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Article
Preparation, Characterization, and Biological
Activities of Topical Anti-Aging Ingredients
in a Citrus junos Callus Extract
Deepak Adhikari † , Vijay Kumar Panthi † , Rudra Pangeni ID
, Hyun Jung Kim * and
Jin Woo Park *
College of Pharmacy and Natural Medicine Research Institute, Mokpo National University,
Muan-gun, Jeonnam 58554, Korea; dpak7adh@gmail.com (D.A.); nepalivijay7@gmail.com (V.K.P.);
capriconpangeni@gmail.com (R.P.)
* Correspondence: hyunkim@mokpo.ac.kr (H.J.K.); jwpark@mokpo.ac.kr (J.W.P.);
Tel.: +82-61-450-2686 (H.J.K.); +82-61-450-2704 (J.W.P.)
† These authors contributed equally to this work.
Abstract: In this study, we prepared and characterized a callus extract from Citrus junos and assessed
its utility as a source of topical anti-aging ingredients. Callus extract was produced by aqueous
extraction from Citrus junos grown on Murashige and Skoog medium with picloram as a growth
regulator. After measuring the total phenolic and flavonoid contents, the major phenolic compound
in calli was identified as p-hydroxycinnamoylmalic acid (1) by spectroscopic analysis. The total
phenol content in the extract was determined to be 24.50 ± 0.43 mg/g of gallic acid equivalents;
however, the total flavonoid content of the extract was not determined. The biological activities
of the callus extract, in terms of skin anti-aging, were assessed by measuring the anti-tyrosinase
activity in, and melanogenesis by, melanoma cells; and proliferation of, and procollagen synthesis
by, human fibroblasts. The callus extract was incorporated into nanoliposomes (NLs) to improve
its percutaneous absorption. Addition of the callus extract resulted in a 1.85-fold decrease in
the melanin content of melanocytes compared with that with arbutin. The extract (500 µg/mL)
significantly promoted the proliferation of, and procollagen synthesis by, fibroblasts (by 154% and
176%, respectively). In addition, the flux through the human epidermis of Citrus junos callus extract
incorporated into NLs was 17.67-fold higher than that of the callus extract alone. These findings
suggest that Citrus junos callus extract-loaded NLs have promise as an anti-aging cosmetic, as well as
having a skin-lightening effect.
Keywords: anti-aging; callus extract; cosmetic ingredients; Citrus junos; plant cell culture;
skin lightening
1. Introduction
Plant tissue culture is a valid alternative method for production of pharmaceutical and
cosmeceutical ingredients; fresh material is always available regardless of the season or the plant
reproductive cycle [1,2]. In addition, growing conditions are readily standardized, affording high-level
consistency from batch to batch, and the extracted components are safe and pure; there is no risk of
pathogenic or environmental contamination. Genetic or biochemical modifications may increase the
concentrations of desired bioactive compounds [3,4].
Citrus junos Siebold ex. Tanaka (Rutaceae), also known as yuja in Korea, is a citrus tree with edible
yellow fruits that has been reported to inhibit oxidative stress and inflammation [5]. In previous studies,
several active compounds, such as rutin, quercetin, tangeretin, naringin, and hesperidin were found in the
fruit; such anti-oxidants prevent or retard the oxidation of other molecules by interrupting the initiation
or propagation of oxidizing chain reactions [6,7]. In addition, such metabolites exhibit significant
protective biological activities, including anticancer, anti-inflammatory, and antiviral properties [8,9].
Furthermore, citrus peel extract is an effective anti-oxidant, anti-acne, and skin-whitening agent, as it is
very rich in phenolic compounds including phenolic acids and flavonoids [10,11].
Phenolic compounds are widely accepted as effective scavengers of reactive oxygen species (ROS)
like singlet oxygen, the superoxide anion radical, the hydroxyl radical, and hydrogen peroxide [12].
Previous studies found that persistent exposure to ROS triggered skin aging via destruction of the
anti-oxidant system, wrinkle formation, and melanogenesis [13,14]. Free radicals can trigger the
formation of collagen cross-links, causing the skin to lose elasticity and thus initiating aging [15].
However, phenolic compounds serve as reducing agents, hydrogen donors, and singlet oxygen
quenchers. These qualities render them extremely useful in cosmetic formulations that seek to prevent
or retard oxidative damage to collagen caused by free radicals. Therefore, antioxidants containing
a variety of bioactive components from natural sources have been used to prevent aging [16].
The physicochemical profiles of promising bioactive compounds must be considered in terms
of successful transport of the materials to the desired site of action across the barrier of the
stratum corneum (SC). Thus, many studies have tried to improve the potency, solubility, and skin
permeability of selected compounds, or to encapsulate them in colloidal nanocarriers, such as solid lipid
nanoparticles, nanostructured lipid carriers, nanoemulsions, nanoparticle suspensions, and polymeric
nanoparticles, to facilitate skin permeation and protect against degradation [17–19].
In this study, we focused on the preparation and characterization of a callus extract from
Citrus junos and assessed its 2,2-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity.
Next, we evaluated the skin-lightening and regeneration efficacies of the extract by measuring its
capacity to inhibit tyrosinase activity and melanin biosynthesis, and to stimulate the proliferation of,
and procollagen synthesis by, human fibroblasts; no report on the biological utility of Citrus junos
callus extract, in terms of skin anti-aging, has yet appeared. Finally, the callus extract was incorporated
into nanoliposomes (NLs) and delivered topically to enhance percutaneous absorption and improve
the skin anti-aging effects.
Figure 1. HPLC
Figure profile
1. HPLC of the
profile extract
of the fromCitrus
extractfrom Citrus junos callusand
junos callus andthe
the structure
structure of major
of its its major phenolic
phenolic
compound,
compound, p-hydroxycinnamoylmalicacid
p-hydroxycinnamoylmalic acid(1).
(1).
Cinnamic acids are aromatic carboxylic acids (C6–C3) that naturally occur in all green plants,
Cinnamic acids are aromatic carboxylic acids (C6–C3) that naturally occur in all green
being intermediates in the biosynthetic pathways leading to phenyl propanoids, flavonoids,
plants, being intermediates in the biosynthetic pathways leading to phenyl propanoids, flavonoids,
isoflavonoids, coumarins, lignans, anthocyanins, spermidines, stilbenes, aurones, and tannins [22,23].
isoflavonoids, coumarins,
These secondary lignans,play
metabolites anthocyanins, spermidines,
essential physiological stilbenes,
roles in plant aurones, and tannins
development, growth, [22,23].
Thesereproduction,
secondaryand metabolites play essential
disease resistance [24,25]. Thephysiological roles
biological activities in plantcinnamic
of phenolic development,
acids includegrowth,
reproduction,
antioxidant,and disease resistance
anti-inflammatory, [24,25].
antitumor, and The biological
antimicrobial activities
activities; of phenolic
multiple cinnamic
cytoprotective effectsacids
thatantioxidant,
include ameliorate neuroinflammation
anti-inflammatory,inantitumor,
neurodegenerative diseases; anti-hypertensive
and antimicrobial activities; multipleactivities; and
cytoprotective
anti-hyperlipidemic effects that minimize oxidation of low-density lipoprotein
effects that ameliorate neuroinflammation in neurodegenerative diseases; anti-hypertensive activities; (LDL) [22,26–29]. In
addition, phenolic acids can directly absorb and neutralize free radicals,
and anti-hyperlipidemic effects that minimize oxidation of low-density lipoprotein (LDL) [22,26–29]. and inhibit enzymes
associated with ROS generation pathways (such as xanthine oxidase, NADPH oxidase, and
In addition, phenolic acids can directly absorb and neutralize free radicals, and inhibit
myeloperoxidase) [29,30]. Phenolic acids also enhance the activities of human antioxidant enzymes,
enzymes associated with ROS generation pathways (such as xanthine oxidase, NADPH oxidase,
including superoxide dismutase and catalase [29,31].
and myeloperoxidase)
The total phenol [29,30]. Phenolic
content of theacids alsowas
extract enhance
24.50 the activities
± 0.43 mg/g of ofhuman antioxidant
gallic acid enzymes,
equivalents.
including superoxide
However, dismutase
flavonoids could notandbecatalase
detected [29,31].
by the corresponding AlCl3 method. Moreover, the
The total phenol
HPLC-PDA profilecontent of theextract
of the callus extractdid was
not24.50
exhibit±an0.43
LCmg/g of gallic
peak profile acid of
typical equivalents. However,
Citrus flavanone
flavonoids could
glycosides, fornot be detected
example, by (UV
hesperidin the corresponding
λmax 280 nm) andAlCl naringin
3 method.
(UV λ Moreover,
max 282 nm), the
which HPLC-PDA
are the
major components of the fruits of Citrus junos. These results suggested remarkable
profile of the callus extract did not exhibit an LC peak profile typical of Citrus flavanone glycosides, differences in the
major secondary metabolite composition of callus and plant extracts
for example, hesperidin (UV λmax 280 nm) and naringin (UV λmax 282 nm), which are the majorof Citrus junos.
components As aof
linear correlation
the fruits between
of Citrus phenolic
junos. Theseconcentration and radical-scavenging
results suggested remarkable differencesactivity has beenmajor
in the
reported in the extracts of many plants, vegetables, and fruits, we expected that the Citrus junos callus
secondary metabolite composition of callus and plant extracts of Citrus junos.
extract would exhibit strong antioxidant activity [30,31]. Figure 2 shows the DPPH radical-scavenging
As a linear correlation between phenolic concentration and radical-scavenging activity has been
activity of the extract. Both ascorbic acid (positive control) and the extract exhibited dose-dependent
reported in the extractsactivity.
radical-scavenging of many At plants, vegetables,
1000 μg/mL, andexhibited
the extract fruits, we expected
68.5 ± 9.49% that the Citrus junos callus
free radical-scavenging
extract
activity, equivalent to that of 125 μM ascorbic acid (62.3 ± 1.78%), due principally radical-scavenging
would exhibit strong antioxidant activity [30,31]. Figure 2 shows the DPPH to its phenolic
activity of the extract.
components. Boththe
However, ascorbic acid activity
antioxidant (positive of control)
the callusand the was
extract extract
lowerexhibited
than thatdose-dependent
of ascorbic
radical-scavenging
acid or cinnamic activity. At 1000
acid. Thus, µg/mL,
further theare
studies extract exhibited
required 68.5 ±the
to optimize 9.49% free conditions
culture radical-scavenging
and
extraction
activity, techniques
equivalent to increase
to that of 125 µMthe functional
ascorbic compound
acid (62.3 content
± 1.78%),of the callus
due extract to enhance
principally its
to its phenolic
antioxidant
components. activity. the antioxidant activity of the callus extract was lower than that of ascorbic
However,
acid or cinnamic acid. Thus, further studies are required to optimize the culture conditions and
Molecules 2017, 22, 2198 4 of 15
extraction techniques to increase the functional compound content of the callus extract to enhance its
Molecules activity.
antioxidant 2017, 22, 2198 4 of 15
(%) (%)
100
110
activity
90
100
80
activity
90
scavenging 70
80
60
70
scavenging
50
60
40
50
radical
30
40
radical
20
30
Free Free
10
20
0
10
500 250 125 50 25 1000 500 200 100 10
0
Ascorbic
500
acid (μΜ)25
250 125 50 Citrus
1000 500 junos
200 100 10
callus extract (μg/mL)
Citrus junos Ascorbic acid (μΜ)
callus extract (μg/mL)
Figure 2. 2,2-Diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity
Figure 2. 2,2-Diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity of Citrus
of Citrus junosjunos
calluscallus
extract. Values are mean ± standard deviation (n = 4 per group).
extract. Values are mean ± standard deviation (n = 4 per group).
Figure 2. 2,2-Diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity of Citrus junos callus
extract. Values are mean ± standard deviation (n = 4 per group).
2.2. Inhibition of Tyrosinase Activity andMelanin
MelaninBiosynthesis
Biosynthesis
2.2. Inhibition of Tyrosinase Activity and
To assess the effects of Citrus junos callus extract on melanogenesis, we examined its ability to
2.2.assess
Inhibition
To theofeffects
Tyrosinase Activityjunos
of Citrus and Melanin Biosynthesis
callus extract on melanogenesis, we examined its ability to
inhibit tyrosinase activity. As shown in Figure 3a, the extract inhibited L-3,4-dihydroxyphenylalanine
inhibit tyrosinase
To assessactivity.
the effects Asofshown
Citrus in Figure
junos callus3a, the extract
extract inhibited L-3,4-dihydroxyphenylalanine
on melanogenesis, we examined its ability to
(L-DOPA) oxidation in a dose-dependent manner; at 500 μg/mL, the magnitude of inhibition was
inhibit
(L-DOPA) tyrosinase
oxidation activity. As shown in Figure 3a, the extract inhibited -3,4-dihydroxyphenylalanine
that ofinthea control.
dose-dependent manner; at 500 aµg/mL, the magnitude
effect onof inhibition
L
25.2 ± 5.57% Moreover, the extract exerted greater inhibitory melanin
(L-DOPA)
was 25.2 ± oxidation in the
a dose-dependent manner;the at 500 μg/mL, the magnitude of inhibition effect
was
synthesis in B16F10 melanoma cells than did arbutin at the same concentrations; inhibition of on
5.57% that of control. Moreover, extract exerted a greater inhibitory
25.2synthesis
melanin ± 5.57% that in of the control.
B16F10 Moreover,
melanoma the extract
did exerted aat
greater inhibitory effect on melanin
melanin synthesis in cells treated with cells than
50 μg/mL arbutin
extract was 208% the
thatsame concentrations;
of cells inhibition
treated with arbutin
synthesis in B16F10 melanoma cells than did arbutin at the same concentrations; inhibition of
of melanin synthesis
(16.3 ± 5.17%) (Figurein 3b).
cellsTreatment
treated with 50 extract
with the µg/mL extract
at 1000 μg/mLwasresulted
208% in that of cellsinhibition
maximum treated with
melanin synthesis in cells treated with 50 μg/mL extract was 208% that of cells treated with arbutin
arbutin (16.3 ± synthesis
of melanin (59.3 ± 4.20%),
5.17%) (Figure which was
3b). Treatment 1.85-fold
with greateratthan
the extract 1000 that of arbutin
µg/mL (1000in
resulted μg/mL)-
maximum
(16.3 ± 5.17%) (Figure 3b). Treatment with the extract at 1000 μg/mL resulted in maximum inhibition
treated
inhibition cells.
of melanin synthesis ± 4.20%),
(59.3 which which was 1.85-fold
of melanin synthesis (59.3 ± 4.20%), was 1.85-fold greater than thatgreater than
of arbutin thatμg/mL)-
(1000 of arbutin
(1000treated
µg/mL)-treated
cells. cells.
50
(%) (%)
50
oxidation
40
oxidation
40
***
30
of L-DOPA
*** ***
30
of L-DOPA
20
*** ***
20
Inhibition
*** ***
10
Inhibition
***
10
0
0 50 100 250 500
0
0
Concentration
50 100
(μg/mL)
250 500
(a)
Concentration (μg/mL)
(a)
Figure 3. Cont.
Molecules 2017, 22, 2198 5 of 15
Molecules 2017, 22, 2198 5 of 15
100
70 ###
### ### ***
60 ### *** ***
***
50
40
*** ***
30
***
***
20
10
0
0 50 100 500 1000
Concentration (μg/mL)
(b)
Figure 3. Inhibitory effects of Citrus junos callus extract on tyrosinase activity and melanin
Figure 3. Inhibitory effects of Citrus junos callus extract on tyrosinase activity and melanin biosynthesis.
biosynthesis. (a) Relative mushroom tyrosinase activity assayed via L-3,4-dihydroxyphenylalanine
(a) Relative mushroom tyrosinase activity assayed via L-3,4-dihydroxyphenylalanine (L-DOPA)
(L-DOPA) oxidation in the absence or presence of the indicated concentrations of the extract; (b) melanin
oxidation in of
content theB16F10
absence or presence
melanoma cells.ofData
the indicated
are mean ±concentrations of the
standard deviation extract; (b)ofmelanin
percentages content
the control
of B16F10 melanoma cells. Data are mean ± standard deviation
(n = 4 per group). *** p < 0.001 compared with the untreated control. percentages
### p < 0.001 compared with(n = 4
of the control
per group). *** p < 0.001 compared with the untreated control. ### p < 0.001 compared with arbutin at
arbutin at the same concentration.
the same concentration.
Lee et al. [32] reported that cinnamic acid and its derivatives inhibit tyrosinase activity because
of their marked antioxidant effects; these materials consistently scavenge diverse ROS, including the
Lee et al. [32] reported that cinnamic acid and its derivatives inhibit tyrosinase activity because
hydroxyl radical, the peroxyl radical, the superoxide anion, hypochlorous acid, and peroxynitrite [33].
of their marked antioxidant effects; these materials consistently scavenge diverse ROS, including the
Kong et al. [34] also showed that cinnamic acid derivatives potently inhibit melanin production in
hydroxyl radical,by
melanocytes thereducing
peroxyl both
radical, the superoxide
tyrosinase anion,
activity and hypochlorous
expression. acid,
This effect wasand peroxynitrite
associated with [33].
Konglow-level
et al. [34]cytotoxicity, resulting in depigmentation of UV-B-induced hyperpigmentation (brown in
also showed that cinnamic acid derivatives potently inhibit melanin production
melanocytes by reducing both tyrosinase activity and expression. This effect was associated with
guinea pig skin).
low-level In this study, resulting
cytotoxicity, the callus in extract at concentrations
depigmentation >5000 μg/mL hyperpigmentation
of UV-B-induced inhibited tyrosinase activity;
(brown atguinea
5000 μg/mL the extract inhibited tyrosinase activity by 23.1 ± 2.36% (35.5% lower than that by
pig skin).
5000
In μg/mL
this arbutin
study, [65.0 ± 5.03%])
the callus extract (data not shown). However,
at concentrations the callusinhibited
>5000 µg/mL extract inhibited L-DOPA
tyrosinase activity;
auto-oxidation in a dose-dependent manner and exerted a greater inhibitory effect on melanin
at 5000 µg/mL the extract inhibited tyrosinase activity by 23.1 ± 2.36% (35.5% lower than that
biosynthesis than arbutin. Although further mechanistic studies are required, the anti-melanogenic
by 5000 µg/mL arbutin [65.0 ± 5.03%]) (data not shown). However, the callus extract inhibited
effect of the callus extract may not be caused by direct inhibition of tyrosinase activity, such as
L-DOPA auto-oxidation in a dose-dependent manner and exerted a greater inhibitory effect on melanin
copper chelation at the active site, but by inhibition of α-glucosidase and glycosylation of tyrosinase
biosynthesis
in melanomathancells
arbutin. Although further mechanistic studies are required, the anti-melanogenic
[35,36].
effect of the callus extract may not be caused by direct inhibition of tyrosinase activity, such as copper
2.3. Skin-Regeneration
chelation Activities
at the active site, but ofbyCitrus junos Callus
inhibition Extract
of α-glucosidase and glycosylation of tyrosinase in
melanoma cells [35,36].
2.3.1. Effects of the Extract on Fibroblast Proliferation and Procollagen Synthesis
2.3. Skin-Regeneration Activities
To evaluate the of Citrus junos
skin-regenerating Callus
activity Extract
of Citrus junos callus extract, we evaluated its effects
on fibroblast proliferation. As shown in Figure 4a, human fibroblasts significantly proliferated after
2.3.1.the
Effects of the
addition of Extract on Fibroblast
>50 ng/mL Proliferation
recombinant and Procollagen
human transforming Synthesis
growth factor-β (rhTGF-β, positive
control)
To and, the
evaluate at 500 ng/mL rhTGF-β,activity
skin-regenerating proliferation wasjunos
of Citrus 128 ±callus
13.6%extract,
greater we
thanevaluated
in the control.
its effects
However, proliferation was not observed following treatment with <100 μg/mL callus extract.
on fibroblast proliferation. As shown in Figure 4a, human fibroblasts significantly proliferated
Maximum fibroblast proliferation with minimal toxicity was caused by treatment with 500 μg/mL
after the addition of >50 ng/mL recombinant human transforming growth factor-β (rhTGF-β,
callus extract (154 ± 8.88%). However, the extract at 10,000 μg/mL severely compromised fibroblast
positive control) and, at 500 ng/mL rhTGF-β, proliferation was 128 ± 13.6% greater than in the
viability due to its cytotoxic effect. Thus, the extract at <1000 μg/mL is well tolerated and may not
control. However, proliferation
exert toxic effects on the skin.was not observed following treatment with <100 µg/mL callus extract.
Maximum fibroblast proliferation with minimal toxicity was caused by treatment with 500 µg/mL
callus extract (154 ± 8.88%). However, the extract at 10,000 µg/mL severely compromised fibroblast
viability due to its cytotoxic effect. Thus, the extract at <1000 µg/mL is well tolerated and may not exert
toxic effects on the skin.
Molecules 2017, 22, 2198 6 of 15
180
***
160 ***
Relative cell proliferation (%)
# #
***
## *** ***
*** ** ***
140
###
###
###
120
100
80
60
40 ###
***
20
0
Untreated 10 50 100 500 1000 1 10 100 200 500 1000 10,000
control
rhTGF-β (ng/mL) Citrus junos
callus extract (μg/mL)
(a)
800
***
700 *** ***
Procollagen type I C-peptide
***
##
concentration (ng/mL)
600 * ##
*
500
400
300
200
100
0
Untreated 10 100 1000 100 200 500
control
rhTGF-β (ng/mL) Citrus junos
callus extract (μg/mL)
(b)
Figure 4. (a) Relative proliferation of and (b) procollagen type I C-peptide synthesis by CCD-986sk
Figure 4. (a) Relative proliferation of and (b) procollagen type I C-peptide synthesis by CCD-986sk
cells after treatment with recombinant human transforming growth factor-β (rhTGF-β) or Citrus junos
cells after treatment
callus with
extract for 24 h.recombinant
Values are meanhuman transforming
± standard deviation growth
(n = 6 perfactor-β
group). *(rhTGF-β)
p < 0.05, ** por Citrus junos
< 0.01,
callus extract for 24
*** p < 0.001 h. Values
compared arethemean
with ± standard
untreated control. deviation
# p < 0.05, p(n< =0.01,
## 6 per pgroup).
### * p < 0.05,with
< 0.001 compared ** p < 0.01,
500 μg/mL
*** p < 0.001 (final concentration)
compared Citrus junos
with the untreated # p < 0.05, ## p < 0.01, ### p < 0.001 compared with
callus extract.
control.
500 µg/mL (final concentration) Citrus junos callus extract.
2.3.2. In Vitro Scratch Wound Recovery Effect of Citrus junos Callus Extract
Treatment
2.3.2. In Vitro Scratchwith Citrus
Wound junos callus
Recovery extract
Effect significantly
of Citrus junosaccelerated wound closure compared
Callus Extract
with the control. An in vitro scratch wound recovery assay showed a 161% higher recovery rate at 24
Treatment with Citrus
h after treatment junos
with 500 callus
μg/mL extract
Citrus junos significantly accelerated
callus extract compared withwound closure
that in the controlcompared
(Figure with
5a). InAn
the control. addition,
in vitrothescratch
callus extract
wound(500 μg/mL final
recovery assayconcentration)
showed a 161%increased fibroblast
higher recoveryproliferation
rate at 24 h after
treatment with 500 µg/mL Citrus junos callus extract compared with that in the control (Figure 5a).
In addition, the callus extract (500 µg/mL final concentration) increased fibroblast proliferation and
migration by 15% compared with rhTGF-β (100 ng/mL); the recovery rate was similar to that afforded
by 500 ng/mL rhTGF-β (Figure 5b).
Molecules 2017, 22, 2198 7 of 15
and migration by 15% compared with rhTGF-β (100 ng/mL); the recovery rate was similar to that7 of 15
Molecules 2017, 22, 2198
afforded by 500 ng/mL rhTGF-β (Figure 5b).
Control
70 rhTGF-β (100 ng/mL)
rhTGF-β (500 ng/mL)
40
30
20
10
0
0 4 8 12 16 20 24
Time (h)
(a)
(b)
Figure 5. Relative scratch wound recovery of CCD-986sk cells after incubation with rhTGF-β or
Figure 5. Relative
Citrus scratch
junos callus wound
extract for 24 recovery of CCD-986sk
h. (a) Time-course cellsscratch
of relative after incubation with(area)
wound recovery rhTGF-β
of or
CitrusCCD-986sk
junos callus
cells;extract for 24 h. (a)
(b) representative Time-course
micrographs of relative
of scratch woundsscratch
treated wound recovery
with rhTGF-β (100 (area)
and of
CCD-986sk cells;or(b)
500 ng/mL) representative
Citrus micrographs
junos callus extract of scratch
(500 μg/mL). Valueswounds
are means treated withdeviation
± standard rhTGF-β(n(100
= 6 and
500 ng/mL) or Citrus
per group). *** p < junos callus extract
0.001 compared with (500 ValuesScale
µg/mL).control.
the untreated are means ± standard
bar in (b): 300 μm. deviation (n = 6
per group). *** p < 0.001 compared with the untreated control. Scale bar in (b): 300 µm.
2.4. Characterization of NLs Containing Citrus junos Callus Extract
2.4. Characterization
To improveof NLs
the skinContaining Citrus
permeability junos
of Citrus Callus
junos Extract
callus extract, extract-loaded NLs were prepared
according to the formulations in Table 1. The average sizes, polydispersity indices (PDIs), and zeta
To improve the skin permeability of Citrus junos callus extract, extract-loaded NLs were prepared
potentials of liposomes produced by emulsification following microfluidization are summarized in
according
Table to the formulations
2. The in Table
liposomes exhibited ~80%1. mean
The average sizes, of
encapsulation polydispersity indices
the callus extract, and (PDIs),
presentedand
as zeta
potentials
nano-sized liposomes 44–147 nm in diameter with PDIs < 0.48. Except for CC-NL#2, all extract-loaded in
of liposomes produced by emulsification following microfluidization are summarized
TableNLs
2. The
hadliposomes exhibited
a highly negative ~80%
surface meanattributable
charge, encapsulation
to theofincorporation
the callus extract, andP75-3.
of Lipoid presented
The as
nano-sized liposomes
mean particle size 44–147 nm inwas
of CC-NL#4 diameter
146.8 ± with
12.66 PDIs < 0.48.
nm, with Exceptsize
a narrow fordistribution
CC-NL#2, (PDI
all extract-loaded
= 0.419).
NLs had a highly negative surface charge, attributable to the incorporation of Lipoid P75-3. The mean
particle size of CC-NL#4 was 146.8 ± 12.66 nm, with a narrow size distribution (PDI = 0.419). The zeta
potential was −65.8 ± 2.29 mV. Transmission electron microscopy (TEM) revealed that all NLs were
well-defined spheres of sizes similar to those determined by particle size analysis (Figure 6).
Molecules 2017, 22, 2198 8 of 15
(a) (b)
Figure 6. (a) Representative particle size distribution and (b) transmission electron micrograph of
(a) Representative
Figure 6. nanoliposomes particle
containing sizecallus
Citrus junos distribution and (b)
extract (CC-NL#4). transmission
Scale bar in (b): 200 electron
nm. micrograph of
nanoliposomes containing Citrus junos callus extract (CC-NL#4). Scale bar in (b): 200 nm.
2.5. In Vitro Skin Permeability of Citrus junos Callus Extract-Loaded NLs
The Permeability
2.5. In Vitro Skin artificial skin permeability of theCallus
of Citrus junos Citrus Extract-Loaded
junos callus extract
NLswas significantly increased by
encapsulation into NLs, with the exception of formulation CC-NL#2 (Figure 7a). The skin permeability of
The artificial
the extractskinwas permeability
influenced by the of the
lipidCitrus junosofcallus
composition the NLsextract
(Tablewas
1 andsignificantly increased by
Figure 7a). The
permeabilities of NLs prepared using Lipoid P75-3 were higher than those of formulations
encapsulation into NLs, with the exception of formulation CC-NL#2 (Figure 7a). The skin permeability of containing
the extractLipoid P100-3 (CC-NL#1 vs. CC-NL#2). In addition, the skin permeability of the callus extract
was influenced by the lipid composition of the NLs (Table 1 and Figure 7a). The permeabilities
increased as the cholesterol content of the NLs increased (CC-NL#1 vs. CC-NL#4). The maximum
of NLs prepared using Lipoid
skin permeability P75-3 were higher than
of p-hydroxycinnamoylmalic acid those
from theof extract-loaded
formulationsNLs containing
was fromLipoid
the P100-3
(CC-NL#1formulation
vs. CC-NL#2).CC-NL#4,In theaddition,
permeability the skin permeability
of which of the
was 5.11-fold greater thancallus
that of extract increased
the extract alone as the
cholesterol(9.34 ± 0.35 ×of
content 10−6
thevs. NLs
1.83 ±increased
0.14 × 10−6 cm/s).
(CC-NL#1 vs. CC-NL#4). The maximum skin permeability
In vitro skin permeation of Citrus junos callus extract across a human epidermal layer is shown
of p-hydroxycinnamoylmalic acid from the extract-loaded NLs was from the formulation CC-NL#4,
in Figure 7b. The cumulative penetrated concentration of p-hydroxycinnamoylmalic acid from NLs
the permeability of which was 5.11-fold greater than that of the extract alone (9.34 ± 0.35 × 10−6 vs.
1.83 ± 0.14 × 10−6 cm/s).
In vitro skin permeation of Citrus junos callus extract across a human epidermal layer is shown in
Figure 7b. The cumulative penetrated concentration of p-hydroxycinnamoylmalic acid from NLs of the
CC-NL#4 formulation at 6 h was 5.84-fold greater than that of Citrus junos callus extract alone, resulting
in a 17.67-fold increase in the skin flux of p-hydroxycinnamoylmalic acid after 24 h (6.89 ± 2.11 vs.
0.39 ± 0.55 µg/cm2 /h).
Molecules 2017, 22, 2198 9 of 15
of the CC-NL#4 formulation at 6 h was 5.84-fold greater than that of Citrus junos callus extract alone,
resulting in 22,
Molecules 2017, a 2198
17.67-fold increase in the skin flux of p-hydroxycinnamoylmalic acid after 924 h
of 15
(6.89 ± 2.11 vs. 0.39 ± 0.55 μg/cm2/h).
12
8 ***
6
***
***
4
2
*
0
os L#
1
L#
2
L#
3
L#
4
L#
5
s jun ract -N -N -N -N -N
r u x t C C C C C C C C C C
Cit lus e
l
ca
(a)
40 Citrus junos callus extract
CC-NL#4
Permeated concentration (μg/mL)
35
30
25 ***
20
15
10
0 3 6 9 12 15 18 21 24
Time (h)
(b)
Figure 7. In vitro permeation of Citrus junos callus extract-loaded NLs across artificial skin (Skin
Figure 7. In vitro permeation of Citrus junos callus extract-loaded NLs across artificial skin (Skin PAMPA)
PAMPA) and a human epidermal layer. (a) Effective permeability values of the callus extract or callus
and a human epidermal layer. (a) Effective permeability values of the callus extract or callus
extract-loaded NLs through Skin PAMPA; (b) time-course of cumulative permeated concentrations of
extract-loaded NLs through Skin PAMPA; (b) time-course of cumulative permeated concentrations of
p-hydroxycinnamoylmalic acid from callus extract solution or callus extract-loaded NLs (CC-NL#4)
p-hydroxycinnamoylmalic acid from callus extract solution or callus extract-loaded NLs (CC-NL#4)
through human epidermis layer. Values are mean ± standard deviation (n = 6 per group). * p < 0.05,
through human epidermis layer. Values are mean ± standard deviation (n = 6 per group). * p < 0.05,
*** p < 0.001 compared with the Citrus junos callus extract.
*** p < 0.001 compared with the Citrus junos callus extract.
Cholesterol enhances the rigidity and physical stability of liposomal membranes, thereby
Cholesterol
decreasing enhances
the leakage the rigidity and physical
of liposome-loaded stability of liposomal
active compounds membranes,
[37]. Furthermore, thereby decreasing
cholesterol increases
the fluidity of phospholipids in the bilayer at temperatures lower than their melting point (Tfluidity
the leakage of liposome-loaded active compounds [37]. Furthermore, cholesterol increases the m), but
of phospholipids
decreases in the bilayer
their fluidity at temperatures
if the bilayer lower thanabove
is at temperatures their their
melting
Tm point (Tm ), but decreases
[38]. Therefore, the skin
their fluidity if
permeability ofthe bilayer
callus is atfrom
extract temperatures
NLs composedaboveof their Tm [38]. Therefore,
hydrogenated the skin permeability
phosphatidylcholine (HPC) and of
callus extract
cholesterol mayfrombe NLs composed
enhanced becauseof hydrogenated phosphatidylcholine
the Tm of phospholipids (HPC)
in the bilayer wasandlowercholesterol
than the may
skin
be enhanced because the T m of phospholipids in the bilayer was
temperature, creating a lipid-fluidizing effect and facilitating lipid incorporationlower than the skin temperature,
into the
creating a lipid-fluidizing
intercellular domain and effect and facilitating
modification of skin lipid
lipids.incorporation
Thus, Citrusinto thecallus
junos intercellular domain NLs
extract-loaded and
modification
may of skin
be used as lipids. Thus,
an anti-aging Citrusthat
cosmetic junos callus
also exertsextract-loaded NLs may
a skin-lightening be used as an anti-aging
effect.
cosmetic that also exerts a skin-lightening effect.
Molecules 2017, 22, 2198 10 of 15
3.1. Materials
Murashige and Skoog (MS) medium, picloram, 6-benzyladenine (BA), and 2,4-dichlorophen
oxyacetic acid (2,4-D) were purchased from Duchefa Bohemia B.V. (Haarlem, The Netherlands).
DPPH, ascorbic acid, L-tyrosine, L-3,4-dihydroxyphenylalanine (L-DOPA), tyrosinase, arbutin,
and recombinant human transforming growth factor-β (rhTGF-β) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Hydrogenated lecithin (Lipoid P75-3) and hydrogenated phosphatidylcholine
(HPC; Lipoid P100-3) were obtained from Lipoid (Ludwigshafen, Germany). Other chemicals were
purchased from Merck KGaA (Darmstadt, Germany) and Thermo Fisher Scientific Inc. (Waltham,
MA, USA).
where A is the absorbance at 475 nm of the control, and B is the absorbance of the test sample at
475 nm.
3.7. Statistics
All data are expressed as means ± standard deviation. A p-value <0.05 was considered to reflect
statistical significance when the t-test was used to compare two mean values of unpaired data, or on
one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test when more
than three mean values of unpaired data were compared.
4. Conclusions
In this work, Citrus junos plant callus was produced by transferring the explants into MS medium
supplemented with picloram as a growth regulator. The total phenol content in callus extracts was
24.50 ± 0.43 mg/g, and the level of p-hydroxycinnamoylmalic acid, the major phenolic compound
was found as 51.49 ± 0.01 mg/g in the extract. The DPPH free radical-scavenging efficacy of the
callus extract (1000 µg/mL) was equivalent to that of 125 µM ascorbic acid. The callus extract
also exhibited anti-tyrosinase activity, and both L-DOPA oxidation and melanin biosynthesis were
significantly reduced by treatment with the extract at >50 µg/mL. Fibroblasts proliferation and
their procollagen synthesis were significantly promoted (by 154% and 176%, respectively) after
treatment with 500 µg/mL of callus extract. In addition, the callus extract significantly accelerated
scratch wound recovery in vitro compared with the control. The flux of Citrus junos callus extract
incorporated into NLs through the human epidermis was 17.67-fold greater than that of the callus
extract alone. Thus, Citrus junos callus extract-loaded NLs may serve as an anti-aging cosmeceutic
with a skin-lightening effect.
Acknowledgments: This research was supported by the Ministry of Trade, Industry, and Energy (MOTIE);
Korea Institute of Advancement of Technology (KIAT), through the Encouragement Program for the Industries of
Molecules 2017, 22, 2198 14 of 15
Economic Co-operation Region (R0004540). We thank BIO-FD&C Co. Ltd. (Hwasun-gun, Jeonnam, Republic of
Korea) for their technical support of this research.
Author Contributions: Hyun Jung Kim and Jin Woo Park conceived and designed the experiments; Jin Woo Park
led the project; Deepak Adhikari, Vijay Kumar Panthi, and Rudra Pangeni performed the experiments;
Deepak Adhikari, Vijay Kumar Panthi, Rudrfa Pangeni, and Hyun Jung Kim analyzed the data; Hyun Jung Kim
and Jin Woo Park wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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