Environmental Toxicology and Pharmacology 46 (2016) 270–276

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Environmental Toxicology and Pharmacology

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

Preventive effects of cedrol against alopecia in

cyclophosphamide-treated mice
Shan-Shan Chen a , Yan Zhang a , Qiu-Li Lu a , Zhe Lin a , Yuqing Zhao a,b,∗
a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
b
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s
Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Although numerous hypotheses have been proposed to prevent chemotherapy-induced alopecia (CIA),
Received 30 March 2016 effective pharmaceuticals have yet to be developed. In our study, the back hairs of C57BL/6 mice were
Received in revised form 16 July 2016 factitiously removed. These mice were then treated with cedrol or minoxidil daily. Mice with early-stage
Accepted 29 July 2016
anagen VI hair follicles were treated with cyclophosphamide (CYP, 125 mg/kg) to induce alopecia. The
Available online 30 July 2016
CYP-damaged hair follicles were observed and quantified by using a digital photomicrograph. The results
demonstrated that the minoxidil-treated mice suffered from complete alopecia similar to the model
Keywords:
6 days after CYP administration. Simultaneously, the cedrol-treated (200 mg/kg) mice manifested mild
Alopecia
Chemotherapy
alopecia with 40% suppression. Histological observation revealed that anagen hair follicles of the cedrol-
Cyclophosphamide pretreated mice (82.5%) likely provided from damage compared with the sparse and dystrophic hair
Cedrol follicles of the model mice (37.0%). Therefore, the use of topical cedrol can prevent hair follicle dystrophy
Hair follicle and provide local protection against CIA.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction Multiple agents isolated from Chinese medicinal herbs or bio-

Although various cancer treatments have been reported,
anti-cancer agents, such as doxorubicin, daunorubicin, cyclophos-
phamide (CYP) and etoposide, are preferred for cancer prevention
or treatment (Susan et al., 2012). However, these agents elicit neg-
ative effects. For instance, chemotherapy-induced alopecia (CIA)
is a serious and distressing adverse effect of cancer therapy. In
general, human scalp hair comprises anagen, catagen and telogen
hair follicles. Anagen hair follicles actively accumulate cytochrome
and rapidly produce hair shaft. Telogen hair follicles are incapable
of producing neonatal hair shafts until these follicles mature into
anagen hair follicles (Hayumi and Tatsuya, 2001). During cancer
chemotherapy, hair follicles shift to the subsequent telogen phase
in advance as a consequence of anti-cancer drug-induced apo-
ptosis. Thus, cancer chemotherapy causes serious hair loss and
inhibits hair follicle proliferation because of damaged hair matrix
keratinocytes that divide cellular subsets or because of disrupted
follicular structure and function (Lindner et al., 2012; Roh et al.,
2005).
∗ Corresponding author.
E-mail addresses: zyq4885@126.com, yqz2016@163.com (Y. Zhao).
logics have elicited modulatory effects on CIA in various models
(Satish et al., 2014; Ranjitha et al., 2014; Cheryl and William,
2015; D’Agostini et al., 2013), although not all have anti-apoptotic
activity. Several trial approaches, such as scalp cooling during
chemotherapy, prevent hair loss in substantial patients (Grevelman
and Breed, 2005; van den Hurk et al., 2012). However, cancer scalp
metastasis may occur because of tumor drug uptake reduced by
scalp cooling (Lemieux et al., 2009; Lemieux et al., 2011). Fur-
thermore, minoxidil can induce hair regeneration and shorten the
telogen phase of hair follicles after CIA and alopecia areata are
treated; however, this treatment ineffectively prevents or allevi-
ates CIA (Duvic et al., 1996). Therefore, effective CIA prevention
agents should be further developed.
Cedrol has been extensively investigated as an anti-
inflammatory constituent, anti-microbial ingredient and antibiosis
agent (Jantan et al., 2005; Oh et al., 2011; Umeno et al., 2008). This
substance is a crystal-type compound found in the volatile oil of
Thuja orientalis. Cedrol is also the major ingredient of Platycladus
orientalis (L.) Franco essential oil. With cedrol, the extracts and
volatile oil of P. orientalis can promote hair growth and alleviate
hair loss by protecting hair follicles from chemotherapy-induced
damage (Shan et al., 2013). The extract of T. orientalis as a tradi-
tional herb has also been used to inhibit hair loss and stimulate
hair growth (Zhang and Park, 2013). Therefore, we supported the

http://dx.doi.org/10.1016/j.etap.2016.07.020
1382-6689/© 2016 Elsevier B.V. All rights reserved.
 S.-S. Chen et al. / Environmental Toxicology and Pharmacology 46 (2016) 270–276
main component cedrol may prevent or alleviate hair loss in CIA.
To investigate the effects of cedrol on CIA, mature C57BL/6 mice
with anagen hair follicle were treated with CYP. Then the effects
of topically applied cedrol on alopecia prevention and hair follicle
protection were examined. Our study elucidated cedrol may have
preventive or alleviative effects on CIA by mitigating dystrophic
follicle atrophy.
2. Materials and methods
2.1. Preparation of herbal extracts
The specimens of P. orientalis leaves (150228) used in our
experiment were purchased from the Traditional Chinese Medicine
Market of Bozhou City in Anhui Province (Anhui China), identified
and authenticated by Prof. Jincai Lu in the school Shenyang Phar-
maceutical. First, the leaves were crushed to powder and soaked
in water at 40 ◦ C for 60 min. Volatile oils were acquired from plant
materials via a steam distillation method under the following con-
ditions: distillation temperature of 150 ◦ C, total distillation time of
3 h, and material-to-solvent ratio of 1 g: 12 mL. Cedrol was sepa-
rated from the volatile oils via a freezing method at 2 ◦ C for 120 h
and recrystallised from ethanol (Yang et al., 2009). Cedrol was dis-
solved in 85% ethanol before the trial was performed.
2.2. Experimental animals
Sixty adult female C57BL/6 mice (certification: No.
211002300005208) aging 6 weeks and having an average body
weight of approximately 20 g were housed in standard cages and
fed with basal feed. Mice were maintained on 12-h light/dark cycle
with ad lib water and lab chow. They were acclimatized for at least
one week prior to experimentation. The temperature of the animal
room was 23 ± 2 ◦ C with the suitable humidity. All experiments
and procedures were conducted in accordance with the protocol
of the Shenyang Pharmaceutical University.
2.3. Grouping of animals
After acclimatization, the healthy mice were randomly divided
into the following six groups (Table 1). Each group was consisted
of 10 mice.
2.4. Induction of anagen
Catagen or telogen hair follicles in the treated skin patches of the
mice should mature into the subsequent anagen phase to assess the
efficacy of protecting mouse neonatal hair (Paus et al., 1990). The
mice were anesthetised by intraperitoneally injecting 4% chloral
hydrate (1 mL/100 g). A 6 cm2 area (longitudinal length, 3 cm; hor-
izontal length, 2 cm) of telogen-phase hair was removed carefully
by using an animal clipper from the dorsal portion of 6-week-old
C57BL/6 N mice before topical drugs were applied to their back.
The skin of the mice appeared pink, which suggested that the hair
follicles were in anagen I to anagen III.
2.5. Induction of alopecia
Freshly dissolved CYP in saline (125 mg/kg body weight) was
administered by single intraperitoneal injection to the mice except
the normal mouse group on the 9th day of depilation when the hair
follicles reached the early stage of anagen VI (Paus et al., 1994a). The
normal mouse group received equal volumes of saline.
271
2.6. Experimental studies with cedrol
Acute toxicity, skin irritation and skin sensitization tests of
cedrol were conducted before the experiment, which suggested
that cedrol had hardly toxic and side effects. The dosage of cedrol
was determined according to the tests described above. Three dif-
ferent doses of cedrol prepared in 85% ethanol were spread on the
dorsal skin of mice 2 days after depilation for several consecutive
days until the mice were all sacrificed under an ether atmosphere
to evaluate whether topical treatment with cedrol may prevent
or alleviate CYP-induced alopecia. The treatment for each group
is summarized in Table 1.
2.7. Mice alopecia and hair regrowth assays
After CYP was injected to the mice, their back skin was exam-
ined daily to determine alopecia symptoms. Hair loss was visually
quantified using the photographs obtained daily. Alopecia scores
were assessed for 5 consecutive days when the mice experienced
hair loss. Hair loss scores were graded as follows: Grade 0, with-
out hair loss; Grade 1, with 0–20% hair loss; Grade 2, with 20–40%
hair loss; Grade 3, with 40–60% hair loss; Grade 4, with 60–80%
hair loss; Grade 5, with 80–100% hair loss; and Grade 6, with com-
plete hair loss. The animals were not sacrificed until they reproduce
hair shaft. Taking the thickness, shade and length of the hair grow
back as indexes, the hair regrowth score was assigned as reported
previously (Zhang et al., 2016).
2.8. Histological analysis of hair follicles
After the mice were treated with different solutions for 15 con-
secutive days when the hair follicle in anagen VI or in catagen was
damaged by CYP, four mice from each group were sacrificed in an
ether atmosphere. Representative skin samples were excised and
maintained in 10% formalin fixative to prepare permanent paraffin
sections. Longitudinal sections were observed and photographed
by using a digital photomicrograph to examine follicle morphology.
Anatomical location in the transverse sections was quantitatively
examined to assess the percentages of hair follicles in anagen or
catagen (Paus et al., 1994b).
3. Results
3.1. Effects of cedrol on alopecia prevention
Blackened skin areas, especially in Groups E and F were observed
9 days after shaving. Hair production continued within the first
3 days. All of the mice presented an almost full coat hair 3 days after
CYP administration (data not shown). Fig. 1, time scheme, illus-
trates the models of the C57BL/6 N female mice with alopecia and
subjected to topical treatments.
Macroscopic assessment and alopecia score (Fig. 2) showed that
complete alopecia could be significantly observed 6 days after CYP
administration in minoxidil and model groups (p > 0.05). By con-
trast, the cedrol-treated mice manifested incomplete hair loss with
approximately 10%, 20% or 40% coat hair. This finding indicated that
high dose cedrol was associated with protective response against
CYP alopecia. The efficacy of topical cedrol in inhibiting CIA was
dose dependent when the cedrol solution was initially titrated from
50 mg/kg to 200 mg/kg (C, D and E in Fig. 2).
3.2. Effects of cedrol on protecting hair follicle from damage
The numbers of relatively dystrophic hair follicles with dis-
rupted melanin granules were observed in the longitudinal section
of the treated skin patches (empty circles shown as solid lines or
272 S.-S. Chen et al. / Environmental Toxicology and Pharmacology 46 (2016) 270–276
Table 1
Animal grouping and treatment of the experiment.
Group n Treatment
Normal (A) 10
Model (B) 10
Low dose cedrol (C) 10
Medium dosage cedrol (D) 10
High dose cedrol (E) 10
Minoxidil (F) 10
Fig. 1. The treatments and results schedule of the experiment in the mice alopecia model.
Fig. 2. (I): Macroscopic effects among the groups. (B) Model group; (C) 50 mg/kg-
cedrol group; (D) 100 mg/kg-cedrol group; (E) 200 mg/kg-cedrol group; (F)
Minoxidil group (Photos were taken on day 13, 14 and 15 of the experiment respec-
tively). (II): Alopecia score of the mice after CYP administration.
dotted circles in Fig. 3). In particular, numerous hair follicles the
mice treated with minoxidil were severely damaged 6 days after
CYP administration (representative pictures in Fig. 3B), which indi-
cated anagen termination and catagen induction. These findings are
similar to those in the model group and thus suggested that irregu-
lar hair shift, distorted hair bulbs and incontinent melanin granules
occurred (Fig. 3B and F). In Fig. 3D, histopathological observa-
tion showed disruption of melanin granules; we can still see less
melanin granules shafted to hair bulb. However, nearly undistorted
follicles presented along with hair follicles in Fig. 3D and E, which
was less dystrophy than others except normal hair follicles. Espe-
Saline (i.p.) + 85% ethanol (topically)
125 mg/kg CYP (i.p.) + 85% ethanol (topically)
125 mg/kg CYP (i.p.) + 50 mg/kg cedrol (topically)
125 mg/kg CYP (i.p.) + 1000 mg/kg cedrol (topically)
125 mg/kg CYP (i.p.) + 200 mg/kg cedrol (topically)
125 mg/kg CYP (i.p.) + 62.5 mg/kg cedrol (topically)
cially, nearly normal modality of hair follicles can be observed in
the high cedrol dose group (Fig. 3E).
Morphometry exhibited a noticeable loss of hair follicles in ana-
gen (Fig. 4). The percentage of anagen hair follicle (37%) decreased
significantly because of the injected CYP. However, high-dose
cedrol-treated mice with 82.5% anagen hair follicles showed rel-
atively healthier hair follicles than the model mouse group did
(p < 0.001).
3.3. Ability of cedrol to promote hair regrowth after CIA
Alopecia, along with hair regeneration, can be reversed after a
given period. The main regions of hair regeneration were marked
in Fig. 5. In our study, 11 days were required for the mice to repro-
duce hair shaft rapidly, as induced by cedrol and minoxidil after
maximum alopecia. As opposed to model mice, whose dorsal hair
with depigmented shaft thinly spread. These results were more evi-
dent in the treated groups than in the model group. In the model
group, hair growth recovery was achieved sparsely (representative
pictures in Fig. 5).
4. Discussion
This study demonstrated that chemotherapy-damaged hair fol-
licle can be suppressed after cedrol is topically administered. As
a result, alopecia in the majority of the treated mice was rela-
tively well inhibited. Mature female C57BL/6 mice were utilized to
assess follicular integrity and hair loss in our study because these
mice undergo hair follicle cycles similar to the human hair cycle
(Wikramanayake et al., 2012). In human scalp, up to 90% of hair
follicles are in the anagen phase; in adult mice, less than 10% of
hair follicles are in the anagen phase (Jankovic and Jankovic, 1998).
The melanin content of the hair follicles was generated in anagen.
As a consequence, the dorsal skin of the mice appeared pink in
the telogen and early anagen phases (Chase, 1954). Telogen hair
follicles in adult mice also mature into anagen hair follicles facti-
tiously preceded by a trial, which simulates human conditions. The
mice with pigmented terminal anagen VI hair follicles experienced
diffuse and reversible alopecia after they were treated with CYP.
Stimulators were topically applied daily to C57BL/6 mice to deter-
mine the alleviative effects of different treatments on CYP-induced
alopecia.
CYP is associated with serious side effects, including gas-
trointestinal disturbances, bone marrow suppression and alopecia
(Kiebert et al., 1990). Minoxidil can effectively promote hair growth
 S.-S. Chen et al. / Environmental Toxicology and Pharmacology 46 (2016) 270–276 273

Fig. 3. Histology skin sections (magnification, 100×) of mice model received on day 15 of the experiment. (A) Normal group; (B) Model group; (C) 50 mg/kg-cedrol group;
(D) 100 mg/kg-cedrol group; (E) 200 mg/kg-cedrol group; (F) Minoxidil group.

Fig. 4. The percentages of hair follicles (Original magnification ×40): (A) Normal group; (B) Model group; (C) 50 mg/kg-cedrol group; (D) 100 mg/kg-cedrol group; (E)
200 mg/kg-cedrol group; (F) Minoxidil group; (*** = P < 0.001 vs. the Normal group; ### = P < 0.001, ## = P < 0.01, vs. the Model group).
274 S.-S. Chen et al. / Environmental Toxicology and Pharmacology 46 (2016) 270–276
Fig. 5. Comparison of hair regrowth in each group at day 27 of the experiment. (A) Normal group; (B) Model group; (C) 50 mg/kg-cedrol group; (D) 100 mg/kg-cedrol group;
(E) 200 mg/kg-cedrol group; (F) Minoxidil group (*** = P < 0.001 compared with the Normal group; ### = P < 0.001, compared with the Model group).
(Messenger and Rundegren, 2004). In our study, the mice in minox-
idil group presented severe alopecia even complete hair loss after
CYP was administered. This finding demonstrated that minoxidil
enhanced normal hair growth but failed to elicit protective effects
on CIA. Similarly, the model group suffered from total alopecia. By
comparison, the cedrol-treated mice manifested mild alopecia. In
Fig. 2(I) E, the high-dose topical administration of cedrol was asso-
ciated with 40% suppression of hair loss. This finding indicated that
cedrol provided a highly consistent protective effect against CYP
alopecia.
CIA is usually characterised by increased antitumor drug-
induced cytotoxicity accompanied with hair follicle damage
(D’Agostini et al., 2007). CYP treatment exacerbates matrix dete-
rioration; as a result, premature catagen is formed and telogen
shedding rate is increased (Paus et al., 2013a). Cedrol commonly
exists in the essential oils of P.orientalis and Thuja orientalis. The
extracts of these medicinal herbs are capable to promote hair
growth by inducing hair follicle in resting anagen phase (Shan et al.,
2013; Zhang and Park, 2013). In our study, the histological analysis
results of the high-dose cedrol group revealed nearly normal hair
follicles with regular hair shafts and relatively regular hair bulb
with concentrated melanin granules. These results suggested that
hair follicle could be protected by cedrol from CYP-induced damage.
In addition, anagen hair follicle loss was also inhibited. This finding
implied that cuticle atrophy with a decrease in the number of ana-
gen hair follicles can be prevented by high cedrol dose. Therefore,
cedrol possibly exhibited a correspondingly significant alleviative
effect against chemotherapy-induced dystrophic changes. On the
basis of morphological observation results and the percentage of
anagen hair follicle, we found that the therapeutic ability of pre-
venting CYP-induced alopecia was dose dependent because of the
gradient dose of cedrol. Our microscopic skin analyses revealed that
cedrol may mitigate apoptosis and dystrophic follicle atrophy in
certain skin areas with alopecia.
After chemotherapy is discontinued, recovery from alopecia
for most patients usually requires several months (Tsunoda et al.,
2001). CYP-induced alopecia was also reversible in the current
study. Furthermore, cedrol can promote hair reproduction. Hair
regrowth occurred within 11 days after complete alopecia man-
ifests. Thus, cedrol promote hair regeneration and shorten the
telogen phase of hair follicles after alopecia occurs. This finding
is similar to that involving minoxidil shown in previous studies
(Duvic et al., 1996). On the basis of these data, we concluded that
cedrol may possess a latent ability of preventing damage to initia-
tory hair follicle and restoring the morphological characteristics of
follicles after they have been damaged by chemotherapy.
The apoptosis of hair follicles during chemotherapy is largely
caused by the redundancy of the transcriptional protein p53,
which plays an inhibitory role on modulating hair follicle cells
(Botchkarev, 2003; Paus et al., 2013b). The p53 protein combines
diverse pathways, including apoptosis, cell cycle and differenti-
ation (Sarig et al., 2010). In detail, the amount of p53 increases
markedly after DNA in follicle cells is damaged by tumour drugs
within 1 h; as a result, hair follicle apoptosis occurs because of the
increased transcription level of p53 target genes (Botchkarev et al.,
2000). The apoptosis of hair matrix keratinocytes caused by p53
is also implicated in CIA pathogenesis. Therefore, cedrol may be
an efficient inhibitor to block redundant pathways or coordinate a
multitude of hair follicle cellular processes; as a consequence, p53
may be suppressed.
Many antitumor drugs have toxic effects on the blood vessel
network of hair follicles, which lead to angiogenesis inhibition.
This damage may play a major role in hair dystrophy and alope-
cia (Amoh et al., 2005; Amoh et al., 2007). Platelet-activating factor
(PAF) has been reported to be a potent glycerophospholipid medi-
ator, causing platelet aggregation, inflammation, and changes in
vascular permeability (Vargaftig and Braquet, 1987; Mallet and
Cunningham, 1985). Moreover, platelet aggregation is involved in
acute arterial thrombosis (Ross, 1993). Cedrol is a PAF antagonist
that can reduce or inhibit platelet aggregation, skin inflammation,
and vascular disorders (Singh et al., 2013; Moharam et al., 2012;
Moharam et al., 2010). In addition, vasculogenesis and angiogenesis
are stimulated by vascular endothelial growth factor (VEGF), which
can be activated by MAPK (Maxwell and Ratcliffe, 2002). MAPK is
considered to act as a regulator to protect vascular smooth muscle
cells from cell death. Given that cedrol acts as an intracellular sig-
nal modulator to promote MAPK activation (Jin et al., 2012a), cedrol
may be involved in toxicity reduction in hair follicle blood vessel
network. Cedrol exerts a protective effect against CIA by increas-
 S.-S. Chen et al. / Environmental Toxicology and Pharmacology 46 (2016) 270–276
ing the blood flow surrounding the hair bulbs and enhancing the
delivery of oxygen and nutrients to hair follicle cells.
Cyclophosphamide treatment enhances systemic inflammatory
reaction, thus causing regression of sebaceous glands (Lagrange and
Gonçalves Da Costa, 1996). The connective tissue sheath surround-
ing the outside of the hair follicles sustains and regenerates the
dermal papilla, which is a necessary component for hair regener-
ation. Sebaceous gland function disorders are associated with hair
loss in some animal models of alopecia (Porter et al., 2002; Stenn,
2001). In addition, cedrol has been linked to activation of p38 MAPK,
and p42/44 ERK and Akt have an effect in collagen and elastin pro-
duction in dermal fibroblasts (Jin et al., 2012b). Collagen plays a
key role in dermis protection, while P38 MAPK and p42/44 ERK are
involved in apoptosis prevention and are important in determining
whether a cell undergoes apoptosis (Xia et al., 1995; Allen et al.,
2005). These findings support that cedrol may also inhibit CYP-
induced fibroblast apoptosis in MAPK-dependent manner, thereby
causing preventive effects on CIA.
In summary, our study enhanced our comprehension of cedrol
function. We discovered that cedrol is an effective inhibitor of CIA.
As such, cedrol can be used as a new agent to manage CIA with a
predictive value. A convenient and efficient strategy of cedrol top-
ical delivery to patients receiving chemotherapy in clinics should
be elucidated to improve the management of alopecia. However,
the specific mechanisms of the bioefficacy of cedrol in preventing
CYP-induced alopecia have yet to be completely described. There-
fore, the molecular mechanism of cedrol activity and the use of its
integrated information should be further investigated.
Conflict of interest
None.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.etap.2016.07.020.
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