanged in the HSCE condition. In contrast, Group II comprised 92 genes that
showed up-regulation in both HSPE and HS-All conditions, but their expression levels
were not significantly altered in the HSCE condition. In the HSCE condition, we identified
five up-regulated and one down-regulated gene (Figure 7B).
Int.
Int.J. J.Mol.
Mol.Sci. 2023,
Sci. 2023,24,
24,x 10853
FOR PEER REVIEW 9 of 2121
9 of

Figure6.6.GOGO
Figure slimslim classification
classification of and
of up- up- down-regulated
and down-regulatedgenesgenes by HSPE
by HSPE according
according to threetofunc-
three
functional
tional categories:
categories: biological
biological process
process (red),
(red), cellular
cellular component
component (blue),
(blue), andandmolecular
molecularfunction
function
(green).
(green).(A)
(A)Number
Numberofofgenes
genesassigned
assignedtotoeach
eachGOGOterm
termfor
forup-regulated
up-regulatedgenes.
genes.(B) (B)Number
Numberofof
genes
genesassigned
assignedtotoeach
eachGOGOterm
termfor
fordown-regulated
down-regulatedgenes.
genes.The
Thenumbers
numbersindicate
indicatethe
thecount
countofofgenes
genes
assigned
assignedtotothe
thecorresponding
correspondingGO GOterm.
term.

2.7. AFunctional
total of 12 Categorization
genes were of Genes Up-Regulated
assigned to known GO by terms
HSPE as down-regulated genes by
HSPE We treatment
performed(Figure
GO6B).
termAccording
enrichment to the biological
analysis process,
to identify thethe metabolic
enriched processof
functions
(10
51genes) and biological
up-regulated regulationby
genes regulated (nine
HSPE genes) were
(Table S2).frequently
Among the identified, whereas
45 enriched only
GO terms
five genes were
associated withassigned to the response
the biological process, to stimulus.
seven Manyincluding
GO terms down-regulated genes were
the response to hy-
targeted to three cellular
poxia (GO:0001666), components:
carboxylic cytosol (seven
acid metabolic processgenes), nucleus (seven
(GO:0019752), genes), and
oxidation-reduction
membrane-enclosed
process (GO:0055114), lumen (seven genes).
angiogenesis As compared
(GO:0001525), andto female
up-regulated genes,
pregnancy down-reg-
(GO:0007565)
ulated genes were
were found to behighly identified
significantly in the cytoskeleton
enriched (Table S3 and (five genes)
Figure 8A).and extracellular
Eight space
genes—EGLN1,
LOXL2,
(four EGLN3,
genes). ADM,toSTC2,
According NDRG1, HK2,
the molecular PDK1,
functions, and BNIP3—were
down-regulated genesinvolved
were mostlyin re-
sponse to with
associated hypoxia, while
protein six genes—HDAC5,
binding EGLN1, PTPRB,
(nine genes), nucleotide binding LOXL2,
(sevenITGA5,
genes),ADM, and
and ion
HK2—were associated
binding (six genes). with angiogenesis (Table S3). In terms of cellular components,
31 GO terms
Next, wewere enriched,
performed with many clustering
hierarchical up-regulated genes targeted
analysis to synapse
of 111 genes (GO:0045202),
in three different
sarcoplasmThe
conditions. (GO:0016528), mitochondrion
heatmap revealed that 111(GO:0005739),
genes could be perinuclear
categorized region of cytoplasm
into two distinct
(GO:0048471),
groups: Group Iand andtertiary
Group granule (GO:0070821)
II (Figure 7A). Group (Table S3 and
I consisted ofFigure
19 genes8B).that
For showed
instance,
seven genes—STXBP1,
down-regulation in bothEGLN1,
HSPE andHAP1, ITGA5,
HS-All SAMD4A,but
conditions, NDRG1, and BNIP3—were
their expression tar-
levels re-
geted to synapse, while FABP3 and HK2 were associated with sarcoplasm
mained unchanged in the HSCE condition. In contrast, Group II comprised 92 genes that (Table S3). Of
the identified 48 enriched GO terms according to molecular function,
showed up-regulation in both HSPE and HS-All conditions, but their expression levels GO terms associated
withnot
were monosaccharide binding
significantly altered (GO:0048029),
in the HSCE condition. carbohydrate
In the HSCE binding (GO:0030246),
condition, we identified and
carboxylic acid binding (GO:0031406), and
five up-regulated and one down-regulated gene (Figure 7B).translation repression activity (GO:0030371)
Int. J. Mol. Sci. 2023, 24, 10853 10 of 21

were significantly enriched (Figure 8C). Five genes—EGLN1, EGLN3, P4HA1, HK2, and
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 21
SLC2A3—were involved in monosaccharide binding, while NANOS1 and SAMD4A were
involved in translation repressor activity.

Hierarchicalclustering
Figure7.7.Hierarchical
Figure clusteringofof111
111differentially
differentiallyexpressed
expressedgenes
genesidentified
identifiedfrom
fromHSPE
HSPEtreat-
treatment.
ment. (A) Heatmap
(A) Heatmap displaying
displaying twotwo distinct
distinct clustersofof111
clusters 111differentially
differentially expressed
expressedgenes,
genes,defined
defined by
by hierarchical
hierarchical clustering
clustering using
using thethe average
average linkage
linkage method,
method, in in three
three different
different conditions.
conditions. (B)(B) Ex-
Expression
pression levels of the identified genes in three different conditions: HSPE, HSCE, and HS-All.
levels of the identified genes in three different conditions: HSPE, HSCE, and HS-All.
Furthermore,
2.7. Functional we identified
Categorization of Genes10 enriched KEGG
Up-regulated by HSPEpathways, among which the path-
wayWe associated with glycolysis and gluconeogenesis
performed GO term enrichment analysis to identify (hsa00010) was thefunctions
the enriched most significantly
of 51
enriched (Table S2). Using the Panther database, we found three
up-regulated genes regulated by HSPE (Table S2). Among the 45 enriched GO terms pathways, including
as-
fructosewith
sociated and galactose metabolism
the biological process, (P02744),
seven GOglycolysis (P00024),
terms including theand the hypoxia
response response
to hypoxia
via HIF activation
(GO:0001666), (P00030).
carboxylic Additionally,
acid metabolic we(GO:0019752),
process identified nine enriched functions
oxidation-reduction pro-using
the (GO:0055114),
cess Reactome database, with glycolysis
angiogenesis (GO:0001525),(R-HSA-70171), elastic fiber
and female pregnancy formationwere
(GO:0007565) (R-HSA-
1566948),
found to beand collagenenriched
significantly formation (R-HSA-1474290)
(Table being
S3 and Figure 8A). significantly
Eight genes—EGLN1, enriched.
LOXL2, Finally,
using the
EGLN3, ADM,WikiPathway
STC2, NDRG1, database, we found
HK2, PDK1, seven enriched
and BNIP3—were functions,
involved including
in response glycol-
to hy-
ysis and
poxia, whilegluconeogenesis
six genes—HDAC5, (WP534)
EGLN1, andPTPRB,
photodynamic therapy-induced
LOXL2, ITGA5, HIF-1 survival
ADM, and HK2—were
signaling (WP3614).
associated with angiogenesis (Table S3). In terms of cellular components, 31 GO terms
were enriched, with many up-regulated genes targeted to synapse (GO:0045202), sarco-
plasm (GO:0016528), mitochondrion (GO:0005739), perinuclear region of cytoplasm
(GO:0048471), and tertiary granule (GO:0070821) (Table S3 and Figure 8B). For instance,
seven genes—STXBP1, EGLN1, HAP1, ITGA5, SAMD4A, NDRG1, and BNIP3—were tar-
geted to synapse, while FABP3 and HK2 were associated with sarcoplasm (Table S3). Of
 the identified 48 enriched GO terms according to molecular function, GO terms associated
with monosaccharide binding (GO:0048029), carbohydrate binding (GO:0030246), and
carboxylic acid binding (GO:0031406), and translation repression activity (GO:0030371)
were significantly enriched (Figure 8C). Five genes—EGLN1, EGLN3, P4HA1, HK2, and
Int. J. Mol. Sci. 2023, 24, 10853 11 of 21
SLC2A3—were involved in monosaccharide binding, while NANOS1 and SAMD4A were
involved in translation repressor activity.

Figure 8.
Figure 8. Enriched
Enriched GO terms associated
associated with
with 51
51 up-regulated
up-regulatedgenes
genesby
byHSPE
HSPEtreatment.
treatment.Directed
Directed
acyclic graphs (DAGs) show the most significant GO terms for up-regulated genes accordingtoto
acyclic graphs (DAGs) show the most significant GO terms for up-regulated genes according
biological process
biological process (A),
(A), cellular
cellular component
component(B),
(B),and
andmolecular
molecularfunction
function(C).
(C).The
TheDAG
DAGdisplays
displaysthe
the
hierarchical relationship of identified GO terms. The blue colored boxes indicate the most significant
hierarchical relationship of identified GO terms. The blue colored boxes indicate the most significant
GO terms. The significance is indicated by the color intensity.
GO terms. The significance is indicated by the color intensity.
Furthermore,
2.8. Functional we identified
Classification 10 enriched
of Genes KEGG pathways,
Down-Regulated among which the pathway
by HSPE Treatment
associated with glycolysis and gluconeogenesis (hsa00010) was the most significantly en-
We performed GO term enrichment analysis to identify enriched functions of 12 down-
riched (Table S2). Using the Panther database, we found three pathways, including fruc-
regulated genes in response to HSPE. Only three enriched GO terms associated with
tose and galactose metabolism (P02744), glycolysis (P00024), and the hypoxia response via
the biological process were identified, including the small-molecule metabolic process
HIF activation (P00030). Additionally, we identified nine enriched functions using the Re-
(GO:0044281), gland development (GO:0048732), and microtubule cytoskeleton orga-
actome database, with glycolysis (R-HSA-70171), elastic fiber formation (R-HSA-1566948),
nization (GO:0000226) (Table S4 and Figure 9A). On the other hand, 43 enriched GO
and collagen formation (R-HSA-1474290) being significantly enriched. Finally, using the
terms were identified according to the cellular component. Among them, four genes,
WikiPathway database, we found seven enriched functions, including glycolysis and glu-
namely NME1, CENPE, KIF20A, and AURKA, were involved in polymeric cytoskeletal
coneogenesis (WP534) and photodynamic therapy-induced HIF-1 survival signaling
fiber (GO:0099513), while SLC7A11 was associated with astrocyte projection (GO:0097449)
(WP3614).
(Table S5 and Figure 9B). Regarding molecular functions, 41 enriched GO terms were
identified (Table S4). Among them, genes associated with retinal dehydrogenase activ-
ity (GO:0001758), sulfur amino acid transmembrane transporter activity (GO:0000099),
arginine N-methyltransferase activity (GO:0016273), and ATP binding (GO:0005524) were
down-regulated (Figure 9C). Of these, AKR1B10 and ALDH1A1 were involved in retinal
dehydrogenase activity, while five genes, namely NME1, CENPE, KIF20A, EARS2, and
AURKA, were involved in ATP binding (Table S5).
 (Table S5 and Figure 9B). Regarding molecular functions, 41 enriched GO terms were
identified (Table S4). Among them, genes associated with retinal dehydrogenase activity
(GO:0001758), sulfur amino acid transmembrane transporter activity (GO:0000099), argi-
nine N-methyltransferase activity (GO:0016273), and ATP binding (GO:0005524) were
down-regulated (Figure 9C). Of these, AKR1B10 and ALDH1A1 were involved in retinal
Int. J. Mol. Sci. 2023, 24, 10853 12 of 21
dehydrogenase activity, while five genes, namely NME1, CENPE, KIF20A, EARS2, and
AURKA, were involved in ATP binding (Table S5).

Figure
Figure 9.
9. Enriched GO terms
Enriched GO terms associated
associatedwith
with1212down-regulated
down-regulatedgenes
genesininresponse
responsetoto HSPE
HSPE treat-
treatment.
ment. Directed acyclic graphs (DAGs) illustrate the most significant GO terms for down-regulated
Directed acyclic graphs (DAGs) illustrate the most significant GO terms for down-regulated genes in
genes in terms of biological process (A), cellular component (B), and molecular function (C). The
terms of
DAGs biological
show process (A),
the hierarchical cellular component
relationship (B), and
of the identified GOmolecular function
terms, with (C). Theboxes
blue colored DAGsindi-
show
the hierarchical relationship of the identified GO terms, with blue colored boxes indicating
cating the most significant GO terms. The color intensity represents the significance level. the most
significant GO terms. The color intensity represents the significance level.
In
In addition, we identified
addition, we identified seven
seven metabolic
metabolicpathways
pathwaysenriched
enrichedby byHSPE
HSPEtreatment
treatmentus-
using
ing thethe KEGG
KEGG database,
database, including
including folate
folate biosynthesis
biosynthesis (hsa00790)
(hsa00790) andand galactose
galactose metab-
metabolism
olism (hsa00052) (Table S4). According to the Panther pathway, five pathways
(hsa00052) (Table S4). According to the Panther pathway, five pathways were enriched, were en-
riched, including de novo pyrimidine ribonucleotides biosynthesis (P02740)
including de novo pyrimidine ribonucleotides biosynthesis (P02740) and heme biosyn- and heme bi-
osynthesis (P02746). Using the Reactome database, a total of 25 pathways were
thesis (P02746). Using the Reactome database, a total of 25 pathways were identified, identified,
including
including kinesins
kinesins(R-HSA-983189),
(R-HSA-983189), COPI-dependent
COPI-dependent Golgi-to-ER
Golgi-to-ERretrograde traffictraffic
retrograde (R-
HSA-6811434),
(R-HSA-6811434), and and
fructose catabolism
fructose (R-HSA-70350).
catabolism AmongAmong
(R-HSA-70350). the 14 pathways identi-
the 14 pathways
fied by thebyWikiPathway,
identified the WikiPathway,we found folate-alcohol
we found and and
folate-alcohol cancer pathway
cancer hypotheses
pathway hypothe-
ses (WP1589), endoderm differentiation (WP2853), and phytochemical activity on NRF2
transcriptional activation (WP3).

2.9. Identification of Differentially Expressed RNAs Associated with HSCE

We identified six differentially expressed RNAs (DERs) after HSCE treatment. Inter-
estingly, all five up-regulated DERs were 18S ribosomal RNAs (N1 to N5) (Table 4). Only a
single gene, IFI6 encoding interferon alpha inducible protein 6, was identified as a DER.
Table 4. RNAs differentially expressed following HSCE treatment.

Gene Name NCBI ID Description Padj log2 FC

IFI6 NM_022873.2 interferon alpha inducible protein 6 0.000925 −1.26271
RNA18SN2 NR_146146.1 18S ribosomal N2 1.27 × 10−6 1.072622
RNA18SN4 NR_146119.1 18S pre-ribosomal N4 9.39 × 10−7 1.109612
RNA18SN1 NR_145820.1 18S ribosomal N1 6.30 × 10−8 1.116593
RNA18SN5 NR_003286.4 18S ribosomal N5 6.30 × 10−8 1.121973
RNA18SN3 NR_146152.1 18S ribosomal N3 1.27 × 10−6 1.136712
Abbreviations: Adjusted p-value (padj), Log2 -fold change (log2 FC).
Int. J. Mol. Sci. 2023, 24, 10853 13 of 21

2.10. Confirmation of RNA Sequencing Results by Real Time RT-PCR

To validate the results obtained from RNA sequencing, we performed real-time
RT-PCR analysis on 10 selected genes that exhibited differential expression (refer to
Table 5). Among these genes, five were up-regulated by HSPE treatment, while one
gene was up-regulated by HSCE treatment. Additionally, four genes, namely ALDH1A1,
NME1, AKR1B10, and IFI6, were found to be down-regulated.
Table 5. Selected genes for real-time RT-PCR analysis and RNA sequencing results.

Condition Gene Name NCBI Reference ID Description Padj log2 FC

HSPE EGLN3-AS1 XR_943736.2 EGLN3 antisense RNA 1, ncRNA 9.76 × 10−38 2.832
HSPE BNIP3 NM_004052.3 BCL2 interacting protein 3 2.64 × 10−114 2.533
HSPE RNASE7 NM_032572.3 ribonuclease A family member 7 1.85 × 10−12 2.225
HSPE MIR210HG NR_038262.1 MIR210 host gene, long non-coding RNA 1.27 × 10−39 2.017
HSPE SAMD4A NM_015589.5 sterile alpha motif domain containing 4A 1.77 × 10−10 1.771
HSPE ALDH1A1 NM_000689.4 aldehyde dehydrogenase 1 family member A1 5.40 × 10−6 −1.3
HSPE NME1 NM_000269.2 NME/NM23 nucleoside diphosphate kinase 1 1.56 × 10−17 −1.36
HSPE AKR1B10 XM_011516416.1 aldo-keto reductase family 1 member B10 1.04 × 10−7 −1.61
HSCE IFI6 NM_022873.2 interferon alpha inducible protein 6 0.0009255 −1.26
HSCE RNA18SN3 NR_146152.1 18S ribosomal N3 1.27 × 10−6 1.137
Abbreviations: Adjusted p-value (padj), Log2 -fold change (log2 FC).

Comparative analysis between real-time RT-PCR and RNA sequencing results revealed
that the expression patterns of all nine genes were consistent, except for the ALDH1A1
gene (Figure 10). Interestingly, the ALDH1A1 gene showed down-regulation in the RNA
sequencing data, whereas it was up-regulated in the real-time RT-PCR results. Furthermore,
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 14 of 21
out of the 10 genes analyzed, real-time RT-PCR results indicated significant differences
between the control and treatment groups for eight genes, as supported by statistical tests.

Figure 10. Real-time RT-PCR results showing the expression levels of 10 selected genes. Among
these genes, eight (indicated by blue dotted lines) were found to be differentially expressed by HSPE,
Figure 10. Real-time RT-PCR results showing the expression levels of 10 selected genes. Among
while two (indicated by green dotted lines) were differentially expressed by HSCE. The presence of
these genes, eight (indicated by blue dotted lines) were found to be differentially expressed by
‘***’ indicates
HSPE, while two a p-value less than
(indicated 0.001,
by green indicating
dotted statistical
lines) were significance
differentially in the comparison
expressed by HSCE. Thebetween
pres-
the
encecontrol
of ‘***’and treatment
indicates groups.
a p-value less than 0.001, indicating statistical significance in the comparison
between the control and treatment groups.

3. Discussion
In this study, we generated a callus from Roselle leaves and further subjected it to
Int. J. Mol. Sci. 2023, 24, 10853 14 of 21

3. Discussion
In this study, we generated a callus from Roselle leaves and further subjected it to
mass production using a bioreactor. The cultured Roselle plant cells were extracted using
water and heat. Using HPLC, diverse in vitro assays, and transcriptome analyses, we
characterized the effects of Roselle plant cell extracts.
Previous studies have generated calluses from Roselle seedlings and demonstrated
the enrichment of anthocyanins in Roselle calluses [17,18]. Hormone combinations play
particularly important roles in callus growth and anthocyanin synthesis, which are inversely
correlated [17]. Moreover, a previous study demonstrated that yeast extract induced
anthocyanin production in the callus generated from Roselle seedlings [18]. It is also
possible to select the highly anthocyanin-producing callus line with a red and purple
aggregate from the Roselle cell line [17]. In this study, we established a mass cell culture
of a generated Roselle callus using a bioreactor for the first time, which can be useful for
commercial applications.
The most commonly identified metabolites from HSPE and HSCE were guanine,
phenylalanine, and tryptophan. Guanine is one of the four main nucleobases that con-
stitute nucleic acids such as DNA and RNA. Phenylalanine and tryptophan are aromatic
amino acids required for protein synthesis. For example, phenylalanine is involved in the
synthesis of several secondary metabolites such as plant pigments including flavonoids,
isoflavonoids, and proanthocyanidins, while tryptophan is related to the synthesis of
vitamins and plant hormones [19].
In this study, we used water and heat to obtain extracts from the Roselle plant and
callus since the main purpose of this study is to apply the HSCE for cosmetics and phar-
maceutical purposes. Normally, only 1% of extracts derived from diverse plant cells are
commercially utilized. However, in this study, we investigated the effects of three dif-
ferent concentrations, specifically 1%, 5%, and 10%. The results from the cell viability
assay conducted on HaCaT cells indicate that the water extract of roselle cells exhibited no
cytotoxicity, irrespective of the HSCE concentration used. Furthermore, the water extract
showed lower cytotoxicity compared to the control group treated with sterile water. Inter-
estingly, the cell viability remained unaffected by the concentration of HSCE in the water
extract of roselle cells. Moreover, HSCE showed strong anti-melanogenic effects by repress-
ing the expression of α-melanocyte stimulating hormone (α-MSH). As the concentration of
HSCE increased, the melanin inhibition activity increased.
FLG protein functions in the skin’s barrier. Thus, the high expression of the FLG gene
suggests the high ability of the skin barrier. The expression of FLG was increased by the
treatment of 5% and 10% of HSCE on HaCaT cells. The antioxidant activity of HSCE on
HaCaT cells was measured by the expression of SOD1, which is an antioxidant enzyme
catalyzing superoxide breakdown. It was observed that only the highest concentration
of HSCE (10%) resulted in a significant difference (1.47) when compared to the control
group. This implies that the 10% HSCE treatment exhibited a more pronounced antioxidant
effect in terms of SOD1 gene expression. However, it is worth noting that there was an
overall increase in SOD1 expression after treatment with both 5% and 10% HSCE, albeit
without reaching statistical significance. These results suggest that HSCE, particularly at a
concentration of 10%, has the potential to induce antioxidant effects through the modulation
of SOD1 gene expression in HaCaT cells.
To date, no studies have investigated the expression profiles of HaCaT cells in response
to Roselle extracts. Our transcriptome analyses revealed that HSPE has a greater impact
on HaCaT cells compared to HSCE. This result suggests that the difference in components
between HSPE and HSCE, as demonstrated by HPLC analysis, may be responsible for the
differential effects observed. However, we cannot determine exactly which component
in the Roselle extract plays an important role in the change in HaCaT transcriptome.
Hierarchical clustering analysis with 111 differentially expressed genes by HSPE showed
that the expression of most differentially expressed genes by HSPE was not changed by
Int. J. Mol. Sci. 2023, 24, 10853 15 of 21

HSCE treatment. This result suggests that some components exclusively present in HSPE
might have a strong impact on the transcriptional changes in HaCaT cells.
The most significantly enriched functions for up-regulated genes by HSPE were
associated with the response to hypoxia, the carbohydrate derivative biosynthetic process,
the oxidation-reduction process, and angiogenesis. In fact, these four biological processes
are highly connected. For example, hypoxia is defined as a condition in which there
is a lack of adequate oxygen supply at a specific region of the body at the tissue level.
It is known that many hypoxia-induced genes are involved in angiogenesis, which is a
physiological process that forms new blood vessels from preexisting vasculature and occurs
during development and tissue modeling [20]. For example, two hypoxia-inducible factors,
EGLN1 and EGLN3, which were up-regulated genes by HSPE in this study, participate in cell
survival and adaptation upon diverse environmental oxygen levels by functioning in many
biological processes such as angiogenesis, erythropoiesis, and tumorigenesis [21]. Other up-
regulated genes involved in angiogenesis, such as HDAC5, PTPRB, LOXL2, ITGA5, ADM,
and HK2, might also be involved in wound healing, growth, and female pregnancy [22,23].
For example, PTPRB, which encodes the receptor-type tyrosine phosphate beta (VE-PTP),
is an enzyme specifically expressed in endothelial cells. VE-PTP plays important roles
in vasculogenesis and blood vessel development [24] as well as for regulating adherens
junction complex [25]. Furthermore, LOXL2, encoding lysyl oxidase homolog 2, is a
secreted enzyme participating in extracellular matrix (ECM) crosslinking and in regulating
angiogenesis through collagen IV scaffolding [26].
Three genes, namely EGLN1, LOXL2, and HK2, function in both angiogenesis and
oxidation-reduction processes. It is known that those redox-signaling-associated genes play
important roles in vascular angiogenesis [27]. Four up-regulated genes, namely ITGA5,
PSG5, ADM, and STC2, in HSPE treatment, are involved in female pregnancy. Of these
genes, STC2, encoding Stanniocalcin-2, is a secreted glycoprotein hormone family that is
induced by diverse stresses, including hypoxia and nutrient deprivation [28]. Moreover,
other enrichment analyses using different databases indicate that many up-regulated
genes by HSPE are involved in hypoxia-inducible factor 1 (HIF-1) signaling pathway and
glycolysis, such as fructose and mannose metabolism, as shown by GO term enrichment
analysis. The activation of HIF-1-related signaling genes induced by HSPE suggests that
HSPE can participate in cell survival, proliferation, angiogenesis, invasion, metastasis,
cancer stemness, and metabolic reprogramming in human skin cells [29]. Furthermore,
several studies suggest that natural products can affect the glycolysis pathway and can be
used to inhibit cancer cells [30]. However, the possible role of HSPE with the suggested
functions should be further characterized.
Many genes, including COX6B2, ANKZF1, STXBP1, HAP1, DEPP1, P4HA1, HK2,
PDK1, and BNIP3, were up-regulated by HSPE, and they are targeted to the mitochondrion.
COX6B2 encodes cytochrome c oxidase subunit 6B2, which is involved in the metabolic
reprogramming of several tumor cells by enhancing oxidative phosphorylation. Moreover,
the expression of COX6B2 is induced by hypoxia [31,32]. Seven up-regulated genes, such as
STXBP1, EGLN1, HAP1, ITGA5, SAMD4A, NDRG1, and BNIP3, were targeted to synapses,
which are structures that enable a nerve cell to pass an electrical or chemical signal to
another neuron cell [33].
In comparison to HSPE, only a few genes, such as five 18S ribosomal genes and IFI6,
were up-regulated by HSCE. The up-regulation of genes encoding components of the 18S
ribosome indicates that HSCE promotes the synthesis of diverse proteins. IFI6 encodes
interferon alpha-inducible protein 6, which is induced by ionizing radiation and provides
protection to skin cells that were injured by ionizing radiation [34,35]. Therefore, the
treatment of HSCE in HaCaT cells might enhance protein synthesis and the healing of
radiation-induced skin injury.
Significant differences were observed in the findings obtained from real-time RT-PCR
analysis and transcriptome analysis using RNA sequencing when examining the effects of
Roselle plant cell extracts. Specifically, real-time RT-PCR analysis yielded specific results
Int. J. Mol. Sci. 2023, 24, 10853 16 of 21

related to cytotoxicity, FLG expression, SOD1 expression, and α-MSH expression following
HSCE treatment. In contrast, transcriptome analysis provided comprehensive insights into
the impact of HSPE and HSCE on HaCaT cells, highlighting variations in gene expression
patterns and their connections to diverse biological processes. Real-time RT-PCR and RNA
sequencing are complementary techniques, each with their own strengths and limitations.
Due to disparities in sensitivity, a targeted versus unbiased approach, technical variability,
data analysis, and biological variability, the specific outcomes derived from real-time
RT-PCR may not directly align with those obtained through transcriptome analysis via
RNA sequencing.
Taken together, HPLC analyses indicate that the components of HSPE and HSCE are
different, although some components are commonly present in both extracts. Moreover,
the differences in components between the two extracts resulted in different transcriptome
profiles of HSPE and HSCE. Functional analyses of differentially expressed genes by
the two extracts indicate that many up-regulated genes by HSPE were associated with
hypoxia, angiogenesis, and glycolysis, while HSCE treatment induced five genes that were
components of the 18S ribosome and IFI6 gene. Diverse in vitro assays indicate possible
applications of HSCE for cosmetics associated with skincare. However, the potential
functional roles of the identified genes associated with Roselle extracts should be further
examined in the near future.

4. Materials and Methods

4.1. Induction and Propagation of Plant Cells from Roselle Leaves
The Roselle seeds used in this study (registration number Je-Chilgok-2017-1—01 ho)
were purchased from an online shop called 1000 Seeds in 2019. To sterilize the seeds,
25 seeds were treated with 70% EtOH for 1 min, followed by 2.5% NaOCl for 20 min. This
method is carefully designed to minimize any potential interference with the viability and
activity of the extracts. Additionally, Tween-20 can be added to enhance the sterilization
effect. After sterilization, the seeds are thoroughly rinsed with sterilized water multiple
times to ensure the complete removal of the sterilization solution. The sterilized seeds were
placed on filter paper to remove excess moisture and then germinated on a medium in a
growth chamber at 24 ◦ C ± 2 ◦ C and 1000 Lux. The resulting seedlings were cut into pieces
of 5 to 8 mm and transferred to Murashige and Skoog (MS) medium (Duchefa, Haarlem,
The Netherlands) (Cat. No. M0222) containing MS (4.4 g/L), sucrose (30 g/L) (Duchefa)
(Cat. No. S0809), gelrite (2.3 g/L) (Duchefa) (Cat. No. G1101), and auxin and cytokinin
at pH 5.8. The initial explants were subcultured every two weeks on the same medium.
After eight weeks, callus tissues derived from various media were evaluated based on their
color, size, and growth rate. To induce callus formation, MS medium supplemented with
4.5 µM 2,4-D (Dichlorophenoxyacetic acid) (Duchefa) (Cat. No. D0911) was used. Cell lines
suitable for suspension culture were selected by repeating the subculture process under the
same conditions. Finally, a bioreactor at the Anti-Aging Research Institute of BIO-FD&C
Co., Ltd., Incheon, Republic of Korea, was used for mass production of the Roselle cells.

4.2. Preparation of Two Different Roselle Extracts through Heat Extraction Method
To prepare two different Roselle extracts, HSPE was obtained from dried plant leaves,
while HSCE was obtained from plant cells. Roselle leaves were first dried at 60 ◦ C for
two days, and then HSPE was extracted from the dried leaves using distilled water through
heat extraction at 121 ◦ C for 15 min. For HSCPE preparation, plant cells (callus) were
induced from Roselle leaves and suspension cultured for six days. The plant cells were then
harvested using a non-woven fabric filter and subjected to heat extraction using distilled
water at 121 ◦ C for 15 min. In both extract preparations, we used 2 g/L of the sample for
extraction. After heat extraction, we removed the solids by filtration through a mesh.
Int. J. Mol. Sci. 2023, 24, 10853 17 of 21

4.3. HPLC Analysis of Water Extracts from Hibiscus sabdariffa

To compare the chromatographic data of the water extracts from HSPE and HSCE, we
performed HPLC using an Agilent 1260 Infinity II system. Separation was achieved using a
Shim-pack GIS C18 column (5 µm, 4.6 × 250 mm) (Shimadzu, Kyoto, Japan) maintained at
30 ◦ C. The mobile phase was a mixture of solvent A (water containing 0.1% trifluoroacetic
acid) and solvent B (acetonitrile containing 0.1% trifluoroacetic acid), which were pumped
under the following gradient conditions: 0→5 min, 0% B→0% B; 5→75 min, 0% B→75% B;
75→77 min, 75% B→95% B; 77→82 min, 95% B→95% B; 82→84 min, 95% B→0% B and
84→95 min 0% B→0% B. The flow rate was set at 1.0 mL/min, and the injection volume
was 20 µL. Chromatographic data were collected using a diode array detector (DAD) within
the wavelength range of 200–600 nm, and the results were extracted at three characteristic
wavelengths (210, 255, and 305 nm).

4.4. Culture and Treatment of Human Keratinocyte Cells with Roselle Extracts
To cultivate and differentiate human cells, primary cultured keratinocytes were iso-
lated from human epidermal tissue and then seeded onto cell culture plates. Mean-
while, human HaCaT keratinocytes, which were obtained from American Type Culture
Collection, were cultured in a dermal cell basal medium supplemented with 10% heat-
inactivated fetal bovine serum (FBS) from Gibco (Carlsbad, CA, USA) and a 100 U/mL
penicillin/streptomycin mixture from Lonza (Walkersville, MD, USA) at 37 ◦ C in a hu-
midified atmosphere containing 5% CO2 . We conducted the experiments using HaCaT
cells that had undergone passages ranging from 10 to 15 or fewer. Once the cells reached
80–90% confluence, they were cultured in the medium. The HaCaT cells were treated with
HSCE, an extract derived from Roselle calluses, at concentrations of 1%, 5%, and 10% for a
duration of 24 h. In this case, the 10% concentration corresponds to a final concentration
of 0.02% when prepared at a ratio of 2 g/L. Evaluating the effects at this concentration
allows for a suitable assessment of the extract’s potential effects while considering the
practical application and potential dilution factors. The experiments included a control
group treated with distilled water.

4.5. Assessment of HSCE Cell Cytotoxicity Using the CCK-8 Assay

The study aimed to assess the impact of HSCE on human skin cell (HaCaT) growth,
propagation, and survival by conducting a CCK-8 assay. HaCaT cells were seeded at a
density of 5 × 104 cells per well in a 96-well plate and incubated for 24 h. The cells were
then treated with final concentrations of 1%, 5%, and 10% of HSCE for 24 h, with sterile
water serving as the control. After HSCE treatment, 1X CCK-8 solution (Cat. No. CCK-3000,
Donginbio, Seoul, Republic of Korea) was added to each well, and the cells were incubated
for 3 h. A Thermo Scientific Multiskan GO Microplate Spectrophotometer (Fisher Scientific
Ltd., Vantaa, Finland) was used to measure the wavelength absorbance at 450 nm. The
percentage of cell viability was calculated using the following formula: (absorbance of
treated cells/absorbance of control cells) × 100.

4.6. Quantificiation of Melanin Content following HSCE Treatment

B16F1 cells (mouse melanoma cell line) were seeded at a density of 1 × 105 cells per
well in 6-well plates and incubated under cell culture conditions for 24 h. Afterward, the
cells were treated with HSCE and cultured for an additional 72 h. A positive control of
100 ppm Kojic acid was also included, and 1 µM α-MSH was added to each sample to
stimulate melanin production. After incubation, the top layer of the medium was removed,
and the cells were harvested using 1X Trypsin-EDTA. A 300 µL solution of 1 N NaOH was
added to the cell pellet, and the melanin was dissolved by boiling at 100 ◦ C for 30 min. The
dissolved melanin solution was then partially transferred onto a 96-well plate to measure
the absorbance at 405 nm. The absorbance value of each sample was quantified based
on the amount of protein, and the melanin production inhibition rate was expressed as
a percentage. The calculation for the melanin production inhibition rate was as follows:
Int. J. Mol. Sci. 2023, 24, 10853 18 of 21

Melanin production inhibition rate (%) = [1 − (test group melanin amount/α-MSH treated
control melanin amount)] × 100.

4.7. Expression Analysis of FLG and SOD1 Marker Genes by Real-Time Reverse Transcription (RT-PCR)
To evaluate the impact of HSCE treatment on the expression levels of FLG and SOD1
genes, HaCaT cells were seeded at a density of 5 × 104 cells per well and treated separately
with three different concentrations of HSCE (1%, 5%, and 10%), with sterile water serv-
ing as the control. After treatment, RNA isolation and cDNA synthesis were performed
using the SuperPrep™ Cell Lysis & RT Kit for qPCR (Cat. No. SC-101, Toyobo, Osaka,
Japan) according to the manufacturer’s instructions. Real-time reverse transcription PCR
(RT-PCR) was conducted using known primers for filaggrin (FLG) (Cat. No. QT02448138,
Qiagen, Hilden, Germany) as a skin barrier marker and for superoxide dismutase 1 (SOD1)
(Cat. No. QT01008651, Qiagen, Hilden, Germany) as an antioxidant marker. The Thunder-
bird SYBR qPCR Mix Kit (Toyobo, Osaka, Japan) was used for real-time RT-PCR following
the manufacturer’s instructions. Furthermore, we performed real-time RT-PCR on ten
selected genes to validate the findings of RNA sequencing. To facilitate this process, we
designed new primers, as detailed in Table S6. For normalization purposes, the GAPDH
gene was used as a control.

4.8. Total RNA Isolation, Library Preparation, and RNA Sequencing for Transcriptome Analysis
The TRIzol reagent (Invitrogen, Waltham, MA, USA) was used to extract total RNA
from cells following the manufacturer’s protocol. The extracted total RNA’s quality was
assessed using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Total RNA with an
RNA Integrity Number (RIN) value of 7 or more was considered for library preparation.
Library preparation was performed using the TruSeq Stranded mRNA LT Sample Prep Kit
following the manufacturer’s instructions (Illumina, San Diego, CA, USA). The HiSeq X
system (Illumina) was used to pair-end sequence nine libraries with their respective indices.

4.9. Identification of Differentially Expressed Genes through Mapping of RNA-seq Reads

The sequence data obtained in this study have been deposited in the NCBI SRA
database under accession numbers, SRR24775354–SRR24775367. Raw reads were mapped
to the GRCh38 reference genome using BBMap with default settings. The number of reads
mapped to each transcript was used to identify differentially expressed genes (DEGs) using
DESeq2 in DEBrowser v1.24.1 [36]. We compared the Roselle-extract-treated HaCaT cells
with those treated with distilled water (control). The number of reads was normalized
using the MRE normalization method without any correction. By applying a fold change
(FC) more than twice and p-values less than 0.05, we identified DEGs for each comparison.
For the HS-All comparison, all six datasets from the Roselle-extract-treated samples were
compared to the three control datasets.

4.10. Functional Characterization of Differentially Expressed Genes (DEGs) by Gene Ontology

(GO) Enrichment Analysis
The up-regulated and down-regulated DEGs resulting from HSPE and HSCE treat-
ment were subjected to GO enrichment analysis using the WebGestalt program [37]. This
program utilizes an overrepresentation analysis (ORA) against the gene ontology (GO)
functional database, which is categorized into three main groups: biological process, cellu-
lar component, and molecular function. First, we determined the number of genes mapped
to GO Slim. From this, we selected genes annotated to specific functional categories and
performed enrichment analysis of the human genome using the following parameters: a
minimum number of IDs in a category of 5, and a maximum of 2000 IDs. We used the
Bonferroni method for the FDR, and the significance level was set at p-value < 0.05 with a
top 100 cut-off. Finally, we only considered identified functional gene sets with a p-value
less than 0.05.
Int. J. Mol. Sci. 2023, 24, 10853 19 of 21

5. Conclusions
In this study, we found that water extracts derived from Roselle plants (HSPE) and
callus cells (HSCE) have different components, as demonstrated by HPLC analysis. The
most common components from both extracts were nucleic acids and metabolites such as
phenylalanine and tryptophan. The cell viability assay showed that HSCE is not cytotoxic
to HaCaT cells and has strong anti-melanogenic effects. HSCE also has functions for
skin barrier and antioxidant activity, but the concentration should be more than 10%
to obtain a positive result. Additionally, we conducted transcriptome profiles of HSPE-
and HSCE-treated human skin cells for the first time. Our results revealed significant
transcriptome differences between human skin cells treated by the two extracts of Roselle.
HSPE has more of an effect on human skin cells compared to HSCE. The up-regulated
genes by HSPE were induced by hypoxia and function in angiogenesis, the oxidation-
reduction process, and glycolysis. In contrast, the up-regulated genes by HSCE encode
ribosome proteins of 18S ribosome and IFI6 functioning in the healing of radiation-injured
skin cells. Thus, we showed that the two different water extracts from Roselle have
different components and effects on human skin cells. Therefore, the commercial uses of
the two different water extracts from Roselle for cosmetic and pharmaceutical purposes
should be applied differently.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/ijms241310853/s1.
Author Contributions: Conceptualization, S.H.M.; data curation, W.K.C.; formal analysis, S.-Y.K.,
S.J.J., S.L., H.-I.K. and E.K.; funding acquisition, S.S.C. and S.H.M.; investigation, S.J.J.; methodology,
S.J.J., S.L., H.-I.K. and E.K.; project administration, J.H.L., S.S.C. and S.H.M.; resources, S.-Y.K.;
Supervision, W.K.C. and J.H.L.; validation, S.J.J., S.L., H.-I.K. and E.K.; writing—original draft,
W.K.C.; writing—review and editing, W.K.C. and S.H.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was funded by Daesang Holdings, Technology Innovation Program (Biorisk
Assessment Renovation Project) (20015900) and Advanced Technology Center Plus (ATC+, 20017936)
funded by the Ministry of Trade, Industry and Energy (MOTIE, Republic of Korea).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data were deposited in the NCBI SRA database with the
following accession numbers: SRR24775354–SRR24775367.
Conflicts of Interest: The authors declare no conflict of interest. Soo-Yun Kim, Sung Joo Jang, Sak
Lee, Hyein Kim, Euihyun Kim, Jeong Hun Lee, and Sang Hyun Moh were employed by the company
BIO-FD&C Co., Ltd., while Sung Soo Choi was employed by the company Daesang Holdings. The
remaining authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest. The company had no
role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of
the manuscript; or in the decision to publish the results.

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