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Wendy Ouriel
Bio 505T
Research Proposal Second Draft
Project Title: The effect of oat beta glucan on elastin production in skin
Investigator: Wendy Ouriel
Project Summary: The purpose of this project is to investigate oat beta glucans ability to
stimulate elastin production in the skin when applied topically. Oat beta glucan has been
shown to have anti-aging effects, such as reducing fine lines and wrinkles in mature skin.
Presently, it is known that oat beta glucan’s anti-aging properties are due in part to its
ability to stimulate collagen production in the skin. Collagen is a key component of the
dermis, and its synthesis is necessary for maintaining youthful skin. However, elastin
plays an important role in skin health as well. As of yet, there have not been any studies
that have examined the role of oat beta glucan in elastin production. The specific aims for
this paper are first to determine if oat beta glucan can penetrate the epidermal and dermal
layers of the skin. The second specific aim is to determine if topically applied oat beta
glucan stimulates elastin production in the skin. The findings of this study will be of great
importance for cosmetic research, as it will identify oat beta glucan as a powerful tool for
anti-aging products.
Detailed Budget: $300,000 and 3 years maximum. Fill in direct costs only
and provide justification.
DIRECT COSTS
BUDGET CATEGORY TOTALS
INITIAL
BUDGET
PERIOD
2ND
ADDITIONAL
YEAR OF
SUPPORT
3RD
ADDITIONAL
YEAR OF
SUPPORT
PERSONNEL (Salary and fringe
benefits.)
CONSULTANT COSTS
EQUIPMENT
SUPPLIES
TRAVEL
OTHER EXPENSES
TOTAL DIRECT COSTS:
TOTAL DIRECT COSTS FOR THE ENTIRE PROPOSED PROJECT
PERIOD
FACILITIES AND ADMINISTRATIVE COSTS
(Do not have to be included in the $300,000
maximum-AM)
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Justification: The costs detailed for this project are necessary to carry out the
proposed experiments for the research described below.
Background:
All organs of the body undergo aging as a consequence of the passage of time.
Skin is no exception. In fact, it may undergo aging at a faster rate than other organs since
it is in direct contact with the environment. Researchers have long searched for a ‘holy
grail’ of skin products: a fountain of youth that will prevent the signs of aging, and
reverse those that have already manifested. The term “anti-aging” is used in the cosmetic
industry to describe any product that purportedly reverses and prevents the various forms
of skin damage that come with age.
Many anti-aging products have made their way to the cosmetic counters over the
years, but there has been little peer-reviewed research to support the anti-aging claims
stated by their manufacturers. In fact, there are only a handful of ingredients that have
held up to their claims when tested in the laboratory. In 1969, Tretinoin, a carboxylic
acid type of vitamin A, received widespread attention for its ability to treat acne, and to
reduce the appearance of stretch marks and wrinkles when applied topically. Since its
introduction onto the skincare market, many other retinoids have been developed for
skincare purposes.
Tretinoin and other retinoids have been studied more than most other
cosmeceutical ingredients for their ability to reverse and prevent the signs of skin aging.
Studies have shown that one of the ways in which retinoids work to reverse skin aging is
by stimulating collagen and elastin production in the skin (Mukherjee, Schwartz). A large
portion of age-associated skin changes occurs in the dermis layer of the skin, which thins
as one ages. The fibroblasts undergo senescence, and as a result, produce collagen and
elastin at a diminished pace. Collagen and elastin give skin its structure, and also play a
role in regeneration. When their growth is slowed, the skin begins to wrinkle and sag.
The breakdown of collagen and elastin are the primary reasons for skin aging, and
environmental factors, such as sun exposure and cigarette smoking can accelerate this
process. Ultraviolet radiation from the sun and cigarette smoking has been shown to
activate the Matrix Metalloproteinase (MMP) enzymes collagenase and elastase, whose
function is to break down collagen and elastin (Overbeek). Prolonged elevations of
MMPs as a result of long-term exposure to UV radiation and cigarette smoke manifests
itself in the form of clumped collagen and elastin. Visibly, this has the appearance of
wrinkled, sagging skin.
Although retinoids have been shown to have anti-aging properties, there are still
some drawbacks to their use. Retinoids increase one’s sensitivity to the sun, and can
cause skin to burn more easily with sun exposure. Users have also reported a lightening
or darkening of the skin, and skin swelling. Those with sensitive skin may also find
retinoids to be too harsh; therefore other substances should be examined for their role in
anti-aging skin care.
Oat beta glucan is a linear polymer of glucose that can be found in the cell wall of
baker’s yeast, plant cellulose, bran, fungi, and mushrooms. Beta glucans are glucose-only
polysaccharides, and exist in the form of connected six-sided D-glucose rings. They play
an important biological role by activating the immune system (Miura) by binding with
receptors on innate immune cells, causing the immune cells to interpret them as a ‘non-
self’ cell (Brown).
Oat beta glucan was first studied in the 1990s for its ability to revitalize skin’s
appearance (Byron). Researchers noted that like retinol, beta glucan also reduces the
typical signs of aging in mature skin. One reason possible reason for its effectiveness is
due to its ability to penetrate the epidermis and dermis of the skin, despite the molecule’s
large size (Pillai). A study on 27 subjects found that after 8 weeks of treatment with
topically applied oat beta glucan, those using the beta glucan treatment showed
significant reduction in fine lines and wrinkles (Pillai). In 2002 a study was published
that found that oat beta glucan stimulates human dermal fibroblast collagen biosynthesis.
This is due to the presence of glucan receptors on human dermal fibroblasts, which
suggests that glucan can directly stimulate collagen synthesis (Wei). However, there
have yet to be any studies performed that investigates oat beta glucan’s ability to
stimulate elastin production in the skin.
Specific Aims
Oat beta glucan has been studied for its ability to prevent and reverse the signs of
skin aging. Studies have shown that it is capable of stimulating collagen production in the
skin, causing fine lines and wrinkles to be reduced. The purpose of this experiment is to
further investigate the anti-aging properties of oat beta glucan on skin. Specifically this
research project aims to examine the effect oat beta glucan has on elastin production
when applied topically to the skin. The main research question that this proposal
addresses is, will oat beta glucan increase elastin production in the skin? The hypothesis
that this research proposal posits is that when applied topically, oat beta glucan will
increase elastin production in the skin. This hypothesis is based on previous research that
has shown oat beta glucan to be a powerful and effective anti-aging ingredient. It has
been noted in past studies that oat beta glucan, when applied topically, has been able to
reduce fine lines and wrinkles in aged skin. Further research found that oat beta glucan
stimulated collagen production. Since skin aging is caused by a combination of reduced
collagen and elastin production, it is likely that oat beta glucan stimulates the growth of
both proteins, and not just collagen alone. Based on these findings, the experimental
focus of this proposal is on the elastin production of skin as a result of topical application
of oat beta glucan
_______________________________________________________________________
_________________________
Aim 1: Demonstrate that oat beta glucan penetrates the epidermis and dermis layers
of the skin. In order to affect collagen and elastin production, oat beta glucan must be
able to penetrate the epidermis and dermis layers of the skin. The dermis sits right below
the epidermis, and is primarily composed of collagen and elastin fibers. Showing that the
molecule can reach the dermis is the first step in supporting that this molecule is an
effective anti-aging ingredient. If skin penetration does not occur, then it will not have an
effect on collagen or elastin production, and will wash off when the skin gets wet. The
goal when looking for potential anti-aging ingredients is to find something that will
readily absorb into the skin, which will allow for long-lasting benefits. These findings
will be significant because it will show that oat beta glucan has the ability to affect
collagen and elastin by penetrating to the level of skin where they are found, and that it
will provide long-lasting effects due to its ability to absorb into the skin, and not just sit
on top.
Specific Question: Will beta glucan penetrate the dermis and epidermis layers of the
skin?
Innovation: This experiment is novel because no study has conclusively shown that oat
beta glucan is able to penetrate the epidermal and dermal layers of the skin. Studies such
as Pillal et al. have merely stated that this occurs, but provided no support for this
statement.
Approach
Samples: Animal skin samples will be used for this portion of the experiment. Yucatan
hairless micropig (HMP) skin has been found to be very similar to humans. The
epidermis of HMP and humans has a similar structure, and the organization of the dermal
extracellular matrix, including collagen and elastin fibers is consistent with that of human
skin (Lavker). These animals can be obtained from Charles River Laboratories (Boston,
MA). Mature (older than 15 years) pigs will be used. Oat beta glucan will be used
topically. This can be obtained from Makingcosmetics (CAS# 9041-22-9). Biopsied
skin will be obtained by punch biopsy, as described by Zeuber. This type of biopsy will
allow a skin specimen containing the epidermis, and the dermis, and yields a sample of 3-
4mm.
Conditions: Biopsied skin samples will be maintained on Nunc Skin graft cell culture
dish. 0.5% of oat beta glucan solution will be applied with a cotton swab and rubbed into
an area at a dose of 5mg per cm2 as described in Pillal et al. Beta glucan will be applied
to the skin of the pig, and then biopsied after the allotted time. Samples A will have one
topical application, and biopsied after 1 hour. Samples B will have topical applications at
1 and 2 hours. Samples C will have topical applications at 1, 2, and 3 hours. Samples D
will have applications at 1,2,3, and 6 hours. Samples E will have applications at 1, 2, 3, 6,
and 12 hours. And samples F will have applications at 1, 2, 3, 6, 12, and 24 hours. There
will be a control running alongside each sample. The control will be deionized water
applied with a cotton swab, and rubbed into the skin. Biopsies will be taken at the same
time intervals as the experimental samples.
Assay: The penetration depth of beta glucan into the epidermis and dermis layers of the
skin will be measured. This will be measured via a dermal/cutaneous penetration assay.
For each condition, the samples will be assayed according to the protocol stated in Gysler
et al. and Antille et al. This assay will require biopsied skin to be mounted on a Franz
diffusion cell (Fig 1). These cells contain two compartments, the donor compartment and
the receptor compartment. Topical beta glucan will be applied at a dosage of 5mg per cm2
in water-based cream containing 0.5% beta glucan to cells within the donor compartment,
and the receptor compartment will be filled with 50 ml phosphate-buffered solution (pH
7.0), and kept at a temperature of 37oC (Oh). The skin will be incubated in the diffusion
cell chamber at the time intervals mentioned above, at a temperature of 37oC. Following
the incubation period, the epidermis will be collected and analyzed.
To carry out analysis of beta glucan’s ability to penetrate the skin, HPLC will be
performed as described in Antille et al. The skin will be collected from the diffusion
apparatus and the dermis will be heat separated (PBS 56oC, 45 seconds) from the dermis.
The epidermis will be chopped up with scissors in 1.92 ml of HPLC buffer (400 l μ
acetate buffer 50 mM pH 4, 1.5 ml isopropanol:tetrahydro- furan (1:1) with 200 μM
butylated hydroxytoluene and 20 l retinyl acetate 10 ÌμM at 4oC) in dim lighting, and
homogenized using a Polytron PT homogenizer in 1.92 ml. Following homogenization,
the homogenate will be collected and sonicated for 10 seconds at 50 watt power and
centrifuged for 10 minutes at 12,000g. The supernatant will be collected with hexane
(4ml). Hexane removal will be carried out via nitrogen evaportation (Nevins). When
hexane has been eliminated, a sample of 100 l will be injected into the HPLC. μ
Predicted Results: I predict that samples A-E will all show penetration of beta glucan
into the dermis and epidermis of the skin. I predict that penetration of beta glucan will
occur quickly, and that there will not be a significant difference between penetration
levels between sample A, which will be taken after one hour, and sample E, which will
be assayed after 24 hours. The control will show that the assay is correctly measuring
beta glucan penetrance. The reason for this prediction is due to previous studies that have
found glucose to have skin permeability (Ghosn).
Conclusions- If beta glucan is shown to penetrate the dermis and epidermis of the skin,
then this means that it is a viable substance to be studied for its use in skin care products.
Since the goal of this experiment is to determine oat beta glucans ability as an anti-aging
ingredient, it is necessary to show that it is able to penetrate the layers of the skin.
Otherwise, it will be washed off and have no long lasting effect. This finding will also
give oat beta glucan an advantage over collagen in terms of topical application, as
collagen cannot penetrate the epidermis of the skin. This is the major reason why
cosmetics containing collagen for topical application are widely ineffective in the long-
term. The collagen merely sits atop the skin, but does not get absorbed into the epidermal
and dermal layers.
_______________________________________________________________________
__________________________
AIM 2: Measure elastin production after applying oat beta glucan topically on skin.
Studies have already shown that oat beta glucans anti-aging properties are due in part to
its ability to stimulate collagen production in the skin. Collagen breakdown is a major
component to wrinkling, sagging, and other physical signs of aged skin. However, elastin
is also a crucial component in skin, and plays an important role in maintaining its
youthful appearance. This experiment will be novel because no research has been
conducted on beta glucans role in stimulating elastin production. The specific question
that will be addressed in this aim is, will applying oat beta glucan topically to skin cause
an increase in elastin production? The hypothesis for this research is that upon topical
application of oat beta glucan to the skin, there will be a significant increase in the
amount of elastin produced in the skin’s dermal layers. This result will be most
pronounced over a period of several months, as previous studies have shown that
collagen and elastin production takes a few months for any marked effect to be observed.
Approach
Samples: Animal skin samples will be used for this portion of the experiment. Skin
samples from the Yucatan hairless micropig (HMP) will be used. These pigs can be
obtained from Charles River Laboratories (Boston, MA). Mature pigs (older than 15
years) will be used. Oat beta glucan will be used topically. This can be obtained from
MakingCosmetics Co. (CAS# 9041-22-9). Biopsied skin will be obtained by punch
biopsy, as described by Zeuber. This type of biopsy will allow a skin specimen
containing the epidermis, and the dermis, and yields a sample of 3-4mm.
Conditions: Previous studies on the effects of retinol on elastin production found that
increases in elastin production after topical application of retinol occurred after the first
day of treatment, and steadily increased over a 12-day period (Rossetti). This experiment
will focus on the short-term effects of topical beta glucan on elastin production. 0.5% of
oat beta glucan solution will be applied with a cotton swab and rubbed into an area at a
dose of 5mg per cm2 as described in Pillai et al. over an allotted time period, and then
biopsied for analysis. Sample A will have twice daily (12 hour intervals) oat beta glucan
applications for 1 day, followed by a biopsy. Additional samples will be taken in the
same manner, but at different time intervals according to the following chart:
Sample Daily Beta Glucan Application Biopsy
0 0 Beginning of experiment
A 2 1 day
B 2 2 days
C 2 3 days
D 2 4 days
E 2 5 days
F 2 6 days
G 2 7 days
H 2 8 days
I 2 9 days
J 2 10 days
K 2 11 days
L 2 12 days
Controls will be taken with each sample. The control will be a clean cotton swab
rubbed onto a designated area of skin. After the allotted time for the particular sample, a
biopsy will also be taken for analysis.
Additional controls must also be considered for this portion of the experiment for
both experimental and control group pigs. All pigs must be kept on the same diet to
eliminate any dietary factors that may influence elastin production. The animals must
also be kept in the same sort of living condition, where temperature, humidity, and light
(including sunlight) are consistent for all pigs. Such environmental factors influence skin
health, therefore must be kept consistent for all test subjects.
Assay: This experiment will measure the amount of elastin in skin of both young and old
pigs, then determine if applying oat beta glucan topically has any effect on increasing
elastin production. Elastin production will be measured using immunohistochemistry. To
do this, the fibroblasts must be isolated from the treated skin samples. This will be done
in the manner described in Seluanov et al.
Similarly to the protocol described in Rossetti et al. an elastin antibody staining
protocol will be followed to measure elastin production at the various time points (Fig 2).
Fibroblasts will be isolated, dehydrated, and embedded in paraffin. Sections will be cut at
a thickness of 5µm at intervals of 150 µm. The sections will then be stained with Luna
staining, which selectively stains for elastin fibers. Images of these sections will be taken
with a microscope camera.
To measure the amount of elastin quantitatively, QPCR will be performed. To do
this, RNA will be extracted from the skin samples, and purified via Arcturus Paradise
Plus RNA Extraction and Isolation Kit (Life Technologies, CAS# KIT0312-I). The RNA,
once purified, will be converted to cDNA via Superscript III reverse transcriptase (Life
Technologies, CAS # 18080085). QPCR will be performed by using a template made
with primers for elastin and 18s RNA. The reaction mixture will contain 12.5 µL qPCR
Mastermix (Sabio Sciences, CAS# 330520), 11µL cDNA, and 1.5 µL of water.
Predicted Results: I predict that elastin production will significantly increase over the 12
days of this experiment. I predict that elastin production will be highest at the 12th day
mark. Previous research has shown that elastin and collagen synthesis takes time, and that
prolonged exposure to topical treatments has an increased effect the more they are used.
Therefore I believe that the same effect will be observed in this experiment. Elastin
production will be highest with the longest amount of exposure. For future studies it
would be interesting to look at elastin production over the course of many years. A
human model would be interesting for this sort of experiment to determine if topical
application of oat beta glucan can maintain elastin production in people of advanced age.
Conclusion: If the predicted results are true, then this experiment supports beta glucan to
be a powerful ingredient for anti-aging skin care products. Previous studies have shown
that beta glucan stimulates collagen, but no studies have looked at elastin. Both collagen
and elastin are critical components of the skin, and maintaining a youthful appearance. If
beta glucan stimulates elastin as well as collagen, then it can be used as an effective anti-
aging ingredient.
Figures
Antille et al.
Figure 1. Model of the Franz cell apparatus. A skin sample is placed between two glass
chambers and sealed. For this experiment, beta glucan will be applied to the surface of
the skin, and the top glass chamber is closed by an air permeable cap to allow for air flow
but to prevent microorganism contamination. The recipient chamber is filled with a
medium (in this case PBS), and sealed.
Rossetti et al.
Figure 2. Staining of elastin fibers using the Luna staining method.
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