Journal of Cosmetics, Dermatological Sciences and Applications, 2012, 2, 151-157
http://dx.doi.org/10.4236/jcdsa.2012.23030 Published Online September 2012 (http://www.SciRP.org/journal/jcdsa)
151
The Melanin Biosynthesis Stimulating Compounds Isolated
from the Fruiting Bodies of Pleurotus citrinopileatus
Tian-Xiao Meng1, Chao-Feng Zhang1,2, Tomofumi Miyamoto3, Hiroya Ishikawa4, Kuniyoshi Shimizu1*,
Shoji Ohga1, Ryuichiro Kondo1
1Department of Agro-Environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan; 2Research Department of
Pharmacognosy, China Pharmaceutical University, Nanjing, China; 3Graduate School of Pharmaceutical Sciences, Kyushu University,
Fukuoka, Japan; 4Department of Nutrition and Health Science, Faculty of Human Environmental Science, Fukuoka Women’s University,
Fukuoka, Japan.
Received June 14th, 2012; revised July 19th, 2012; accepted July 30th, 2012
ABSTRACT
In the course to find a stimulating compound for melanin biosynthesis, which should be useful for a gray and a white
hair-preventive agent or tanning agent, we evaluated the effects of the methanol extract from mushroom of Pleurotus
citrinopileatus on melanin production in B16 melanoma cells without theophylline. Activity-guided fractionation led to
isolate myo-inositol (3) and D-mannitol (4) as the stimulating compounds on melanin production in B16 melanoma cells.
Also, ergosterol (1), uracil (2), and D-glucose (5) have been isolated from the methanol extract of P. citrinopileatus and
showed no effect on melanin production in B16 melanoma cells. These results indicated that myo-inositol (3) and D-
mannitol (4) are potential candidates that could be useful such as a gray and a white hair-preventive agent or tanning
agent.
Keywords: Pleurotus citrinopileatus; myo-Inositol; D-Mannitol; Melanin Stimulating Activity; White Hair-Preventive
Agent; Tanning Agent
1. Introduction
Mushrooms are a nutritionally functional food and a source
of physiologically beneficial medicines. Fruiting bodies
of some wild and cultivatable mushrooms contain medi-
cinal compounds that are used in traditional medicines
and cosmetics. There are numerous potential medicinal
products from mushrooms that could be used in cosme-
ceuticals (products applied topically, such as creams, lo-
tions, and ointments) or nutricosmetics (products that are
ingested orally). But, there are numerous mushroom spe-
cies that are untested, undescribed, or not yet cultiva-
table and that have huge potential for use in the cosmetic
industry. Some fungi are also used in bio-transformation,
and products such as lactic acid and ceramides could po-
tentially be used in cosmetics [1,2].
Pleurotus citrinopileatus is an edible mushroom (Sy-
nonymy: P. cornucopiae, P. cornucopiae var. citrinopilea-
tus) belonging to the genus Pleurotus, Pleurotaceae fam-
ily. The name of this mushroom in English is golden
oyster mushroom, tamogitake in Japanese, yuhuangmo in
Chinese, goldenseed in Korean, and weishenga limonaya
in Russian. A half dozen recent studies have focused on
the cultivation of P. citrinopileatus for its numerous mul-
tifunctional biological activities, such as melanin bio-
synthesis inhibitory activity, antioxidant, antibacterial,
and antihyaluronidase activities [2,3]. However, there are
a limited number of previous studies on the chemical
composition, and there have been a few of reports iden-
tifying the lectin, peptide and protein from water extracts
of P. citrinopileatus [4,5].
Skin pigmentation results from melanin synthesis by
melanocytes and is caused by exposure to UV radiation.
Tyrosinase is a key enzyme of melanin synthesis that ca-
talyzes three different reactions: the hydroxylation of ty-
rosine to 3,4-dihydroxyphenylalanine (DOPA), the oxi-
dation of DOPA to DOPA-quinone, and the oxidation of
5,6-dihydroxyindole (DHI) to indole-quinone [6]. In the
absence of thiols DOPA-quinone changes to DOPA chro-
me and then to DHI or indole 5,6-quinone 2-carboxy-lic
acid (DHICA). Broadly, there are two further steps in
this melanogenic pathway, one involves tyrosinase rela-
ted protein-2 (TRP-2; DOPA chrome tautomerase) which
catalyzes the conversion of DOPA chrome to DHICA,
and the other involves TRP-1 (DHICA oxidase) which
catalyzes the oxidation of DHICA [7,8]. There are seve-
ral signal pathways for enhancing melanin production.
*Corresponding author.

The Melanin Biosynthesis Stimulating Compounds Isolated from the Fruiting Bodies of Pleurotus citrinopileatu 152
The cAMP-mediated pathway is a well known melanin
synthesis cascade. α-melanocyte stimulating hormone (α-
MSH), prostgland-in E2 (PGE2), and adrenocorticotropic
hormone (ACTH) activate the cAMP-mediated pathway
[9,10].
Human skin is repeatedly exposed to ultraviolet radia-
tion (UVR) that influences the function and survival of
many cell types and is regarded as the main causative
factor in the induction of skin cancer. It has been tradi-
tionally believed that skin pigmentation is the most im-
portant photo-protective factor, since melanin, besides
functioning as a broadband UV absorbent, has anti-oxi-
dant and radical scavenging properties. Besides, many
epidemiological studies have shown a lower incidence
for skin cancer in individuals with darker skin compared
to those with fair skin. Skin pigmentation is of great cul-
tural and cosmetic importance. In light of the increasing
incidence for UV induced skin cancer and the progres-
sive depletion of the ozone layer, which contrasts to pub-
lic perception of a tan as being healthy, a better under-
standing of the role of melanin in preventing UV induced
DNA damage and malignant transformation of skin cells
would be more than desirable [11]. Melanin containing
tissues have been located in various parts of the human
body outside the skin complex, including in the heart,
lungs, liver, brain [12], lymphocytes [13], and inner ear
[14]. Melanin is a pigment that gives color to the skin,
eyes, and hair. Lack of melanin pigmentation occurs prin-
cipally due to regional lack of melanocytes (e.g. piebald-
ism or vitiligo) or to the genetic transmission of muta-
tions in pigment related genes that give rise to hypopig-
mentation (e.g. albinism) when inherited in a homozy-
gous form. There are several forms of oculocutaneous al-
binism [15]. Skin lightening or whitening (leukoderma,
hypopigmentation) is most commonly the result of de-
creased melanin content in the skin (hypomelanosis) [16].
Increase of epidermal turnover can also induce hypome-
lanosis. Hypomelanosis may affect hair color. Canities
means a generalized loss of hair color, whereas poliosis
refers to localized hypomelanosis involving a tuft of hair
or a few hairs in the eyebrows or eyelashes [16].
In our preliminary screening, we have found that the
methanol extract of P. citrinopileatus showed the stimu-
lating effects on melanin formation in B16 melanoma
cells. We investigate the melanin biosynthesis stimulatory
effect of the methanol extract from the mushroom of P.
citrinopileatus on B16 melanoma cells in order to identify
potential melanin producing candidates, which are useful
such as skin-tanning and white hair-preventive cos metics.
2. Materials and Methods
2.1. General Experimental Procedure
Column chromatography was performed by silica gel
(Wakogel C-200 particle size 75 - 150 μm; Wako Pure
Chemical Industries, Co., Ltd., Japan). Thin layer chro-
matography (TLC) was carried out using Merck pre-
coated silica gel 60 F254 plates (0.25 mm, Merck & Co.,
Inc., Darmstadt, Germany) and spots were detected with
I2 detection and under UV light. The compound 1 was
isolated by preparative high performance liquid chroma-
tography (HPLC) using a Waters TM 600 Controller,
Waters TM 486 Tunable Absorbance Detector and Wa-
ters 600 Multi-solvent Delivery System (Japan Water Co.,
Ltd., Japan). The absorbance was measured by Tecan
Spectra microplate reader (Tecan Japan Co., Ltd., Japan)
and UV/VIS Spectrometer V-530 (JASCO Co., Japan).
Preparative column using Inertsil preparative ODS col-
umn (20 mm i.d. × 250 mm) from GL Sciences (GL
Sciences Inc., USA).
2.2. Chemicals
Dimethylsulfoxide (DMSO), potassium hydroxide solu-
tion (NaOH), hydrochloric acid (HCl) and sodium hy-
drogen carbonate (NaHCO3) were purchased from Wako
(Wako Pure Chemical Industries, Ltd., Japan). Thiazolyl
blue tetrazolium bromide (MTT) was obtained from
Sigma (Sigma-Aldrich Co., USA). Qualified fetal bovine
serum (FBS) was obtained from Gibco® (Life Technolo-
gies Co., USA). Ethylene diamine tetraacetic acid (EDTA)
was obtained from Dojindo (Dojindo Molecular Techno-
logies, Inc., Japan). Trypsin was obtained from Nihon
Pharmaceutical (Nihon Pharmaceutical Co., Ltd., Japan).
Eagle’s minimal essential media (EMEM) and Glutamine
were purchased from Nissui (Nissui Pharmaceutical Co.,
Ltd., Japan). Theophylline was obtained from Sigma (Sig-
ma Chemical Co., USA).
2.3. Mushroom Materials
Fresh fruiting bodies of P. citrinopileatus were obtained
from Tamogitake Pharmaceutics Co., Ltd. (Nagano, Ja-
pan). The fruiting bodies were cleaned to remove any re-
sidual materials and then freeze-dried. The milled freeze-
dried P. citrinopileatus (900.0 g) were extracted with
methanol (2 × 9.0 L) at room temperature for one week
and then filtered. The methanol extract was concentrated
by a rotary evaporator. The yield of the methanol extract
was 114.0 g (12.7%).
2.4. Extraction and Isolation
A portion of the methanol extract (100.0 g) was applied
to a silica gel column (Wakogel C-200, 4.0 kg; 19 cm i.d.
× 50 cm) and eluted with n-hexane/chloroform (7:3, 5:5,
3:7, 0:10), ethyl acetate, acetone, ethanol and methanol
(each 8.0 L), followed by methanol/water (50:1, 6.0 L;
11:1, 2.0 L; 8:1, 3.0 L), affording eight fractions (Fr.1 to

The Melanin Biosynthesis Stimulating Compounds Isolated from the Fruiting Bodies of Pleurotus citrinopileatu 153
Fr.8). Based on TLC analysis, using n-hexane/chloro-
form (1:9), Fr.1 (Rf = 0, 0.21, 0.34, 0.41, 0.62, 0.72,
0.82), Fr.2 (Rf = 0, 0.17, 0.28, 0.34, 0.62, 0.69) and Fr.3
(Rf = 0, 0.17, 0.28, 0.34, 0.62, 0.69) were combined to
yield Fr.1' (7.0 g). A portion of the Fr.1' (4.6 g) was ap-
plied to a silica gel column chromatography (Wakogel C-
200, 900 g; 5.5 cm i.d. ×120 cm) and eluted with n-hex-
ane/ethyl acetate (20:1, 15:5, 10:10, 0:20, each 1.5 L),
ethyl acetate/methanol (20:1, 15:5, 10:10, 0:20, each 1.5
L) to give eight fractions (Fr.1'-1 to Fr.1'-8). Fr.1'-4-2
(494 mg) was recrystallized from methanol to give com-
pound 1 (401 mg). Fr.1'-7 (344 mg) was fractionation by
preparative HPLC (Inertsil Prep-ODS column, 20 mm i.d.
× 250 mm) and retention time was 16.5 min to give com-
pound 2 (36 mg). Fr.1'-8 (1.0 g) was recrystallized from
methanol to give compound 3 (42 mg). Fr.4 and Fr.5
were combined to Fr.4' (4.5 g), which was fractionated
by silica gel column chromatography (Wakogel C-200,
4.0 kg; 19 cm i.d. × 50 cm) to obtain six fractions (Fr.4'-1
to Fr.4'-6). Fraction 4’-6 (1.0 g) of by silica gel column
chromatography (120 cm × 6.0 cm i.d.; n-hexane/chlo-
roform 2:8, 1:9, 0:10; chloroform/methanol 9:1, 8:2, 7:3,
6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, each 1400 mL) to obtain
seven fractions (Fr.4'-6-1 to Fr.4'-6-7). Fraction 4'-6-5
(182 mg) was recrystallized from methanol to give com-
pound 4 (54 mg). Fr. 6 (3.0 g) was fractionated by silica
gel column chromatography (120 cm × 6.0 cm i.d.; chlo-
roform/methanol 2:8, 1.5:8.5, 1:9, 0.5:9.5, 0:10, each
1400 ml) to obtain five fractions. Fr.6-3 (2.4 g) was re-
crystallized from methanol to give compound 5 (2.3 g).
The nuclear magnetic resonance (NMR, 400MHz, JEOL
Ltd., Japan) and gas chromate-graphy mass spectrometry
(GC-MS) (GC-17A/QP5050; Shimadzu Corporation Ltd.,
Japan) data of the isolating compounds (Figure 1) were
compared with those of authentic samples and reference
[17-24].
2.5. Inhibitory Effect on Melanogenesis Using
Cultured B16 Melanoma Cells
2.5.1. Determination of Melanin Content
B16 melanoma, a mouse melanoma cell line producing
melanin, was obtained from RIKEN Cell Bank. The cells
were maintained in EMEM supplemented with 10% (v/v)
fetal bovine serum (FBS) and 0.3 mg/ml glutamine. The
cells were incubated at 37˚C in a humidified atmosphere
of 5% CO2. Confluent cultures of B16 melanoma cells
were rinsed in phosphate buffered saline (PBS) and re-
moved from the plastic using 0.25%trypsin/0.02%EDTA.
The cells were placed 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, the media were replaced
with 900 μL (998 μL) of fresh media and 100 μL (2 μL)
of water (DMSO) was added with or without (control)
HO
HH
NH
ON
H
O
HO
HO
OH OH
OH
OH
HO
OH
OH
OHOH
OH
O
HO
HO
OH
OH
OH
1 2
3 4
5
Figure 1. Structures of compounds 1 - 5.
the test sample at various concentrations and its repli-
cates were three times. The cells were incubated for an
additional 48 h, and then the medium was replaced with
fresh medium containing each sample. After 24 h, re-
moving the medium and washing the cells, the cell pellet
was dissolved in 1.0 mL of 1 N NaOH. The crude cell
extracts were assayed using a micro plate reader at 405
nm to determine melanin content. The results from the
samples were analyzed as a percent of the control culture.
Theophylline was used as a positive control.
2.5.2. Cell Viability
Cell viability was determined by use of the microculture
tetrazolium technique (MTT assay). A culture was initi-
ated, and after incubation, 50 μL of MTT in phosphate
buffered saline (5 mg/mL) was added to each well. The
plates were incubated for 4 h. After removing the me-
dium, formazan crystals were dissolved in 1.0 mL of
0.04 N HCl and the absorbance was measured at 570 nm
relative to 630 nm.
3. Results
In present study, we evaluate the effect of the methanol
extracts of the fruiting bodies of P. citrinopileatus on
melanin production in B16 melanoma cells without theo-
phylline. To search for melanin production stimulating
compounds, we modified the assay using B16 melanoma
cells. It should be noted that theophylline is usually
added to medium for stimulating melanin production in
B16 melanoma cells. Theophylline is known as an ana-
logue of cAMP, which is a second messenger for mela-

The Melanin Biosynthesis Stimulating Compounds Isolated from the Fruiting Bodies of Pleurotus citrinopileatu 154
nin biosynthesis [25]. So, in our modified assay, theo-
phylline was not added into medium, which is for finding
melanin production stimulating compound. An index such
as “mean of melanin content (%)/mean of cell viability
(%)” called as MC/CV value was applied for evaluating
the stimulating activity of melanin production per cell in-
duced by samples.
The methanol extracts were assayed by using B16 me-
lanoma cells in order to evaluate the stimulation of mela-
nin production and cell viability. The stimulating effect
of methanol extracts on melanin production in B16 mela-
noma cells was shown at various concentrations (Table
1). At the concentration of 5.0 mg/mL, the methanol ex-
tract showed melanin production stimulating activity per
cell with MC/CV value of 1.5.
The methanol extract was subjected to a silica gel col-
umn (Wakogel C-200, 4.0 kg; 19 cm i.d. × 50 cm), gra-
dient eluted with n-hexane/chloroform (7:3, 5:5, 3:7,
0:10), ethyl acetate, acetone, ethanol and methanol (each
8.0 L), followed by methanol/water (50:1, 6.0 L; 11:1,
2.0 L; 8:1, 3.0 L), affording eight fractions (Fr.1 to Fr.8).
Fr.3 and Fr.4 showed (Table 2) higher melanin produc-
tion stimulating activity with MC/CV value of 4.0 and
4.4, respectively. Activity-guided fractionation of Fr.3
and Fr.4 led to the isolation of uracil (2), myo-inositol (3)
and D-mannitol (4) as main components. Activity-guided
fractionation of Fr.4 led to the isolation of D-mannitol as
dominant component. Both of myo-inositol (3) (12 mg/
ml) and D-manntiol (4) (18 mg/ml) showed (Tables 3
and 4) potential melanin production stimulating activity
with MC/CV value of 1.6. Also, another weak activity-
guided fractionation of Fr.2 and Fr.6 led to ergosterol (1)
and D-glucose (5). However, ergosterol (1), uracil (2),
and D-glucose (5) showed no effect on melanin produc-
tion in B16 melanoma cells (data not show). These re-
Table 1. The effect of methanol prepared extract from the
fruiting bodies of P. citrinopileatus on B16 melanoma cells.
Concentration
(mg/mL)
Melanin content
(% vs. control)
Cell viability
(% vs. control) MC/CVb
0.0 100.0 ± 1.5 100.0 ± 3.2 1.0
0.3 92.6 ± 5.5 92.7 ± 4.8 1.0
0.6 96.5 ± 3.6 97.9 ± 0.5 1.0
1.3 86.0 ± 2.4** 93.6 ± 4.7 0.9
2.5 95.3 ± 0.7* 77.3 ± 6.1* 1.2
5.0 88.3 ± 1.8** 60.4 ± 4.4** 1.5
Theophyllinea
(0.01) 138.6 ± 3.4** 96.1 ± 3.4 1.4
Data presented as means ± R.S.D. (n = 3); *p < 0.05, **p < 0.01, Signifi-
cantly different from control group. aPositive control for melanin stimulating
activity. bMC/CV indicate “mean of melanin content (%)/mean of cell vi-
ability (%)”.
Table 2. The effect of each fractions obtained from metha-
nol extract from the fruiting bodies of P. citrinopileatuson
B16 melanoma cells.
Samples Concentration
(mg/mL)b
Melanin
content
(% vs.
control)
Cell
viability
(% vs.
control)
MC/CVc
Control 0.0 100.0 ± 2.1 100.0 ± 2.71.0
Fr.1 0.4 103.5 ± 7.4 87.9 ± 6.0*1.2
Fr.2 1.9 86.1 ± 7.5 35.3 ± 1.8** 2.4
Fr.3 1.7 67.2 ± 7.9* 16.8 ± 3.4** 4.0
Fr.4 1.9 73.6 ± 0.9* 16.6 ± 1.5** 4.4
Fr.5 2.6 78.1 ± 5.2* 26.8 ± 0.8** 2.9
Fr.6 3.1 121.4 ± 4.6* 58.5 ± 4.5** 2.1
Fr.7 5.6 50.8 ± 2.2** 40.2 ± 2.9** 1.3
Fr.8 4.9 51.7 ± 0.7** 18.3 ± 1.0** 2.8
Theophyllinea0.01 138.6 ± 3.4** 96.1 ± 3.41.4
Data presented as means ± R.S.D. (n = 3); *p < 0.05, **p < 0.01, Signifi-
cantly different from control group. aPositive control for melanin stimulating
activity. bThe concentration of each sample with maximum solubility were
selected. cMC/CV indicate “mean of melanin content (%)/ mean of cell via-
bility (%)”.
Table 3. The effect of myo-inositol isolated from the fruiting
bodies of P. citrinopileatus on B16 melanoma cells.
Samples (mg/mL) Melanin content
(% vs. control)
Cell viability
(% vs. control)MC/CVb
0 100.0 ± 2.2 100.0 ± 4.0 1.0
1 106.3 ± 5.7 92.8 ± 2.5* 1.2
2 103.8 ± 2.9 91.5 ± 0.9** 1.1
5 101.9 ± 2.9 89.2 ± 1.1** 1.1
9 110.1 ± 3.8* 76.8 ± 0.41** 1.4
18 111.4 ± 5.8* 70.8 ± 4.7** 1.6
Theophyllinea (0.01) 140.5 ± 5.0** 102.6 ± 6.7 1.4
Data presented as means ± R.S.D. (n = 3); *p < 0.05, **p < 0.01, Signifi-
cantly different from control group. aPositive control for melanin stimulating
activity. bMC/CV indicate “mean of melanin content (%)/mean of cell vi-
ability (%)”.
sults indicated that myo-inositol (3) and D-mannitol (4)
are potential candidates that could be useful, as a tanning
and a white hair-preventive agent.
4. Discussion
As described above, skin pigmentation results from me-
lanin synthesis by several enzymes such as tyrosinase in
melanocytes and is caused by exposure to UV radiation.
Outside of them, there are several pathways for enhanc-

The Melanin Biosynthesis Stimulating Compounds Isolated from the Fruiting Bodies of Pleurotus citrinopileatu 155
Table 4. The effect of D-mannitol isolated from the fruiting
bodies of P. citrinopileatus on B16 melanoma cells.
Sampes (mg/mL) Melanin content
(% vs. control)
Cell viability
(% vs. control) MC/CVb
0 100.0 ± 2.5 100.0 ± 1.7 1.0
2 101.3 ± 10.1 88.5 ± 7.5 1.1
3 112.9 ± 4.3 104.0 ± 2.0** 1.1
6 111.2 ± 3.5 94.4 ± 5.1** 1.2
12 116.1 ± 6.3* 86.0 ± 2.8** 1.3
25 106.3 ± 2.8* 66.0 ± 3.1** 1.6
Theophyllinea (0.01) 137.9 ± 1.3** 100.3 ± 0.7 1.4
Data presented as means ± R.S.D. (n = 3); *p < 0.05, **p < 0.01, Signifi-
cantly different from control group. aPositive control for melanin stimulating
activity. bMC/CV indicate “mean of melanin content(%)/ mean of cell viabi-
lity (%)”.
ing melanin production. The cAMP-mediated pathway is
a well-known melanin synthesis cascade and α-MSH, pro-
staglandin E2 (PGE2), and adrenocorticotropic hormone
(ACTH) activate the cAMP-mediated pathway [9,10].
On the other hand, a cGMP-mediated pathway can
also increase melanin production. This pathway is acti-
vated by nitric oxide (NO), which is released by kerati-
nocytes following UV-B irradiation. Protein kinase C
(PKC) can activate tyrosinase. UV light might activate
cell membrane bound phospholipase C, and augmented
diacylglycerol (DAG) can activate PKC [26].
Skin is a major candidate target of oxidative stress
caused by reactive species (RS), including reactive oxy-
gen species and reactive nitrogen species. RS are major
and significant contributors to skin hyper pigmentation
and skin aging. It is generally believed that agents having
antioxidant activity show anti-aging, whitening, and anti-
inflammatory activities [27]. If free radicals are inappro-
priately processed in melanin synthesis, hydrogen per-
oxide (H2O2) is generated, leading to the production of
hydroxyl radicals (HO‧) and other reactive oxygen spe-
cies (ROS) [28]. Oxidative stress may be induced by in-
creasing the generation of ROS and other free radicals.
UV radiation can induce the formation of ROS in skin
such as singlet oxygen and superoxide anions, promoting
biological damage in exposed tissues via ironcatalyzed
oxidative reactions. These ROS enhance melanin bio-
synthesis, damage DNA, and may induce proliferation of
melanocytes [29]. Yamakoshi et al. [29] found evidence
for a role of oxidative stress in the pathogenesis of skin
disorders. It is known that ROS scavengers or inhibitors
such as antioxidants may reduce hyperpigmentation. Ad-
ditionally, superoxide dismutase (SOD, EC 1.15.1.1), which
catalyzes the dismutation of the superoxide anion into
hydrogen peroxide and molecular oxygen, is one of the
most important antioxidative enzymes.
The myo-inositol (3) and D-mannitol (4) were known
as hydroxyl radical scavengers [30], so should be con-
cerned in radical pathway. Further, the two compounds
showed no activity against ORAC, SOD like assay and
DPPH (data not show). Considering the role of ROS and
their effects against ROS, their mechanisms of the mela-
nin production stimulating activity in B16 melanoma
cells should be related with other factor such as cAMP
signaling rather than their effects on ROS.
According to the increase of the elderly population,
many people are afflicted with white hair. Thus, the mar-
ket for hair-dye and anti-white hair agents are growing.
White hair is caused by a genetic predisposition, aging,
decrement of melanocytes by environmental stress, and
decrement of the biosynthesis of melanin pigment, or
melanogenesis [31,32]. Hair-dye agents are used for the
treatment of white hair, and some anti-white hair agents
are under development. However, there remain some
problems with these agents, such as insufficient activity
and side effects due to the dyes. Thus, there is a need for
safer anti-white hair agents exhibiting satisfactory mela-
nogenesis activity and white hair prevention [33]. Since a
melanocyte reservoir exists in the human hair follicle
[31], it is considered that stimulation and/or activation of
melanocyte in the hair follicle is a prospective means to
prevent white hair [33].
In addition, inositol 1,4,5-trisphosphate (IP3) releases
calcium from intracellular stores [34,35], signal transduc-
tion, stress, protection, hormonal homeostasis and cell
wall biosynthesis in plants [36]. The functions and roles
of myo-inositol in humans have been linked to bipolar
disorder [37], production of L-chiro-inositol and D-chiro-
inositol in insulin action [38], multiple sclerosis [39], Al-
zheimer’s disease [40] and regulation of the sorbitol path-
way in diabetic patients [41]. Mannitol is used as a sweet-
tasting bodying and texturing agent [42]. The complex of
boric acid with mannitol is used in the production of dry
electrolytic capacitors. It is an extensively used polyol
for production of resins and surfactants [43]. Mannitol is
used in medicine as a powerful osmotic diuretic (to in-
crease the formation of urine in order to prevent and treat
acute renal failure and also in the removal of toxic sub-
stances from the body) and in many types of surgery for
the prevention of kidney failure (to alter the osmolarity
of the glomerular filtrate) and to reduce dye and brain
oedema (increased brain water content). Hypertonic man-
nitol can enhance the transport of drugs across the blood-
brain barrier for the treatment of life-threatening brain
diseases [44]. Inhaled mannitol improves the hydration
and surface properties of sputum in patients with cystic
fibrosis [45]. Mannitol hexanitrate is a well-known vaso-
dilator, used in the treatment of hypertension [46]. Man-
nitol is also a scavenger of hydroxyl radicals [30].

The Melanin Biosynthesis Stimulating Compounds Isolated from the Fruiting Bodies of Pleurotus citrinopileatu 156
5. Conclusion
In this study, we found a new facet of biological activity
of myo-inositol (3) and D-mannitol (4) isolated from P.
citrinopileatus, stimulating activity of melanin produc-
tion. Therefore, these compounds are potential candidates
that could be useful as a gray and a white hair-preventive
agent or a tanning agent.
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