Anti‐Wrinkle Therapy: Significant New Findings in the Non‐Invasive Cosmetic Treatment of Skin Wrinkles with Beta‐Glucan

Abstract

Oat beta-glucan is a water soluble, linear polymer of glucose consisting of 1,4 (70%) and 1,3 (30%) linkages with an average molecular weight of 1 × 106 Da. Scientific reports indicate beta-glucan is a film-forming moisturizer, a biological response modifier, and a promoter of wound healing. Our objective was to study the penetration of oat (1,4:1,3) beta-glucan in human skin models and to evaluate clinically its efficacy for reducing fine-lines and wrinkles. Penetration studies performed on human abdominal skin used a single application of 0.5% beta-glucan solution at a dose of 5 mg per cm2. The results showed that beta-glucan, despite its large molecular size, deeply penetrated the skin into the epidermis and dermis. A clinical study of 27 subjects was performed to evaluate the effects of beta-glucan on facial fine-lines and wrinkles. After 8 weeks of treatment, digital image analysis of silicone replicas indicated a significant reduction of wrinkle depth and height, and overall roughness.
This work is the first ex vivo and in vivo demonstration of the physiological effects of beta-glucan in the penetration and restructuring of human tissue. The study supports the use of oat beta-glucan in the care and maintenance of healthy skin and the cosmetic treatment of the signs of aging.

Full text

Reprint
VOLUME 8, NUMBER 1 JANUARY / MARCH 2005
magazine
The Global Publication of the International Federation of Societies of Cosmetic Chemists
R. Pillai, M. Redmond, J. Röding
Anti-Wrinkle Therapy: Significant New Findings in the
Non-Invasive Cosmetic Treatment of Skin Wrinkles with
Beta-Glucan

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IFSCC Magazine – Reprint
INTRODUCTION
Oat has a long history of safe use to pro-
vide fast, temporary relief of the itching,
redness, and pain associated with many
minor skin irritations such as poison ivy/
oak/sumac, insect bites, and allergy [1]. In
the cosmetic application of beta-glucan,
consumers have described various bene-
fits including excellent, sustained mois-
turization properties together with an im-
proved, smoother appearance of the skin.
In recent years new wound product appli-
cations for beta-glucan have been found
in the management of partial thickness
burns, shallow abrasions, and laser treat-
ment [2, 3]. It has been reported that topi-
cal glucan administration enhances
wound healing by increasing macrophage
infiltration into the wound milieu, stimulat-
ing tissue granulation, collagen deposi-
tion, and re-epithelialization, together
with increasing the tensile strength of the
recovered wound [4, 5].
Laboratory experiments using beta-glu-
can from cereal (1,4; 1,3 linear glucose
polymer) and fungi (1,3; 1,6 branched glu-
cose polymer) indicated that all beta-glu-
cans are biological response modifiers,
with oat beta-glucan producing the great-
est cytokine induction activity in macro-
phages [6, 7].
The mechanism by which glucan or glu-
can-induced immunomodulators enhance
wound repair has remained elusive. We
do know that beta-glucan receptors exist
on mammalian macrophages and fibrob-
lasts [4, 8]; the effect of glucan on wound
repair is speculated to involve macro-
phage release of wound growth factors
with the further direct and indirect modu-
lation of fibroblast activity, including col-
lagen biosynthesis.
In the case of wounds, the disruption of
the dermal barrier gives clear and open
access of macrophages and fibroblasts to
topically applied beta-glucan. It remained
to be determined if cellular effects could
be achieved through normal, intact skin
and if the structure of aged skin could be
affected positively through the cosmetic
application of beta-glucan.
EXPERIMENTAL
Oat (1,4; 1,3) beta-glucan
Beta-glucan is the soluble fiber found in
the cell walls of oat kernels
(Figure 1)
.
Structurally, oat beta-glucan is a linear
polymer of glucose consisting of 1,4 (70%)
and 1,3 (30%) glycosidic linkages
(Figure
2)
.
The beta-glucan used in the present stud-
ies was extracted from oat and supplied
as a clear, viscous, 1% solution (Symrise
Inc., New Jersey). Briefly, the extraction
method comprised the aqueous, mild al-
kali (pH 9.2) extraction of beta-glucan from
oat bran, followed by the removal of pro-
tein, and ultrafiltration through a 0.1 mi-
Anti-Wrinkle Therapy: Significant New Findings
in the Non-Invasive Cosmetic Treatment of
Skin Wrinkles with Beta-Glucan
Ravi Pillai1, Mark Redmond2, Joachim Röding3
1Symrise Inc., 10 Gordon Drive, Totowa, New Jersey, USA
2Ceapro Inc. 1008 RTF University of Alberta, Edmonton, Alberta, Canada
3Symrise GmbH & Co KG., Bleichenbrücke 10, 20354 Hamburg, Germany
Corresponding author – email: ravi.pillai@symrise.com
Abstract
Oat beta-glucan is a water soluble, linear polymer of glucose consisting of 1,4 (70%) and 1,3 (30%) linkages with an
average molecular weight of 1x106Da. Scientific reports indicate beta-glucan is a film-forming moisturizer, a biolog-
ical response modifier, and a promoter of wound healing. Our objective was to study the penetration of oat (1,4 :1,3)
beta-glucan in human skin models and to evaluate clinically its efficacy for reducing fine-lines and wrinkles.
Penetration studies performed on human abdominal skin used a single application of 0.5% beta-glucan solution at a
dose of 5 mg per cm2. The results showed that beta-glucan, despite its large molecular size, deeply penetrated the
skin into the epidermis and dermis.
A clinical study of 27 subjects was performed to evaluate the effects of beta-glucan on facial fine-lines and wrinkles.
After 8 weeks of treatment, digital image analysis of silicone replicas indicated a significant reduction of wrinkle depth
and height, and overall roughness.
This work is the first
ex vivo
and
in vivo
demonstration of the physiological effects of beta-glucan in the penetration
and restructuring of human tissue. The study supports the use of oat beta-glucan in the care and maintenance of
healthy skin and the cosmetic treatment of the signs of aging.
Keywords: Beta-glucan, skin penetration, wrinkles, anti-aging

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IFSCC Magazine – Reprint
cron filtration system. The resulting solu-
tion was double precipitated with ethanol
and resuspended to a final concentration
of 1%. The ultrafiltration of the beta-glu-
can solution produced a clear solution
confirmed by a low turbidity (<40 Neph-
elometric Turbidity Units: NTU). The mole-
cular weight range of the beta-glucan was
determined to be 0.5 x 106– 1.0x 106Da as
measured by the method of Wood [9].
Skin penetration study
In the first study we examined the dermal
penetration of (1,4; 1,3) beta-glucan into
sections of surgically-removed, human
abdominal skin. The penetration of beta-
glucan was visualized using Calcofluor
White, a beta-glucan specific fluorescent
stain. The use of Calcofluor White also
allowed semi-quantitative measurement
of beta-glucan penetration with fluores-
cence densitometry [10, 11].
Sections of abdominal tissue were re-
moved surgically without the
subcutaneous fat. The skin was
sliced to fit a penetration and
deposition chamber based
on a Franz Diffusion Cell. The
skin sections were first deep
frozen by liquid nitrogen and
sterilized by gamma-radiation,
which destroyed all yeast and
fungal elements that could in-
terfere with the assay. After ir-
radiation, the skin-section was
thawed and the specimens
were inspected for integrity be-
fore use with a pressure test.
Next, the skin section was con-
ditioned with respect to surface
temperature and moisture con-
tent. This condition was achiev-
ed by pre-heating the liquid
medium in the test chamber and
adjusting the air flow through the cham-
ber’s ventilation channel. A macroscopic
and physical examination of the skin spec-
imen was carried out before the test to
ensure suitability, and the area of the test
application site was 10cm2for all sam-
ples. During testing, the skin specimen
was supplied with a uniformly circulated
nutrient medium, which rinsed its lower
surface. The experimental conditions
were non-occlusive.
The test procedure involved one applica-
tion of 0.5% (w/w) beta-glucan solution
using a micro dose applicator at a dose of
5 mg per cm2of skin. After 8 hours of in-
cubation, the skin tissue was deep frozen.
It was then cut into thin slices and air
dried. Then the skin was cut from lower to
higher possible concentration, meaning
deeper dermis to horny layer.
The specimens were placed on thin glass
slides and allowed to dry. One drop of
Calcofluor White (BactidropTM, Remel,
Lenexa, KS, USA) was added and stained
for 30 seconds. The excess stain was re-
moved by washing with deionized water.
The specimens were then examined using
a fluorescent microscope with an excita-
tion wavelength ranging between 400 –
500 nm and a peak of 440 nm. Untreated
skin was used as the control.
The tests were done simultaneously with
two samples and one control for each vol-
unteer skin. All tests were repeated with
the skin of five volunteers.
Anti-aging study
In the second study, we performed a clin-
ical evaluation of the capacity of beta-
glucan to alleviate the extrinsic signs of
aging. The study was conducted in Col-
orado during the winter months to provide
a dry environmental challenge together
with a high exposure rate to UV.
The test was conducted on a panel of 27
subjects, with two carbomer gel formula-
tions; one contained 0.1% (w/w) (1,4; 1,3)
beta-glucan and the other was placebo.
The subjects applied the randomly as-
signed products twice daily, using a half-
face design. The subjects observed a
3-day conditioning period immediately
prior to baseline measurements. Each of
the 27 subjects treated their left and right
sides of the face, twice daily for eight
weeks. After 8 weeks of treatment, the
skin was evaluated for changes from
baseline values of various parameters
including fine lines, wrinkles and rough-
ness.
The clinical study included subjective and
objective assessments which were rec-
orded at baseline and 2, 4, and 8 weeks.
For the evaluation of fine-lines and wrin-
kles, silicone replicas of the outer canthus
of the eye area (crow’s feet) were sub-
jected to digital image analysis by expert
graders. Macrophotography was also
Figure 1: Fluorescent stained section of an oat
kernel. The beta-glucan present in the cell wall of
the oat fluoresces a brilliant blue when stained
with Calcofluor White.
Figure 2: Chemical structure of oat beta-glucan showing the beta 1,4 and beta 1,3 glycosidically linked glucose polymer structure.

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IFSCC Magazine – Reprint
used to evaluate the changes in fine lines
and wrinkles.
RESULTS AND DISCUSSION
The results of the skin penetration study
showed that (1,4; 1,3) beta-glucan had
penetrated the skin into the epidermis and
dermis
(Figures 3 and 5)
. No fluorescence
staining occurred in the control skin sec-
tion which was not treated with beta-glu-
can
(Figure 4)
. Quantitative assay of the
fluorimetric staining indicated that a sig-
nificant portion of the product (28.5% of
the applied beta-glucan) had entered the
skin
(Figure 6)
.
The clinical trial results indicated a higher
incidence of improvement with (1,4; 1,3)
beta-glucan than with the placebo.
Fig-
ure 7
shows the average percentage
change of the selected parameters from
the baseline, compared to the treatment
with placebo. The silicone replicas after
the test period demonstrated a smoothing
of the cutaneous surface after 8 weeks
of treatment with (1,4; 1,3) beta-glucan.
Macrophotography of the left and right
sides of the face also showed a reduction
in lines and wrinkles.
These results represent remarkable new
findings which will contribute to our un-
derstanding of the interaction of skin with
beta-glucan, and the ability of beta-glucan
to penetrate the skin deeply and elicit cel-
lular changes.
In the past, the potential ability of beta-
glucan to penetrate the skin was disre-
garded because it was thought that the
high molecular weight (> 0.5x106Da) of
the compound would prevent it from pen-
etrating into the epidermis and dermis,
and it would therefore be unable to inter-
Figure 3: Photograph of dermis skin sec-
tion treated with 0.05% (w/w) (1,4; 1,3) be-
ta-glucan solution (magnification x125).
Figure 4:Photograph of the control dermis
skin section (magnification x125).
Figure 5: High magnification photograph
of epidermis skin section treated with a
0.05% (w/w) solution of beta-glucan. Note
that beta-glucan staining is associated
with the inter-cellular matrix indicating
that the beta-glucan permeates the skin
by passing between cells rather than
passing through cells directly (magnifica-
tion x250).
stratum corneum epidermis dermis
Percentage of beta-glucan penetrated
Figure 6: Graphical analysis of the fluorimetric data obtained from the
skin penetration study. The blue bars represent the beta-glucan treat-
ed skin and the green bars represent the control skin. The results indi-
cate that beta-glucan is able to penetrate into the lower levels of the
skin, and therefore is able to interact with the fibroblasts and other
structural elements.
deep wrinkle all wrinkle average peak roughness
reduction reduction reduction reduction
Average percentage change from baseline
Figure 7: Graphical analysis of the facial results of the clinical trial obtained
through digital picture analysis after 8 weeks. The blue bars represent the
beta-glucan treated skin and the green bars represent the control skin.

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IFSCC Magazine – Reprint
act with macrophages and fibroblasts.
Beta-glucan is able to adopt a number of
conformations and is typically extracted
in the form of aggregate particles > 1µm
which are clearly visible under a light
microscope. It is understandable that
such large particles may not be expected
to enter the skin and the effects of beta-
glucan were thought to be limited to the
skin’s surface.
However, the beta-glucan used in the
present penetration study was subject to
sub-micron filtration to produce an aggre-
gate-free, low-turbidity solution with no
particles visible under the light micro-
scope. Examination of the micrographs in
Figure 5
shows that the beta-glucan used
in our study does not enter the skin by
direct passage through the cells of the
epidermis and dermis, but instead works
its way into the skin by passing through the
inter-cellular matrix. Such a process may
be facilitated by a diffusion gradient and
by lipid and phospholipid interactions. In-
teractions of beta-glucan with lipids are
known and are the basis of the health-en-
hancing, lipid-controlling properties rec-
ognized by the FDA [12].
Having penetrated the skin to the dermis,
beta-glucan is able to interact with spe-
cific cells, namely macrophages and
fibroblasts. Results of
in vitro
experiments
have demonstrated that beta-glucan in-
teracts with macrophages to induce the
production of IL-1, which indirectly pro-
motes the production of procollagen by
fibroblasts. In addition, beta-glucan has
been shown to interact with fibroblast re-
ceptors, which results directly in the pro-
duction of procollagen [13, 14]. The con-
version of procollagen to collagen and its
incorporation into collagen bundles would
result in the type of effects noted in our
clinical study, specifically the facial skin
tightening leading to a reduction of fine
lines and wrinkles.
Questionnaires and subject follow-ups
indicated that the effects on fine lines
and wrinkles associated with use of beta-
glucan treatment were long-lived but not
permanent. With normal cellular turnover,
there was an appearance of fine lines. It
may be speculated that the continued use
of products containing beta-glucan would
result in a sustained improvement of ap-
pearance.
The results presented in the present study
offer a cosmetic alternative to other more
invasive treatments aimed at the reduc-
tion of fine lines and wrinkles in an aging
population. Injectable fillers like collagen
– either from human, bovine, or porcine
sources – are common, and recently
hyaluronic acid fillers have also been
introduced. Such fillers produce tempo-
rary, soft tissue with effects that last on
average for 3 to 4 months. With a similar
duration of effect, the cosmetic use of
Botulinum
toxin type A has been reported
to have increased multifold since 1997
[15]. Actives like retinoic acid and coen-
zyme Q10 are also used for the treatment
of wrinkles [16-18]. The regular and fre-
quent use of cosmetics containing oat (1,4;
1,3) beta-glucan is a new and exciting tool
in the fight against the signs of aging.
CONCLUSION
Oat (1,4; 1,3) beta-glucan is a natural
active ingredient offering significant per-
formance-enhancing properties for per-
sonal care applications. Our studies have
shown that the molecule, despite its con-
siderable molecular weight, is able to en-
ter the stratum corneum and epidermis
and penetrate deep into the dermis. The
observed effects of beta-glucan on tissue
restructuring and wrinkle reduction are
most likely effects mediated by fibroblast
stimulation and collagen deposition in the
dermis. These unique properties make oat
beta-glucan a promising and effective
ingredient for cosmetics.
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