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Journal of the Korean Society of Aesthetic Science6Kwon Je2like2010

The Journal of Cosmetological Science 6(2), 121~129(2010)

C57BL/6macroscopic of rosemary oil in mice,

Histological hair growth promoting effect

Kim Mi-hyangOne, seaweed steelOne,*

Keimyung School
One School Public Health Department

The Promoting Effect of Rosemary Oil on Hair Growth by

Gross and Histological Observation in C57BL/6 Mice

Mi Hyang KimOneand Young Chul KimOne,*

OneDepartment of Public Health, Graduate School, Keimyung University, Daegu 704-701, Korea
(Received May 1, 2010; Revised June 1, 2010; Accepted June 5, 2010)

ABSTRACT

This study was carried out to investigate the effect of rosemary essentialoil on hair growth in a shaving animal model of
C57BL/6 mice. Five weeks old male mice were divided into four groups including normal group (N; saline), vehicle control
group (VC; jojoba oil), positive control group (PC; 3% minoxidil) and experimental group (E; 3% rosemary oil) and they were
topically applied with an amount of 100 μL once a day, 6 days a week, for 4 weeks. At 4-week application, hair regrowth on
dorsal skin in E (42%) and PC (55%) groups was much faster than N (3%) and VC (20%) groups in gross observation. Hair
follicle number, follicle depth and dermal thickness of E and PC groups were significantly higher (p⁄ 0.05) than N group in
histological observation. And number of mast cells in dermis and hypodermis of E and PC groups were significantly lower (p⁄
0.05) than N and VC groups. These results indicate that rosemary oil effectively stimulated hair growth in an animal model.

Keywords:C57BL/6 mice, Hair follicle, Hair regrowth, Rosemary oil

Because the perception of stress increases along with the expression, and

west theory social and emotional disorders such as difficulties in relationships with people

or the opposite sex (Kim Hwi-Jun et al.,2001; Paus & Cotsarelis, 1999)Hair loss

Human hair has a cosmetic role in addition to the primary role of is considered a health problem.

protecting the hair from sunlight, cold, and external shock absorption ( The number of people with hair loss in Korea continues to increase every year, and

Randall, 1994).With the improvement of the national economy and recently, not only middle-aged men but also young people and women are experiencing hair

increasing interest in appearance, hair loss is a major disease. loss in general (Hyo-Jeong Bang, Hyo-Jeong Bang,2003).Currently in Korea

Rather than being regarded only as a concomitant symptom, it is now recognized as There are two types of hair loss treatment on the market: Minoxidil, a hair growth

an independent disease (Hyojun Kang et al.,2004).Alopecia sufferers are agent for transdermal application.®and finated, an oral hair growth agent®there is In

psychologically associated with depression, shame, social isolation and anger. the case of minoxidil2005annual sales of approx.44in billions2007about a year

* Correspondence should be addressed to Dr. Young Chul Kim, Department of Public Health, Graduate School, Keimyung University, Daegu 704-701, Korea. Ph.:
(053) 580-5931, Fax: (053) 588-5233, E-mail: yckim@kmu.ac.kr
122 The Journal of Cosmetological Science Vol. 6, No. 2, 2010

1,000increase to 100 million won, and2007Annual sales are approx.31 It was 100 all(Tobin et al., 1998).Therefore, in this study, rosemary oil, which has vasodilation,

million won (Daily Farm,2007;Medifarms Today,2007).There are several known muscle relaxation, and antibacterial effects to promote hair growth, was used in this

causes of hair loss, such as genetic factors, excess of male hormones, mental stress, study.C57BL/6on the back of the mouse4Including weekly percutaneous application,

blood circulation disorders, sheep disorders, endocrine disorders, aging, US-FDAThe purpose of this study is to investigate the practical potential of rosemary

autoimmune diseases, environmental pollution, dyeing, etc.Mitsui et al., 1997). oil as a hair loss prevention and hair growth promoter by comparing and analyzing

Among them, the main causes are the loss of volume supply and male hormones the effect of promoting hair growth visually and histologically with minoxidil, a

due to the circulation disorders of the blood vessels surrounding the dermal papilla certified product.

and hair follicles.Messenger & Rundegren, 2004).

Currently in the United StatesFood and Drug Administration

(US-FDA)The drug that promotes hair growth is finasteride. Materials and Methods

(finasteride)and minoxidil (minoxidil)There is (Trueb, 2001).

Minoxidil is known to cause several side effects when used for a One.Reagents and Instruments

long time. It can cause dermatitis with itching, erythema,

As a test substance, rosemary oil is certified by an organic
peeling and dryness, and allergic contact dermatitis and
certification organization (ECOCERT-F-32600) 100%Pure natural
seborrheic dermatitis (Hagemann et al., 2005; Suzuki et al.,
essential oilSanoflorebuy(France)product, jojoba oil (jojoba oil)silver
2002),It has also been reported to cause rapid weight gain,
Desert Whalebuy(USA)use the product The components of
edema, increased heart rate, angina pectoris and
rosemary oil and jojoba oil used in this experiment wereTable 1and
hematological side effects. (Peters et al., 1996; Ellenhorn &
Table 2 same as Positive assistant, hair regrowth3%Purchase and
Barceloux, 1988).Because of these side effects, interest in
use minoxidil (current drug).
natural products has recently increased.
rosemary oil (rosemary oil)silver rosemary (Rosmarinus officinalisL.)
2.Laboratory animals and treatment

Extracted by steam extraction from the upper part of the flower of

monoterpenesRyuwaoxidesRyuga70% occupies more than Rosemary oil 5spirit murmurC57BL/6Mouse Co. from a biolink
has been used as a flavoring agent for food, beverages, and cosmetics
Table 1.Composition of rosemary oil
since ancient times (Al-Sereitia et al., 1999),antibacterial and antifungal (

Genena, 2008; Kabouche et al., 2005),anticancer (Cheung & Tai, 2007;

Ingredients %

Leal et al., 2003),antithrombotic (Naemura et al., 2008)Studies have been α-pinene 11.15
camphene 4.17
reported to be effective. In addition, oral administration of rosemary oil β-pinene 8.06
to male rats myrcene 1.46
para-cymene 1.04
As the dose increases whentestosteronedecreased the number (
limonene 2.15
Nusier et al., 2007), nerve growth factor (NGF)stimulated the production 1-8 cineole 46.96

(Kosaka & Yokoi, 2003),Antispasmodic effect in the gastrointestinal tract

γ-terpinene 0.54
terpinolene 0.40
and bile ducts (Al-Sereitia et al., 1999)and liver detoxification (Sotelo- linaloi 0.55
Félix et al., 2002)It has been reported that there is On the one hand, so camphre 8.38
borneol 2.87
farrosemary oilAs a study on the hair growth effect ofrosemary oilclass α-terpineol 1.75
lavender oilPrevious studies using individual and mixed oils of2007) β-caryophyliene 5.28
the others 5.24
However, there are deficiencies that are biased only on visual

observation.

Experimental animals used for hair growth and hair loss prevention
Table 2.Composition of jojoba oil
research are well known for their genetic characteristics.6From the age
Ingredients %
of one, the hair cycle is at rest (telogen)going intoC57BL/6I use the
docosenyl elcosenoate 41.53
mouse a lot.C57BL/6Mice are characterized by spontaneous hair loss. elcosenyl elcosenoate 27.65
melanocyteis limited to the hair folliclesmelaninThe synthesis coincides elcosenyl docosenoate 10.33
tetracosanyl elcosanate 6.27
well with the hair growth cycle, so the hair growth cycle can be elcosenyl oleate 5.38
controlled only by observing the skin color. docosenyl stearate 3.21
5.63
It is the target of hair research
 Kim MH & Kim YC : The Promoting Effect of Rosemary Oil on Hair Growth 123

Received and in the nurseryOneAfter acclimatization for a week, the skin color on the back straight cut10%It is fixed in formalin solution and used for
side of the hair removedpink colorsign6week-old mice by oocyte method4divided into histological observation.
groups of dogs per group11by each4Experiment with weekly percutaneous application
3.Preparation and application of samples
C,phase
All. Animal breeding room temperature22±One� humidity50±5%,light
Cycle12Day and night were maintained for each hour, and feed and water were freely REOThe sample is a solventJOto3%It is manufactured by mixing with an electric

supplied during the entire period of the experiment. Experimental animals were in the hair removal device for animals (electric clipper)as2 cm×4 cmAfter hair removal with

normal group (N): Saline Application group, solvent group (VC): Jojoba oil (JO)application OneWorkOneepisode100 μLeach week6work,4Weekly transdermal is also included.

group, positive group (PC): 3% minoxidil (MXD)Application group, experimental group (E):

3% rosemary essential oil (REO)gunnery, gun4classified into dog groups. experimental

4.visual observation
animal water1, 2, 4each week3, 3, 5Treat each animal one by one,

After the start of the experiment, during the experiment

period to visually observe the state of hair growth.5
Table 3.Comparison of hair regrowth effect in an alopecia model of
C57BL/6 mice applied with test compounds topically for 4 weeks episode(0, 1, 2, 3, 4After light anesthesia with ether, take a
Groups picture with a digital camera. The hair growth effect of each
Weeks
N VC PC E group wasscoring systemEach animal is visually judged by
One - - - - in the motherhood follow0~19% (-), 20-39% (±),40-59% (+),
2 - - ± ± 60-79% (++), 80-100% (+++) to set All.
3 - - + +
4 - ± + + 5.histological observation

N: Saline application group, VC: JO application group, PC: 3% MXD

1) Hematoxylin and Eosin (H&E)Light microscopic observation after staining
application group, E: 3% REO application group, Hair growth scoring
index: 0-19% (-), 20-39% (±),40-59% (+), 60~79% (++), 80-100% (+++). exam1, 2, 4The skin tissue cut in the week10%neutral formalin

Groups
Weeks
N VC PC E

One

Figure 1.Comparison of hair g compounds topically for 4 weeks.

124 The Journal of Cosmetological Science Vol. 6, No. 2, 2010

in solution24After fixing the time, it is embedded in paraffin after washing with water, dehydration, Compared to the normal group, the group190%Significantly many (Figure 2). 2 In parking,

transparency, and penetration according to the usual method. embedded tissue4 μmmake a section the positive group compared with the normal group.337%was significantly higher, and the

in thicknessH&Eafter dyeing100The number of hair follicles was counted with a magnification light experimental group132%noticeably a lot4In parking, the positive group compared with the

microscope, and the changes in the thickness of the dermis and the depth of the hair follicles were normal group.374%Significantly many observations were made, and the experimental

evaluated using the microscope program.scale barto measure using group340%Significantly many observations were made, and both the experimental group

All. and the positive control group were each in the solvent group.56%, 68%appeared

significantly (p⁄0.05).
2) Toluidine blueLight microscopic observation after staining
Dermal skin thickness (μm)cast100at magnificationscale barMeasurement result
Cut the skin tissue at room temperature10%in neutral formalin solution24 After fixing
usingOneIn parking, the positive group compared with the normal group.40% was
the time, it is washed with water, dehydrated, transparent, and penetrated in the usual way,
significantly higher,2In parking, the positive group compared with the normal
and then embedded with paraffin.4 μmmake a section in thicknessToluidine blueAfter
group.39%was significantly higher (Figure 3).and4In parking, the positive control
staining, the number of mast cells in the dermis and subcutaneous layer was counted.200
group was stronger than the normal group.36%was significantly higher, and the
Count with a magnification light microscope.
experimental group42%was significantly higher (p⁄0.05).

6.Statistical processing

60
SPSS 14.0 for windows (SPSS Inc., USA)One-way ANOVA using N VC PC E
c c
statistical program (one-way ANOVA)to conduct a homogeneity 50

analysis. To test the difference between each groupDuncan's

40 b
Follicle number. .

multiple range testis used to perform post-hoc analysis. c

Statistical significance test is α=0.05carried out in 30

20 b

b b ab a
Results and Discussion 10 a
a a

0
One.physical change One 2 4
Weeks
The result of visually observing the condition of hair growth on the
Figure 2.Changes of hair follicle number in an alopecia model of C57BL/
back Table 3, Figure 1same as ExperimentOneThe hair did not grow on
6 mice applied with test compounds for 4 weeks. Values are mean±SD
the first week, but the experiment2From the first week, hair growth was of 3, 3, 5 mice at 1, 2, 4 week, respectively. Values with different
superscripts in the same week are significantly different (p⁄ 0.05) by
observed on the back side of the positive group and the experimental
ANOVA and Duncan's multiple range test.
group. In the solvent group, hair did not grow, but a part of the skin

turned black. Experiment3On the first week, hair growth was observed

on the back side of mice in all groups except the normal group. 400
N VC PC E c
Experiment4During the week, in the experimental group,42%,About in bc
b
320 b ab
the positive group 55%,About in the solvent group20%Looking at the
Dermal thickness (μm)

hair growth of the normal group,3%see slight hair growth. through this ab a
240 ab a
a
REO was confirmed to have a hair growth promoting effect. Lee Yong- a ab
woo (2007)rosemary oil in ethanol5%Wow15%In the hair growth 160

experiment using diluted with20on each day54%Wow77%reported the

80
hair growth rate of , which indicates that the concentration of rosemary

oil used3%)This is thought to be due to the higher

0
One 2 4
Weeks
2.histological changes
Figure 3.Changes of dermal thickness in an alopecia model of
1) Hematoxylin and Eosin (H&E)Light microscopic observation after staining C57BL/6 mice applied with test compounds topically for 4 weeks.
Values are mean±SD of 3, 3, 5 mice at 1, 2, 4 week, respectively.
100As it was visually confirmed at the magnification, the
ripts in the same week are significantly A
experimental group was better than the normal group.210% and Duncan's multiple range test.
 Kim MH & Kim YC : The Promoting Effect of Rosemary Oil on Hair Growth 125

hair follicle depth (μm)in100at magnificationscale barMeasurement result group compared to the normal group238%was significantly higher, and the

usingOneIn parking, the positive group compared with the normal group. experimental group111%was significantly higher.4In parking, the positive group

130% was significantly higher (Figure 4). 2positive group in parking compared with the normal group.220%was significantly higher in the experimental

group than in the normal group.137%significantly higher (p⁄0.05).

Characteristics of the growth phase are the thickening of the dermal layer, the increase
600
in the size of the hair follicles, the deepening of the hair follicles into the dermis and
N VC PC E
500 c subcutaneous fat, and the start of melanin synthesis (Slominski et al., 1991).growth stage (
c
b
Follicle depth (μm)

400 anagen I-VI) In the hair follicle length increases and in the catagen stage (catagen I-VII) In
b
the hair follicle length is reduced. from rest to growthIIUntil the stage of growth, hair
300
b b follicles are located in the dermis and do not reach the subcutaneous fat layer.IIIIn this

200 a
a stage, the hair follicles descend into the subcutaneous fat layer. This is the growth stage of
a
a a a
100 dermal fibroblastsI-IIBecause it surrounds the hair follicle during the phase (Müller-Röver et

al., 2001). The continuous growth of hair follicles in the anagen phase and the degeneration
0
One 2 4 of hair follicles in the anagen phase are associated with periodic changes in the length and
weeks
thickness of the dermis and subcutaneous tissue (Fig.Chase et al., 1953).growing seasonII

Figure 4.Changes of follicle depth in an alopecia model of C57BL/6 mice Until the stage, the thickness of the epidermal layer increases andIIIFrom this stage, it

applied with test compounds topically for 4 weeks. Values are mean±SD decreases and the dermal layer and subcutaneous fat layer thicken (Moffat, 1968).The
of 3, 3, 5 mice at 1, 2, 4 week, respectively. Values with different
increase in epidermal thickness is related to keratinocytes (keratinocytes)due to an increase
superscripts in the same week are significantly different (p⁄ 0.05) by
ANOVA and Duncan's multiple range test.

a b

c d

Figure 5.Histological observation on h test compounds topically in week 4. H n

& E stain,×100. 5a: N group, 5b: VC g hair follicle number, dermal thickness
and follicle depth than N and VC groups
126 The Journal of Cosmetological Science Vol. 6, No. 2, 2010

a b

c d

Figure 6.Histological observation on hair growth in an alopecia model of C57BL/6 mice applied with test compounds topically in week 4. H & E stain,×
100. 6a: N group, 6b: VC group, 6c: PC group, 6d: E group. E and PC groups promoted elongation of hair follicles from subcutaneous to epidermis.

(Movérare et al., 2002),Keratinocytes in the telogen phaseII It 12

c
increases up to the stage and decreases after that (Oh & Smart, 10
Number of mast cells.s

b
1996). 8 a
a
This study period (4Note) As time passes, the number of follicles, the thickness of the dermis, and 6
the depth of the follicles increase.4During the week, the number of hair follicles was significantly 4
higher in the experimental group and the positive group compared to the normal group and the
2
solvent group, and the depth and dermal thickness of the hair follicles also increased (Figures 5, 6).
0
N VC PC E
Moffat (1968)Also reported the results of a study that the thickness of the dermis and subcutaneous

fat was thickened in hair follicles during the growth phase. On the other hand, in the experimental
group, as in the positive control group,3From week onwards, hair follicles in the growth phase
continue to exist in the subcutaneous fat layer.REOIt is thought to have the effect of delaying the
transition to the catagen phase by continuing the induced growth phase, but this needs to be
Figure 7.Comparison in the number of mast cells in an alopecia
model of C57BL/6 mice applied with test compounds topically in
week 4. Each value represents mean±SD of 5 mice. Values with
different superscripts in the same week are significantly different (p⁄
0.05) by ANOVA and Dunca's multiple range test.

confirmed through additional research according to the mother cycle by extending the experimental

period.
The specific action of the has not yet been elucidated. (Lotti et al., 1995). Paus

2) Toluidine blueLight microscopic changes after staining et al. (1994)The number of mast cells is the least observed in the early stage of

Mast cells are involved in a defense mechanism against hypersensitivity reactions, inflammatory growth, and increases in the late stage of growth and declines.

diseases, and various microorganisms in normal skin. When hair follicles enter the growth phase again,
 Kim MH & Kim YC : The Promoting Effect of Rosemary Oil on Hair Growth 127

a b

c d

Figure 8.Histochemically detectable mast cells (arrows) in an alopecia model of C57BL/6 mice applied with test compounds topically in week 4.
Toluidine blue stain,×200. 8a: N group, 8b: VC group, 8c: PC group, 8d: E group. E and PC groups showed decreasing number of mast cell as hair grows.

Report that the number of mast cells has a decreasing cycle. In a recent study, After birth to find out as6ghostly maleC57BL/6Note on the back of the mouse

Lee Moon-won (2008)Also reported the difference in the number of mast cells 6every time, every time100 μLeach4In the weekly application test, the

according to the growth cycle. following results were obtained.

Based on this research report, the skin tissueToluidine blueObserve the existingUS-FDAcertified product3%In comparison and observation of minoxidil and gross

number of mast cells by staining.200The number of mast cells observed hair growth promoting effect, experiment4positive control group in the week (approx.55%)

under the light microscope of the embryo was in the experimental group ( and the experimental group (approx.42%)is the normal group (approx.3%)and solvent

5.81±1.47)and positive group (5.40±1.07)This normal group (9.90±0.70)and groups (approx.20%)Compared to that, it has a distinct hair growth promoting effect.

solvent group (7.70 ±0.42)less significantly observed (Figure 7). 4It was In order to examine the changes in the hair follicle shape according to the hair cycle, the skin

observed that the number of mast cells decreased as the amount of hair tissue wasH&EAs a result of checking the number of hair follicles, dermal thickness and hair follicle

growth increased in parking (Figure 8).Experiment4At week week, the depth by staining, the number of hair follicles, dermal thickness, and depth of hair follicles increased

experimental group and the positive group showed a significant decrease in over time during the experiment period.4positive with the experimental group in the week

the number of mast cells compared to the normal group and the solvent The control group had significantly more hair follicles than the normal group, and the depth and

group, confirming that the hair follicles were in the growth phase. dermal thickness of the hair follicles also increased significantly.

In order to check the difference in the hair growth stage, the skin tissue isToluidine blueAs a result of

observing the number of mast cells by staining, the experiment4At week, the experimental group and the

texture theory positive control group showed a significant decrease in the number of mast cells compared to the normal group

and the solvent control group, so that the hair follicles are in the growth phase.

3%The hair growth promoting effect of rosemary oil

128 The Journal of Cosmetological Science Vol. 6, No. 2, 2010
Summarizing the above experimental results,3%rosemary oil is C57BL/6existing
in the mouseUS-FDAApproved hair growth promoter for transdermal application3%
Although it is somewhat inferior to minoxidil, it is expected to be useful as a hair
growth promoting agent and hair loss prevention agent, as it has a visually distinct
hair growth promoting effect and histologically similar increases in the number of
hair follicles, hair follicle depth, and dermal thickness.
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