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Int. J. Mol. Sci. 2017, 18, x; doi: www.mdpi.com/journal/ijms
Article
The Potential of Triterpenoids from Loquat Leaves
(Eriobotrya japonica) for Prevention and Treatment of
Skin Disorder
Hui Tan, Tamrakar Sonam and Kuniyoshi Shimizu *
Department of Agro-environmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan; thth229@gmail.com (H.T.); tamrakar.snm@gmail.com (T.S.)
* Correspondence: shimizu@agr.kyushu-u.ac.jp; Tel.: +81-092-642-3002
Academic Editor: Maurizio Battino
Received: 25 February 2017; Accepted: 6 May 2017; Published: date
Abstract: The leaves of loquat (Eriobotrya japonica) possess high medicinal value and have been
used as traditional medicines. However, there are no evidence-based studies on the skin-care
effects of E. japonica leaves. To explore new biological activities of E. japonica leaves against skin
disorder and to gain a better understanding of the chemical components associated with
bioactivities, we evaluated 18 triterpenoids from E. japonica leaves on anti-melanogenesis, anti-acne,
anti-allergy and anti-aging activities. Our results revealed that eight compounds showed
anti-melanogenesis activity, of which ursolic acid (1) and maslinic acid (7) were the most potent
with the similar selective index to that of arbutin. Structure–activity relationship and possible
mechanism of active compounds were proposed. Twelve compounds exhibited anti-acne effect;
ursolic acid (1), maslinic acid (7), corosolic acid (8) and euscaphic acid (12) showed highest
activities against P. acnes. Four compounds displayed anti-allergy and anti-inflammatory activity;
3-epicorosolic acid (9) and euscaphic acid (12) showed marked activity against β-hexosaminidase
release. Finally, ursolic acid (1), pomolic acid (10), colosolic acid (8) and its methylated derivative
(6) exhibited the highest anti-aging activity by stimulating collagen and hyaluronic acid (HA)
production. Our findings provide valuable evidence that E. japonica leaves have potential
applications as ingredients of function foods or cosmetics for health benefits and a number of
triterpenoids may play an important role in these bioactivities.
Keywords: Eriobotrya japonica; loquat leaves; triterpenoids; skin disorder; anti-melanogenesis;
anti-acne; anti-allergy; anti-aging
1. Introduction
The skin accounts for 15% of a human being’s total body weight, and it directly confronts the
external environment serving as a primary defense system [1]. It plays a crucial role in not only
preventing harmful substances, such as ultraviolet radiation, microbial pathogens and antigens
from entering the body, but also in the maintenance of body homeostasis. Skin generally consists of
a three-layer structure: the epidermis, the dermis and subcutaneous tissues. The epidermis is the
outermost layer of the skin and keratinocytes are the most abundant cell type. Keratinocytes act via
their capacity to produce UV-absorbing molecules, inflammatory mediators and antimicrobial
peptides [2]. The dermis is the structure beneath the epidermis, its three primary structural
components are collagen, elastin and glycosaminoglycans, which have been the subjects of the
majority of anti-aging research [3].
Plant-derived natural, bioactive compounds have shown potential in beauty enhancement
along with health benefits against disorders, such as photo-aging, inflammation, oxidation, aging,
bacterial infections and so on [4]. These bioactive compounds continue to gain popularity based on
Int. J. Mol. Sci. 2017, 18, x 2 of 13
their several advantages, which include often having fewer side effects, showing better patient
tolerance, and being relatively less expensive and acceptable due to a long history of use compared
to synthetic ingredients [5]. In the search for effective drug leads from herbs used against skin
abnormalities, bioactive compounds such as aloin [6], curcumin [7], resveratrol [8], gallic acid [9],
quercetin, genistein and catechins [10] have been found.
Loquat (Eriobotrya japonica), a subtropical evergreen tree belonging to the Rosaceae family, is
widely cultivated for its edible fruits. The leaves are well-known traditional Chinese medicine, used
for the treatment of diabetic patients and for soothing effects on the digestive and respiratory
systems. In Japan, loquat leaves are sold as a tasty herbal tea with health benefits. It has been
reported that E. japonica leaves have great potential for the prevention of inflammation [11] and
obesity [12]. In addition, loquat leaves may ameliorate Alzheimer’s disease, since E. japonica leaves
protect against oxidative stress and cognitive deficits induced by the Aβ peptide [13]. Our previous
studies showed that E. japonica have the potential to suppress ovariectomy-induced bone mineral
density deterioration [14], and isolated triterpenoids as the major bioactive compounds suppress
osteoclast differentiation [15]. As a powerful indigenous therapy, loquat leaves have also been used
for skin-care application such as for sunburn, dermatitis, and skin aging. However, to the best of our
knowledge, no study has investigated this bioactivity and desired chemical components from loquat
leaves. Pentacyclic triterpenoids are present at their highest concentration in loquat leaves, and they
have been reported to be related to various bioactivities. Therefore, in the present study we
evaluated the potential involvement in skin-related bioactivity of a series of triterpenoids from E.
japonica leaves (Figure 1), including anti-melanogenesis, anti-acne, anti-allergy, and anti-aging
activity.
Compound
R1
R2
R3
R4
R5
R6
R7
R8
1
β–OH
H
H
COOH
Me
H
H
Me
2
β–OH
H
H
COOH
H
H
Me
Me
3
β–OH
H
H
CH2OH
Me
H
H
Me
5
β–OH
OH
H
COOCH3
H
H
Me
Me
6
β–OH
OH
H
COOCH3
Me
H
H
Me
7
β–OH
OH
H
COOH
H
H
Me
Me
8
β–OH
OH
H
COOH
Me
H
H
Me
9
α–OH
OH
H
COOH
Me
H
H
Me
10
β–OH
H
H
COOH
Me
OH
H
Me
11
β–OH
OH
H
COOH
Me
OH
H
Me
12
α–OH
OH
H
COOH
Me
OH
H
Me
13
=O
H
H
COOH
Me
H
H
Me
14
=O
H
OH
COOH
Me
OH
H
Me
15
=O
OH
H
COOH
Me
OH
H
Me
17
β–trans-p-coumaroyl
OH
H
COOH
Me
OH
H
Me
18
β–cis-p-coumaroyl
OH
H
COOH
Me
OH
H
Me
Figure 1. The structures of the 18 tested triterpenoids from E. japonica leaves.
Int. J. Mol. Sci. 2017, 18, x 3 of 13
2. Results and Discussion
2.1. Anti-Melanogenesis Effect of Triterpenoids
Excessive ultraviolet (UV) radiation stimulates the production of melanin, which leads to
various skin injuries, including inflammation, age spots, melasma and freckles [16]. We found
previously that the methanol extract of E. japonica leaves exhibited significant and dose-dependent
melanogenesis inhibition in B16 melanoma cells (Figure S1). In the present study, we focused on the
potential anti-melanogenesis effect of 18 triterpenoids from E. japonica leaves. We tested various
concentrations’ inhibitory effects on melanin synthesis and cell viability (Tables S1 and S2). The
CC50 values for cell viability and the IC50 values against the melanogenesis of B16 cells are listed in
Table 1. The relative effectiveness of the compounds for inhibiting melanogenesis compared to
inducing cell death is defined as the selectivity index (S.I.). It is desirable to have a high selectivity
index (S.I. > 1.0), which indicate the potential for selective melanogenesis inhibition. Our results
demonstrated that eight of the compounds, namely ursolic acid (1), oleanolic acid (2), betulinic acid
(4), methyl maslinate (5), methyl corosolate (6), maslinic acid (7), corosolic acid (8) and tormentic
acid (11), showed melanin synthesis inhibitory activity with less cytotoxicity (S.I. > 1.0). Among
them, ursolic acid (1) and maslinic acid (7) showed the highest S.I. values at 1.9, which is the same as
the S.I. of arbutin, a skin-lightening ingredient commonly used in the cosmetic industry.
Table 1. Anti-melanogenesis effect of triterpenoids from E. japonica leaves.
No.
CC50 a (μM)
IC50 b (μM)
S.I c
No.
CC50 (μM)
IC50 (μM)
S.I e
1
9.4
4.8
1.9
10
10.5
14.0
0.8
2
39.9
26.8
1.5
11
23.3
18.5
1.3
3
>45.2
>45.2
n.d. d
12
25.6
34.0
0.7
4
16.7
11.8
1.4
13
8.9
18.0
0.5
5
17.5
12.8
1.4
14
10.9
14.8
0.7
6
18.7
16.1
1.2
15
>42.3
>42.3
n.d.
7
29.3
18.5
1.9
16
16.5
21.6
0.8
8
25.8
18.7
1.4
17
24.0
>30.9
n.d.
9
>42.3
>42.3
n.d.
18
19.5
>30.9
n.d.
a The CC50 value is defined as the cytotoxic concentration causing death in 50% of viable cells, and is
calculated from the corresponding dose–response curves (for the individual concentrations see Table
S1); b The IC50 value is defined as the concentration required for 50% inhibition of melanin synthesis,
and is calculated from the corresponding dose–response curves (for the individual concentrations
see Table S2); c The selectivity index (S.I.) was calculated as the ratio of the concentration of the
compound that is required to reduce the cell viability to 50% to the concentration of the compound
needed to inhibit melanogenesis to 50% of the control value (i.e., the CC50 value/IC50 value); d n.d.:
non-detectable; e Arbutin was used as a positive control, its S.I was calculated as 1.9.
In addition, in our comparison of the bioactive chemical structures with those of inactive
compounds, we found that the beta-form of the hydroxyl group at the R1 position may play an
important role in the melanin synthesis inhibition activity, because there was a loss in this activity
when the beta-form of the hydroxyl group was replaced by an alpha-form, (9) and (12), a ketone,
(13)–(15), or another substituent group, (17) and (18). In addition, the carbonyl group at R4 position
may partly contribute to the activity because uvaol (3), via the presence of a hydroxymethyl group
at R4, loses its activity. The methyl esterification of the carboxyl group causes a slight loss of activity,
since (5) and (6) showed weaker activity than (7) and (8).
Tyrosinase is the rate-limiting enzyme for controlling the production of melanin, it can catalyze
the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to
dopaquinone [17]. From dopaquinone, the melanin synthesis pathways diverge to produce either
black-to-brown eumelanin or red-to-yellow pheomelanin. In the present study, we also evaluated a
potential mechanism of the active compounds against melanin synthesis by performing a tyrosinase
inhibitory assay. We use either L-tyrosine or L-DOPA as the substrates. The results revealed that
Int. J. Mol. Sci. 2017, 18, x 4 of 13
compounds (1), (2), (4) and (7) showed moderate inhibitory activity on tyrosinase with L-tyrosine as
substrate at relatively high concentrations, but no inhibitory effect in the case with L-DOPA as
substrate (Figure 2). This result suggests that observed melanogenesis inhibition effect among eight
active compounds, compounds (1), (2), (4) and (7) are at least partly contributed by tyrosinase
inhibitory activity. Our study offers preliminary evidence for selecting potential candidates that
may be used as skin-whitening agents and for further clearly exploring the mechanism of each
compound, melanogenesis-related enzymes including melanocortin type 1 receptor (MC1R),
tyrosinase-related proteins 1 and 2 (TRP1 and TRP2), and protein kinase regulators of
melanogenesis-related signaling pathways (MAPK, PKA signaling pathways) need to be further
discussed.
Figure 2. The effect of active compounds on tyrosinase activity (with respect to L-tyrosine). The
inhibition curve of each compound was calculated under their maximum solubility of 330 μg/mL.
Kojic acid is positive control.
2.2. Anti-Bacterial Activity of Triterpenoids
Propionibacterium acnes (P. acnes) is an anaerobic gram-positive bacterium located primarily in
the pilosebaceous unit of the skin. Together with the sebaceous gland, it plays an important role in
the development of acne [18]. In our study, the antibacterial activity of triterpenoids against P. acnes
was evaluated. Among these triterpenoids, 12 compounds, i.e., ursolic acid (1), oleanolic acid (2),
methyl corosolate (6), maslinic acid (7), corosolic acid (8), pomolic acid (10), tormentic acid (11),
euscaphic acid (12), 6β,19-dihydroxyurs-12-en-3-oxo-28-oic acid (14), hyptadienic acid (16),
3-O-trans-p-coumaroyltormentic acid (17) and 3-O-cis-p-coumaroyltormentic acid (18), were active
against P. acnes at their maximum solubility. The minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) values of the active compounds are shown in Table 2.
The results showed that ursolic acid (1), maslinic acid (7), corosolic acid (8) and euscaphic acid (12)
showed the highest activities against P. acnes with an MIC value of 50 μg/mL and MBC values
ranging from 50 to 150 μg/mL.
Int. J. Mol. Sci. 2017, 18, x 5 of 13
Table 2. Anti-acne effect of triterpenoids from E. japonica leaves.
No.
Concentration
μg/mL
Inhibition
Rate %
MIC a/MBC b
μg/mL
No.
Concentration
μg/mL
Inhibition e
Rate %
MIC/MBC
μg/mL
1
100
109.8 ± 0.5
50/100
10
100
84.4 ±1.2
n.d.
2
100
100.4 ± 0.8
100/200
11
100
102.8 ± 2.1
100/200
3
50
– c
–
12
100
98.5 ± 1.3
50/50
4
100
–
–
13
100
–
–
5
100
–
–
14
100
87.6 ± 7.4
n.d.
6
100
77.9 ± 1.6
n.d. d
15
100
–
–
7
100
104.1 ± 3.1
50/150
16
100
103.9 ± 3.1
100/200
8
100
105.9 ± 0.4
50/100
17
100
101.4 ± 2.1
100/200
9
100
–
–
18
100
103.5 ± 2.2
100/200
a The minimum inhibitory concentration (MIC) is the lowest concentration of an anti-bacterial agent
required to completely inhibit the growth of a particular bacterium; b The minimum bactericidal
concentration (MBC) is the lowest concentration of an antibacterial agent required to kill the
bacterium; – c : no inhibition; n.d. d : non-detectable; e Benzalkonium chloride was used as the
positive control and showed 100% inhibition of bacterial growth at the conc. of 200 μg/mL.
Although the triterpenoids showed low hydrophilicity with logP values ranging from 5 to 8,
they also exhibited high levels of antibacterial activity in the present study. However, the exact
mode of action of these triterpenes on bacteria, especially against P. acnes, is still unknown.
Antibiotic therapy (such as that with tetracyclines, erythromycin and clindamycin) directed against
P. acnes has been a mainstay of acne treatment for many years [19], but the drug resistance associated
with these antibiotics is an increasing concern [20]. Our present findings may contribute to the
development of new natural cosmetic products that can be used to prevent acne, and offer an
alternative plant-derived antibacterial compounds against the growth of P. acnes.
2.3. Anti-Allergic and Anti-Inflammatory Effects of Triterpenoids
Allergy and inflammation can manifest as hyper-responsiveness to allergenic environmental
substances in various target organs of the body (the skin, nose, lungs, gastrointestinal tract, etc.) [21].
In the case of skin as the primary interface between the body and the environment, inappropriate or
misdirected immune activity results in inflammations such as skin rash, itching or eczema [22].
Allergic reactions are triggered when allergens cross-link preformed immunoglobulin E (IgE) bound
to the high-affinity receptor (FcεRI) on mast cells [23]. The mast cells then degranulate and release
vasoactive amines (mainly histamine), mediators (nitric oxide, prostaglandins and cysteinyl
leukotrienes), chemokines and other cytokines [24].
Here we examined the anti-allergic effects by checking the release of β-hexosaminidase from
IgE-stimulated RBL-2H3 mast cells. First, we selected several working concentrations for all tested
compounds and the result demonstrated that four triterpenoids exhibited significant inhibitory
activity at 25 μg/mL (p < 0.01; 25 μg/mL is maximum solubility) as shown in Table S3. Three
triterpenoids in particular—ursolic acid (1, inhibitory rate 72.5%), 3-epicorosolic acid (9, inhibitory
rate 54.4%) and euscaphic acid (12, inhibitory rate 74.5%)—significantly suppressed the release of
β-hexosaminidase at a non-cytotoxic concentration. Maslinic acid (7) showed a suppression rate of
66% on β-hexosaminidase with 84.4% cell viability.
We also determined the IC50 values against the anti-allergy and anti-inflammatory activity of
active compounds. As shown in Table3, compounds (1), (7), (9) and (12) possessed inhibitory activity
against β-hexosaminidase release with the IC50 value of 39.5, 22.86, 14.2 and 15.8 μM, respectively.
Int. J. Mol. Sci. 2017, 18, x 6 of 13
Table 3. Anti-allergy and anti-inflammatory effects of triterpenoids from E. japonica leaves.
No.
IC50 (μM)
25 μg/mL
12.5 μg/mL
5 μg/mL
1 μg/mL
Inhibition Rate (%)
1
39.5
72.5 ± 3.0 **
37.6 ± 3.0 *
0.0 ± 11.5
n.d.
7
22.8
66.0 ± 2.6 *
58.2 ± 3.0 *
9.5 ± 6.6
n.d.
9
14.2
54.4 ± 4.9 *
50.7 ± 3.2 *
33.7 ± 6.9
8.9 ± 5.2
12
15.8
74.5 ± 1.3 **
67.0 ± 1.8 **
37.7 ± 3.9 *
10.4 ± 10.8
Quercetin was used as the positive control and showed the inhibition rate of β-hexosaminidase
release of 72.5 ± 3.0% without cytotoxicity at 82 μM. n.d.: non-detectable. * p < 0.05 and ** p < 0.01.
Our results are consistent with previous studies [25,26] showing that compounds (1), (7), (9) and
(12) have significant inhibitory activity against inflammation induced by 12-O-tetradecanoylphorbol-
13-acetate (TPA) in mice and TPA-induced Epstein–Barr virus early antigen activation. In the
present study, we reveal a probable alternative mechanism to account for the anti-allergy and
anti-inflammatory activity of the active compounds.
2.4. Skin Type I Collagen Production-Promoting Activity of Triterpenoids
Skin aging is characterized by atrophy of the dermal–epidermal junction with a decrease in
mechanical tension and loss of elasticity, due mainly to marked reduction of the dermal
extracellular matrix, including collagen, elastin, and hyaluronic acid [27,28]. Collagen secreted by
normal human dermal fibroblasts (NHDFs) plays important roles in cell–cell adhesion, cell
proliferation and cell differentiation, and thus the functional properties of the skin depends on the
quality and condition of the collagen present in the dermis [29].
In our method, both the collagen production activity (CP) and cell viability (CV) were
calculated individually at several concentrations for each compound. We then determined the
CP/CV ratio (value > 1.0), which represents the “ratio of the mean of collagen production (%) to the
mean of cell viability (%)” as an index of the stimulating activity of collagen production per cell. As
shown in Figure 3, our results demonstrated that some triterpenoids can significantly enhance the
dermal fibroblast collagen production per cell. Of the 18 test triterpenoids, ten induced type I
collagen production at a concentration of 10 μg/mL, with methyl corosolate (6) and corosolic acid
(8) showing especially potent stimulation of up to 3.4- and 3.6-fold that of the control, and the rest
of the compounds showed a moderate promoting effect. At 5 μg/mL, 16 compounds triggered
increased collagen production, with ursolic acid (1) and betulinic acid (4) increasing the collagen
production up to 3.8- and 3.0-fold over the control level, respectively. Six compounds showed
collagen-promoting activity at low concentrations. Concentrations higher than 10 μg/mL are not
shown because of cytotoxicity.
Int. J. Mol. Sci. 2017, 18, x 7 of 13
Figure 3. Collagen production (CP)—promoting effects of triterpenoids from leaves of E. japonica
leaves. Data are the means ± SD of three independent tests, and all data were calculated as the
percentage compared with the control value. Ascorbic acid (100 μM) was used as a positive control.
* The promoting ratio (or CP/CV value) represents the mean of collagen production (%)/the mean of
cell viability (CV) (%).
Our results may contribute to the development of new cosmeceuticals and formulas from
natural resources that can facilitate both the repair of skin wrinkles and fight against skin aging
[30].
2.5. Stimulating Effect of Triterpenoids on Hyaluronic Acid Synthesis
The loss of the skin’s high water content is one of the hallmarks of skin aging. The key
molecule involved in skin moisture is hyaluronic acid, which is a high molecular weight
glycosaminoglycan with a unique capacity to retain water [31]. Increasing amount of hyaluronic
acid is also important for the wound healing process [32].
We therefore evaluated the potential effect of triterpenoids on hyaluronic acid production
(Figure 4). At 10 μg/mL, ten compounds exhibited a potent stimulatory effect on hyaluronic acid
production in human dermal fibroblasts, with the compounds corosolic acid (8) and pomolic acid
(10) showing the strongest hyaluronic acid stimulating activity of 5.9- and 5.8-fold that of the control,
respectively; methylation derivative (6) also showed a high stimulating effect. At 5 μg/mL, six
compounds stimulated hyaluronic acid production, and it is worth noting that ursolic acid (1)
achieved a 4.7-fold increase in hyaluronic acid (HA) production. At a low concentration (1 μg/mL),
however, only betulinic acid (4) had a promoting effect. Concentrations higher than 10 μg/mL are
not shown because of cytotoxicity.
Int. J. Mol. Sci. 2017, 18, x 8 of 13
Figure 4. Stimulatory effects of triterpenoids from E. japonica leaves on hyaluronic acid (HA)
production. Data are the means ± SD of three independent tests, and all data were calculated as
percentages compared with the control values. Ascorbic acid (100 μM) was used as a positive control.
* The promoting ratio (or HA/CV value) represents the mean of hyaluronic acid (%)/the mean of cell
viability (%).
Our present findings did not reveal a direct reason for the accumulation of collagen and
hyaluronic acid by triterpenoids, but a pair of studies have shown that triterpenes extracted from the
medicinal herb Centella asiatica induced changes in hyaladherin TNF alpha induced protein 6
(TNFAIP6) and cytokine expression, and both of these changes were expected to lower the rate of
proteolysis in the extracellular matrix and support accumulation collagen, the triterpenes did not
directly affect the expression levels of collagen gene [33,34]. It seems that the stimulation of
hyaluronic acid synthesis by triterpenoids could also account for the inhibitory effect on
hyaluronidase, an enzyme that degrades hyaluronic acid by cleaving the N-acetylglucosamidic
bonds of hyaluronic acid via a β-elimination process to produce hyaluronic acid oligosaccharides
[35].
3. Materials and Methods
3.1. Chemicals
Triterpenoids isolated from E. japonica leaves for biological activities were obtained as follow:
Ursolic acid (1) was obtained from Wako Pure Chemical Industries (Osaka, Japan); Oleanolic acid (2)
was from Cayman Chemicals (Ann Arbor, MI, USA); Uvaol (3) was obtained from Extrasynthese
(Genay Cedex, France); Betulinic acid (4) was obtained from Focus Biomolecules (Plymouth
Meeting, PA, USA); Maslinic acid (7) was obtained from Cayman Chemicals; Pomolic acid (10) was
obtained from Quality Phytochemicals (East Brunswick, NJ, USA); Corosolic acid (8), 3-epicorosolic
acid (9), tormentic acid (11), euscaphic acid (12), 3-oxours-12-en-28-oic acid (13),
Int. J. Mol. Sci. 2017, 18, x 9 of 13
6β,19-dihydroxyurs-12-en-3-oxo-28-oic acid (14), hyptadienic acid (16), 3-O-trans-p-coumaroyl
tormentic acid (17) and 3-O-cis-p-coumaroyl tormentic acid (18) were obtained from Chemfaces
(Wuhan, China). Methyl maslinate (5), methyl corosolate (6) and 2α,
19α-dihydroxy-3-oxo-12-ursen-28-oic acid (15) were previously isolated from E. japonica leaves. The
purity of all tested compounds was above 95% as verified by analytical high-performance liquid
chromatography (HPLC).
3.2. Anti-Melanogenesis Assay
This assay was performed as described by Arung et al. [17]. The mouse melanoma cell line B16
was obtained from the Riken Bioresource Center Cell Bank (Tsukuba, Japan). The B16 melanoma
cells were maintained in Eagle’s Minimal Essential Medium (EMEM, Nissui Chemical, Tokyo)
supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 0.09 mg/mL
theophylline (Sigma-Aldrich, St. Louis, MO, USA). The cells were placed in 24-well plastic culture
plates at a density of 1 × 105 cells/well and incubated for 24 h in media prior to being treated with the
samples. After 24 h, test samples were dissolved in DMSO (<0.2% per well) and sample solution
were added to each well. Arbutin (Tokyo Kasei Kogyo, Tokyo) was used as a positive control. The
cells were incubated for an additional 48 h, and then the medium was replaced with fresh medium
containing each sample again. After 24 h, the melanin content and the cell viability were measured
using a microplate reader (BioTek-ELX800, BioTek Instruments, Winooski, VT, USA) at 405 nm and
570 nm, respectively. Cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) method.
3.3. Tyrosinase Inhibition Assay Assay
Tyrosinase inhibition activity was evaluated by using L-tyrosine and L-DOPA as substrates, as
described [36]. Briefly, 333 μL of 2.5 mM L-tyrosine or L-DOPA solution (Sigma-Aldrich) was mixed
with 600 μL of 0.1 M phosphate buffer (pH 6.8) and incubated at 25 °C . Then, 33 μL of the sample
solution (dissolved in DMSO in various concentrations) and 33 μL of 1380 units/mL mushroom
tyrosinase (Sigma-Aldrich) were added to the mixture. The optical density was measured at 475 nm
up to the appropriate time. Kojic acid (Sigma-Aldrich) was used as the positive control; the final
concentration is 3.3 μg/mL.
3.4. Anti-Bacterial Assay
The antibacterial assay was based mostly on the methods described by Tanaka et al. with slight
modification [37]. Propionibacterium acne (NBRC 107605) was used for the antibacterial assay. A
single colony of the test strain was taken and 5 mL of Gifu anaerobic medium (GAM) broth (Nissui,
Tokyo) was added to it. This culture was incubated at 37 °C for 30 h. It was then added to the
bacterial suspension to prepare a bacterial concentration at 105 CFU/mL. The bacterial solution was
used for the subsequent antibacterial assay. Each compound was dissolved in DMSO, and
benzalkonium chloride (Wako) was used as a positive control. The plate was incubated at 37 °C for
30 h. Finally, bacterial growth was measured by a microplate reader at 630 nm. The MIC and MBC
were evaluated by the standard broth microdilution method.
3.5. Anti-Allergic and Anti-Inflammatory Assay
The anti-allergy assay was based mostly on the methods described by Zhu et al. [38] and Yun et
al. with slight modification [39]. Rat basophilic leukemia (RBL-2H3) cells were obtained from the
Riken Bio-resource Center Cell Bank (Tsukuba, Japan). RBL-2H3 cells were incubated in EMEM
medium containing 10% FBS and 1% antibiotics–antimycotics at 37 °C. The cells were seeded on a
96-well plate (2 × 105 cells/well). After 24 h of incubation, the medium was supplemented with 0.5
μg/mL anti-DNP (dinitrophenol) mouse immunoglobulin E (IgE) (Sigma-Aldrich). After an
additional 24 h of incubation, the medium was replaced with 100 μL of Tyrode buffer. The sample
dissolved in DMSO (<0.5% per well) was added, and the cell plate was incubated again for 30 min,
Int. J. Mol. Sci. 2017, 18, x 10 of 13
then replaced with 100 μL of tyrode buffer containing 50 μg/mL DNP-bovine serum
albumin (BSA) (Invitrogen, Carlsbad, CA, USA). After 1 h of incubation, 50 μL of the supernatant
was collected from each well and mixed with 50 μL of substrate solution
(p-nitrophenyl-N-acetyl-β-glucosaminide, 2 mM; Sigma-Aldrich) in a new 96-well plate. The mixture
was incubated at room temperature on a shaker for 3 h. Finally, 100 μL of stop solution was added,
and the absorbance was read at 405 nm. A parallel MTT experiment was performed to measure the
cell viability. The MTT experiment was done exactly as described [40].
3.6. Skin Collagen Synthesis Promoting Assay
The assay for measuring the promotion of skin collagen synthesis was described by Kishikawa
et al. [41]. Adult NHDF cells (Lonza, Tokyo) were routinely maintained in 10% FBS and 1%
antibiotic-antimycotic (Invitrogen) in Dulbecco’s modified Eagle’s medium (DMEM, Wako). After
growing to confluence, cells at a density of 5 × 103 cells/well were seeded on 96-well plates for 24 h
before the treatment. The medium was replaced with a mixture of 1 μL of sample solution (DMSO <
0.5% per well) and 99 μL of DMEM supplemented with 0.5% FBS. After 72 h of cultivation, the
amount of collagen in the medium was measured using a human type I collagen enzyme-linked
immunosorbent assay (ELISA) kit (ACEL, Kanagawa, Japan). The amount of collagen production
was calculated using a standard curve prepared by measurement by the same ELISA. The cells
remaining in the 96-well plate were subjected to an MTT assay. Ascorbic acid (100 μM, Wako) was
used as a positive control.
3.7. Hyaluronic Acid Production Assay
NHDF-Ad cells were seeded on 96-well plates (5 × 103 cells/well) for 24 h before the treatment.
The medium was replaced with a mixture of 1 μL of sample solution (DMSO < 0.5% per well) and 99
μL of DMEM supplemented with 0.5% FBS. After 72 h cultivation, the amount of hyaluronic acid in
the medium was measured using a QnE Hyaluronic acid ELISA kit (Biotech Trading Partners,
Encinitas, CA, USA). The amount of hyaluronic acid production was quantitatively calculated from
the standard curve. Cell viability was measured by the MTT method, and ascorbic acid was used as a
positive control.
3.8. Statistical Analysis
All values from three independently repeated experiments are presented as the mean ±
standard deviation. The significance of differences between means for two groups was determined
by Student’s t-test. Differences were considered significant at * p < 0.05 and ** p < 0.01. The IC50 and
CC50 values were calculated from the corresponding dose–response curves in at least four
concentrations.
4. Conclusions
By establishing a complete skin-related bioactivity evaluation method and using previous
studied secondary metabolites from the leaves of E. japonica, we have obtained a better
understanding of the multiple functions of E. japonica leaves, and provided an evidence-based study
that E. japonica leaves can be used to prevent skin-related problems. It is clear that triterpenoids from
E. japonica leaves exhibit powerful skin-protecting effects, such as the inhibition of melanogenesis, P.
acnes growth and allergies, and the promotion effects for collagen/hyaluronic acid production. In
contrast to the previously identified anti-melanogenesis, anti-acne, anti-allergy, and anti-aging
triterpenoids, newly identified and more potent inhibitors were identified in our study.
In terms of anti-melanogenesis activity, we identified eight compounds that have melanin
synthesis inhibitory activity. By means of a structure–activity relationship study, we deduced the
functional groups that contribute to activity. The mechanism underlying four active compounds’
melanogenesis inhibition effect is partly associated with their tyrosinase-inhibitory activities. There
has been very little reported research about P. acne growth inhibition by triterpenoids, and here we
Int. J. Mol. Sci. 2017, 18, x 11 of 13
found twelve compounds that were active against P. acnes at their maximum solubility. These
compounds could be considered an alternative source to combat antibiotics resistance. However,
the mechanism of action of each active component remains unknown and further research of this
chemical class is needed. With regards to anti-allergy activity, our present findings revealed an
alternative mechanism of active triterpenoids from E. japonica leaves by suppressing
β-hexosaminidase. In addition, treatment using triterpenoids from the leaves of E. japonica can
efficiently restore collagen and hyaluronic acid synthesis, as we observed a 3- to 4-fold increase in
the level of collagen content and a 5- to 6-fold increase in hyaluronic acid synthesis.
Triterpenes represent a promising class of multi-target agents. In this study, the compounds
ursolic acid (1) and maslinic acid (7) showed multiple activities against different skin-related
problems. The mechanism underlying their multi-targets is interesting. The synergistic effects of
these triterpenoids should also be considered. The results of this study indicate that E. japonica
leaves have great potential to be used as ingredients in function food for increasing nutritional value
and in cosmetics for skin-care effect.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/link.
Acknowledgments: We thank the technical assistance from Scarpa Co. Ltd. (Nagasaki, Japan).
Author Contributions: Hui Tan and Kuniyoshi Shimizu conceived, designed the experiments; Hui Tan
performed the experiment, analyzed the data and wrote the paper; Tamrakar Sonam helped revise the paper
and did additional experiments during revision; Kuniyoshi Shimizu supervised the experiments. All authors
read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
CV
Cell viability
CP
Collagen production activity
HA
Hyaluronic acid
MIC
Minimum inhibitory concentration
MBC
Minimum bactericidal concentration
S.I.
Selectivity index
References
1. Lee, S.H.; Jeong, S.K.; Ahn, S.K. An update of the defensive barrier function of skin. Yonsei Med. J. 2006, 47,
293–306.
2. Xue, M.; Jackson, C.J. Novel functions of the anticoagulant activated protein C in maintaining skin barrier
integrity to impact on skin disease. Pathobiology 2015, 82, 100–106.
3. Ganceviciene, R.; Liakou, A.I.; Theodoridis, A.; Makrantonaki, E.; Zouboulis, C.C. Skin anti-aging
strategies. Dermatoendocrinology 2012, 4, 308–319.
4. Ganesan, P.; Choi, D.-K. Current application of phytocompound-based nanocosmeceuticals for beauty
and skin therapy. Int. J. Nanomed. 2016, 11, 1987–2007.
5. Tabassum, N.; Hamdani, M. Plants used to treat skin diseases. Pharmacogn. Rev. 2014, 8, 52–60.
6. Liu, F.-W.; Liu, F.-C.; Wang, Y.-R.; Tsai, H.-I.; Yu, H.-P. Aloin protects skin fibroblasts from heat
stress-induced oxidative stress damage by regulating the oxidative defense system. PLoS ONE 2015, 10,
e0143528.
7. Hwang, B.-M.; Noh, E.-M.; Kim, J.-S.; Kim, J.-M.; You, Y.-O.; Hwang, J.-K.; Kwon, K.-B.; Lee, Y.-R.
Curcumin inhibits UVB-induced matrix metalloproteinase-1/3 expression by suppressing the
MAPK-p38/JNK pathways in human dermal fibroblasts. Exp. Dermatol. 2013, 22, 371–374.
8. Ndiaye, M.; Philippe, C.; Mukhtar, H.; Ahmad, N. The grape antioxidant resveratrol for skin disorders:
Promise, prospects, and challenges. Arch. Biochem. Biophys. 2011, 508, 164–170.
9. Su, T.-R.; Lin, J.-J.; Tsai, C.-C.; Huang, T.-K.; Yang, Z.-Y.; Wu, M.-O.; Zheng, Y.-Q.; Su, C.-C.; Wu, Y.-J.
Inhibition of melanogenesis by gallic acid: Possible involvement of the PI3K/Akt, MEK/ERK and
Wnt/β-catenin signaling pathways in B16F10 cells. Int. J. Mol. Sci. 2013, 14, 20443–20458.
Int. J. Mol. Sci. 2017, 18, x 12 of 13
10. Li, N.; Deng, L.; Xiang, L.; Liang, Y. Photoprotective effect of tea and its extracts against ultraviolet
radiation-induced skin disorders. Trop. J. Pharm. Res. 2014, 13, 475.
11. Cha, D.S.; Eun, J.S.; Jeon, H. Anti-inflammatory and antinociceptive properties of the leaves of Eriobotrya
japonica. J. Ethnopharmacol. 2011, 134, 305–312.
12. Shih, C.-C.; Lin, C.-H.; Wu, J.-B. Eriobotrya japonica improves hyperlipidemia and reverses insulin
resistance in high-fat-fed mice. Phytother. Res. 2010, 24, 1769–1780.
13. Kim, M.-J.; Lee, J.; Seong, A.-R.; Lee, Y.-H.; Kim, Y.-J.; Baek, H.-Y.; Kim, Y.J.; Jun, W.J.; Yoon, H.-G.
Neuroprotective effects of Eriobotrya japonica against β-amyloid-induced oxidative stress and memory
impairment. Food Chem. Toxicol. 2011, 49, 780–784.
14. Tan, H.; Furuta, S.; Nagata, T.; Ohnuki, K.; Akasaka, T.; Shirouchi, B.; Sato, M.; Kondo, R.; Shimizu, K.
Inhibitory effects of the leaves of loquat (Eriobotrya japonica) on bone mineral density loss in
ovariectomized mice and osteoclast differentiation. J. Agric. Food Chem. 2014, 62, 836–841.
15. Tan, H.; Ashour, A.; Katakura, Y.; Shimizu, K. A structure-activity relationship study on antiosteoclastogenesis
effect of triterpenoids from the leaves of loquat (Eriobotrya japonica). Phytomedicine 2015, 22, 498–503.
16. Choi, M.-H.; Shin, H.-J. Anti-melanogenesis effect of quercetin. Cosmetics 2016, 3, 18.
17. Arung, E.T.; Furuta, S.; Ishikawa, H.; Kusuma, I.W.; Shimizu, K.; Kondo, R. Anti-melanogenesis properties
of quercetin- and its derivative-rich extract from Allium cepa. Food Chem. 2011, 124, 1024–1028.
18. Beylot, C.; Auffret, N.; Poli, F.; Claudel, J.-P.; Leccia, M.-T.; Del Giudice, P.; Dreno, B. Propionibacterium
acnes: An update on its role in the pathogenesis of acne. J. Eur. Acad. Dermatol. Venereol. 2014, 28, 271–278.
19. Eady, E.A.; Gloor, M.; Leyden, J.J. Propionibacterium acnes resistance: A worldwide problem. Dermatology
2003, 206, 54–56.
20. Walsh, T.R.; Efthimiou, J.; Dréno, B. Systematic review of antibiotic resistance in acne: An increasing
topical and oral threat. Lancet Infect. Dis. 2016, 16, 23–33.
21. Juckmeta, T.; Thongdeeying, P.; Itharat, A.; Juckmeta, T.; Thongdeeying, P.; Itharat, A. Inhibitory effect on
β-hexosaminidase release from RBL-2H3 cells of extracts and some pure constituents of benchalokawichian,
a Thai herbal remedy, used for allergic disorders. Evid. Based Complement. Altern. Med. 2014, 2014, 1–8.
22. Kupper, T.S.; Fuhlbrigge, R.C. Immune surveillance in the skin: Mechanisms and clinical consequences.
Nat. Rev. Immunol. 2004, 4, 211–222.
23. Charles A Janeway, J.; Travers, P.; Walport, M.; Shlomchik, M.J. Effector Mechanisms in Allergic Reactions;
Garland Science: New York, NY, USA, 2001.
24. Larché, M.; Akdis, C.A.; Valenta, R. Immunological mechanisms of allergen-specific immunotherapy. Nat.
Rev. Immunol. 2006, 6, 761–771.
25. Banno, N.; Akihisa, T.; Tokuda, H.; Yasukawa, K.; Taguchi, Y.; Akazawa, H.; Ukiya, M.; Kimura, Y.;
Suzuki, T.; Nishino, H. Anti-inflammatory and antitumor-promoting effects of the triterpene acids from
the leaves of Eriobotrya japonica. Biol. Pharm. Bull. 2005, 28, 1995–1999.
26. Banno, N.; Akihisa, T.; Tokuda, H.; Yasukawa, K.; Higashihara, H.; Ukiya, M.; Watanabe, K.; Kimura, Y.;
Hasegawa, J.-I.; Nishino, H. Triterpene acids from the leaves of Perilla frutescens and their
anti-inflammatory and antitumor-promoting effects. Biosci. Biotechnol. Biochem. 2004, 68, 85–90.
27. Taihao Q. Molecular Mechanisms of Skin Aging and Age-Related Diseases; Quan, T., Ed.; CRC Press: New York,
NY, USA, 2016.
28. Decorps, J.; Saumet, J.L.; Sommer, P.; Sigaudo-Roussel, D.; Fromy, B. Effect of ageing on tactile
transduction processes. Ageing Res. Rev. 2014, 13, 90–99.
29. Matsui, Y.; Sugiyama, K.; Kamei, M.; Takahashi, T.; Suzuki, T.; Katagata, Y.; Ito, T. Extract of passion fruit
(Passiflora edulis) seed containing high amounts of piceatannol inhibits melanogenesis and promotes
collagen synthesis. J. Agric. Food Chem. 2010, 58, 11112–11118.
30. Mukherjee, P.K.; Maity, N.; Nema, N.K.; Sarkar, B.K. Bioactive compounds from natural resources against
skin aging. Eur. J. Integr. Med. 2011, 19, 64–73.
31. Papakonstantinou, E.; Roth, M.; Karakiulakis, G. Hyaluronic acid: A key molecule in skin aging.
Dermatoendocrinology 2012, 4, 253–258.
32. Prosdocimi, M.; Bevilacqua, C. Exogenous hyaluronic acid and wound healing: An updated vision.
Panminerva Med. 2012, 54, 129–135.
33. Maquart, F.X.; Bellon, G.; Gillery, P.; Wegrowski, Y.; Borel, J.P. Stimulation of collagen synthesis in
fibroblast cultures by a triterpene extracted from Centella asiatica. Connect. Tissue Res. 1990, 24, 107–120.
Int. J. Mol. Sci. 2017, 18, x 13 of 13
34. Coldren, C.D.; Hashim, P.; Ali, J.M.; Oh, S.-K.; Sinskey, A.J.; Rha, C. Gene expression changes in the
human fibroblast induced by Centella asiatica triterpenoids. Planta Med. 2003, 69, 725–732.
35. Abdullah, N.H.; Thomas, N.F.; Sivasothy, Y.; Lee, V.S.; Liew, S.Y.; Noorbatcha, I.A.; Awang, K.
Hyaluronidase inhibitory activity of pentacylic triterpenoids from Prismatomeris tetrandra (Roxb.) K.
Schum: Isolation, synthesis and QSAR study. Int. J. Mol. Sci. 2016, 17, 143.
36. Wang, K.H.; Lin, R.D.; Hsu, F.L.; Huang, Y.H.; Chang, H.C.; Huang, C.Y.; Lee, M.H. Cosmetic applications
of selected traditional Chinese herbal medicines. J. Ethnopharmacol. 2006, 106, 353–359.
37. Tanaka, A.; Zhu, Q.; Tan, H.; Horiba, H.; Ohnuki, K.; Mori, Y.; Yamauchi, R.; Ishikawa, H.; Iwamoto, A.;
Kawahara, H.; et al. Biological activities and phytochemical profiles of extracts from different parts of
bamboo (Phyllostachys pubescens). Molecules 2014, 19, 8238–8260.
38. Zhu, Q.; Nakagawa, T.; Kishikawa, A.; Ohnuki, K.; Shimizu, K. In vitro bioactivities and phytochemical
profile of various parts of the strawberry (Fragaria × ananassa var. Amaou). J. Funct. Foods 2015, 13, 38–49.
39. Yun, S.-S.; Kang, M.-Y.; Park, J.-C.; Nam, S.-H. Comparison of anti-allergenic activities of various
polyphenols in cell assays. J. Appl. Biol. Chem. 2010, 53, 139–146.
40. Mira, A.; Tanaka, A.; Tateyama, Y.; Kondo, R.; Shimizu, K. Comparative biological study of roots, stems,
leaves, and seeds of Angelica shikokiana Makino. J. Ethnopharmacol. 2013, 148, 980–987.
41. Kishikawa, A.; Ashour, A.; Zhu, Q.; Yasuda, M.; Ishikawa, H.; Shimizu, K. Multiple biological effects of
olive oil by-products such as leaves, stems, flowers, olive milled waste, fruit pulp, and seeds of the olive
plant on skin. Phytother. Res. 2015, 29, 877–886.
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