European Journal of Pharmaceutical Sciences 192 (2024) 106664

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Preparation of paeoniflorin-glycyrrhizic acid complex transethosome gel

and its preventive and therapeutic effects on melasma
Yaoyao Xiao a, b, c, d, e, 1, Lele Zhou a, b, c, d, e, 1, Wenkang Tao a, b, c, d, e, Xuan Yang a, b, c, d, e
, Junying Li a, b, c, d, e, Rulin Wang a, b, c, d, e, Yanan Zhao f, *, Can Peng a, b, c, d, e, *, Caiyun Zhang a, b
, c, d, e, **

a
Anhui Province Key Laboratory of Pharmaceutical Preparation Technology and Application, Center for Xin’an Medicine and Modernization of Traditional Chinese
Medicine of IHM, School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
b
Anhui Provincial Department of Education, Engineering Technology Research Center of Modern Pharmaceutical Preparation, China
c
Anhui Genuine Chinese Medicinal Materials Quality Improvement Innovation Collaborative Center, Hefei 230012, China
d
Institute of Pharmaceutics, Anhui Academy of Chinese Medicine, Hefei 230012, China
e
Anhui Key Laboratory of Compound Chinese Materia Medica, Hefei 230012, China
f
Hefei Cancer Hospital, Chinese Academy of Sciences, Hefei 230000, China

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

Keywords: Paeoniflorin (PF) and glycyrrhizic acid (GL) have skin beautifying effects of anti-inflammation, anti-oxidation,
Paeoniflorin inhibition of melanin formation, and reduction of skin pigmentation. To improve the transdermal permeability of
Glycyrrhizic acid PF and GL in transdermal drug delivery system (TDDS) and enhance their anti-melasma efficacy, PF-GL trans­
Ethanol injection method
ethosome (PF-GL-TE) was prepared by ethanol injection method, and finally gelled with carbomer-940 to form
Transethosome
PF-GL-TE gel. Consequently, the obtained PF-GL-TE is small and uniform, with an average particle size and a PDI
Gel
Melasma value of about 167.9 nm and 0.102. PF-GL-TE gel showed sustained release behavior and high transdermal
permeability in vitro release and transdermal tests. Meanwhile, PF-GL-TE gel played significant preventive effects
on melasma induced by progesterone injection and ultraviolet radiation B (UVB) irradiation. According to the
results of H&E staining and Masson staining of rat skin, PF-GL-TE gel can alleviate the skin inflammation of and
reduce the loss of collagen fibers of back skin in the melasma model rats. Compared with the PF-GL mixture gel,
PF-GL-TE gel significantly attenuated the oxidative damage of liver and skin by increasing the activity of SOD
and reducing the content of MDA. The results of Western blot showed that PF-GL-TE gel might down-regulate
melanin-related proteins expressions of MITF/TYR/TRP1 and TRP2 to prevent and treat melasma. These find­
ings indicate that PF-GL-TE gel is an effective TDDS for delivering PF and GL into the skin, providing a promising
preparation for effective prevention and treatment of melasma.

1. Introduction
Melasma is a common acquired pigmentation disorder that affects up
to 30 % of reproductive women in some areas (Passeron et al., 2018).
Facial skin pigmentation is mostly caused by pigment metabolism dis­
order, which brings many troubles and pains to patients in life and spirit
(Kang et al., 2014). Clinical and scientific studies have shown that
melasma is mainly caused by ultraviolet radiation B (UVB) exposure and
hormonal effects (Achar et al., 2011). Hormones have a great impact on
of melasma, and pregnancy, oral contraceptives, and other hormonal
treatments increase its incidence rate (Filoni et al., 2019; Guinot et al.,
2010; Tamega Ade et al., 2013). Melasma is a kind of skin aging, which
is partly because the increase of free radicals destroys the cellular
function in skin tissue (Kim et al., 2022).
Pigmentation disorder is caused by excessive production of melanin
by melanocytes and excessive deposition of melanin in epidermal

* Corresponding authors.
** Corresponding author at: Anhui Province Key Laboratory of Pharmaceutical Preparation Technology and Application, Center for Xin’an Medicine and
Modernization of Traditional Chinese Medicine of IHM, School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China.
E-mail addresses: yananzhao888@126.com (Y. Zhao), pengcan@ahtcm.edu.cn (C. Peng), cyzhang6@ustc.edu (C. Zhang).
1
Yaoyao Xiao and Lele Zhou contributed to equally to this paper and should be regarded as co-first authors.

https://doi.org/10.1016/j.ejps.2023.106664
Received 25 July 2023; Received in revised form 15 November 2023; Accepted 4 December 2023
Available online 5 December 2023
0928-0987/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Y. Xiao et al.
keratinocytes (Lin et al., 2007). Melanin is synthesized by the hydrox­
ylation of phenylalanine to L-tyrosine or directly initiated by L-tyrosine,
and then hydroxylation of L-tyrosine to L-dihydroxyphenylalanine
(Kumari et al., 2018). Tyrosinase (TYR), tyrosinase-associated protein 1
(TRP1), and tyrosinase-associated protein 2 (TRP2) are involved in the
melanin synthesis pathway (Lu et al., 2021). The
microphthalmia-associated transcription factor (MITF) is a transcription
factor containing a basic helix-loop-helix-leucine zipper structure,
which mainly regulates the development, survival, and function of
melanocyte (La Spina et al., 2020). When MITF is activated, the
expression of TYR, TRP1, and TRP2 increases, ultimately promoting
melanin synthesis in melanocytes (Sun et al., 2017).
Skin is the largest organ of the body, primarily consisting of the
epidermis, dermis, and hypodermis, which is the first barrier against
external factors (Ventrelli et al., 2015). Transdermal drug delivery sys­
tems (TDDS) have garnered significant attention for avoiding the
first-pass effect, improving patient compliance, expanding the scope of
action, and reducing the frequency of administration (Durand et al.,
2012; Kassem et al., 2018). Transdermal patches, gel, emulsions, and
liposomes are common dosage forms of TDDS (Mao et al., 2019).
However, these carriers exhibit low skin permeability and transdermal
flux (Elsayed et al., 2007). To address these challenges, a new vesicular
carrier, ethosome has been developed (Abdallah et al., 2021). Ethosome
is a soft and malleable vesicular system, mainly composed of phospho­
lipid (PL), water, and ethanol (Limsuwan et al., 2017). Compared with
classical liposomes, the unique characteristics of ethosomes is their high
ethanol content (20–45 %), which improves the stability of TDDS vesicle
(Natsheh et al., 2019). Additionally, the high deformability of etho­
somes enhances the skin permeability, thereby enhancing the trans­
dermal performance of both hydrophilic and hydrophobic drugs (Li
et al., 2012). Ethanol is a well-known osmotic enhancer that gives ly­
sosomes unique properties, including high elasticity and deformability,
allowing them to penetrate deeply into the skin, enhancing drug pene­
tration and deposition (Paiva-Santos et al., 2021). Transethosome (TE),
considered as the next generation ethosome vesicular system, is an
elastic nanovesicle based on PL, ethanol, water, and edge surfactants
(Song et al., 2019). The edge surfactants include sodium cholate, sodium
deoxycholate, Tween 80, dipotassium glycyrrhizinate, etc., which can
disturb the bilayer structure of PL, increase the fluidity and flexibility of
TE, and can pass through the stratum corneum to the dermis through
extrusion deformation after external use (Romero et al., 2013). Vera
Pérez et al. (2022) proposed TE as a potential TDDS for an extract of
Chenopodium murale to overcome its low solubility and inadequate
molecular size in skin therapy.
Paeonia lactiflora Pall and Glycyrrhiza uralensis Fisch are commonly
used traditional Chinese medicine (TCM) pairs in many classic pre­
scriptions (Liang et al., 2020). Their active ingredients, Paeoniflorin (PF)
and Glycyrrhizic acid (GL), are respectively present in high concentra­
tions in the classic whitening TCM compound preparation ’San-Bai
Decoction’ (Xiao et al., 2022). Paeonia lactiflora Pall has been used as a
traditional whitening TCM for thousands of years, which can brighten
skin and alleviate skin disorders (Nie et al., 2020). PF is the main
component extracted from the dried root of Paeonia lactiflora Pall (Ma
et al., 2015). As a monoterpene glycoside compound (Fig. 1A), PF is the
main active ingredient for reducing skin pigmentation (Qiu et al., 2016).
However, the poor lipophilicity of PF limits its transdermal delivery and
permeable absorption (Liu et al., 2006). Zhang et al. (2017) prepared a
novel glycerosome carrier containing essential oils for transdermal PF
delivery, which enhanced the skin permeability of PF and improved the
drug absorption synovium.
Glycyrrhiza uralensis Fisch can neutralize and remove toxic sub­
stances from the skin, making it a classic TCM whitening skin care
(Castangia et al., 2015). When used in combination with other medi­
cines, Glycyrrhiza uralensis Fisch will enhance their synergistic effects
(Luo et al., 2021). As, a pentacyclic triterpenoid saponin compound
(Fig. 1B), GL has whitening skin, anti-inflammatory, anti-allergic, and
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European Journal of Pharmaceutical Sciences 192 (2024) 106664
immunomodulatory activities that can enhance enzymatic antioxidation
and reduce oxidative stress (Sutticha et al., 2021; Wang et al., 2021b).
With its an amphiphilic structure, GL can improve the solubility (Wang
et al., 2021b), and bioavailability (Liu et al., 2006) of other combination
drugs (Nokhodchi et al., 2002; Wang et al., 2016). However, the high
molecular weight (822.93) and low skin permeability of GL hinder its
efficacy in skin treatment (Barone et al., 2020; Yamamoto et al., 2013).
Shen et al. (2021b) improved the skin permeability and
anti-inflammatory effect of GL by developing pluronic
F127/D-a-tocopherol polyethylene glycol 1000 succinate mixed
micelles-based hydrogel.
Based on the structural and properties advantages of TE in increasing
skin permeability, PF-GL TE was prepared here for the first time to
improve the transdermal absorption and anti-melasma efficacy of PF
and GL. Meanwhile, PF-GL TE was gelated with carbomer-940 to in­
crease its adhesion to the skin surface and control drug release. After
formula optimization, we conducted in vitro drug release and in vitro skin
penetration test. Further, we investigated in vivo efficacy of PF-GL TE gel
on the melasma model rats induced by progesterone injection and UVB
irradiation, detected the melanin-related proteins expressions by West­
ern blotting to explore the potential anti-melasma mechanism of PF-GL
TE gel. In this study, a comprehensive in vitro-in vivo evaluation of PF-GL
TE gel was performed to provide a promising nano-TDDS for effective
transdermal delivery of PF and GL and their prevention and treatment of
melasma.
2. Materials and methods
2.1. Materials
Paeoniflorin (PF, batch number: ZLSW201128–1, purity ≥ 98 %) was
purchased from Xi’an Zelang Biotechnology Co., Ltd (Xi’an, China).
Glycyrrhizic acid (GL, batch number:20122305, purity ≥ 98 %) refer­
ence substance was purchased from Chengdu Man Site Biological
Technology Co., Ltd (Chengdu, China). Phosphotungstic acid was pur­
chased from Shanghai Macleans Biochemical Technology Co., Ltd
(Shanghai, China). Glycyrrhizic acid (GL, batch number: N09GS166788,
purity ≥ 95 %) was purchased from Shanghai Yuanye Biotechnology
Co., Ltd (Shanghai, China). Phospholipid (PL, purity > 98 %) was gained
from Shanghai Tywei Pharmaceutical Co., Ltd (Shanghai, China). Hy­
droquinone cream (Batch Number 20211205, specification: 10 g/piece)
was produced by Guangdong Renrenkang Pharmaceutical Co., Ltd
(Guangdong, China). Progesterone injection (Batch No. 220114, speci­
fication: 50 mg/mL) was gained from Shanghai Quanyu Biotechnology
(Zhumadian) Animal Pharmaceutical Co., Ltd (Shanghai, China). So­
dium cholate (Batch Number 1216S021, purity ≥ 95 %) was obtained by
Beijing Solibor Technology Co., Ltd (Beijing, China). High performance
liquid chromatography (HPLC) grade acetonitrile and methanol were
obtained from Merck (Darmstadt, Germany). Carbomer-940 (batch
number: C12550651) was purchased from Shanghai Macleans
Biochemical Technology Co., Ltd (Shanghai, China). Triethanolamine
(batch number: 20201005) was purchased from Tianjin Damao Chem­
ical Reagent Factory (Tianjin, China). LOGAN Transdermal diffusion
SYSTEM 918-6/24 (USA). Superoxide dismutase (SOD) and malondial­
dehyde (MDA) kit (Nanjing Jiancheng Institute of Bioengineering).
Narrow spectrum 311 UV lamp (Philips, Model: PL-S9W/01/2P).
The antibodies used in this research were rabbit anti-Tyrosinase
(Jiangsu Philogeneic Biology Research Center Co., Ltd., Jiangsu,
China), anti-TRP2 (Wuhan Sanying Biotechnology Co., Ltd., Wuhan,
China), anti-MITF (Abcam, Cambridge, UK), anti-TRP1 (Abcam, Cam­
bridge, UK), and mouse anti-GAPDH (ZSJQ Biotechnology Co., Ltd.,
Beijing, China).
Y. Xiao et al. European Journal of Pharmaceutical Sciences 192 (2024) 106664

Fig. 1. Chemical structures of (A) paeoniflorin and (B) glycyrrhizic acid; Appearance of (C) PF-GL-TE and (D) PF-GL-TE gel; (E) The particle size and (F) TEM image
of PF-GL-TE.

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Y. Xiao et al. European Journal of Pharmaceutical Sciences 192 (2024) 106664

2.2. Methods Table 2

Orthogonal design and results.
2.2.1. Preparation of PF-GL-TE and PF-GL-TE gel NO. A (mg/ B C D PF EE GL EE Total EE
PF-GL-TE was prepared by the ethanol injection method. A certain mL) (mg) (◦ C) (%) (%) (%)
amount of sodium cholate, GL, and PF were dissolved in PBS, and PL was 1 1 1 1 1 62.55 90.05 76.3
completely dissolved in ethanol. Under certain temperatures and stirring 2 1 2 2 2 62.8 86.93 74.87
conditions, PF-GL-TE was formed by dropping ethanol solution into PBS 3 1 3 3 3 24.96 49.74 37.35
solution. In the optimization of prescription, the single-factor experi­ 4 2 1 2 3 34.64 70.71 52.67
5 2 2 3 1 44.76 50.58 47.67
ment was firstly conducted to investigate the effects of PL concentration 6 2 3 1 2 40.72 24.41 32.56
(A), amount of sodium cholate (B), and temperature (C) on PF-GL-TE. 7 3 1 3 2 22.72 39.96 31.34
After that, the optimal three levels were selected for each factor 8 3 2 1 3 27.57 13.34 20.46
(Table 1), and the L9 (34) orthogonal design of three factors with three 9 3 3 2 1 30.71 19.96 25.34
K1 62.84 53.44 43.11 49.77
levels (Table 2) was carried out to study the influence on the encapsu­
K2 44.30 47.67 50.96 46.26
lation rate (EE%) of PF-GL-TE. K3 25.71 31.75 38.79 36.83
To prepare PF-GL-TE gel, carbomer-940 aqueous solution with three R 37.13 21.69 12.17 12.94
concentrations (20 mg/mL, 40 mg/mL, 60 mg/mL) was added to PF-GL-
Note: A: Concentration of PL. B: Amount of sodium cholate. C: Temperature. D:
TE in batches and continuously ground in this addition process. Finally, Blank column.
PF-GL-TE gel was gelated by adjusting the pH value of 6.8 with K: The average of the sum of the response values of each factor at each level.
triethanolamine. R: The range value between the response values of the same factor at different
The preparation of blank TE gel is consistent with the above steps, levels.
except that PF and GL are not added.
As a control, PF-GL mixture gel was prepared by directly dispersing 2 rotary rheometer (TA company, USA). A lamina with a diameter of 20
sodium cholate, GL, PF, ethanol, and PL in PBS, and then simply mixed mm and a cone angle of 1.017◦ was selected for the upper fixture, and
with carbomer-940 aqueous solution. the appropriate sample (about 200 mg) was placed in the center of the
lower fixture. The sensor was adjusted to narrow the distance between
2.2.2. Characterization of PF-GL-TE and PF-GL-TE gel the upper and lower fixtures to achieve the required geometric gap for
measurement. At 37 ◦ C, rheological properties of PF-GL-TE gel were
2.2.2.1. Average particle size and polydispersion index (PDI) of PF-GL- evaluated by analyzing its viscosity and elasticity.
TE. The particle size and PDI of PF-GL-TE were measured by the dy­
namic laser scattering (DLS) method on Malvin Zetasizer 3000HS 2.2.4. In vitro release of PF-GL-TE gel
(Malvin Instrument, Malvern, UK), with three parallel data for each. The in vitro drug release behaviors of PF-GL-TE gel and PF-GL
mixture gel were investigated by the dialysis bag method. PF-GL-TE
2.2.2.2. EE % of PF-GL-TE gel. The EE % of PF-GL-TE was determined gel (10 g) and PF-GL mixture gel (10 g) with equal amounts of PF and
by ultrafiltration method according to our previous studies (Wang et al., GL were respectively placed in three dialysis bags (≤12 kDa, China). The
2021a). Briefly, PF-GL-TE gel (0.5 mL) were placed into the upper release medium was phosphate buffered solution (PBS), with a release
chamber of ultrafiltration tubes (3 kDa, Millipore Corporation, Billerica, temperature and stirring speed of 37 ± 0.5 ◦ C and 100 rpm, respectively.
MA, USA) and centrifuged at 5000 rpm for 10 min. The total content of At certain intervals (0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 h), 0.2 mL of release
PF and GL were measured after extraction with methanol in an ultra­ medium was taken out for HPLC analysis (Huang et al., 2021), while
sonic bath and the free drug was collected from the sample recovery supplementing with an isothermal amount of fresh PBS.
chamber, which were determined by high-performance liquid chroma­
tography (HPLC). The EE% was calculated using the following equation. 2.2.5. In vitro skin penetration study
( / )
EE% = 1 − Wt Wf × 100%
2.2.5.1. Fabrication of isolated rat skin. Female SD rats (200±20 g) were
where Wt is the total amount of PF or GL in PF-GL-TE gel and Wf is the purchased from Anhui Experimental Animal Center (2021089, China).
amount of free drug of PF or GL unencapsulated in PF-GL-TE gel. All The experiment was conducted under controlled conditions (20–25 ◦ C,
samples were performed in triplicate. 60–70 % humidity, 12:12 h light/dark cycle). Rats were adaptively fed
for a week and anesthetized with 1.2 g/kg of urethane. After removing
hair from the abdominal skin and cutting off their necks, the abdominal
2.2.2.3. The morphology of PF-GL-TE. A small amount of PF-GL-TE with
skin was taken with blunt scissors. Finally, the subcutaneous fat and
appropriate concentration was dropped onto the copper wire with
tissue were removed and cleaned with normal saline to obtain the skin
supporting film. Then, 2 % of phosphotungstic acid (W/V) was dropped
for transdermal test.
and dyed. After drying for a while, its morphology was observed by
transmission electron microscopy (TEM, Tecnai G2 SpirtBiotwin,
Thermo, USA) and the photos were recorded. 2.2.5.2. Transdermal penetration test. The rat abdominal skin was fixed
on the diffusion pool of the LOGAN transdermal instrument (LOGAN
2.2.3. Rheological property study of PF-GL-TE gel company, USA) with an effective diffusion area of 1.76 cm2. The cuticle
The rheological properties of PF-GL-TE gel were analyzed by a DHR- faced the administration chamber, and the dermis faced the acceptance
chamber. The chamber volume was 12 mL, and the receiving solution
was PBS solution (pH = 7.4). The temperature and the speed are 37±0.5
Table 1 ◦
C and 600 r⋅min− 1, respectively. 1.5 g PF-GL-TE gel and PF-GL mixture
Single-factor factors and levels for orthogonal experiment. gel were injected into the administration chamber and evenly coated on
Factors Levels the surface of the rat skin. At 1, 2, 4, 6, 8, 10, 12, and 24 h, the liquid in
1 2 3 the receiving chamber was filtered by 0.22 µm microporous membrane.
Finally, the drug concentration was determined by HPLC to compare the
A 15 mg/mL 20 mg/mL 25 mg/mL
B 2 mg 4 mg 6 mg
cumulative transdermal amount of PF-GL mixture gel and PF-GL-TE gel
C 30 ◦ C 40 ◦ C 50 ◦ C at different times.

4
Y. Xiao et al.
2.3. Animals experimental design
Healthy female SD rats (200 ± 20 g) were procured from Anhui
Medical University with license number SCXK (Wan) 2017–001. All
animal experimental treatments were approved by the Ethics Committee
of Anhui University of Chinese Medicine (AHUCM-rats-2022147). After
adaptive feeding for one week, the SD rats were randomly assigned to six
groups (n = 6): the control group, the melasma model group, blank TE
gel group, PF-GL mixture gel group, PF-GL-TE gel group, and the posi­
tive group (hydroquinone cream).
According to the Refs. Krishnaraj et al. (2020), Miao et al. (2017), the
melasma model rat was established with progesterone injection com­
bined with UVB irradiation. Briefly, the back hair of SD rats was
removed with an electric razor and 8 % Na2S aqueous solution, and the
back skin was fully exposed about 3 cm × 3 cm. Then, SD rats were
intramuscularly injected with progesterone at a dose of 20 mg /kg/d,
and the exposed back skin was irradiated under UVB (290–320 nm; 72
mJ/cm2) once a day to induce pigmentation. The melasma model rat
was established after 30 days of combined treatment. Comparably, the
controls group was injected with normal saline.
Simultaneously, each group was given treatment medication on the
back skin for 30 days while modeling. The back skin of the control group
was not treated, and the positive drug group was treated with hydro­
quinone cream. The back skin of the other three groups were respec­
tively treated with blank TE gel, PF-GL mixture gel, and PF-GL-TE gel
once a day for 30 days.
2.4. Effect of PF-GL-TE gel on the activities of MDA and SOD in skin and
liver tissues of melasma model rats
Protein levels in the skin and liver were determined by BCA protein
quantification kit (Beyotime, Haimen, China). The prevention effect of
melasma was evaluated by measuring the activities of MDA and SOD in
skin and liver tissues, detected by commercial kits (Nanjing Jiancheng
Biological Engineering Research Institute, China). All procedures were
followed by the manufacturer’s instructions.
2.5. H&E and Masson staining of skin specimen
At the end of the experiment, all rats were sacrificed, and skin
samples were collected, fixed with 10 % formalin, and embedded in
paraffin. The paraffin blocks were then cut into 5~8 μm thin slices with
an ultra-thin machine. The skin sections were stained with H&E, and the
collagen in the skin sections was stained with Masson trichromatic
staining (Kuo et al., 2015; Yuan et al., 2022) and examined under a
microscope.
2.6. Detection of the expression levels of melanin-related proteins
Skin tissue (100 mg) was washed with precooled PBS, dried, then
transferred to liquid nitrogen for grinding, and placed in EP tubes con­
taining RIPA lysates. After the tissue was cleaved on ice for more than
30 min, it was finally centrifuged at 4 ◦ C at 12,000 rpm for 10 min. Part
of the supernatant was collected and the protein concentration was
determined with the BCA protein quantification kit. The remaining su­
pernatant was added to 5x loading buffer (supernatant: loading buffer =
4:1) and boiled at 100 ◦ C for 10 min. Proteins (30 μg) were separated by
8 %− 12 % SDS-PAGE, transferred to PVDF membranes (0.45 μm), and
closed with 5 % skim milk. After incubating with specific primary an­
tibodies (MITF 1:200, TYR 1:1000, TRP1 1:1000, TRP2 1:1000, GAPDH
1:1000) at 4 ◦ C overnight, the membranes were soaked in TBST three
times for 10 min each (2 L TBS buffer + 1 mL Tween20). Subsequently,
the membranes were incubated in secondary antibody (1:10,000 or
1:20,000) at about 25 ◦ C for 1.5 h. Finally, the incubated antibody bands
were uniformly coated with enhanced chemiluminescence solution
(solution A: solution B = 1:1) and detected in the chemiluminescence
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European Journal of Pharmaceutical Sciences 192 (2024) 106664
system (ProteinSimple, Silicon Valley, USA). Image J software (version
1.45S, NIH, USA) was used for specific bands.
2.7. Statistical analysis
GraphPad Prism 8.0.2 software and Origin 2018 were used for sta­
tistical analysis, and one-way analysis of variance (ANOVA) and Bon­
ferroni post hoc test were used for statistical comparison. Data were
expressed as mean ± SD, and P < 0.05 was considered a statistically
significant difference.
3. Results
3.1. Characterization of PF-GL-TE and PF-GL-TE gel
According to the optimal three levels of each factor (Table 1) and the
range analysis in Orthogonal experiment in Table 2, the order of influ­
ence on total EE (%) is PL concentration (A) > amount of sodium cholate
(B) > temperature (C). Based on the EE% value, the best process con­
dition is A1B1C2. Briefly, a certain amount of PL was dissolved in
ethanol to obtain a 15 mg/mL of solution. Then, this solution was
dropped into 20 mL of PBS (pH = 7.4) containing 2 mg of sodium
cholate, 4 mg of GL, and 4 mg of PF. The mixture was stirred at 40 ◦ C and
300 rpm for 60 min, and PF-GL-TE with blue opalescence was obtained
(Fig. 1C). PF-GL-TE and carbomer-940 aqueous solution (c = 60 mg/mL)
were mixed and ground evenly, and the PF-GL-TE gel was obtained by
adjusting the pH value to about 6.8 with triethanolamine (Fig. 1D). The
final compositions of PF-GL-TE gel was shown in Table 3 with PF-GL
mixture gel as the control.
Fig. 1E showed that the average particle size and the PDI of PF-GL-TE
were 167.9 ± 4.7 nm and 0.102 ± 0.009, respectively, indicating that its
particle size was small and uniform. The morphology of PF-GL-TE
observed under TEM was a uniform distributed spherical shape (Fig. 1F).
3.2. Rheological tests of PF-GL-TE gel
To study the rheological behavior of PF-GL-TE gel in the shear rate
range of 0.1–200 s− 1, the rheometer was set to a flow-sweep measure­
ment mode for steady shear test. For comparison, PF-GL-TE was gelled
with carbomer-940 at three concentrations of 20 mg/mL, 40 mg/mL,
and 60 mg/mL to obtain different viscosities of PF-GL-TE gels. The
rheological curves of three PF-GL-TE gels were shown in Fig. 2A. It can
be observed that the viscosity of three PF-GL-TE gels decreases with the
increase of shear rate. This shear-thinning property indicates that three
PF-GL-TE gel belong to the pseudo-plastic fluid in the non-Newtonian
fluid category.
In the Oscillation-Amplitude mode, the linear viscoelastic region of
three PF-GL-TE gels was monitored at a fixed frequency of 10 rad⋅s− 1
and a strain range of 0.01–50 %. The results (Fig. 2B–D) showed that
their G’ values were consistently greater than their G’’ values with solid
properties, and their linear viscoelastic region appeared in the strain
range of 0.05 %~0.5 %.
Based on the results of the linear viscoelastic region of three PF-GL-
TE gels, a fixed stress of 0.1 % was selected for frequency scanning tests.
In the Oscillation-Amplitude mode, the frequency range was set to
0.01–100 Hz to further investigate the variation of the modulus with
frequency. As shown in Fig. 2E–G, their G’ values were consistently
greater than the G’’ values, indicating that three PF-GL-TE gels were
elastic systems.
Fig. 2H displays the scanning diagram between the composite vis­
cosity and frequency of three PF-GL-TE gels with different concentra­
tions of carbomer-940. By comparing their viscosities, PF-GL-TE gel
gelated with carbomer-940 at a concentration of 60 mg/mL is most
suitable for TDDS. Therefore, PF-GL-TE gel gelated with 60 mg/mL of
carbomer-940 aqueous solution was selected as the final gel condition
for the subsequent studies.
Y. Xiao et al. European Journal of Pharmaceutical Sciences 192 (2024) 106664

Table 3
Compositions of PF-GL-TE gel and PF-GL mixture gel.
Formulation sodium cholate (mg) GL (mg) PF (mg) ethanol (mL) PL (mg) carbomer-940 (mg/mL) PBS (mL)

PF-GL-TE gel 2 4 4 5 70 60 20
PF-GL mixture gel 2 4 4 5 70 60 20

Fig. 2. (A) Rheology curves for three concentrations of carbomer; The linear viscoelastic region scanning of three concentrations of carbomer: (B) 20 mg/mL
carbomer, (C) 40 mg/mL carbomer, (D) 60 mg/mL carbomer; The variation of storage modulus G’ and loss modulus G’’ with frequency: (E) 20 mg/mL carbomer, (F)
40 mg/mL carbomer, (G) 60 mg/mL carbomer; (H) The variation of complex viscosity with frequency.

3.3. In vitro drug release of PF-GL-TE gel
The in vitro release curve of PF and GL in PF-GL-TE gel was shown in
Fig. 3A and B. The release rates of PF in PF-GL-TE gel (Fig. 3A) at 12 h
and 48 h were found to be 46.26 % and 70.47 %, respectively. In
contrast, the release rates of PF in PF-GL mixture gel at 12 h and 48 h
were 70.76 % and 92.92 %, respectively. For GL, the release rates in PF-
GL-TE gel were 41.68 % and 65.91 % at 4 h and 12 h, respectively, while
the corresponding release rates in PF-GL mixture gel were 61.76 % and
98.36 %, respectively (Fig. 3B). The results showed that PF-GL-TE gel
had a better sustained-release effect compared with PF-GL mixture gel
(Fig. 3A, 3B).
3.4. In vitro skin penetration test of PF-GL-TE gel
The cumulative skin penetration results of PF-GL-TE gel and PF-GL
mixture gel at different times were shown in Fig. 4. In PF-GL-TE gel,
the skin penetration percentages of PF at 12 h and 24 h were 41.46 μg/
cm2 and 65.56 μg/cm2, respectively. At the corresponding time points,
the skin penetration percentages of GL were 59.23 μg/cm2 and 88.79
μg/cm2, respectively (Fig. 4A). In PF-GL mixture gel, the skin penetra­
tion percentages of PF were at 12 h and 24 h 30.84 μg/cm2 and 50.49
μg/cm2, respectively, and the penetration percentages of GL were 48.42
μg/cm2 and 57.11 μg/cm2, respectively (Fig. 4B). These results indicated
that PF-GL-TE gel had better skin permeability than PF-GL mixture gel at
the same time points.
3.5. Preventive and therapeutic effects of PF-GL-TE gel on the skin of
melasma model rats
After 30 days of treatment, the appearance of the back skin in each
group was observed in Fig. 5. Obviously, some melasma pigmentation

Fig. 3. Release profiles of (A) PF and (B) GL in PF-GL-TE gel and PF-GL mixture gel in PBS (pH=7.4) at 37 ± 0.5 ◦ C (n = 3).

6
Y. Xiao et al. European Journal of Pharmaceutical Sciences 192 (2024) 106664

Fig. 4. In vitro skin penetration and transdermal absorption of (A) PF and (B) GL in PF-GL-TE gel and PF-GL mixture gel (n = 3).

Fig. 5. (A) Skin appearance of rats in each group, (B) Typical images of Hematoxylin and eosin (H&E) stained skin slices of rats in each group (scale bars: 200 μm),
and (C) Representative images of Masson-stained skin slices of rats in each group (scale bars: 200 μm).
Note: (a) control group; (b) model group; (c) blank TE gel; (d) PF-GL mixture gel group; (e) PF-GL-TE gel group; (f) hydroquinone cream group.

appeared on the back skin of the model group (Fig. 5(A), b), indicating PF-GL mixed gel group, the treatment did not effectively reduce the
that progesterone injection combined with UVB irradiation is an effec­ pigmentation (Fig. 5(A), c, d). Like the control group, no obvious
tive method for melasma modeling. In the blank TE gel group and the pigmentation was observed on the back skin in the PF-GL-TE gel group

7
Y. Xiao et al.
(Fig. 5(A), e) and the positive group (hydroquinone cream) (Fig. 5(A), f).
Interestingly, the skin in the PF-GL-TE gel group was smooth and moist,
suggesting that PF-GL-TE gel can effectively fade out pigments and
melasma, and whiten and moisturize skin.
3.6. Histochemical staining
According to the results of hematoxylin and eosin (H&E) staining in
Fig. 5(B), there were many inflammatory cells observed in the melasma
model group, indicating that the combined model had inflammatory
damage to the rats’ skin. In the blank TE gel group and PF-GL mixture
gel group, inflammatory cells were infiltrated in the dermis and sub­
cutaneous tissues. Fortunately, there was no obvious skin inflammation
observed in PF-GL-TE gel group and hydroquinone cream group.
Compared with the control group, the amount of collagen in the
dermis stained blue by Masson’s tricolor in the model group was
significantly decreased, indicating that the combined modeling resulted
in severe loss of skin collagen (Fig. 5C). After 30 days of treatment, the
content and density of collagen in PF-GL-TE gel group and hydroquinone
cream group recovered significantly. However, the loss of collagen in the
blank TE gel group and PF-GL mixture gel group was not well alleviated.
Fig. 6. The activity of SOD in (A) liver and (B) skin of rats; MDA content in (C) liver and (D) skin. Data are presented as the mean ± SD (n = 6). Significant dif­
ferences compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001; compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001; compared
with the PF-GL mixture gel group, &p < 0.05, &&p < 0.01, &&&p < 0.001.
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European Journal of Pharmaceutical Sciences 192 (2024) 106664
3.7. Determination of the activities of SOD and MDA in the liver and skin
of rats
In this study, the activities of SOD and MDA in the liver and skin of
rats were detected to determine the oxidative injury in each group. Fig. 6
showed that the MDA content in the liver and skin of the melasma model
group was significantly higher than that of the normal control group,
while the SOD activity was lower than that of the control group, indi­
cating that the liver and skin of the melasma model rats were damaged
by oxidation during modeling. Compared with the model group, the
activities of SOD in liver and skin of PF-GL-TE gel group and hydro­
quinone cream group were relatively increased, and the corresponding
MDA contents were obviously decreased. These results demonstrate that
PF-GL-TE gel improves the activity of SOD and reduces the content of
MDA in the melasma model rats, meaning it can prevent and alleviate
oxidative damage in the liver and skin.
3.8. Western blotting assay on the expressions of MITF/TYR/TRP1 and
TRP2 proteins
To explore the potential mechanism of PF-GL-TE gel in preventing
and treating melasma, the effects of PF-GL-TE gel on the expression of
MITF/TYR/TRP1 and TRP2 proteins were analyzed by Western blotting
(Fig. 7). The results showed that the expressions of MITF/TYR/TRP1 and
Y. Xiao et al.
Fig. 7. Western blotting assay on the expression of MITF/TYR/TRP1 and TRP2 proteins. (A) Immunoblot assays of MITF, TYR, TRP1, and TRP2. Relative densities of
(B-E) MITF, TYR, TRP1, and TRP2. Data are presented as the mean ± SD (n = 6). Significant differences compared with the control group, #p < 0.05, ##p < 0.01,
###
p < 0.001; compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with the PF-GL mixture gel group, &p < 0.05, &&p < 0.01, &&&p
< 0.001.
TRP2 proteins in the model group were significantly increased by about
1.4–1.9 times compared to the control group. Compared with the model
group, the proteins expressions of MITF/TYR/TRP1 and TRP2 in the PF-
GL-TE gel group were significantly down-regulated, and the corre­
sponding proteins expressions were about 0.5–0.7 times lower than
those in the model group. These results suggest that PF-GL-TE gel may
prevent and treat melasma through downregulating the expression of
MITF/TYR/TRP1 and TRP2 proteins.
4. Discussion and conclusion
Melasma is an acquired pigmentation disorder that commonly occurs
on the face. It is more prevalent in women and dark skin types, mainly
due to UV exposure and hormonal influences (Ogbechie-Godec et al.,
2017). According to the occurrence of endocrine-related melasma, the
increase of estrogen and progesterone levels can stimulate melanocytes
to produce pigmentation (Konisky et al., 2023). The formation of mel­
asma is primarily due to the enhanced activity and function of
melanin-producing cells, which secrete more melanin particles (Cayce
et al., 2004). UVB irradiation can activate the function of melanocytes to
increase the production of melanin (Jian et al., 2014). Therefore, the
combination of progesterone injection and UVB irradiation was used to
simulate the melasma model rat in this study. Our existing experimental
results showed that the back skin of the model rats had obvious
pigmentation, collagen loss, the inflammatory reaction caused by
oxidative damage, and increased expressions of melanin-related pro­
teins, which indicated that the combined modeling could successfully
construct the melasma model rats.
PF and GL are whitening compounds capable of reducing skin
pigmentation, enhancing enzymatic antioxidation, and reducing the
content of oxidative stress markers, which are the main effective com­
ponents of ’Shaoyao-Gancao Decoction’ and ’San-Bai Decoction’ (Ding
et al., 2016; Sutticha et al., 2021). However, there is currently no suit­
able dosage form to transdermal deliver them simultaneously and
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European Journal of Pharmaceutical Sciences 192 (2024) 106664
enhance their skin permeabilities. Based on the advantage of TE (an
elastic nanovesicle) that can improve skin penetration rate of drugs and
extend drug release time (Lalit et al., 2020), PF-GL-TE gel prepared in
this study overcame their TDDS challenge and improved their
anti-melasma efficacy. Through orthogonal experiment optimization,
we prepared uniform PF-GL-TE nanovesicle (167.9 ± 4.7 nm) with
spherical morphology (Fig. 1), which is beneficial to transdermal de­
livery. By the sustained-release effect of TE and gelated carbomer-940,
PF-GL-TE gel showed a lower in vitro drug release than PF-GL mixed
gel (Fig. 3). Meanwhile, the results of in vitro skin penetration experi­
ments showed the skin permeation of PF-GL-TE gel was higher than that
of PF-GL mixture gel (Fig. 4). Specifically, the first reason for the higher
skin permeability of new nano PF-GL-TE gel is that TE, as a new type of
super-deformable nanovesicle, can effectively promote drugs through
the epidermis of the skin with its high deformability, elasticity, and
nano-size effect (Raj et al., 2023). Seconly, fully dispersed and dissolved
ethanol and phospholipid in TE can change the regular arrangement of
lipid molecules in the stratum corneum, enlarge the cell gap, increase
lipid fluidity, and better promote drug penetration (Akhtar et al., 2022;
Andleeb et al., 2021; Huang et al., 2021). Thirdly, the released GL from
PF-GL-TE gel can serve as a penetration enhancer with its amphiphilic
structure to further improve the skin penetration of the drugs by
combining with TE (Rajan et al., 2012; Shen et al., 2021a). Finally,
PF-GL-TE gel has good viscosity, which increased its adhesion with the
skin surface and effectively promoted skin penetration.
In PF-GL mixed gel, the drugs (PF and GL), PL, ethanol, and sodium
cholate were directly mixed with carbomer-940 aqueous solution,
leading to coarse dispersion between components, poor drug encapsu­
lation, rapid drug release, and low skin permeability. In addition, the
direct mixing of ethanol with 60 mg/mL of carbomer-940 reduced the
viscosity of mixed gel, further resulting in poor skin adhesion and
permeability of PF-GL mixed gel.
When the body generates excessive oxygen free radicals, the activity
of antioxidant enzymes (such as SOD) will be reduced, resulting in
Y. Xiao et al.
membrane lipid peroxidation to form lipid peroxides (LPO) (He et al.,
2018). LPO is unstable and rapidly decomposes into aldehydes, with the
final product being MDA. The increase of MDA will attack phospholipids
and proteins of pigment cells to cause oxidative damage and promote
the oxidation reaction of tyrosinase. This above process will increase
melanin and its significant accumulation in the basal layer of skin, which
is one of the pathogenesis of pigmented melasma (Jian et al., 2014). Our
experimental results indicate that PF-GL-TE gel can significantly in­
crease the activity of SOD in the liver and skin tissue, reduce the content
of MDA, and inhibit the tyrosinase activity of melanocytes and the for­
mation of melanin, which is effective in treating melasma.
Melanin synthesis is regulated by genes, proteins, and enzymes, and
melanin-related proteins mainly include MITF, TYR, TRP1, and TRP2
(Alam et al., 2017; Cichorek et al., 2013). In this study, the regulatory
effect of PF-GL-TE gel on the expressions of MITF, TYR, TRP1, and TRP2
proteins were comprehensively evaluated by western blotting. In this
study, we evaluated the influence of PF-GL-TE gel on the expressions of
MITF, TYR, TRP1 and TRP2 proteins by Western blot. The experimental
results showed that PF-GL-TE gel significantly inhibited the expression
of these proteins to varying degrees, and the potential mechanism might
involve inhibiting of MITF, down-regulating the levels of TYR, TRP1 and
TRP2, and ultimately preventing the synthesis and accumulation of
melanin.
In summary, PF-GL-TE was optimized through an orthogonal
experiment, and further gel to obtain PF-GL-TE gel as a new TDDS
formulation. The pharmacodynamic experiment results showed that PF-
GL-TE gel could significantly prevent and treat melasma and alleviate
the inflammation and collagen loss in the melasma model rats. PF-GL-TE
gel exhibited antioxidant effects by increasing the activity of SOD and
decreasing the content of MDA in the liver and skin. Furthermore, the
results of western blot indicated that PF-GL-TE gel effectively inhibited
the expressions of melanin-related proteins MITF/TYR/TRP1 and TRP2,
which revealed the potential mechanism of its prevention and treatment
of melasma.
CRediT authorship contribution statement
Yaoyao Xiao: Investigation, Writing – original draft, Writing – re­
view & editing. Lele Zhou: Investigation, Writing – original draft,
Writing – review & editing. Wenkang Tao: Data curation, Writing –
review & editing. Xuan Yang: Data curation, Writing – review & edit­
ing. Junying Li: Data curation, Writing – review & editing. Rulin
Wang: Writing – review & editing. Yanan Zhao: Investigation, Super­
vision, Writing – original draft, Writing – review & editing. Can Peng:
Investigation, Supervision, Writing – original draft, Writing – review &
editing. Caiyun Zhang: Investigation, Supervision, Writing – original
draft, Writing – review & editing.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Data availability
Data will be made available on request.
Acknowledgments
This research was funded by the Project of National Natural Science
Foundation of China (51303006), Provincial Natural Science Founda­
tion of Anhui Province (202203a07020031, KJ2021ZD0065,
KJ2019A0314, and KJ2018ZD031), and Center for Xin’an Medicine and
Modernization of Traditional Chinese Medicine of IHM ‘Jie Bang Gua
Shuai’ Project (2023CXMMTCM014)
10
European Journal of Pharmaceutical Sciences 192 (2024) 106664
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