Antioxidants 12 00314

antioxidants
Article
Structural Optimization of Cannabidiol as Multifunctional
Cosmetic Raw Materials
Xuelian Chen, Jie Su, Runan Wang, Rui Hao, Chenggong Fu, Jingjing Chen, Jiazhong Li * and Xin Wang
School of Pharmacy, Lanzhou University, 199 West Donggang Rd., Lanzhou 730000, China
* Correspondence: lijiazhong@lzu.edu.cn; Tel.: +86-93-1891-5686
Abstract: Cannabidiol (CBD), derived from the plant cannabis, can be used in the cosmetics industry
for its antioxidant, anti-inflammatory, anti-wrinkle and whitening effects. However, CBD is purified
from the hemp plant extract, its source is very limited and under strict control. So in this study,
computational and experimental methods were combined to search for novel CBD substitutes with
high biology potencies. The action mode between CBD and target protein cannabidiol receptor 1 was
studied to find the key skeleton, which was used to virtually screen a natural products database
to search for compounds with 70% similarity. The hit compounds with high docking scores were
selected for the ABTS and DPPH free radical scavenging experiments for antioxidant evaluation. The
effects on the expressions of nitric oxide (NO), interleukin-6 (IL-6), COX-2 and iNOS in RAW264.7
cell line were detected to demonstrate their anti-inflammatory abilities. The effect of anti-wrinkle
ability were evaluated by detecting the extracellular matrix, such as collagen, elastin, fibronectin and
reactive oxygen species (ROS) in HFF-1. The effects on melanin production and tyrosinase activity in
Bb16F10 were also detected. As a result, two compounds were found to be superior to cannabidiol, in
terms of antioxidant, anti-wrinkle and whitening efficacy with a lower cytotoxicity.
Keywords: cannabidiol; virtual screening; anti-oxidation; anti-inflammation; anti-wrinkle; whitening
Citation: Chen, X.; Su, J.; Wang, R.; 1. Introduction

Hao, R.; Fu, C.; Chen, J.; Li, J.; Wang,
The appearance of skin is a major factor used to roughly estimate a person’s facial
X. Structural Optimization of
attractiveness [1]. When affected by exogenous factors (ultraviolet, dust particles, drugs,
Cannabidiol as Multifunctional
Cosmetic Raw Materials. Antioxidants
etc.) [2–4] or endogenous factors (ischemia, metabolic processes, hormones, etc.) [5], skin
2023, 12, 314. https://doi.org/
may be infected with some pathogens. In response to these infections, the body will produce
10.3390/antiox12020314 oxidative stress, which induces the body’s immune system [6]. Macrophages start to work
by activating nuclear factor kappa-B (NF-κB) channels. Overprotection of the immune
Academic Editor: Alessandra
system is responsible for the development of skin problem, such as dermatitis, winkling
Napolitano
and skin dryness [1]. Oxidative stress can also lead to the formation of reactive oxygen
Received: 19 December 2022 species (ROS) in the skin, which result in the melanin biosynthesis and DNA damage [7–10].
Revised: 17 January 2023 Furthermore, ultraviolet (UV) irradiation can also promote the production of a large amount
Accepted: 19 January 2023 of ROS in the dermis and epidermis, because UV is a high-energy light wave, which can
Published: 29 January 2023 promote the oxidative stress reaction of some skin biomolecules containing chromophores.
UV irradiation appears to be a key factor in skin aging and fibroblast apoptosis.
The reactive oxygen species (ROS) are essential to life, as they are involved in several
biological functions. However, when produced at high levels, ROS become highly harmful,
Copyright: © 2023 by the authors. leading to the cellular damage. ROS can promote the expression of activator protein 1 (AP-1)
Licensee MDPI, Basel, Switzerland.
and its upstream signal transduction proteins, including mitogen-activated protein kinases
This article is an open access article
(MAPK) [11]. The MAPKs family, including p38 MAPK, c-jun amino terminal kinase (JNK)
distributed under the terms and
and extracellular signal regulated kinase (ERK) [12], induces the expression of matrix
conditions of the Creative Commons
metalloproteinases (MMPs). MMPs further catalyze the decomposition of extracellular
Attribution (CC BY) license (https://
matrix (ECM) [2,5,13,14], such as collagen, elastin and fibronectin [5,15], which widely exist
creativecommons.org/licenses/by/
4.0/).
in the dermis [16], maintaining skin firmness, strength, smoothness and elasticity [2]. In
Antioxidants 2023, 12, 314. https://doi.org/10.3390/antiox12020314 https://www.mdpi.com/journal/antioxidants

Antioxidants 2023, 12, 314 2 of 22
addition, ROS can lead to the collagen degradation and reduce its production by inhibiting
the proliferation of fibroblasts. Interaction of ROS with biomacromolecules, such as DNA,
lipids and proteins [17], can accelerate the senescence of the fibroblasts, which in turn
generates more ROS. It is a vicious cycle [18]. Furthermore, the occurrence of inflammation
can also promote the generation of MMPs [5,19], leading to the degradation of ECM [20].
Additionally, ultraviolet light induces keratinocytes in the epidermis to secrete
α-melanocyte-stimulating hormone (α-MSH). α-MSH binds to melanocortin-1 receptor (MC1R)
of melanocytes activating microphthalmia transcription factor (MITF) via the MAPK [21] or
cAMP-PKA-CREB signaling pathway [7,11,22,23]. MITF can modulate melanocyte differentia-
tion and melanogenesis. Furthermore, MITF can regulate the expression of tyrosinase and other
melanozymes. Tyrosinase, which is considered as a rate-limiting enzyme, catalyzes the first two
steps of melanogenesis, hydroxylates tyrosine to 3,4-dihydroxyphenylalanine (DOPA) [24] and
the further oxidation of DOPA into DOPA quinone [15,24]. Tyrosinase-related protein (TRP-1
and TRP-2) resides in melanosome can also regulate melanin generation [25,26]. Following
a couple of reactions, L-dopaquinone converts into 5,6-dihyroxyindole, which eventually be-
comes melanin, catalyzed also by tyrosinase [21]. This process is a signal cascade reaction of
α-MSH-MITF [11,24].
It is reported that cannabidiol (CBD), derived from the plant cannabis, can enhance the
activities of antioxidant enzymes, such as glutathione peroxidase (GSH-Px) and superoxide
dismutase (SOD) [27–30], thereby reducing the content of reactive oxygen species. In
2019, a study showed that CBD has whitening and brightening effects [31]. Actually, CBD
has many biological characteristics, such as regulating immune response and treating
depression, excellent anti-inflammatory effect by inhibiting the generation of TNF-α, IL-1β,
nitric oxide (NO), iNOS and COX-2 [28,30,32,33]. CBD can also inhibit the activity of
MMPs, which further prevent the ECM from being degraded, thus maintaining the level of
collagen, elastin and fibronectin. However, the main source of CBD is purified from hemp
plant extracts, which results in its strict supervision by the government. Hemp seed fruit,
hemp seed oil, hemp leaf extract and CBD are listed as banned ingredients in cosmetics in
many countries.
So, this study aims to find CBD substitutes owning similar or even better anti-
inflammatory, antioxidant, anti-wrinkle and whitening effects that can be used in the
cosmetics. At first, the action mode between CBD and the target protein canadiol receptor 1
(CB1) [20] was investigated to find the key skeleton related to its biological characteristics.
The key skeleton was placed into a natural products database to search for compounds with
a 70% structural similarity. Then molecular docking was used to fish out compounds with
similar binding modes with CBD. As the anti-wrinkle, anti-inflammatory and whitening
abilities of the compounds are closely related to the antioxidant ability, it is necessary to
test the antioxidant capacity. The antioxidant capacity of the hit compounds was tested by
DPPH, ABTS radical scavenging experiments and fluorescent stained flow cytometry, The
MTT assay, western blotting and ELISA, etc., are also used to evaluate the skin care efficacy
of these compounds.
2. Materials and Methods

2.1. Materials and Chemicals
Complete medium for RAW264.7 was bought from Procell (Procell, Wuhan, China).
Griess reagents were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing
Jiancheng Bioengineering Institute, Nanjing, China). IL-6 kits were bought from Elabscience
(Elabscience, Wuhan, China). iNOS antibody and COX-2 antibody were purchased from Sig-
nalway Antibody (Signalway Antibody, College Park, MA, USA) CBD and the hit compounds
were purchased from TargetMol (TargetMol, Shanghai, China). Collagen kit, elastin kit and
fibrin kit were bought from mlbio (mlbio, Shanghai, China). Apoptosis kit was bought from
MultiSciences (MultiSciences, Zhejiang, China). Lipopolysaccharide (LPS) was purchased
from Sigma-Aldrich (St. Louis, MO, USA). DPPH, ABTS, MTT and 2, 7-dichlorofuorescin
and the hit compounds were purchased from TargetMol (TargetMol, Shanghai, China).
Collagen kit, elastin kit and fibrin kit were bought from mlbio (mlbio, Shanghai, China).
Apoptosis kit was bought from MultiSciences (MultiSciences, Zhejiang, China). Lipopol‐
ysaccharide (LPS) was purchased from Sigma‐Aldrich (St. Louis, MO, USA). DPPH,
Antioxidants 2023, 12, 314 ABTS, MTT and 2, 7‐dichlorofuorescin diacetate (DCFH‐DA) were bought from Solarbio 3 of 22
Science & Technology (Solarbio, Beijing, China). α‐MSH was bought from APExBIO
(APExBIO, Houston, TX, USA).
diacetate (DCFH-DA) were bought from Solarbio Science & Technology (Solarbio, Beijing,
2.2. Computer‐Aided
China). Virtual Screening
α-MSH was bought from APExBIO (APExBIO, Houston, TX, USA).
Virtual screening is a common, efficient and economical method for drug design. The
2.2. Computer-Aided
virtual VirtualofScreening
screening process this study is shown in Figure 1.
Virtual screening is a common, efficient and economical method for drug design. The
virtual screening process of this study is shown in Figure 1.
Figure 1. Virtual
Figure 1. Virtual filtering
filtering process
process in
in this
this study.
study.
2.2.1. CBD Substitutes Obtained and Primary Docking Screening
2.2.1. CBD Substitutes Obtained and Primary Docking Screening
The cannabinoid receptor CB1, a member of the endocannabinoid system, is the target
protein of CBD [34]. The CB1 receptor is involved in various physiological functions
of the skin, such as inflammation, regulation of immune responses, proliferation and
apoptosis [35]. The crystal structure of CB1 (PDB entry: 5TGZ) was obtained from the RCSB
(PDB; https://www.rcsb.org/) (accessed on 17 October 2020) [36]. Accelrys Discovery
Studio 2.5 software (DS 2.5) (Accelrys Software Inc., San Diego, CA, United States) was
used to prepare the protein receptor. The location of antagonist AM6538 in the CB1 crystal
structure was set as the active binding site. The structure of CBD was prepared using
ligands tools. Then, the Glide XP (extra Precision) docking was implemented to explore the
interaction between CBD and the target protein. Based on the docking results, the key CBD
structural skeleton was determined. Compounds with 70% similarity to the key skeleton
structure were searched in the Specs database and the InterBioScreen database CDOCKER
protocol was used for the binding ability of the compounds to CB1. Compounds with better
scores than CBD were retained for further analysis.
2.2.2. Drug Likeness Evaluation

Lipinski’s rule of five and Veber’s rules were used to screen the drug likeness evalua-
tion of the compounds. If a small molecule drug has less than ten hydrogen bond acceptors,
five hydrogen bond donors, the calculated LogP (CLogP) <5 and a molecular weight (MWT)
<500, it may show better pharmacokinetic properties. Veber’s rules refer to a compound
with a rotatable bond ≤10 and a polar surface area of ≤140 Å2 . If the compound meets the
Veber’s rules, it has a higher drug likeness [26].
2.2.3. High Precision Screening

We used Schrödinger’s Glide XP (extra precision) to dock these compounds with
CB1. These compounds are given an OPLS_2005 force field. We set the pH to 7.0 + 2 to
convert the compounds to the charged state. Schrödinger’s LigPrep module is used to
generate different tautomers. Compounds with a docking score less than −7 were selected
for further analysis [37].
2.2.4. Similarity Searching

In order to obtain compounds with diverse structures, these compounds were clustered
using hierarchical clustering in canvas module (Canvas, Schrödinger, LLC, New York, NY,
USA). The cluster similarity threshold was set to 0.98. Selecting the candidate compounds
required a combination of the following factors, receptor-ligand binding mode, docking
score, molecular skeleton, N-heterocyclic compound, hydrocarbon group, amino and amide,
halogen, etc. [38].
2.3. Antioxidant Capacity Evaluation

Following the virtual screening, the DPPH free radical scavenging assay and ABTS
free radical scavenging assay were used to test their antioxidant effects.
2.3.1. DPPH Radical Scavenging Activity

CBD and its derivative solution (0.015625–1.0 mg/mL) were prepared with dis-
tilled water. First, 40 µL each sample and 160 µL of 0.1 mg/mL ethanol solution of
DPPH were mixed and reacted for 30 min in the dark [39]. The absorbance was mea-
sured at 520 nm [39]. The DPPH radical scavenging rate was calculated using the fol-
lowing formula: Y% = (A0 − A1 )/A0 × 100, where A0 is the absorbance of the blank
group (distilled water + DPPH), and A1 is the absorbance of the sample reaction (sam-
ple + DPPH).
2.3.2. ABTS Radical Scavenging Activity

ABTS working solution was generated by mixing equal volumes of 2.45 mM potas-
sium persulfate and 7 mM ABTS. The reaction was carried out in the dark for 12–16 h.
Then, the radical solution obtained was diluted with PBS of pH 7.4 to an absorbance of
0.70 ± 0.05, measured at 405 nm. CBD and its derivatives solution (0.015625–1.0 mg/mL)
were prepared with distilled water. First, 10 µL each sample and 200 µL ABTS working
solution were mixed and reacted for 30 min in the dark. Then, the absorbance was mea-
sured at 405 nm [39]. The ABTS radical scavenging rate is calculated using the following
formula: Y% = (A0 − A1 )/A0 × 100, where A0 is the absorbance of the blank group (dis-
tilled water + ABTS working solution), and A1 is the absorbance of the sample reaction
(sample +ABTS working solution).
2.4. Cytotoxicity Measurement of CBD and Its Derivatives

The RAW264.7 macrophage line, HFF-1 fibroblast and B16F10 melanoma cell were
cultured with high glucose medium containing 13%, 15% and 10% fetal bovine serum (FBS),
respectively, and 1% penicillin–streptomycin (P/S) was added to the medium at 37 ◦ C in a

5% CO2 -humidified air environment [40].
RAW264.7 cells were inoculated into 96-well plates at a density of 5 × 103 cells per
well. Then, 24 h later, the medium containing different concentrations of samples was
added and cultured in a 37 ◦ C cell culture box containing 5% carbon dioxide for another
24 h. Then, 15 µL MTT (5 mg/mL) was added to each well, and 150 µL triple solution was
added after 4 h. We dissolved methylzan crystals overnight (the triad solution was made
of 0.01 M hydrochloric acid solution, 5% isopropyl alcohol and 10% SDS). The absorbance
at 570 nm was measured using an enzyme plate the next day [41]. The cell viability test
procedure of B16F10 cells and HFF-1 cells were the same as RAW264.7 cell, except that
they were cultured for three days after adding medium containing different concentrations
of samples.
2.5. Anti-Inflammatory Activity Test

The Griess method was used to detect the changes in NO released by the cells in
media containing different concentrations of compounds. The ELISA method was used to
exam their effects on the release of IL-6, and the western blot was applied to analyze the
expression level of COX-2 and iNOS.
2.5.1. Measurement of NO and IL-6 Levels

RAW264.7 cells were inoculated into 96-well plates at a density of 2 × 104 cells per
well. Twenty-four hours later, medium containing different concentrations of compounds
was added and cultured in a 37 ◦ C cell culture box containing 5% carbon dioxide. Then,
2 h later, except for the control group, 1.5 µL LPS (final concentration: 1 µg/mL) was
added and cultured for 24 h. Then, the supernatant was collected and treated with Griess
reagent to detect NO levels. The absorbance of the supernatant at 520 nm was detected by a
microplate reader. The expression of IL-6 was detected by ELISA method. The experiment
was carried out according to the kit instructions.
2.5.2. Western Blot Analysis

The cells were inoculated into 6-well plates at a density of 2 × 106 cells per well
and placed in an incubator for 24 h. Medium containing different concentrations of com-
pounds was added. Two hours later, except for the control group, LPS (final concentration:
1 µg/mL) was added and cultured for 24 h. Cells were collected by centrifugal collection
and the cell lysate was added. The mixture was placed on ice and shaken for 0.5 h, fol-
lowed by centrifugation. A Bradford protein assay was used to determine the total protein
concentrations; 10 µg protein from each sample was subjected to SDS-PAGE, transferred to
BioBond nitrocellulose membranes, and then probed with the antibodies against iNOS and
COX-2 (both in the ratio 1:2000). A horseradish peroxidase-conjugated goat anti-rabbit IgG
antibody (1:5000) was used as the secondary antibody. Finally, enhanced chemilumines-
cence was used to visualize the antibody binding. The obtained images were analyzed by
ImageJ software.
2.6. Antiwrinkle Activity Test

The MTT method was used to detect the repair effect of the compounds on HFF-1 cells
damaged by UV radiation, and the ELISA method was used to detect the changes in the
content of collagen, elastin and fibronectin in cells after adding CBD and its derivatives. The 2,
7-dichlorofuorescin diacetate (DCFH–DA, 10 µM) color method was used to detect the contents
of fibroblasts’ intracellular reactive oxygen species. Annexin V-FITC/PI double staining cell
apoptosis assay was used to detect the effect of compounds on the apoptosis of HFF-1.
2.6.1. Repair Ultraviolet Radiation Damage Experiment

The cells were inoculated into 96-well plates at a density of 5 × 103 cells per well and
placed in an incubator for 24 h. Medium containing different concentrations of compounds
was added. Fours hours later, except for the control group, UV intensity of 70 mW/cm was
irradiated for 5 min. Following another 20 h, 15 µL MTT (5 mg/mL) was added to each
well, and 150 µL triple solution was added after 4 h. Once the methylzan crystals were
dissolved overnight (the triad solution was made of 0.01 M hydrochloric acid solution, 5%
isopropyl alcohol and 10% SDS), the absorbance at 570 nm was measured using an enzyme
plate, the next day.
2.6.2. Detection of Collagen, Elastin and Fibronectin Levels

was added. Four hours later, except for the control group, UV intensity of 70 mW/cm
was irradiated for 5 min. Following 20 h of culture, HFF-1 cells were digested and cell
precipitation was obtained by centrifugation. Collagen, elastin and fibronectin in cells were
released by RIPA lysis solution, and the expression of collagen, elastin and fibronectin
was detected by the ELISA method. The experiment was carried out, according to the
kit instructions.
2.6.3. Measurement of the ROS Content

was added. Four hours later, except for the control group, UV intensity of 70 mW/cm
was irradiated for 5 min. Following 20 h of culture, the cells were incubated with probes
2, 7-dichlorofuorescin diacetate (DCFH-DA, 10 µM) at 37 ◦ C in 5% CO2 -humidified air
environment for 45 min. The excess probes were removed by PBS. Finally, the outcome was
detected by flow cytometry.
2.6.4. Detection the Effects on the Apoptosis of HFF-1 Cells

was added. Four hours later, except for the control group, UV intensity of 70 mW/cm was
irradiated for 5 min. Following 20 h of culture, HFF-1 cells were digested, cell precipitation
was obtained by centrifugation. Then, after adding 10 µL PI staining solution, 5 µL Annexin
V-FITC and 100 µL of working solution were added and left for 30 min at room temperature
in the dark. The fluorescence of FITC and PI were measured using a flow cytometer and
quantified using CytExpert software.
2.7. Determination of Melanin Content and Tyrosinase Activity

was added. Four hours later, except for the control group, α-MSH was added and culture
was continued for 48 h, then the medium was removed and washed three times with PBS.
B16F10 melanin content was detected by the cleavage method. Following the addition
of 800 µL 1 M NaOH solution containing 10% DMSO at 80 ◦ C for 1 h, The absorbance at
405 nm was measured.
Tyrosinase activity was detected by the principle that tyrosine and its substrate will
appear in color when combined; 720 µL 1% TritonX-100 was placed in the −80 ◦ C refrigera-
tor for half an hour to release the tyrosinase in B16F10 cells; 80 µL 1 mg/mL LOPA was
added and incubated at 37 ◦ C for 1 h. Then it was centrifuged, the supernatant was taken,
and its absorbance was measured at 492 nm.
2.8. Statistical Analysis

All experiments were repeated three times. All of the data were analyzed by e Graph-
Pad Prism 5.0 statistical software and expressed as means ± standard deviation (SD). Data
ator for half an hour to release the tyrosinase in B16F10 cells; 80 uL 1 mg/ mL LOPA was
and its absorbance was measured at 492 nm.
2.8.
addedStatistical Analysisat 37 °C for 1 h. Then it was centrifuged, the supernatant was taken,
and incubated
and its absorbance
All experiments was measured at 492 nm.
2.8. Statistical Analysis were repeated three times. All of the data were analyzed by e
GraphPad Prism 5.0 statistical software and expressed as means ± standard deviation
2.8. All experiments were repeated three times. All of the data were analyzed by e
(SD).Statistical Analysis
Data analysis used unpaired t test to test whether there was a significance between
Antioxidants 2023, 12, 314 GraphPad Prism 5.0 statistical software and expressed as means ± standard deviation7 of 22
the twoAll groups of data.were
experiments p < 0.05 was considered
repeated three times. statistically significant.
All of the data were analyzed by e
(SD). Data analysis used unpaired t test to test whether there was a significance between
GraphPad Prism 5.0 statistical software and expressed as means ± standard deviation
the two groups of data. p < 0.05 was considered statistically significant.
3. Results
(SD). Data analysis used unpaired t test to test whether there was a significance between
the
3.1. analysis
two
Virtualgroups used unpaired
of data.
Screening t test
p < 0.05 wastoconsidered
test whether there wassignificant.
statistically a significance between the two
3. Results
groups of data. p < 0.05 was considered statistically significant.
3.1. The CB1
Virtual crystal structure (PDB No.: 5TGZ) was downloaded from the PDB database.
Screening
3. Results
The active
3.The
Resultsbinding site was determined, according to the position of the coordinates of the
3.1. Virtual CB1 crystal structure (PDB No.: 5TGZ) was downloaded from the PDB database.
Screening
known ligands in the target protein [42]. Docking CB1 with CBD yielded an excellent
The 3.1.
active Virtual Screening
binding site was determined, according to the position of the coordinates of the
score,The and CB1thecrystal
resultsstructure
are shown (PDBin Tables 1 andwas
No.: 5TGZ) 2. From Table 2, from
downloaded it canthe
be PDB
seen database.
that can‐
known ligands The CB1 incrystal structure
the target protein(PDB No.:
[42]. 5TGZ) CB1
Docking was downloaded
with CBD yielded from theanPDB database.
excellent
nabidiol formed a hydrogen bonding with hydroxyl group of
The active binding site was determined, according to the position of the coordinates residue SER383, and
of the
score,The andactive bindingare
the results siteshown
was determined,
in Tables 1 according
and 2. From to the position
Table of the
2, it can coordinates
be seen that can‐of the
benzene
known ring formed
ligands in thePi–Pi
targetstacking
protein with residue PHE170. Therefore, the skeleton, shown
known
nabidiol ligands
formed in the
a hydrogentarget protein[42].
bonding [42].Docking
with Docking
hydroxylCB1
CB1 with CBD yielded
withofCBD
group
yielded
residue SER383,
anexcellent
an excellent
and thescore,
in Figure
score, and 1,the
wasresults
submitted
are to InterBioScreen
shown 1 and Specs databases toitsearchbefor compounds
and
benzene the
ringresults
formed are shown
Pi–Pi in in
stacking
Tables
Tables and
with1residue
and 2. From
2. From Table
Table
PHE170. 2, it2,can
Therefore,
can
be
theseen
seen
thatthat
skeleton,
can‐
cannabidiol
shown
withformed
70%formed
nabidiol similaritya with
hydrogen
a hydrogen
CBD.
bonding
As
bondinga result,
with
with hydroxyl
422 compounds
hydroxyl group remained.
of residue SER383, and the ring
in Figure 1, was submitted to InterBioScreen andgroup
Specsof residue SER383,
databases to searchand
for the benzene
compounds
benzene ring formed Pi–Pi stacking with residue PHE170. Therefore, the skeleton, shown
withformed
Table 70%
1. The
Pi–Pi stacking
similarity
molecular with withAs
CBD.
structures
residue
andadocking
PHE170.
result, Therefore,
422 compounds
scores of CBD
the skeleton, shown in Figure 1,
andremained.
the selected compounds, by vir‐
in Figure 1, was submitted to InterBioScreen and Specs
was submitted to InterBioScreen and Specs databases to search databases to search for compounds
for compounds with 70%
tual
with screening.
70% similarity with CBD. As adocking
result, 422 compounds
Table similarity
1. The with CBD.
molecular As a result,
structures and 422 compounds
scores of CBD andremained.
remained.
the selected compounds, by vir‐
No.
tual screening. Database Structure Docking Score
Table 1. The molecular structures and docking scores of CBD and the selected compounds, by vir‐
Table 1. The molecular structures and docking scores of CBD and the selected compounds, by
No.
tual screening. Database Structure Docking Score
virtual screening.
CBD
No. InterBioScreen
Database Structure −10.274
Docking Score
No. Database Structure Docking Score
CBD InterBioScreen −10.274
CBD
CBD InterBioScreen
InterBioScreen −10.274
−10.274
S‐88614 InterBioScreen −8.494
Antioxidants 2023, 12, x FOR PEER REVIEWS‐88614

S-88614 InterBioScreen
−8.494
8 of 24
S‐88614
Antioxidants 2023, 12, x FOR PEER REVIEW InterBioScreen −8.4948 of 24
S‐88745
−9.267

S‐92151
−7.285
Antioxidants 2023, 12, x FOR PEER REVIEW 9 of 24

S‐92153
Antioxidants 2023, 12, x FOR PEER REVIEW
−8.219
9 of 24
Table 2. The stereo view and floor plan view of the docking results of CBD and the selected com‐
Table 2. The stereo view and floor plan view of the docking results of CBD and the selected com‐
pounds.
pounds.
Table 2. The stereo view and floor plan view of the docking results of CBD and the selected compounds.
Stereo View Floor Plan View of
No. Stereo View Floor
Floor Plan
Plan View
View of
of the
No.
No. StereoofView
the Docking Results
of the Docking Results the Docking Results
of the Docking Results theDocking
Docking Results
Results
CBD
CBD
CBD
S‐88614
Stereo View Floor Plan View of
No.
of the Docking Results the Docking Results
Antioxidants 2023, 12, 314 CBDCBD

CBD 8 of 22
CBD
CBD
CBD
Table 2. Cont.
Floor Plan View of the

No. Stereo View of the Docking Results
Docking Results
S‐88614
S‐88614
S‐88614
S‐88614
S‐88614
S-88614
S‐88614
S‐88745
S‐88745
S‐88745
S-88745
S‐88745
S‐88745
S‐88745
S‐92151
S‐92151
S-92151
S‐92151
S‐92151
S‐92151
S‐92151
S‐92153
S-92153
S‐92153
As shown in Figure 1, the CDOCK module was used to dock the remaining com‐
AsAsshown
shownin inFigure theCDOCK
Figure 1, the CDOCKmodule
module waswas used
used to dock
to dock the remaining
the remaining com‐
compounds
pounds with CB1, and 357 compounds with a better score than CBD were left. Then,
with CB1,
pounds withandCB1,357and
compounds with a better
357 compounds with ascore than
better CBD
score thanwereCBDleft.were
Then, Lipinski’s
left. Then,
Lipinski’s rule of five and Veber’s rules were further applied to remove the compounds
rule of five
Lipinski’s ruleand Veber’s
of five andrules were
Veber’s further
rules wereapplied
further to remove
applied to the compounds
remove that were
the compounds
that were weak drug likenesses., which resulted in 228 compounds remaining for the fol‐
weak
that drug
were weaklikenesses., which resulted
drug likenesses., in 228 in
which resulted compounds
228 compoundsremaining for the
remaining forfollowing
the fol‐
lowing process. Then, the Glide XP module with higher docking precision was imple‐
process. Then, the Glide XP module with higher docking precision was
lowing process. Then, the Glide XP module with higher docking precision was imple‐ implemented, and
mented, and 153 compounds with a docking score less than −7 were obtained. Via the
153 compounds with a docking score less than − 7 were obtained.
mented, and 153 compounds with a docking score less than −7 were obtained. Via the Via the hierarchical
hierarchical clustering, these compounds were clustered into 10 groups. Within these clus‐
clustering,clustering,
hierarchical these compounds were clustered
these compounds into 10 groups.
were clustered Within Within
into 10 groups. these clusters, four
these clus‐
ters, four compounds
compounds (S‐88614,
(S-88614, S-88745,S‐88745,
S-92151,S‐92151,
S-92153)S‐92153)
owningowning
similar similar binding modes
ters, four compounds (S‐88614, S‐88745, S‐92151, S‐92153) owningbinding
similar modes
bindingwith CBD,
modes
with CBD, as shown in Table 1, were chosen for the subsequent experiments, because the
nitrogen and oxygen atoms of these compounds can form one or two hydrogen bonds
nitrogen and oxygen atoms of these compounds can form one or two hydrogen bonds
with the key residues GLN116 or HIS181, and benzene ring to form Pi–Pi interactions with
with the key residues GLN116 or HIS181, and benzene ring to form Pi–Pi interactions with
the key residues PHE102, PHE170 or HIS178 (Table2. Their rotatable bonds are less than
the key residues PHE102, PHE170 or HIS178 (Table2. Their rotatable bonds are less than
or equal to 10, and their polar surface area is equal to or <140 Å2,2 indicating that these
or equal to 10, and their polar surface area is equal to or <140 Å , indicating that these
pounds with CB1, and 357 compounds with a better score than CBD were left. Then,
Lipinski’s rule of five and Veber’s rules were further applied to remove the compounds
that were weak drug likenesses., which resulted in 228 compounds remaining for the fol‐
lowing process. Then, the Glide XP module with higher docking precision was imple‐
mented, and 153 compounds with a docking score less than −7 were obtained. Via the
Antioxidants 2023, 12, 314 hierarchical clustering, these compounds were clustered into 10 groups. Within these clus‐ 9 of 22
ters, four compounds (S‐88614, S‐88745, S‐92151, S‐92153) owning similar binding modes
nitrogen
as shownand oxygen
in Table atomschosen
1, were of theseforcompounds
the subsequent can form one or two
experiments, hydrogen
because bonds
the nitrogen
with
and the key residues
oxygen atoms ofGLN116 or HIS181, and
these compounds can benzene
form oneringor to
twoform Pi–Pi interactions
hydrogen bonds with with
the
the
keykey residues
residues PHE102,
GLN116 PHE170and
or HIS181, or HIS178
benzene(Table2. TheirPi–Pi
ring to form rotatable bonds are
interactions withless
thethan
key
residues
or equal toPHE102,
10, andPHE170 or HIS178
their polar (Table
surface area2).is Their
equal rotatable
to or <140bonds are less than
Å2, indicating thator these
equal
to 10, and their
compounds havepolar
a goodsurface area is equal
bioavailability [43]. or <140 Å2 , indicating
toFurthermore, cannabidiol thatshows
these antioxidant
compounds
have a good
properties bioavailability
because hydroxyl [43]. Furthermore,
is easy to be oxidized cannabidiol shows
[44], we can antioxidant
deduce properties
that the four com‐
becausemay
pounds hydroxyl is easy
also have to be oxidized
antioxidant [44], we can
performance duededuce that the four
to the functional compounds
group may
of phenolic
also have The
hydroxyl. antioxidant performance
hit compounds due in
are listed to Table
the functional
1. group of phenolic hydroxyl. The
hit compounds are listed in Table 1.
3.2. Extracellular Antioxidant Test
3.2. Extracellular Antioxidant Test
The ABTS and DPPH tests were used, as stated in Section 2.3, to assess the molecular
The ABTS
antioxidant and DPPH
abilities. tests were
The results used, as
indicated stated
that in SectionS‐88745
compounds 2.3, to assess the molecular
and S‐92153 had a
antioxidant abilities. The results indicated that compounds S-88745
stronger antioxidant capacity than cannabidiol, as shown in Figure 2 and Figure 3, while and S-92153 had a
strongerand
S‐88614 antioxidant
S‐92151 were capacity
weakerthan cannabidiol,
(data not shown).asInshown
the DPPHin Figures 2 andexperiment,
scavenging 3, while S-
88614 and S-92151 were weaker (data not shown). In the DPPH scavenging experiment,
the DPPH scavenging rate of S‐88745 was significantly higher than that of CBD when the
the DPPH scavenging rate of S-88745 was significantly higher than that of CBD when
concentration was higher than 0.5 mg/mL (Figure 2A), and the ability of S‐92153 to scav‐
the concentration was higher than 0.5 mg/mL (Figure 2A), and the ability of S-92153 to
enge DPPH free radicals was similar to CBD (Figure 2B). In the ABTS scavenging experi‐
scavenge DPPH free radicals was similar to CBD (Figure 2B). In the ABTS scavenging
ment, the antioxidant capacities of S‐88745 and S‐92153 were significantly stronger than
experiment, the antioxidant capacities of S-88745 and S-92153 were significantly stronger
that of CBD. The maximum scavenging rate of S‐88745 and S‐92153 can reach 80% and
than that of CBD. The maximum scavenging rate of S-88745 and S-92153 can reach 80% and
85%, respectively (Figure 3A,B). Therefore, it is worthy to further test the anti‐inflamma‐
85%, respectively (Figure 3A,B). Therefore, it is worthy to further test the anti-inflammatory,
tory, anti‐wrinkle and whitening effects of S‐88745 and S‐92153.
anti-wrinkle and whitening effects of S-88745 and S-92153.

Figure 2. The graph of DPPH scavenging ability. (A) The DPPH scavenging abilities of CBD and its
Figure 2. The graph of DPPH scavenging ability. (A) The DPPH scavenging abilities of CBD and its
skeletonderivative,
skeleton derivative,S‐88745.
S-88745.(B)
(B)The
TheDPPH
DPPHscavenging
scavengingability
abilityofofCBD
CBDand
andits
itsskeleton
skeletonderivative,
derivative,
S-92153. The
The control group was not treated with CBD, S-88745 and andS-92153, and only contained DPPH
DPPH solution. The experiments were repeated three times. Each bar illustrates the averagecontained
S‐92153. control group was not treated with CBD, S‐88745 S‐92153, and only ± stand‐
solution. The experiments were repeated three
ard deviation (SD) counted from three experiments. times. Each bar illustrates the average ± standard
deviation (SD) counted from three experiments.
Figure 3. The graph of ABTS scavenging ability. (A) The ABTS scavenging abilities of CBD and its
Figure 3. The
skeleton graph ofS-88745.
derivative, ABTS scavenging ability.
(B) The ABTS (A) The ABTS
scavenging abilityscavenging
of CBD andabilities of CBD
its skeleton and its
derivative,
skeleton derivative, S‐88745. (B) The ABTS scavenging ability of CBD and its skeleton derivative,
S-92153. The control group was not treated with CBD, S-88745 and S-92153, and only contained ABTS S‐
92153. The control group was not treated with CBD, S‐88745 and S‐92153, and only contained ABTS
solution. The experiments were repeated three times. Each bar illustrates the average ± standard
3.3. Anti-Inflammatory Activity Test
3.3. Anti‐Inflammatory
3.3.1. Activity Test
Cytotoxicities Measurement
3.3.1. Cytotoxicities Measurement
The cytotoxicity of CBD, S-88745 and S-92153 (0.000256–100 µM) on RAW264.7 cells
wasThe cytotoxicity
measured by MTT of assay.
CBD, S‐88745 andare
The results S‐92153
shown(0.000256–100 uM)where
in Figure 4, from on RAW264.7 cells
we can see that
was measured by MTT assay. The results are shown in Figure 4, from where we can see
that CBD showed a significant cytotoxicity effect on RAW264.7 cells in the range of 20–
100 uM (Figure 4A), while S‐88745 and S‐92153 did not show any cytotoxicity effect on
RAW264.7 cells (Figure 4B,C). In order to study the anti‐inflammatory effect of S‐88745
and S‐92153, the levels of NO, IL‐6, iNOS and COX expression were determined in
3.3. Anti‐Inflammatory Activity Test

3.3.1. Cytotoxicities Measurement
The cytotoxicity of CBD, S‐88745 and S‐92153 (0.000256–100 uM) on RAW264.7 cells
was measured by MTT assay. The results are shown in Figure 4, from where we can see
that CBD showed a significant cytotoxicity effect on RAW264.7 cells in the range of 20–
CBD
100 uMshowed a significant
(Figure 4A), whilecytotoxicity
S‐88745 and effect on RAW264.7
S‐92153 cells in
did not show anythecytotoxicity
range of 20–100
effectµM
on
(Figure 4A), while S-88745 and S-92153 did not show any cytotoxicity effect on
RAW264.7 cells (Figure 4B,C). In order to study the anti‐inflammatory effect of S‐88745 RAW264.7
cells (Figure 4B,C).
and S‐92153, In order
the levels of to
NO,study
IL‐6,theiNOS
anti-inflammatory effect of were
and COX expression S-88745 and S-92153,
determined in
the levels of NO, IL-6, iNOS and COX expression were determined in RAW264.7 cells at
RAW264.7 cells at non‐cytotoxic concentrations (determined as 0.04, 0.4 and 4 uM).
non-cytotoxic concentrations (determined as 0.04, 0.4 and 4 µM).
Figure
Figure 4.4. Cytotoxicity of (A) CBD, (B) S‐88745
S-88745 and (C) S‐92153,
S-92153, on RAW264.7 cells. RAW264.7 cells
were treated with cannabidiol, S-88745 or S-92153
S‐88745 S‐92153 for 24 h, and MTT assay was carried out to detect
the cell viability.
viability. The control group was not treated with CBD, S‐88745
S-88745 and S‐92153.
S-92153. The experiments
experiments
were repeated three times. Each bar illustrates the average ± standard deviation (SD)
were repeated three times. Each bar illustrates the average ± standard deviation (SD) counted
counted from
from
three experiments.
three experiments.
3.3.2. Suppression of NO
NO, an indicator of the inflammatory degree, is synthesized by inducible nitric oxide
synthase (iNOS) from L‐arginine.
L-arginine. NO serves anan important
important pathological
pathological role.
role. Lipopolysac‐
Lipopolysac-
charide (LPS) stimulates inflammatory stress, inducing a large number number of NO [37].
of NO [37]. As
shown in Figure 5, RAW264.7 cells treated by LPS showed a sharp increase
shown in Figure 5, RAW264.7 cells treated by LPS showed a sharp increase in NO in NO produc-
pro‐
tion, but but
duction, CBD, S-88745
CBD, and and
S‐88745 S-92153 significantly
S‐92153 reduced
significantly the NO
reduced thesecretion to 4 µM
NO secretion to 4(36%,
uM
24.7%, 14.5%).14.5%).
(36%, 24.7%,
3.3.3. Inhibitory Effect on IL-6 Production
As can be seen from Figure 6, IL-6 significantly increased after LPS was added to
stimulate inflammation. CBD and the two compounds can counteract the effect in a
concentration dependent manner, as reported [45,46]. CBD (4 µM) reduced the release of IL-
6 induced by LPS (Figure 6A) by 33.4%, S-88745 (4 µM) decreases by 47% (Figure 6B) and S-
92153 (4 µM) decreases by 65% (Figure 6C), compared with the LPS group. Comparing with
CBD, the hit compounds S-88745 and S-92153 can decline IL-6 more efficiently, indicating
their better anti-inflammatory effects.
Figure 5. Inhibition of (A) CBD, (B) S-88745 and (C) S-92153 on LPS-induced nitric oxide (NO) in
Figure 5. Inhibition of (A) CBD, (B) S‐88745 and (C) S‐92153 on LPS‐induced nitric oxide (NO) in
RAW 264.7
RAW 264.7macrophages.
macrophages.The Thecontrol
controlgroup
group was
was not
not stimulated
stimulated byby LPS.
LPS. TheThe LPS-treated
LPS‐treated group
group (‐)
was stimulated by LPS to develop inflammation. The experiments were repeated three times. times.
(-) was stimulated by LPS to develop inflammation. The experiments were repeated three Each
Each
bar bar illustrates
illustrates the average
the average ± standard
± standard deviation
deviation (SD) counted
(SD) counted from from
three three experiments.
experiments. p < was
p < 0.05 0.05
considered
was considered statistically significant.
statistically p < 0.05
significant. p <was
0.05considered statistically
was considered significant.
statistically * p < 0.05
significant. * p <com‐
0.05
pared
comparedto thetoLPS−treated group;
the LPS−treated ** p <**
group; 0.01
p <compared to thetoLPS−treated
0.01 compared group;group;
the LPS−treated **** p <****
0.0001 com‐
p < 0.0001
pared
comparedto thetoLPS−treated group;
the LPS−treated #### p####
group; < 0.0001 compared
p < 0.0001 to theto
compared control group.
the control group.
3.3.3. Inhibitory Effect on IL‐6 Production

As can be seen from Figure 6, IL‐6 significantly increased after LPS was added to
stimulate inflammation. CBD and the two compounds can counteract the effect in a con‐
centration dependent manner, as reported [45,46]. CBD (4 uM) reduced the release of IL‐
6 induced by LPS (Figure 6A) by 33.4%, S‐88745 (4 uM) decreases by 47% (Figure 6B) and
S‐92153 (4 uM) decreases by 65% (Figure 6C), compared with the LPS group. Comparing
stimulate inflammation. CBD and the two compounds can counteract the effect in a con‐
centration dependent manner, as reported [45,46]. CBD (4 uM) reduced the release of IL‐
6 induced by LPS (Figure 6A) by 33.4%, S‐88745 (4 uM) decreases by 47% (Figure 6B) and
S‐92153 (4 uM) decreases by 65% (Figure 6C), compared with the LPS group. Comparing
Antioxidants 2023, 12, 314 with CBD, the hit compounds S‐88745 and S‐92153 can decline IL‐6 more efficiently, 11
indi‐
of 22
cating their better anti‐inflammatory effects.
Effects
Figure6.6.Effects
Figure of (A)
of (A) CBD,
CBD, (B) (B) S-88745
S‐88745 and and (C) S-92153
(C) S‐92153 on IL‐6 on induced
IL-6 induced
by LPS.byInLPS. In addition
addition to an
to an untreated
untreated control control
group and group
the and the LPS−
LPS−treated treatedRAW264.7
group, group, RAW264.7 cells pretreated
cells pretreated with CBD,with CBD,
S‐88745
S-88745
and andatS-92153
S‐92153 0.04, 0.4,atand
0.04, 0.4, for
4 μM and1.5
4 µM forthen
h, and 1.5 h, and then
treated treatedwith
all groups all groups with the
LPS, except LPS, except
control
group for 20 group
the control h. Thefor
levels
20 h.ofThe
IL‐6levels
in theofculture
IL-6 insupernatants of RAW264.7ofmacrophages
the culture supernatants were then
RAW264.7 macrophages
detected
were then by ELISA.
detectedTheby experiments
ELISA. The were repeatedwere
experiments threerepeated
times. Eachthreebar illustrates
times. Each the
baraverage ±
illustrates
standard deviation (SD) counted from three experiments. p < 0.05 was considered
the average ± standard deviation (SD) counted from three experiments. p < 0.05 was considered significant. p <
0.05 was considered statistically significant. ** p < 0.01 compared to the LPS−treated group; *** p <
significant. p < 0.05 was considered statistically significant. ** p < 0.01 compared to the LPS−treated
0.001 compared to the LPS−treated group; **** p < 0.0001 compared to the LPS−treated group; ####
group; *** p < 0.001 compared to the LPS−treated group; **** p < 0.0001 compared to the LPS−treated
p < 0.0001 compared to the control group.
group; #### p < 0.0001 compared to the control group.
3.3.4.
3.3.4.Effects
Effectson
onProtein
ProteinExpression
ExpressionofofiNOSiNOSand
andCOX‐2
COX-2
InInorder
ordertotoevaluate
evaluatethe
theeffects of of
effects CBDCBDand its its
and derivatives on proinflammatory
derivatives on proinflammatory medi‐
me-
ators, the the
diators, protein expressions
protein expressionsof iNOS andand
of iNOS COX‐2COX-2were determined.
were determined.CBD, S‐88745
CBD, andand
S-88745 S‐
92153 can decrease the expression of iNOS protein, especially S‐92153, which has
S-92153 can decrease the expression of iNOS protein, especially S-92153, which has the mostthe most
significant
significanteffect,
effect,decreased
decreasedthetheexpression
expressionofofiNOS
iNOSby by11%,
11%,compared
comparedtotothetheLPS‐treated
LPS-treated
model group. Because NO is synthesized by iNOS from L‐arginine,
model group. Because NO is synthesized by iNOS from L-arginine, we can we can deduce thatthat
deduce S‐
88745
S-88745andand
S‐92153
S-92153maybe
maybereduce
reducethethe
NO content
NO content bybyinhibiting
inhibitingthe
theexpression
expressionofofiNOS
iNOS
(Figures
(Figures55and
and7C).
7C).
The results are shown in Figure 4. It can be seen that CBD (4 µM) decreased the
expression of COX-2 by 22.9% in a concentration dependent manner. S-92153 (4 µM)
decreased the expression of COX-2 by 13.4% also in a concentration dependent manner.
Though S-88745 reduced the expression of COX-2 to some extent, there was no significant
difference, compared to the LPS-treated model group.
3.4. Anti-Wrinkle Activity Test

3.4.1. Cytotoxicity Measurement on HFF-1 Cell Line
Inhibiting the proliferation of fibroblasts will lead to the degradation of collagen,
which is one of the reasons for wrinkles. Therefore, the MTT method was used to detect the
effects of compounds on the proliferation of HFF-1. The results are shown in Figure 8, in
which CBD showed a significant cytotoxicity effect on HFF-1 cells in the range of 20–100 µM
(Figure 8A). On the contrary, S-88745 and S-92153 promoted fibroblast proliferation in the
range of 20–100 µM. These two compounds markedly promoted the proliferation of fibrob-
lasts at 20 µM. The survival rate of S-88745 in 20 µM cells was 188.3%, compared to the
control group (Figure 8B), and that of S-92153 in 20 µM cells was 121.7% (Figure 8C). There-
fore, S-88745 and S-92153 may increase the content of extracellular matrix by promoting
fibroblast proliferation (Figure 8B,C).
3.4.2. Fibroblast Damage Repairment Evaluation

Ultraviolet rays entering the atmosphere are mainly UVA (315–400 nm) and UVB
(280–315 nm) [11,13,23]. UVB is more genotoxic and causes more sunburn. The oxidative
stress in skin induced by UVB radiation generates reactive oxygen species, such as hydrogen
peroxide, superoxide anion and hydroxyl radical, which leads to biological reactions in
the skin, such as inflammation induction, melanin production, photoaging fibroblast
death, etc. [23]. We chose UVB to build the UV irradiation injury model. As shown in
Figure 9A, CBD can promote the proliferation of HFF-1 cells in the concentration of 4 µM,
but it accelerated the damage to the fibroblasts in the range of 20–100 µM. Therefore,
the range of CBD concentrations that can be used to treat the light injury is relatively
narrow. On the contrary, S-88745 and S-92153 can significantly repair the damage caused
by ultraviolet radiation even at high concentrations. The survival rate of HFF-1 cells
treated with S-88745 (4–100 µM) recovered to the level before UV damage (Figure 9B).
S-92153 also has an excellent ability to repair damage caused by UV radiation in 20–100 µM
(Figure 9C). Considering the anti-ultraviolet effect of them, 0.16–4 µM was selected for the
following experiments.
Figure 7. Effects of CBD, S-88745 and S-92153 on the expression of iNOS and COX-2 induced by LPS.
In addition to an untreated control group and the LPS−treated group, RAW264.7 cells pretreated with
CBD, S-88745 and S-92153 at 0.04, 0.4, and 4 µM for 1.5 h, and then treated all groups with LPS, except
the control group for 20 h. The levels of iNOS and COX-2 in the culture supernatants of RAW264.7
macrophages were then detected by western blot. (A) iNOS and COX-2 protein expression levels.
(B) Relative ration analysis of iNOS expression. (C) Relative ration analysis of COX-2 expression. The
experiments were repeated three times. Each bar illustrates the average ± standard deviation (SD)
counted from three experiments. p < 0.05 was considered statistically significant. * p < 0.05 compared
to the LPS−treated group; ** p < 0.01 compared to the LPS−treated group; *** p < 0.001 compared to
the LPS−treated group; **** p < 0.0001 compared to the LPS−treated group; # p < 0.0001 compared to
the control group.
to detect the cell viability. The experiments were repeated three times. Each bar illustrates the aver‐
age ± standard deviation (SD) counted from three experiments.

Antioxidants
Antioxidants 2023, x FOR PEER REVIEWUltraviolet
12, 314
2023, 12, rays entering the atmosphere are mainly UVA (315–400 nm) and 1413UVB
of
of 24
22
stress in skin induced by UVB radiation generates reactive oxygen species, such as hydro‐
gen peroxide, superoxide anion and hydroxyl radical, which leads to biological reactions
in the skin, such as inflammation induction, melanin production, photoaging fibroblast
Figure 9A, CBD can promote the proliferation of HFF‐1 cells in the concentration of 4 uM,
but it accelerated the damage to the fibroblasts in the range of 20–100 uM. Therefore, the
range of CBD concentrations that can be used to treat the light injury is relatively narrow.
On the contrary, S‐88745 and S‐92153 can significantly repair the damage caused by ultra‐
violet radiation even at high concentrations. The survival rate of HFF‐1 cells treated with
S‐88745
Figure 8.(4–100 uM) recovered
Cytotoxicity
Cytotoxicity of
of (A) CBDtoand
(A) CBD theits
and level
its before UV
derivatives,
derivatives, (B)damage
(B) S‐88745 (Figure
S-88745 and
and (C) 9B). S‐92153
(C) S‐92153,
S-92153, on alsocells.
on HFF‐1
HFF-1 has
cells.
an excellent
HFF‐1 ability to repair damage caused
HFF-1 cells were treated with cannabidiol, S‐88745 by UV radiation
S-88745 or S‐92153 in 20–100 uM (Figure
S-92153 for 72 h, and MTT assay was carried 9C).
Considering
to
to detect
detectthe theviability.
thecell
cell anti‐ultraviolet
viability. The effect of
experiments
The experiments them,
were 0.16–4
repeated
were uM
three
repeated was
times.
three selected
EachEach
times. for
barthe following
bar illustrates the aver‐
illustrates the
age ± standard
experiments. deviation (SD) counted from three experiments.
average ± standard deviation (SD) counted from three experiments.

Ultraviolet rays entering the atmosphere are mainly UVA (315–400 nm) and UVB
stress in skin induced by UVB radiation generates reactive oxygen species, such as hydro‐
gen peroxide, superoxide anion and hydroxyl radical, which leads to biological reactions
in the skin, such as inflammation induction, melanin production, photoaging fibroblast
Figure 9A, CBD can promote the proliferation of HFF‐1 cells in the concentration of 4 uM,
Figure
but 9. Cell viability
it accelerated of
theof (A) CBD and
damage its derivatives, (B) S-88745 and (C) S-92153, on HFF-1 cells.
Figure 9. Cell viability (A) CBDtoand theits
fibroblasts
derivatives,in(B)theS‐88745
range andof 20–100 uM. Therefore,
(C) S‐92153, on HFF‐1 cells.the
HFF-1
range cells
of were
CBD treated with
concentrations cannabidiol,
that can S-88745
be used ortoS-92153
treat for
the 4 h,
lightthen UV
injury
HFF‐1 cells were treated with cannabidiol, S‐88745 or S‐92153 for 4 h, then UV intensity of 70 intensity
is of
relatively 70 mW/cm
narrow.
wasthe
On
mW/cm irradiated forS‐88745
contrary, 5 min.
was irradiated for 5Following
and
min.S‐92153another 20 h, MTT
cananother
Following significantly assay
20 h, MTTrepair wasthecarried
assay wasdamage outcaused
carried to
outdetectbythe cell
ultra‐
to detect the
viability.
violet
cell Each
radiation
viability. bar illustrates
Eacheven the average ±
at high concentrations.
bar illustrates standard deviation
The survival
the average ± standard (SD)
deviation ratecounted from
of counted
(SD) HFF‐1 cells three experiments.
fromtreated with
three experi‐
ments.
S‐88745 The experiments
The experiments
(4–100 uM) were repeated
wererecovered
repeated three three
times.
to the times.
levelEach Each
bar
before bar illustrates
illustrates
UV damage the(Figure
average ± standard
the average
9B). ± standard
S‐92153 de‐
deviation
also has
viation (SD)
(SD)excellent
an counted
counted from
ability from
three three experiments.
experiments.
to repair damage p <causedp < 0.05
0.05 wasby was considered
considered
UV radiation significant.
significant. p < 0.05
in 20–100 uMp < 0.05
was was con‐
considered
(Figure 9C).
sidered statistically
statistically
Considering significant.*
significant.* p < 0.05p compared
the anti‐ultraviolet < 0.05 compared
effect the to
oftothem,UV the UV−treated
−treated
0.16–4 wasgroup;
uMgroup; ** p <**0.01
selected p for
< 0.01thecompared
compared to
to the
following
the UV−treated group; *** p < 0.001 compared to the UV−treated group; **** p
UV−treated group; *** p < 0.001 compared to the UV−treated group; **** p < 0.0001 compared to the < 0.0001 compared to
experiments.
the UV−treated group; #### p < 0.0001 compared to the control group.
UV−treated group; #### p < 0.0001 compared to the control group.
3.4.3.
3.4.3. Intracellular
Intracellular Antioxidant
Antioxidant Activity
Activity
ROS isisrelated
ROS relatedtotomany
many pathological
pathological states
states of the
of the skin,skin,
suchsuch as a serious
as a serious loss ofloss of
extra‐
extracellular
cellular matrixmatrix
leadingleading to wrinkles
to facial facial wrinkles [5,18,47].
[5,18,47]. Therefore,Therefore,
we carriedweout
carried out the
the intracel‐
intracellular
lular antioxidantantioxidant
experimentsexperiments
to evaluateto evaluate the screened
the screened compounds. compounds.
As shownAs in shown
Figure
in Figure
10A, 10A, UVcaused
UV radiation radiation caused the
the increase increase in intracellular
in intracellular ROS levels inROS levels
HFF‐1, in HFF-1,
compared to
compared
control to control
group. group. The
The clearance clearance
rate of CBDrate of CBD
reactive reactive
oxygen oxygen
species species
was 61.2% was
at 61.2%
0.8 uM, at
0.8 µM, which was consistent with the literature [36]. The intracellular antioxidant capacity
of S-88745 is better than that of CBD, and the clearance rate of active oxygen is 89.8% at
Figure
0.8 µM.9.Furthermore,
Cell viability of (A)
the CBDofand
level its derivatives,
active oxygen has(B) S‐88745 to
returned and
the(C) S‐92153,
level onUV
before HFF‐1 cells.
damage
HFF‐1
(Figurecells were
10A). treated with
Compound cannabidiol,
S-92153 has the S‐88745 or S‐92153 for
best scavenging 4 h, then
capacity UV intensity ROS,
of intracellular of 70
mW/cm
and thewas irradiatedrate
scavenging for 5of
min.
ROS Following
is 93.6%another
at 0.8 20
µM,h, MTT
which assay was carried
indicates out to detect
that S-92153 has the
an
cell viability. Each bar illustrates the average ± standard deviation (SD) counted from three experi‐
excellent antioxidant capacity (Figure 10B).
ments. The experiments were repeated three times. Each bar illustrates the average ± standard de‐
viation (SD) counted from three experiments. p < 0.05 was considered significant. p < 0.05 was con‐
sidered statistically significant.* p < 0.05 compared to the UV−treated group; ** p < 0.01 compared to
the UV−treated group; *** p < 0.001 compared to the UV−treated group; **** p < 0.0001 compared to
the UV−treated group; #### p < 0.0001 compared to the control group.
3.4.3. Intracellular Antioxidant Activity

ROS is related to many pathological states of the skin, such as a serious loss of extra‐
cellular matrix leading to facial wrinkles [5,18,47]. Therefore, we carried out the intracel‐
lular antioxidant experiments to evaluate the screened compounds. As shown in Figure
10A, UV radiation caused the increase in intracellular ROS levels in HFF‐1, compared to
control group. The clearance rate of CBD reactive oxygen species was 61.2% at 0.8 uM,
which was consistent with the literature [36]. The intracellular antioxidant capacity of S‐
88745 is better than that of CBD, and the clearance rate of active oxygen is 89.8% at 0.8
uM. Furthermore, the level of active oxygen has returned to the level before UV damage
(Figure 10A). Compound S‐92153 has the best scavenging capacity of intracellular ROS,
Antioxidants 2023, 12, 314 and the scavenging rate of ROS is 93.6% at 0.8 uM, which indicates that S‐92153 14 of 22
has an
excellent antioxidant capacity (Figure 10B).
Figure10.
Figure InhibitionofofCBD,
10.Inhibition CBD,S‐88745
S-88745and
andS‐92153
S-92153 on
on UV‐induced
UV-induced ROS ROSin inHFF-1.
HFF‐1.Each
Eachbar
barillustrates
illustrates
theaverage
deviation (SD)
(SD) counted
counted fromfrom three
three experiments.
experiments. (A) ROS
(A) ROS content
content of S-
of S‐88745
88745
and CBD and
(0.8CBD
uM).(0.8 ROS(B)
(B)µM). ROS of
content content of and
S‐92153 S-92153
CBDand (0.8CBD
uM).(0.8
TheµM). Thegroup
control controlwas
group
not was
stim‐
not stimulated
ulated by UV. The byUV‐treated
UV. The UV-treated
group (‐)group (-) produced
produced a lot of reactive
a lot of reactive oxygen after
oxygen species species
UVafter UV
stimula‐
stimulation. The experiments were repeated three times. Each bar illustrates the average ± standard
deviation (SD) counted from three experiments. p < 0.05 was considered statistically significant.
** p < 0.01 compared to the UV−treated group; ## p < 0.01 compared to the control group.
3.4.4. Fibroblast Apoptosis Experiment

Oxidative stress caused by ultraviolet rays produces a large amount of oxygen, which
will break the function of DNA and promote cell apoptosis. It leads to a decrease in
tion. The experiments were repeated three times. Each bar illustrates the average ± standard devia‐
tion (SD) counted from three experiments. p < 0.05 was considered statistically significant. ** p < 0.01
compared to the UV −treated group; ## p < 0.01 compared to the control group.
3.4.4. Fibroblast Apoptosis Experiment

Oxidative stress caused by ultraviolet rays produces a large amount of oxygen, which
will break the function of DNA and promote cell apoptosis. It leads to a decrease in the
number of fibroblasts in the dermis, and finally leads to a decrease in the content of extra‐
the number of fibroblasts in the dermis, and finally leads to a decrease in the content
cellular matrix, resulting in wrinkles. Annexin V‐FITC/PI staining was used to detect the
of extracellular matrix, resulting in wrinkles. Annexin V-FITC/PI staining was used to
effect of the compounds on the proliferation of fibroblasts, and flow cytometry was used
detect the effect of the compounds on the proliferation of fibroblasts, and flow cytometry
to analyze Annexin V‐FITC/PI staining. As shown in Figure 11, the apoptosis rate was
was used to analyze Annexin V-FITC/PI staining. As shown in Figure 11, the apoptosis
increased to 40.71% after UV irradiation. CBD can significantly alleviate cell apoptosis
rate was increased to 40.71% after UV irradiation. CBD can significantly alleviate cell
caused by UV, with the best effect at 0.16 uM. Compared with the UV group, the apoptosis
apoptosis caused by UV, with the best effect at 0.16 µM. Compared with the UV group, the
rate decreased
apoptosis bydecreased
rate 53% (Figure by 11A). S‐8874511A).
53% (Figure and S‐92153
S-88745can
andalso significantly
S-92153 can alsoinhibit cell
significantly
apoptosis, and
inhibit cell the effectand
apoptosis, of the
inhibiting
effect ofcell apoptosis
inhibiting cellbecomes
apoptosismore obvious
becomes morewith the in‐
obvious with
crease of concentration. Compared with the ultraviolet group, the apoptosis rate
the increase of concentration. Compared with the ultraviolet group, the apoptosis rate decreases
bydecreases
64% and 61.5%
by 64%atand
4 uM, respectively,
61.5% for S‐88745 for
at 4 µM, respectively, andS-88745
S‐92153and
(Figure 11B,C).
S-92153 (Figure 11B,C).
Figure 11. Cont.

Figure 11. Inhibition of (A) CBD, (B) S-88745 and (C) S-92153 on UV-induced apoptosis in HFF-1.
Figure 11. Inhibition
The control group wasof (A)
not CBD, (B) S‐88745
stimulated by UV.and
The(C) S‐92153 on
UV-treated UV‐induced
group apoptosis
(-) produced in reactive
a lot of HFF‐1.
The control group was not stimulated by UV. The UV‐treated group (‐) produced a lot of reactive
oxygen species after UV stimulation. The experiments were repeated three times. Each bar illustrates
oxygen species after UV stimulation. The experiments were repeated three times. Each bar illus‐
the average ± standard deviation (SD) counted from three experiments. p < 0.05 was considered
trates the average ± standard deviation (SD) counted from three experiments. p < 0.05 was consid‐
statistically
ered significant.
statistically ** p <**0.01
significant. p < compared to thetoUV
0.01 compared −treated
the group;
UV−treated ### p ###
group; < 0.001 compared
p < 0.001 to the
compared
control group.
to the control group.
3.4.5. Effects Evaluation on Collagen, Elastin and Fibronectin Production

3.4.5. Effects Evaluation on Collagen, Elastin and Fibronectin Production
Collagen, which is responsible for the strength and resilience of skin, provides support
Collagen, which is responsible for the strength and resilience of skin, provides sup‐
for the epidermal structure. Its degradation can cause skin laxity. Elastin fibers maintain
port for the epidermal structure. Its degradation can cause skin laxity. Elastin fibers18main‐
stretch conditions and provide recoil to the tissues. Fibronectin is widely involved
Antioxidants 2023, 12, x FOR PEER REVIEW in
of 24
tain stretch conditions and provide recoil to the tissues. Fibronectin is widely involved in
cell migration, adhesion and tissue repair. We detected the effects of compounds on the
cell migration, adhesion and tissue repair. We detected the effects of compounds on the
intracellular collagen, elastin and fibronectin content in fibroblasts.
intracellular
The
collagen,
results
elastinwere
of collagen
and fibronectin
shown
content infrom
in Figureof12,
fibroblasts.
where
group. CBD
The works
results of only at awere
collagen highshown
concentration
in Figure 12,4 from
uM, where
and theititcollagen
can be seen
can be the
seen
that after
content has
thatgroup.
after
UV irradiation,
increased by the
27.7%, content
which of
is collagen
near the decreased
level by
before UV 34%, compared
damage (Figure to only
12A). UVB
S‐88745 and
UV
CBDirradiation,
works the content of collagen of
decreased
µM, andby the34%, compared tohas
only the UVB
S‐92153 workonly at aconcentrations,
at low high concentration
and the4collagen collagen
content content
at 0.16 increased
uM increased by 19%by
27.7%, which is near the level before UV damage (Figure 12A). S-88745 and S-92153 work
and 40.8%, respectively (Figure 12B,C).
at low concentrations, and the collagen content at 0.16 µM increased by 19% and 40.8%,
respectively (Figure 12B,C).
Figure 12. Production of (A) CBD, (B) S-88745 and (C) S-92153 on UV-stimulated collagen in HFF-
Figure 12. Production of (A) CBD, (B) S‐88745 and (C) S‐92153 on UV‐stimulated collagen in HFF‐
1. The control group was not stimulated by UV. The UV-treated group (-) was stimulated by UV
1. The control group was not stimulated by UV. The UV‐treated group (‐) was stimulated by UV to
to cause
cause ECM ECM degradation.
degradation. The experiments
The experiments were were repeated
repeated three three
times.times. Each
Each bar bar illustrates
illustrates the
the aver‐
average ± standard deviation (SD) counted from three experiments. p < 0.05 was considered
age ± standard deviation (SD) counted from three experiments. p < 0.05 was considered statistically statisti-
cally significant.
significant. * p < compared
* p < 0.05 0.05 compared to the
to the UV−treated
UV−treated group; ** p**<p0.01
group; < 0.01 comparedtotothe
compared UV−treated
theUV−treated
group; # p < 0.05 compared to the control group; ## p < 0.01 compared to the control
group; # p < 0.05 compared to the control group; ## p < 0.01 compared to the control group. group.
As shown in Figure 13, after ultraviolet radiation, the elastin content in fibroblasts
decreased by 22%. It is obvious that CBD, S‐92153 and S‐92153 can alleviate the loss of
elastin caused by UV irradiation, and even restore the skin to the level before UV injury.
However, the abilities of S‐88745 and S‐92153 to improve collagen are negatively corre‐
lated to the concentration, and the effect is best at 0.16 uM (Figure 13B,C). As a result,
Figure 12. Production of (A) CBD, (B) S‐88745 and (C) S‐92153 on UV‐stimulated collagen in HF
1. The control group was not stimulated by UV. The UV‐treated group (‐) was stimulated by UV
cause ECM degradation. The experiments were repeated three times. Each bar illustrates the ave
age ± standard deviation (SD) counted from three experiments. p < 0.05 was considered statistica
significant. * p < 0.05 compared to the UV−treated group; ** p < 0.01 compared to the UV−treat
Antioxidants 2023, 12, 314 group; # p < 0.05 compared to the control group; ## p < 0.01 compared to the control
17 ofgroup.
22
As shown in Figure 13, after ultraviolet radiation, the elastin content in fibroblas
decreased by 22%. It is obvious that CBD, S‐92153 and S‐92153 can alleviate the loss
As shown in Figure 13, after ultraviolet radiation, the elastin content in fibroblasts
elastin caused by UV irradiation, and even restore the skin to the level before UV injur
decreased by 22%. It is obvious that CBD, S-92153 and S-92153 can alleviate the loss of
However, the abilities of S‐88745 and S‐92153 to improve collagen are negatively corr
elastin caused by UV irradiation, and even restore the skin to the level before UV injury.
lated to the concentration, and the effect is best at 0.16 uM (Figure 13B,C). As a resu
However, the abilities of S-88745 and S-92153 to improve collagen are negatively correlated
CBD, S‐88745 and S‐92153 can increase the elastin by 38.8%, 17.9% and 63% at 0.16 uM
to the concentration, and the effect is best at 0.16 µM (Figure 13B,C). As a result, CBD,
compared to the
S-88745 and S-92153 can model
increase group,
the elastinrespectively (Figure
by 38.8%, 17.9% and13).
63% at 0.16 µM, compared
to the model group, respectively (Figure 13).
Figure 13. Production of (A) CBD,of

Figure 13. Production (B) S-88745
(A) andS‐88745
CBD, (B) (C) S-92153 on UV-stimulated
and (C) elastin in HFF-1.
S‐92153 on UV‐stimulated elastin in HFF
The control The
group was not
control stimulated
group was not by UV. The by
stimulated UV-treated group (-) was
UV. The UV‐treated stimulated
group (‐) was by UV to by UV
stimulated
cause ECM degradation.
cause ECM degradation. The experiments
The experiments were repeatedwere
threerepeated threebar
times. Each times. Each bar
illustrates illustrates the ave
the average
± standard age ± standard
deviation (SD)deviation (SD) counted
counted from from three experiments.
three experiments. p < 0.05 was pconsidered
< 0.05 was statistically
considered statistica
significant. significant. * p < 0.05tocompared
* p < 0.05 compared to the UV−treated
the UV−treated group; ** p <group; ** p < 0.01tocompared
0.01 compared to the UV−treat
the UV−treated
group; ## p <group; ## p < 0.01tocompared
0.01 compared the controlto group.
the control group.
The resultsThe results of fibronectin

of fibronectin were shown were shown14.
in Figure in It
Figure 14. It is
is obvious obvious
that UV can that UV can promo
promote
the decomposition
the decomposition of and
of fibronectin, fibronectin, andofthe
the content content of
fibronectin fibronectin
decreases decreases
by 9%, comparedby 9%, com
Antioxidants 2023, 12,
to xthe pared
FORcontrol
PEER to the control
REVIEW
group. group. Unfortunately,
Unfortunately, CBD and S-88745 CBD anddid S‐88745 did not significantly
not significantly promote promo1
fibronectinfibronectin
production.production. Only
Only S-92153 S‐92153
could couldfibronectin
promote promote fibronectin
productionproduction and the effe
and the effect
was best at 0.16 uM, which increased the fibronectin
was best at 0.16 µM, which increased the fibronectin content by 26.7%. content by 26.7%.
Figure 14. Production of (A) CBD, (B) S-88745 and (C) S-92153 on UV-stimulated fibronectin in
Figure 14. Production of (A) CBD, (B) S‐88745 and (C) S‐92153 on UV‐stimulated fibronectin i
HFF-1. The control group
1. The wasgroup
control not stimulated by UV. ThebyUV-treated
was not stimulated group (-) was
UV. The UV‐treated groupstimulated by
(‐) was stimulated by
UV to cause ECM degradation. The experiments were repeated three times. Each bar illustrates
cause ECM degradation. The experiments were repeated three times. Each bar illustrates th
the average ± standard deviation
age ± standard (SD) counted
deviation from three
(SD) counted fromexperiments. p < 0.05
three experiments. p < was
0.05 considered
was considered stati
statistically significant. ** p <
significant. ** 0.01 compared
p < 0.01 to the
compared to the −treated group;
UVUV−treated # p#<p 0.05
group; compared
< 0.05 compared toto
the
the control
control group.
Based on the above results, S‐92153 is expected to rapidly restore extracellular
Based on the above
content, results,
such S-92153elastin
as collagen, is expected to rapidly
and fibrin restore
in a short extracellular
time when dealingma-with UV
trix content, such as collagen,
ation damage. elastin and fibrin in a short time when dealing with UV
irradiation damage.
3.5. Whitening Activity Test
3.5.1. Cytotoxicity Measurements on B16F10
Following the
Following the exploration exploration
of the of the anti‐inflammatory
anti-inflammatory and anti-wrinkleandeffects,
anti‐wrinkle
we con-effects, w
tinued to study the whitening effects of CBD and its derivatives. At first, the cytotox
tinued to study the whitening effects of CBD and its derivatives. At first, the cytotoxicities
on the
on the proliferation ofproliferation of B16F10
B16F10 melanoma cellsmelanoma cellsby
were studied were studied
using MTT. by
Theusing MTT.
results are The resu
shown in Figure 15, from where we can see that CBD can significantly
shown in Figure 15, from where we can see that CBD can significantly inhibit the activity inhibit the a
of B16F10 cells in a concentration‐dependent manner (Figure 15A). Compound S
demonstrates cytotoxicity only at 80 uM (Figure 15B), while S‐92153 was not cytot
B16F10 cells (Figure 15C). Based on the MTT test results, 0.0256–0.64 uM was selec
subsequent experiments.
Following the exploration of the anti‐inflammatory and anti‐wrinkle effects, we con‐
Antioxidants 2023, 12, 314 tinued to study the whitening effects of CBD and its derivatives. At first, the cytotoxicities
18 of 22
on the proliferation of B16F10 melanoma cells were studied by using MTT. The results are
shown in Figure 15, from where we can see that CBD can significantly inhibit the activity
ofofB16F10
B16F10cells
cellsininaaconcentration‐dependent
concentration-dependentmanner
manner(Figure
(Figure15A).
15A).Compound
CompoundS‐88745
S-88745
demonstrates
demonstrates cytotoxicity only at 80 µM (Figure 15B), while S-92153 wasnot
cytotoxicity only at 80 uM (Figure 15B), while S‐92153 was notcytotoxic
cytotoxictoto
B16F10
B16F10cells
cells(Figure
(Figure15C).
15C).Based
Basedononthe
theMTT
MTTtest
testresults,
results,0.0256–0.64
0.0256–0.64uM
µMwas
wasselected
selectedfor
for
Figure 15. Cytotoxicity of (A) CBD and its derivatives, (B) S-88745 and (C) S-92153, on B16F10 cells.
Figure 15. Cytotoxicity of (A) CBD and its derivatives, (B) S‐88745 and (C) S‐92153, on B16F10 cells.
B16F10 cells were treated with cannabidiol, S-88745 or S-92153 for 72 h, and MTT assay was carried
B16F10 cells were treated with cannabidiol, S‐88745 or S‐92153 for 72 h, and MTT assay was carried
totodetect
detectthe
thecell
cellviability.
viability.The
Thecontrol
controlgroup
groupwas
wasnot
nottreated
treatedwith
withCBD,
CBD,S‐88745
S-88745and
andS‐92153.
S-92153.The
The
experiments were repeated three times. Each bar illustrates the average ± standard deviation
experiments were repeated three times. Each bar illustrates the average ± standard deviation (SD) (SD)
countedfrom
counted fromthree
threeexperiments.
experiments.
3.5.2. Whitening Activity Tests

3.5.2. Whitening Activity Tests
In order to detect the whitening effects of CBD, S-88745 and S-92153, α-MSH was
In order to detect the whitening effects of CBD, S‐88745 and S‐92153, α‐MSH was
used to stimulate B16F10 melanoma cells to secrete melanin. Then compounds were added
used to stimulate B16F10 melanoma cells to secrete melanin. Then compounds were
with different concentrations, and the sodium hydroxide lysis method was used to detect
added with different concentrations, and the sodium hydroxide lysis method was used to
the intracellular melanin content. The results are shown in Figure 16. It can be seen that
detect the intracellular melanin content. The results are shown in Figure 16. It can be seen
all three compounds can reduce the intracellular melanin content to some extent, among
Antioxidants 2023, 12, x FOR PEER REVIEW
that all three compounds can reduce the intracellular melanin content to some 20 of 24
extent,
which S-92153 significantly reduced the content of melanin in a concentration dependent
among which S‐92153 significantly reduced the content of melanin in a concentration de‐
manner (Figure 16C).
pendent manner (Figure 16C).
Figure 16. Inhibition of (A) CBD, (B) S-88745 and (C) S-92153 on α-MSH-induced melanogenesis
Figure 16. Inhibition of (A) CBD, (B) S‐88745 and (C) S‐92153 on α‐MSH‐induced melanogenesis in
in B16F10. The control group was not stimulated by α-MSH. The α-MSH -treated group (-) was
B16F10. The control group was not stimulated by α‐MSH. The α‐MSH ‐treated group (‐) was stim‐
stimulated by α-MSH to produce pigmentation. The experiments were repeated three times. Each
ulated by α‐MSH to produce pigmentation. The experiments were repeated three times. Each bar
bar illustrates
illustrates the average
deviation (SD) counted
(SD) counted from three
from three experiments.
experiments. p <was
p < 0.05 0.05con‐
was
considered
sidered statistically
statistically significant.
significant. p <compared
** p <**0.01 0.01 compared
to thetoα‐MSH
the α-MSH −treated
−treated group;group;
*** p < *** p <com‐
0.001 0.001
compared
pared to theto the α-MSH
α‐MSH −treated
−treated group;
group; **** **** p <compared
p < 0.0001 0.0001 compared −treated
to the α-MSHgroup;
to the α‐MSH−treated # p <group;
0.05
compared
# p < 0.05 to the control
compared group;
to the ## pgroup;
control < 0.01 compared
## p < 0.01to the control
compared group;
to the ####group;
control p < 0.0001
####compared
p < 0.0001
tocompared
the control
to group.
the control group.
3.5.3.The
3.5.3. TheEffects
EffectsononTyrosinase
TyrosinaseActivity
Activity
Tyrosinaseplays
Tyrosinase playsan animportant
importantrolerolein inthe
theformation
formationof ofmelanin,
melanin,so sowe
weinvestigated
investigated
whether CBD and its derivatives can affect the activity of tyrosinase.
whether CBD and its derivatives can affect the activity of tyrosinase. As shown As shown inin
Figure 17,
Figure
the ability of CBD to inhibit tyrosinase was negatively correlated with the
17, the ability of CBD to inhibit tyrosinase was negatively correlated with the concentra‐ concentration
(Figure
tion 17A).
(Figure At 0.16
17A). the activity
µM, uM,
At 0.16 of tyrosinase
the activity was 70.9%,
of tyrosinase of theof
was 70.9%, group with only
the group withadded
only
melanin. The ability of CBD to inhibit melanin is also negatively related to the
added melanin. The ability of CBD to inhibit melanin is also negatively related to the con‐ concentration,
because CBD
centration, may reduce
because CBD may the production of melanin by
reduce the production of inhibiting
melanin bythe activity of
inhibiting tyrosinase.
the activity
S-88745 significantly decreased tyrosinase activity at 0.0256–0.64 µM (Figure
of tyrosinase. S‐88745 significantly decreased tyrosinase activity at 0.0256–0.64 uM (Figure 17B). S-92153
decreased the activity of tyrosinase in a concentration dependent manner.
17B). S‐92153 decreased the activity of tyrosinase in a concentration dependent manner. At 0.64 µM,
the activity of tyrosinase was only 58.8% of the model group. The ability
At 0.64 uM, the activity of tyrosinase was only 58.8% of the model group. The ability of S‐ of S-92153 to
92153 to inhibit melanin is also negatively related to the concentration, because S‐92153
may inhibit melanin production by inhibiting the activity of tyrosinase (Figure 17C).
17, the ability of CBD to inhibit tyrosinase was negatively correlated with the conc
tion (Figure 17A). At 0.16 uM, the activity of tyrosinase was 70.9%, of the group with
added melanin. The ability of CBD to inhibit melanin is also negatively related to th
centration, because CBD may reduce the production of melanin by inhibiting the ac
Antioxidants 2023, 12, 314 of tyrosinase. S‐88745 significantly decreased tyrosinase activity at 0.0256–0.64
19 of 22 uM (F
17B). S‐92153 decreased the activity of tyrosinase in a concentration dependent ma
At 0.64 uM, the activity of tyrosinase was only 58.8% of the model group. The ability
inhibit melanin92153 tonegatively
is also inhibit melanin
relatedistoalso
the negatively related
concentration, to the
because concentration,
S-92153 may inhibitbecause S‐
may inhibit melanin production by inhibiting the activity
melanin production by inhibiting the activity of tyrosinase (Figure 17C). of tyrosinase (Figure 17C
Figure 17. Inhibition

Figure of17.
(A)Inhibition
CBD, (B) ofS-88745 and (B)
(A) CBD, (C)S‐88745
S-92153andon α-MSH-induced tyrosinase activity
(C) S‐92153 on α‐MSH‐induced tyrosinase a
in B16F10. The in control
B16F10.group
The was not group
control stimulated by α-MSH.
was not stimulatedTheby α-MSH
α‐MSH. -treated group (-)
The α‐MSH was group (
‐treated
stimulated
stimulated by α-MSH by α‐MSH
to produce to produceThe
pigmentation. pigmentation.
experiments The experiments
were weretimes.
repeated three repeated three times
Each
baraverage
bar illustrates the illustrates the average
± standard ± standard
deviation deviation
(SD) counted (SD)three
from counted from three
experiments. p <experiments.
0.05 was p < 0.0
considered statistically significant. * p < 0.05 compared to the α‐MSH
considered statistically significant. * p < 0.05 compared to the α-MSH −treated group; ** p < 0.01 −treated group; ** p
compared to the α‐MSH −treated group; **** p < 0.0001 compared
compared to the α-MSH −treated group; **** p < 0.0001 compared to the α-MSH−treated group; to the α‐MSH−treated grou
< 0.05 compared to the control group; #### p < 0.0001 compared to the control group.
# p < 0.05 compared to the control group; #### p < 0.0001 compared to the control group.
4. Discussion 4. Discussion
Based on theresults,
Based on the experimental experimental results,
we further we this
explore further explore
study. this study.
Cannabidiol Cannabidiol
is one of
the few cosmetic ingredients
of the with multiple
few cosmetic skincare
ingredients benefits, skincare
with multiple but its usage is limited
benefits, due
but its usage is li
to the limited resources
due to theand rulesresources
limited acting inand
many countries.
rules Therefore,
acting in we intend
many countries. to find we inte
Therefore,
substitutes to CBD that have similar skincare benefits by combining computer-aided
find substitutes to CBD that have similar skincare benefits by combining drugcomputer‐
designs and experimentation
drug designs and methods.
experimentation methods.
We studied the action mode between CBD and target protein CB1 to find the molec-
ular key skeleton, and virtually screened huge natural products databases to search for
compounds with a 70% structural similarity to the skeleton. Following Lipinski’s rule of
five, Veber’s rules screening, and docking screening, 153 compounds with a docking score
of less than −7 and drug-like properties were retained. Via the hierarchical clustering, these
compounds were clustered into 10 groups. Within these clusters, four compounds (S-88614,
S-88745, S-92151, S-92153) were chosen and purchased for the subsequent experiments.
These four compounds have phenolic hydroxyl structures similar to CBD, which may be
the basis of their antioxidant properties.
Then, the determination of extracellular tests (ABTS and DPPH) and intracellular
reactive oxygen species was used to verify the antioxidant properties of the compounds,
and S-88745 and S-92153 showed great potential.
Experimental results show that cannabidiol and its derivatives can reduce the pro-
duction of NO, IL-6, iNOS and COX-2, thereby producing anti-inflammatory effects. Our
research shows that the tested compounds can reduce NO and iNOS expression, and they
may down regulate the content of NO by inhibiting the expression of iNOS. They also can
compensate for the UV damage. The experimental results indicated that these compounds
can increase the content of collagen, elastin and fibronectin, and can affect the apoptosis
of fibroblasts. IL-6 and COX-2 can upregulate the expression of MMPs, thus promoting
the degradation of extracellular matrix. Cannabinol and its derivatives can reduce the
degradation of collagen, elastin and fibronectin caused by ultraviolet radiation, which may
be related to their ability to down regulate the expression of IL-6 and COX-2. In addition, ul-
traviolet radiation can cause fibroblast death, thereby reducing the overall expression of the
extracellular matrix. Compared with the model group, the survival rate of cells treated with
CBD and its derivatives was greatly improved, and the apoptosis rate was also decreased.
Therefore, cannabinol and its derivatives can alleviate the collagen caused by ultraviolet
radiation. The degradation of elastin and fibronectin may be related to their increased cell
survival rate and decreased apoptosis rate after ultraviolet radiation. All of these results
indicated that cannabidiol and its derivatives have anti-wrinkle effects. Furthermore, they
can significantly reduce the activity of tyrosinase and adjust the generation of melanin. It is
worth mentioning that the anti-inflammatory and anti-wrinkle whitening effect of S-88745
and S-92153 are stronger than that of CBD, which is consistent with the antioxidant effect.
Therefore, it can be inferred that inflammation, loss of ECM, and melanin precipitation may
be affected by reactive oxygen species by the common MAPK pathway.
The abundant generation of reactive oxygen species activates the MAPK pathway,
which then excites NF-κB, MMPs and MITF. NF-κB can activate downstream inflammatory
factors and inflammation-related proteins. MMPs promote ECM degradation, leading to
skin laxity and loss of elasticity. MITF activates tyrosinase and other enzymes involved
in melanin synthesis. It can be seen that MAPK channel is related to a variety of skin
problems. Studies suggested that CBD modules the activation of the MAPK path through
JNK and ERK signaling path, and then activate NF-κB Channel [48,49]. In addition, CBD
can significantly reduce the expression level of p38, ERK, JNK mRNA and the expression
of MMPs [50]. At present, the mechanism of reducing melanin content in CBD is not
clear. This study indicated that cannabidiol may have anti-inflammatory, anti-wrinkle
and whitening effects by inhibiting the MAPK pathway. CBD and its derivatives generate
multiple skin care effects through the MAPK pathway; therefore drug design can be carried
out for this pathway.
5. Conclusions
In this study, the structure of CBD was optimized to obtain two compounds with
similar multifunctional skin care efficacies as CBD. Firstly, The action mode between CBD
and target protein CB1 was analyzed, and the action skeleton was put into the natural
products database. Following the molecular docking, drug likeness evaluation and cluster
analysis, four compounds with strong binding abilities to CB1 were obtained. ABTS and
DPPH radical scavenging experiments showed that S-88745 and S-92153 had a stronger
antioxidant capacity than CBD compounds. MTT, ELISA, western blot, flow cytometry and
other experimental methods were used to analyze the anti-inflammatory, anti-wrinkle and
whitening effects of these two compounds. These results proved that the two compounds S-
88745 and S-92153 are superior to CBD, in terms of antioxidant, anti-wrinkle and whitening
efficacy with a lower cytotoxicity. These two compounds can replace cannabinol in the
cosmetics industry, which can solve the problem of insufficient sources and restricted usage
of CBD. This may be a new solution to skin problems.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/antiox12020314/s1.
Author Contributions: Conceptualization and software, X.C.; methodology, X.C. and J.S.; validation,
X.C., X.W. and J.L.; formal analysis, X.C., J.S. and R.W.; writing—review and editing, X.C., C.F., J.C. and
R.H.; supervision, J.L. All authors have read and agreed to the published version of the manuscript.
Funding: This research is supported by the Key Project of the Natural Science Foundation of Gansu
Province, China(Grant No. 21JR7RA439).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in Supplementary Material.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Roh, E.; Kim, J.E.; Kwon, J.Y.; Park, J.S.; Bode, A.M.; Dong, Z.; Lee, K.W. Molecular mechanisms of green tea polyphenols with
protective effects against skin photoaging. Crit. Rev. Food Sci. Nutr. 2017, 57, 1631–1637. [CrossRef] [PubMed]
2. Adamczyk-Grochala, J.; Wnuk, M.; Duda, M.; Zuczek, J.; Lewinska, A. Treatment with Modified Extracts of the Microalga
Attenuates the Development of Stress-Induced Senescence in Human Skin Cells. Nutrients 2020, 12, 1005. [CrossRef] [PubMed]
3. Chen, Y.; Huang, F.; McClements, D.J.; Xie, B.; Sun, Z.; Deng, Q. Oligomeric Procyanidin Nanoliposomes Prevent Melanogenesis
and UV Radiation-Induced Skin Epithelial Cell (HFF-1) Damage. Molecules 2020, 25, 1458. [CrossRef]
4. Wang, L.; Lee, W.; Jayawardena, T.U.; Cha, S.-H.; Jeon, Y.-J. Dieckol, an algae-derived phenolic compound, suppresses airborne
particulate matter-induced skin aging by inhibiting the expressions of pro-inflammatory cytokines and matrix metalloproteinases
through regulating NF-κB, AP-1, and MAPKs signaling pathways. Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc. 2020,
146, 111823. [CrossRef]
5. Lee, E.S.; Lee, E.Y.; Yoon, J.; Hong, A.; Nam, S.J.; Ko, J. Sarmentosamide, an Anti-Aging Compound from a Marine-Derived
Streptomyces sp. APmarine042. Mar. Drugs 2020, 18, 463. [CrossRef] [PubMed]
6. Lorz, L.R.; Yoo, B.C.; Kim, M.Y.; Cho, J.Y. Anti-Wrinkling and Anti-Melanogenic Effect of Pradosia mutisii Methanol Extract. Int.
J. Mol. Sci. 2019, 20, 1043. [CrossRef]
7. Lim, H.Y.; Jeong, D.; Park, S.H.; Shin, K.K.; Hong, Y.H.; Kim, E.; Yu, Y.G.; Kim, T.R.; Kim, H.; Lee, J.; et al. Antiwrinkle and
Antimelanogenesis Effects of Tyndallized Lactobacillus acidophilus KCCM12625P. Int. J. Mol. Sci. 2020, 21, 1620. [CrossRef]
8. Huang, H.C.; Lien, H.M.; Ke, H.J.; Chang, L.L.; Chen, C.C.; Chang, T.M. Antioxidative characteristics of Anisomeles indica extract
and inhibitory effect of ovatodiolide on melanogenesis. Int. J. Mol. Sci. 2012, 13, 6220–6235. [CrossRef]
9. Bourhim, T.; Villareal, M.O.; Gadhi, C.; Isoda, H. Elucidation of Melanogenesis-Associated Signaling Pathways Regulated by
Argan Press Cake in B16 Melanoma Cells. Nutrients 2021, 13, 2697. [CrossRef]
10. Yamakoshi, J.; Otsuka, F.; Sano, A.; Tokutake, S.; Saito, M.; Kikuchi, M.; Kubota, Y. Lightening effect on ultraviolet-induced
pigmentation of guinea pig skin by oral administration of a proanthocyanidin-rich extract from grape seeds. Pigment Cell Res.
2003, 16, 629–638. [CrossRef]
11. Pucci, M.; Pasquariello, N.; Battista, N.; Di Tommaso, M.; Rapino, C.; Fezza, F.; Zuccolo, M.; Jourdain, R.; Finazzi Agro, A.; Breton,
L.; et al. Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid receptor. J. Biol. Chem. 2012, 287, 15466–15478.
[CrossRef] [PubMed]
12. Park, C.-H.; Min, S.-Y.; Yu, H.-W.; Kim, K.; Kim, S.; Lee, H.-J.; Kim, J.-H.; Park, Y.-J. Effects of Apigenin on RBL-2H3, RAW264.7,
and HaCaT Cells: Anti-Allergic, Anti-Inflammatory, and Skin-Protective Activities. Int. J. Mol. Sci. 2020, 21, 4620. [CrossRef]
13. Luczaj, W.; Domingues, M.D.R.; Domingues, P.; Skrzydlewska, E. Changes in Lipid Profile of Keratinocytes from Rat Skin
Exposed to Chronic UVA or UVB Radiation and Topical Application of Cannabidiol. Antioxidants 2020, 9, 1178. [CrossRef]
[PubMed]
14. Zhang, Z.; Xu, Y.; Lai, R.; Deng, H.; Zhou, F.; Wang, P.; Pang, X.; Huang, G.; Chen, X.; Lin, H.; et al. Protective Effect of the
Pearl Extract from Pinctada fucata martensii Dunker on UV-Induced Photoaging in Mice. Chem. Biodivers. 2022, 19, e202100876.
[CrossRef] [PubMed]
15. Jeong, S.; Yoon, S.; Kim, S.; Jung, J.; Kor, M.; Shin, K.; Lim, C.; Han, H.S.; Lee, H.; Park, K.Y.; et al. Anti-Wrinkle Benefits of
Peptides Complex Stimulating Skin Basement Membrane Proteins Expression. Int. J. Mol. Sci. 2019, 21, 73. [CrossRef]
16. Naylor, E.C.; Watson, R.E.; Sherratt, M.J. Molecular aspects of skin ageing. Maturitas 2011, 69, 249–256. [CrossRef]
17. Biernacki, M.; Brzóska, M.M.; Markowska, A.; Gałażyn-Sidorczuk, M.; Cylwik, B.; G˛egotek, A.; Skrzydlewska, E. Oxidative
Stress and Its Consequences in the Blood of Rats Irradiated with UV: Protective Effect of Cannabidiol. Antioxidants 2021, 10, 821.
[CrossRef]
18. Jankovic, A.; Saso, L.; Korac, A.; Korac, B. Relation of Redox and Structural Alterations of Rat Skin in the Function of Chronological
Aging. Oxidative Med. Cell. Longev. 2019, 2019, 2471312. [CrossRef]
19. Mussard, E.; Jousselin, S.; Cesaro, A.; Legrain, B.; Lespessailles, E.; Esteve, E.; Berteina-Raboin, S.; Toumi, H. Andrographis
Paniculata and Its Bioactive Diterpenoids Protect Dermal Fibroblasts Against Inflammation and Oxidative Stress. Antioxidants
2020, 9, 432. [CrossRef]
20. Li, F.M.; Pirzgalska, R.M.; Marques-da-Silva, D.; Ledent, C.; Kofalvi, A.; Carvalho, E. Diabetes and Cannabinoid CB1 receptor
deficiency promote similar early onset aging-like changes in the skin. Exp. Gerontol. 2021, 154, 111528. [CrossRef]
21. Hsu, J.Y.; Lin, H.H.; Li, T.S.; Tseng, C.Y.; Wong, Y.; Chen, J.H. Anti-Melanogenesis Effects of Lotus Seedpod In Vitro and In Vivo.
Nutrients 2020, 12, 3535. [CrossRef] [PubMed]
22. Oh, T.I.; Jung, H.J.; Lee, Y.M.; Lee, S.; Kim, G.H.; Kan, S.Y.; Kang, H.; Oh, T.; Ko, H.M.; Kwak, K.C.; et al. Zerumbone, a Tropical
Ginger Sesquiterpene of Zingiber officinale Roscoe, Attenuates alpha-MSH-Induced Melanogenesis in B16F10 Cells. Int. J. Mol.
Sci. 2018, 19, 3149. [CrossRef]
23. Jeong, D.; Lee, J.; Park, S.H.; Kim, Y.A.; Park, B.J.; Oh, J.; Sung, G.-H.; Aravinthan, A.; Kim, J.-H.; Kang, H.; et al. Antiphotoaging
and Antimelanogenic Effects of Pursh Ethanol Extract due to Antioxidant- and Autophagy-Inducing Properties. Oxidative Med.
Cell. Longev. 2019, 2019, 9679731. [CrossRef] [PubMed]
24. Jin, K.-S.; Oh, Y.N.; Hyun, S.K.; Kwon, H.J.; Kim, B.W. Betulinic acid isolated from Vitis amurensis root inhibits 3-isobutyl-1-
methylxanthine induced melanogenesis via the regulation of MEK/ERK and PI3K/Akt pathways in B16F10 cells. Food Chem.
Toxicol. 2014, 68, 38–43. [CrossRef] [PubMed]
25. Chen, T.; Zhao, B.; Liu, Y.; Wang, R.; Yang, Y.; Yang, L.; Dong, C. MITF-M regulates melanogenesis in mouse melanocytes.
J. Dermatol. Sci. 2018, 90, 253–262. [CrossRef]
26. Li, P.H.; Chiu, Y.P.; Shih, C.C.; Wen, Z.H.; Ibeto, L.K.; Huang, S.H.; Chiu, C.C.; Ma, D.L.; Leung, C.H.; Chang, Y.N.; et al.
Biofunctional Activities of Equisetum ramosissimum Extract: Protective Effects against Oxidation, Melanoma, and Melanogenesis.
Oxidative Med. Cell. Longev. 2016, 2016, 2853543. [CrossRef]
27. Pisanti, S.; Malfitano, A.M.; Ciaglia, E.; Lamberti, A.; Ranieri, R.; Cuomo, G.; Abate, M.; Faggiana, G.; Proto, M.C.; Fiore, D.; et al.
Cannabidiol: State of the art and new challenges for therapeutic applications. Pharmacol. Ther. 2017, 175, 133–150. [CrossRef]
28. Sunda, F.; Arowolo, A. A molecular basis for the anti-inflammatory and anti-fibrosis properties of cannabidiol. FASEB J. 2020, 34,
14083–14092. [CrossRef]
29. Jarocka-Karpowicz, I.; Biernacki, M.; Wronski, A.; Gegotek, A.; Skrzydlewska, E. Cannabidiol Effects on Phospholipid Metabolism
in Keratinocytes from Patients with Psoriasis Vulgaris. Biomolecules 2020, 10, 367. [CrossRef]
30. Gegotek, A.; Atalay, S.; Domingues, P.; Skrzydlewska, E. The Differences in the Proteome Profile of Cannabidiol-Treated Skin
Fibroblasts following UVA or UVB Irradiation in 2D and 3D Cell Cultures. Cells 2019, 8, 995. [CrossRef]
31. Kennedy, P.J. Anti-Aging and Skin Tone Lightening Compositions and Methods For Same. U.S. Patent No 11,337,905, 2019.
32. Grimaldi, C.; Capasso, A. The endocannabinoid system in the cancer therapy: An overview. Curr. Med. Chem. 2011, 18, 1575–1583.
[CrossRef]
33. Baswan, S.M.; Klosner, A.E.; Glynn, K.; Rajgopal, A.; Malik, K.; Yim, S.; Stern, N. Therapeutic Potential of Cannabidiol (CBD) for
Skin Health and Disorders. Clin. Cosmet. Investig. Dermatol. 2020, 13, 927–942. [CrossRef]
34. Pertwee, R.G. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: Delta9-tetrahydrocannabinol,
cannabidiol and delta9-tetrahydrocannabivarin. Br. J. Pharm. 2008, 153, 199–215. [CrossRef]
35. Luo, H.; Rossi, E.; Saubamea, B.; Chasseigneaux, S.; Cochois, V.; Choublier, N.; Smirnova, M.; Glacial, F.; Perrière, N.; Bourdoulous,
S.; et al. Cannabidiol Increases Proliferation, Migration, Tubulogenesis, and Integrity of Human Brain Endothelial Cells through
TRPV2 Activation. Mol. Pharm. 2019, 16, 1312–1326. [CrossRef]
36. Hua, T.; Vemuri, K.; Pu, M.; Qu, L.; Han, G.W.; Wu, Y.; Zhao, S.; Shui, W.; Li, S.; Korde, A.; et al. Crystal Structure of the Human
Cannabinoid Receptor CB. Cell 2016, 167, 750–762.e14. [CrossRef]
37. Shi, Y.; Chen, X.; Qiang, S.; Su, J.; Li, J. Anti-Oxidation and Anti-Inflammatory Potency Evaluation of Ferulic Acid Derivatives
Obtained through Virtual Screening. Int. J. Mol. Sci. 2021, 22, 11305. [CrossRef]
38. Han, W.; Shi, Y.; Su, J.; Zhao, Z.; Wang, X.; Li, J.; Liu, H. Virtual Screening and Bioactivity Evaluation of Novel Androgen Receptor
Antagonists From Anti-PCa Traditional Chinese Medicine Prescriptions. Front. Chem. 2020, 8, 582861. [CrossRef]
39. Nguyen, T.T.; Choi, Y.-J.; Rohmah, Z.; Jeong, S.-B.; Hwang, D.-J.; Choi, B.-D. Antioxidant Activities in Processed Cockle(Tegillarca
granosa) from the Yeosu. J. Agric. Life Sci. 2017, 51, 131–138. [CrossRef]
40. Garcin, G.; Le Gallic, L.; Stoebner, P.-E.; Guezennec, A.; Guesnet, J.; Lavabre-Bertrand, T.; Martinez, J.; Meunier, L. Constitutive
expression of MC1R in HaCaT keratinocytes inhibits basal and UVB-induced TNF-alpha production. Photochem. Photobiol. 2009,
85, 1440–1450. [CrossRef] [PubMed]
41. Voravuthikunchai, S.P.; Kanchanapoom, T.; Sawangjaroen, N.; Hutadilok-Towatana, N. Antioxidant, antibacterial and antigiardial
activities of Walsura robusta Roxb. Nat. Prod. Res. 2010, 24, 813–824. [CrossRef]
42. Syed Haneef, S.A.; Ranganathan, S. Structural bioinformatics analysis of variants on GPCR function. Curr. Opin. Struct. Biol.
2019, 55, 161–177. [CrossRef] [PubMed]
43. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 2001, 46, 3–25. [CrossRef] [PubMed]
44. Almeida, D.L.; Devi, L.A. Diversity of molecular targets and signaling pathways for CBD. Pharmacol. Res. Perspect. 2020, 8, e00682.
[CrossRef] [PubMed]
45. Burstein, S. Cannabidiol (CBD) and its analogs: A review of their effects on inflammation. Bioorganic. Med. Chem. 2015, 23,
1377–1385. [CrossRef] [PubMed]
46. Dos-Santos-Pereira, M.; Guimaraes, F.S.; Del-Bel, E.; Raisman-Vozari, R.; Michel, P.P. Cannabidiol prevents LPS-induced microglial
inflammation by inhibiting ROS/NF-kappaB-dependent signaling and glucose consumption. Glia 2020, 68, 561–573. [CrossRef]
[PubMed]
47. Chung, Y.C.; Ko, J.H.; Kang, H.K.; Kim, S.; Kang, C.I.; Lee, J.N.; Park, S.M.; Hyun, C.G. Antimelanogenic Effects of Polygonum
tinctorium Flower Extract from Traditional Jeju Fermentation via Upregulation of Extracellular Signal-Regulated Kinase and
Protein Kinase B Activation. Int. J. Mol. Sci. 2018, 19, 2895. [CrossRef] [PubMed]
48. Wang, Y.; Wang, X.; Yang, Y.; Quan, Q.; Huo, T.; Yang, S.; Ju, R.; An, Q. Comparison of the in vitro Anti-Inflammatory Effect of
Cannabidiol to Dexamethasone. Clin. Cosmet. Investig. Dermatol. 2022, 15, 1959–1967. [CrossRef] [PubMed]
49. Juknat, A.; Pietr, M.; Kozela, E.; Rimmerman, N.; Levy, R.; Gao, F.; Coppola, G.; Geschwind, D.; Vogel, Z. Microarray and pathway
analysis reveal distinct mechanisms underlying cannabinoid-mediated modulation of LPS-induced activation of BV-2 microglial
cells. PloS ONE 2013, 8, e61462. [CrossRef] [PubMed]
50. Zhang, X. The Effects and Mechanism of Cannabidol on Photoaging Dermal Fibroblasts. Master’s Thesis, Kunming Medical
University, Kunming, China, 2021. [CrossRef]
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antioxidants

Keywords: cannabidiol; virtual screening; anti-oxidation; anti-inflammation; anti-wrinkle; whitening

Citation: Chen, X.; Su, J.; Wang, R.; 1. Introduction

Antioxidants 2023, 12, 314. https://doi.org/10.3390/antiox12020314 https://www.mdpi.com/journal/antioxidants

2. Materials and Methods

2.2.2. Drug Likeness Evaluation

2.2.3. High Precision Screening

2.2.4. Similarity Searching

2.3. Antioxidant Capacity Evaluation

2.3.1. DPPH Radical Scavenging Activity

2.3.2. ABTS Radical Scavenging Activity

2.4. Cytotoxicity Measurement of CBD and Its Derivatives

respectively, and 1% penicillin–streptomycin (P/S) was added to the medium at 37 ◦ C in a

2.5. Anti-Inflammatory Activity Test

2.5.1. Measurement of NO and IL-6 Levels

2.5.2. Western Blot Analysis

2.6. Antiwrinkle Activity Test

2.6.1. Repair Ultraviolet Radiation Damage Experiment

2.6.2. Detection of Collagen, Elastin and Fibronectin Levels

2.6.3. Measurement of the ROS Content

2.6.4. Detection the Effects on the Apoptosis of HFF-1 Cells

2.7. Determination of Melanin Content and Tyrosinase Activity

2.8. Statistical Analysis

S‐88614 InterBioScreen −8.494

Antioxidants 2023, 12, x FOR PEER REVIEWS‐88614

S‐88745 InterBioScreen −9.267

Antioxidants 2023, 12, x FOR PEER REVIEW 9 of 24

Antioxidants 2023, 12, 314 CBDCBD

Floor Plan View of the

Antioxidants 2023, 12, x FOR PEER REVIEW 11 of 24

3.3. Anti‐Inflammatory Activity Test

3.3.3. Inhibitory Effect on IL‐6 Production

3.4. Anti-Wrinkle Activity Test

3.4.2. Fibroblast Damage Repairment Evaluation

3.4.2. Fibroblast Damage Repairment Evaluation

3.4.2. Fibroblast Damage Repairment Evaluation

3.4.3. Intracellular Antioxidant Activity

3.4.4. Fibroblast Apoptosis Experiment

3.4.4. Fibroblast Apoptosis Experiment

Antioxidants 2023, 12, x FOR PEER REVIEW 17 of 24

Figure 11. Cont.

3.4.5. Effects Evaluation on Collagen, Elastin and Fibronectin Production

Figure 13. Production of (A) CBD,of

The resultsThe results of fibronectin

3.5.2. Whitening Activity Tests

Figure 17. Inhibition

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