Professional Documents
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EXPERIMENTAL DERMATOLOGY
ISSN 0906-6705
Introduction
The hair follicle is an epithelial structure that undergoes cycles of active growth (anagen), regression (catagen) and rest (telogen) (1). During
anagen the hair follicle grows down into the dermis, forming a complex layered structure with a
pigmented shaft. During catagen the hair follicle
shortens, and in telogen the epithelial cells are
resting while the hair shaft remains inside the short
follicle. The morphological changes throughout
the hair cycle are well-documented (2), but the
regulation of the different phases of this cycle is
not completely understood. The synchronized hair
growth mouse model (3, 4) has been extensively
utilized to better understand the regulation of hair
growth and cycling.
One main function of mammalian hair is to provide environmental protection. However, that
function has been lost in humans, in whom hair is
kept or removed for social and cosmetic purposes.
Many procedures are used to remove unwanted
hair, from simple inexpensive home treatments like
shaving, plucking and waxing, to expensive and
potentially time-consuming methods like electrolysis, laser, light therapies and therapeutic antiandrogens. These methods differ not only in the duration of hair removal, their price range and their
pain and discomfort levels, but also in their possible undesired effects (reviewed in (5)). Shaving
may result in nicks and cuts in the skins surface,
may increase the risk of infection, may leave a perception of an increase in the rate of hair growth,
and may also leave undesirable stubble. Plucking
causes pain and discomfort, and often results in
poor removal of short hair. While electrolysis may
keep an area free of unwanted hair for a prolonged
period of time, the process is expensive and painful
and may further result in scarring. Promising clinical results have been obtained with laser treatments, but not without changes in skin texture and
pigmentation. Several unwanted side effects, such
as liver toxicity or effects on muscularity often accompany the use of antiandrogens. Alternatives to
hair removal are popular hair cosmetic treatments
like hair dying or bleaching, used to reduce hair
visibility in desired body parts. These treatments,
which are sometimes irritating, are not always satisfying, as the emerging portions of the hair shafts
are always darker than the already treated parts.
The Kunitz-type trypsin inhibitor (soybean trypsin inhibitor, STI), and the BowmanBirk protease
inhibitor (BBI) are two serine protease inhibitors
isolated from soybeans (reviewed in (6, 7)). STI inhibits the proteolytic activity of trypsin (6), and
BBI inhibits trypsin and chymotrypsin (7, 8). Re405
Seiberg et al.
cently we demonstrated that soymilk, STI and BBI
inhibit PAR-2 activation and thus induce skin depigmentation (9). The studies described here were
designed to explore the potential use of these
agents in the inhibition of hair pigmentation. Our
data demonstrate that STI, BBI and soymilk reduce not only hair pigmentation, but also the rate
of hair growth and the final dimensions of the hair
shaft. These data suggest that such agents may
serve as an inexpensive, natural alternative treatment for undesired hair growth.
406
Results
STI and BBI delay hair growth and reduce hair
follicle size and pigmentation
To determine the effect of STI and BBI on hair
growth and pigmentation, we examined their effect
on the mouse hair cycle. Hair growth of C57Bl/6
and C3H mice, ages 89 weeks, was induced by
wax depilation of the animals back fur as described in (4), and animals were treated daily with
STI or BBI (1%, in GDL liposomes), or with liposomes alone. Animals were observed daily for hair
appearance and biopsies were analyzed for histological changes following these treatments. Visual
observations throughout the hair cycle indicated
that both STI and BBI delayed hair growth and
reduced hair shaft length, while the vehicle alone
had no effect. Fig. 1 shows that the hair of STI
(Fig. 1b) or BBI (Fig. 1c) treated C57Bl/6 mice was
shorter and appeared more orderly organized
relative to the untreated control (not shown) or
liposome vehicle treatment (Fig. 1a), when examined at low magnification. Histological analysis
confirmed these observations. Fig. 1df shows that
at day eight of the hair cycle STI (Fig. 1e) and
BBI (Fig. 1f) reduced hair follicle size in C3H mice,
relative to the liposome control (Fig. 1d). The inhibitory effect of STI and BBI was identical in
both mouse strains, but hair appearance was more
homogeneous using STI, relative to BBI.
Figure 1. STI and BBI affect hair growth. C57Bl/6 (ac) and C3H (df) mice were wax depilated to induce a new hair cycle, and
were treated daily with liposome vehicle (a, d), STI (b, e) or BBI (c, f). At day 18 of the hair cycle, a visual difference was observed
in hair appearance, as shown in images of skin biopsies (ac) at 8 magnification. Histological analysis (df, F&M staining)
revealed that STI and BBI treatments lead to reduced hair follicle dimensions (day 8).
407
Seiberg et al.
Soymilk delays hair growth and reduces hair
follicle size and pigmentation
To test the hypothesis that soybean extracts can
induce similar effects, mice induced for a new hair
cycle were treated daily with fresh soymilk and observed as indicated earlier. Soymilk treated mice
showed delayed and reduced hair growth and their
hairs were visibly thinner, more directionally organized, and smoother to touch, relative to untreated controls (Fig. 2a, day 21 of the hair cycle).
The treated mice did not show skin darkness at
days 78 of the hair cycle, as expected, and hair
shafts were not visible at days 1112 as in the control animals. In average, the hair cycle of the soymilk treated mice was delayed by 36 days. Histological analysis confirmed these observations, and
documented a delayed and shorter hair cycle with
2
4
5
20
Soy
Anagen
III
Anagen
IIIIV
Anagen
VVI
198
3
102
235
62
300
161
Catagen
Anagen
III
Anagen
IIIIV
Anagen
VVI
Catagen
139
300
204
96
253
47
27
273
Table 2.
Shaft diameter
Aubers line length
Control (m) SD
Soymilk (m) SD
% decrease
P-value
(t-test)
92.821.8
200.4127.40
53.2614.9
153.3310.70
42.6
23.48
0.001
0.001
soybean-derived phytoestrogens, alone and in combination with soymilk, on hair growth. Since the
soymilk preparations used in this experiment contain about 0.001% isoflavones, we treated mice
throughout the hair cycle with SE flavosterone, a
soybean derived isoflavone extract (at about
0.001% isoflavones), soymilk (containing STI, BBI,
and about 0.001% isoflavones), or a combination
of both (containing STI, BBI and about 0.002%
isoflavones). To ease the application process, these
actives were formulated with a thickener (Polyacrylamide/Laureth-7/C1314 Isoparrafin, 3%). Fig.
5 shows the hair of C57Bl/6 mice following 3 weeks
of topical treatment. Both untreated control mice
and vehicle treated mice have long and randomly
ordered hair (Fig. 5ab). As expected, soymilk reduced hair growth and led to a more uniform, directionally ordered hair appearance (Fig. 5c). Isoflavones (0.001%) reduced hair growth, but not as
effectively as soymilk (Fig. 5d). A heat inactivated
soymilk preparation, with minimal or no trypsin
409
Seiberg et al.
Seiberg et al.
death at the follicular papilla. This death signal,
which is independent of the proteolytic activity of
the protease, resulted in delaying hair growth and
pigmentation (26). Here we show that the trypsin
inhibitors STI and BBI also lead to delayed hair
growth, as well as to reduced follicle dimensions
and reduced pigment deposition within the hair
shaft. Since we could not detect apoptotic cells in
the papillae of soymilk, STI or BBI treated mice
(not shown), we suggest that STI and BBI do not
affect papillae cell death, but exert their effect on
hair growth and size via a different mechanism.
Humans keep or remove hair from different
body parts for social and cosmetic purposes. Unwanted hair is removed using home treatments or
professional services, or is bleached to reduce its
visibility. A safe and effective agent that could reduce hair growth, hair size and hair pigmentation
would add to the available home treatments, which
are not always satisfying. The finding that soymilk
and soymilk-derived serine protease inhibitors delay hair growth and reduce hair shaft dimensions
and pigmentation provides a new concept in hair
growth and management, and could serve as an
effective natural way to manage unwanted hair
growth. The human studies presented here represent a positive proof-of-concept pilot study, which
led us to perform larger double blind studies. Preliminary data of these studies (J.-C. Liu, in preparation) indicate that treatment with soymilk-containing formulations reduced the rate of hair
growth of womens legs, affected the direction of
hair growth to look more homogeneous, and made
the hair softer, finer and less noticeable.
Hirsutism is a relatively frequent condition affecting about 4% of women. Facial hirsutism often
interferes with personal and work activities, and
temporary hair removal is a major component in
the management of hirsute patients. Shaving is the
most frequently used temporary method for facial
hair, as plucking, waxing and depilatories are more
difficult to tolerate and care must be taken to avoid
folliculitis, pigmentary changes, and scarring. Cosmetic cover-ups are usually used to hide cuts and
stubble (27, 28), and electrolysis and thermolysis
are used for permanent hair removal when affordable (29). Daily treatment with soybean-derived protease inhibitors or soymilk products
would be painless and of modest cost and could
serve to reduce hair growth and visibility in these
patients, enhancing their quality of life. Preliminary clinical data (J.-C. Liu, in preparation) indicate that treatment with STI and soymilk-containing formulations significantly reduced womens facial hair growth rate and visibility.
African-type hair is unique in its morphology
a kinky hair shaft with variations in diameter. This
412
complex shaft structure creates the need for specialized grooming products and procedures to ensure that the African-type hair maintains cosmetic
desired properties. The addition of STI, BBI or
soymilk into hair care products could reduce this
complexity and make the African-type hair more
manageable, improving its appearance.
The studies presented here establish soymilk and
the soybean-derived serine protease inhibitors as
effective and inexpensive cosmetic agents for hair
care and management. These agents could reduce
the rate of hair growth, decrease hair shaft dimensions and reduce hair pigmentation, resulting in
slower hair growth, softer and lighter hair shafts,
and more manageable hair care.
Acknowledgement
We would like to thank A. Harmon, A. Johnson and C. Paine
for technical assistance, Dr R. Gallagher for help with imaging
and Dr G. Cauwenbergh for fruitful discussions throughout
this study. Special thanks to Dr V. Hearing for the generous gift
of antibodies to tyrosinase and to TRP-1.
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