Project Title: The effect of beta glucan on elastin production in skin

Investigator:  Wendy Ouriel

Project Summary:

The degradation of elastin is one consequence of the aging process. As people grow older, the skin develops a tired, wrinkled appearance from losing this vital protein. Many products have been developed to slow the effects of time, but most fail to erase the telltale symptoms of maturity. Retinol, a derivative of vitamin A, has shown to eliminate fine lines and wrinkles due in part to its ability to stimulate elastin (Rossetti et al., 2011; Schwartz, and Kligman, 1995;). However, retinol can cause severe side effects in some patients, therefore a viable and comparable alternative should be researched.  Beta glucan, a linear polymer of glucose, was originally used for treating wounds (Wei et al., 2002), but its use expanded to cosmetic applications when patients noticed that it reduced fine lines and wrinkles in the applied area (Pillai, 2005). Currently, no studies have been performed that investigate beta glucan’s elastin-stimulating ability. This study will explore this subject by first determining if beta glucan can penetrate the epidermal and dermal layers of the skin, something that has yet to be conclusively shown. This study will then examine beta glucans ability to stimulate elastin production. The findings of these two experiments will determine beta glucans ability as an effective ingredient for use in anti-aging skin care products. The specific hypothesis of the proposed research is that when applied topically to the skin, beta glucan will penetrate the epidermal and dermal layers, and increase elastin production. The rationale for pursuing this study is the weak body of evidence supporting beta glucans ability to penetrate the epidermis and dermis. The ability of beta glucan to penetrate the epidermis and dermis has merely been assumed, due to its ability to increase collagen production (Wei). However, no dermal penetration studies have been conducted to test this claim. Furthermore, studies that have shown that other topical products that are capable of penetrating to the dermal layer, such as retinol, are capable of stimulating elastin production (Schwartz and Kligman, 1995). It is therefore a logical pursuit to study the potential of beta glucan in stimulating elastin.

Relevance

Anyone that is fortunate to live to old age will manifest the physical signs of aging. Unfortunately, very few products exist that can effectively reduce fine lines and wrinkles by working beyond a superficial level. Retinol is an effective product, but its side effects may outweigh the benefits. Beta glucan is a viable alternative that may offer the same, if not greater benefits, without the negative side effects. Studies have already shown that it is capable of stimulating collagen production in the skin, therefore, we should explore further to better understand the biology behind this potential powerful anti-aging product. This work will provide important new information into the mechanisms for how beta glucan works, and how it may work at the cellular level to mitigate the effects of time.

Research Plan

1.     Specific Aims

AIM 1: Demonstrate that beta glucan penetrates the epidermis and dermis layers of the skin.

In order to affect collagen and elastin production, 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. Showing that the molecule can penetrate the epidermis and 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 elastin cells, 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.

Dermal penetration will be assessed using donated human skin samples. These samples will be exposed to beta glucan at varying time points, followed by biopsy, and analysis via Franz Diffusion cell. High performance liquid chromatography will be used to further assess dermal penetration of the skin samples. Beta glucans penetrance will be compared to that of the control (Retinol) to determine which product penetrates faster and deeper into the dermis.

AIM 2: Measure elastin production after applying beta glucan topically on skin.

Studies have already shown that beta glucans anti-aging properties are due in part to its ability to stimulate collagen production in the skin. Elastin production is a significant component of healthy, youthful looking skin. Therefore, investigating the ability of beta glucan to stimulate elastin production is necessary to support claims of beta glucan’s anti-aging abilities.  Specifically, this aim’s purpose is to answer the question, will beta glucan increase elastin production when topically applied to skin?

Animal skin samples will be used for this portion of the experiment. This experiment will take place over the course of 12 days, where beta glucan will be applied twice daily at 12 hours intervals, followed by a biopsy. Elastin production will be measured via IHC, following an antibody staining protocol. Quantitative measurements for elastin will be carried out using QPCR. QPCR for this experiment will quantify elastin mRNA. The amount of elastin induced by beta glucan will be compared to the amount using retinol to determine which product stimulates more elastin production.

2.     Research Strategy (Significance, Innovation, and Approach)

(A)  Significance

All organs of the body undergo aging as a consequence of the passage of time, and skin is no exception. In fact, skin may undergo aging at a faster rate than other organs since it is in direct contact with the environment. Aging is a universal concern, and has prompted scientists to search 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, and appeals to anyone wishing to regain or retain their youth. 

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 made by their manufacturers.  In fact, there are only a handful of ingredients that have held up to scientific scrutiny 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 into the skincare market, many other retinoids have been developed for cosmetic 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, 2006; Schwartz, 1995). 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, 2013). 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.

Collagen, specifically type I collagen, is the most abundant protein in the human body. It is an extracellular matrix protein whose primary role is to maintain the structural integrity of tissue by interacting with cell surfaces, and other extracellular matrix proteins. Disorders caused by mutations in collagen often affect connective tissue (Di Lullo et al, 2002). One such disorder is Ehlers-Danlos syndrome, which can cause deformities of connective tissue. This syndrome has the potential to be lethal, as it can cause a rupturing of the arteries (Hamel et al, 1998).

Elastin, like collagen, is an extracellular matrix protein that is important for maintaining the integrity of connective tissue. Elastin is especially necessary for tissues that expand and contract during normal function. Elastin gives these tissues their elastic qualities, allowing them to retain their natural shape following expansion or contraction. For example, a hallmark of youthful skin is the ability to poke, pinch, or pull an area, and it will return to normal. This is elastin at work. When skin loses elastin, it may take a longer time to retain its original form. Other tissues that require elastin for normal function include the arteries (Abraham et al., 1982) and lungs (Mercer and Crapo, 1990). The arteries pump large volumes of blood away from the heart. The elasticity of the arteries allows the vessels to accommodate for varying volumes of blood, without the risk of a rupture. The lungs expand and contract thousands of times a day, allowing us to breathe. Elastin prevents the lungs from stretching out, even after millions of breaths taken over a lifetime.

One disease associated with elastin is emphysema (Houghton et al., 2006). For those with emphysema, the lungs have an abnormally large air space near the bronchioles, in addition to an overall breakdown of the lung tissue. The cause for this is an α1-proteinase inhibitor deficiency (Finlay et al., 1996). This proteinase inhibits elastase, a protease that breaks down elastin. Cigarette smoking inhibits this proteinase from carrying out its function, ultimately leading to the breakdown of elastin, and the development of emphysema.

Since it is known that the breakdown of collagen and elastin are the primary causes for the visible signs of skin aging, a logical question to ask is, will using products that stimulates growth of these two proteins can improve the look of aged skin? Studies on retinol have found that it stimulates both collagen and elastin production in skin, with human test subjects reporting diminished fine lines and wrinkles (Davis et al, 1990;McGowan et al., 1997; Mukherjee et al, 2006). 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 et al., 2010). These studies, and many others, all support that the ability to increase collagen and elastin production in the skin can be beneficial in creating a healthier, more youthful appearance.

It is also important to address the rationale for studying other products when Retinol has been thoroughly supported to increase collagen and elastin production in the 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 the skin to burn more easily upon 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. An important question to ask at this point is if there is another product out there that also stimulates collagen and elastin, like retinol, but without the above-mentioned side effects. And if so, could this product possibly do a better job?

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, 1996) by binding to receptors on innate immune cells, causing the immune cells to interpret them as a ‘non-self’ cell (Brown, 2001).

Beta glucan was first studied in the 1990s for its ability to revitalize skin’s appearance. Researchers noted that like retinol, beta glucan also reduces the typical signs of aging in mature skin. In 2002 a study was published that found that beta glucan stimulates human dermal fibroblast collagen biosynthesis (Wei et al., 2002) This is due to the presence of glucan receptors on human dermal fibroblasts, which suggests that glucan can directly stimulate collagen synthesis (Wei et al., 2002).  However, there have yet to be any studies performed that investigates beta glucan’s ability to stimulate elastin production in the skin, or use human skin as a sample. Direct exposure to cells via Franz Diffusion is also novel in this experiment. In addition, there is no strong support for beta glucan’s ability to penetrate the epidermis and the dermis. One study merely states that this is occurring, but provides no methodology, or figures to support their claim (Pillai et al., 2005). The ability of beta glucan to penetrate to the dermal layer is necessary for it to have any effect on elastin, thus necessitating this specific experiment.

This study will examine beta glucans ability to penetrate the skin to the dermal layer, and to stimulate elastin production. The proposed experiments will use retinol as a positive control, providing a means of comparison between the two products.  It will be interesting to determine which product, beta glucan or retinol, penetrates the skin faster and deeper, and stimulates the most elastin production. If beta glucan is more readily absorbed into the skin, and stimulates more elastin production than retinol, then it may hold great promise as the next great ingredient in anti-aging skin care.

(B)  Innovation

This experiment is novel because no study has conclusively shown that beta glucan is able to penetrate the epidermal and dermal layers of the skin. Studies (Pillai et al., 2005). have merely stated that this occurs, but provided no support for this statement. Furthermore, this experiment is unique because no study has examined the ability of beta glucan to stimulate elastin production in the skin. The work done in this experiment will help us gain an understanding for how anti-aging products work at the cellular level.

This study is also novel because it is the first time beta glucan has been studied using retinol as a control. Using retinol as a control will allow for comparative analysis to determine which product is superior in terms of penetration and elastin producing ability.

(C)  Approach

Aim 1: Demonstrate that beta glucan penetrates the epidermis and dermis:

Samples: Abdominal Human skin will be obtained from consenting donors undergoing plastic surgery (UCLA Medical Center, Plastic Surgery, Los Angeles, CA, U.S.A). Beta glucan will be used topically. Biopsied skin will be obtained by punch biopsy (Rossetti, 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 mounted on Franz diffusion cells (Fig 1), an apparatus to allow beta glucan to be delivered into the skin. These cells contain two compartments, the donor compartment and the receptor compartment, and the skin samples will be placed in the donor compartment. Topical beta glucan (0.5%, 1%, or 1.5% concentration) in a water-based cream will be applied at a dosage of 5mg per cm2  (Wei et al., 2002). Beta glucan will be applied to the skin sample, and collected after the allotted time (Table 1).  Samples A will have one topical application, and assayed after 30 minutes. 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. The assay will be performed 30 minutes following the final application. This will be kept constant for all samples. There will be two controls running alongside each sample. The control will be deionized water applied with a cotton swab, and rubbed into the skin, and an oil- in water emulsion of retinol (0.04%) as a positive control, due to its known ability to penetrate the skin. The experiment will be carried out 3 times for each concentration of beta glucan.

Assays:

I. Franz Diffusion Cells.  The penetration depth of beta glucan into the epidermis and dermis layers of the skin will be measured via a dermal/cutaneous penetration assay. For each condition, the samples will be assayed according to the published protocol (Gysler et al., 1999 and Antille et al., 2004). Topical beta glucan (0.5%, 1%, or 1.5% concentration) in a water-based cream will be applied at a dosage of 5mg per cm2  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 et al, 2006). The skin will be incubated in the diffusion cell chamber at the time intervals mentioned in table 1, at a temperature of 37oC. Following the incubation period, the epidermis will be collected and analyzed. The same procedure will be followed for both the DI water and retinol controls.

II. High Performance Liquid Chromatography. To carry out analysis of beta glucan’s ability to penetrate the skin, HPLC will be performed according to protocol (Antille et al., 2004) The skin will be collected from the diffusion apparatus and the dermis will be heat separated (PBS 56oC, 45 seconds) from the dermis. The skin (epidermis and dermis) will be chopped up with scissors in 1.92 ml of HPLC buffer (400 μl acetate buffer 50 mM pH 4, 1.5 ml isopropanol:tetrahydro- furan (1:1) with 200 μM butylated hydroxytoluene and 20 μl retinyl acetate 10 ÌM  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 100μl will be injected into the HPLC.

Predicted Results: I predict that dermal penetration will occur, where beta glucan penetrates through the epidermis to the dermal layer.  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 represents penetration 1 hour after application, and sample F, which represents penetration after 24 hours (Fig. 2). I cannot make a prediction as to the ability of beta glucan to penetrate deeper or faster into the dermis in comparison to retinol. There have not been any prior studies comparing the two in any regard, therefore any prediction would be purely speculative, and would not have any scientific backing.

Potential Pitfalls: The most likely pitfall for this experiment is that dermal penetration does not occur. We may only find glucan present in the epidermis, which is problematic because we want to stimulate elastin production, which necessitates penetration to the dermis. If this occurs, two likely reasons may be time and concentration. Future experiments would require carrying out the experiment over a longer period of time,  such as an additional day, or up to a week. Penetration of beta glucan may be a slow process, so an experiment, such as the one proposed, that only measures over the course of 24 hours, may need to be revised if dermal penetration is not observed. The other potential issue could be the concentration of beta glucan used. For this problem, I would revise the experiment to use a greater variety of concentrations.

AIM 2: Measure elastin production after applying oat beta glucan topically on skin.

Samples: Animal skin samples will be used for this portion of the experiment. The reason for using animal samples in this AIM is due to the timespan that this experiment requires. Human skin, or any excised skin will begin to decay after a few days. This method maintains the freshness of the skin, and will allow for all biological processes to continue unfettered. Skin samples from the Yucatan hairless micropig (HMP) will be used. Numerous studies have used HMP’s as a model for skin study, including studies on permeation (Fujii et al, 1997), skin metabolism (Oh et al., 2002), and skin pathology (Lavker et al., 1991). HMP’s, as these studies suggest, are an accepted model system in the field of cutaneous biology.

These pigs can be obtained from Charles River Laboratories (Boston, MA).  Mature pigs (older than 15 years) will be used. These pigs have an average height of about 38-45 cm, and a width 10-15 cm. This size provides enough surface area for skin samples to be taken.

Oat beta glucan will be used topically on the skin. Biopsied skin will be obtained by punch biopsy, as described in aim 1. For each sample (table 2) the same pig will be used only once, but the same pig will have 2 samples, the experimental and the control. The positive control for this experiment will be an oil-in water emulsion of retinol (0.04%) due to its known ability to promote elastin production.

Conditions:

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 over an allotted time period (Table 2), and then biopsied for analysis.

Controls will be taken with each sample. The control will be retinol (0.04%, 5mg/ cm 2) rubbed onto a designated area of skin with a cotton swab. After the allotted time for the particular sample, a biopsy will also be taken for analysis.

Additional controls will also be used 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. 

Sample A will have twice daily (12 hour intervals) oat beta glucan applications for 1 day, followed by a biopsy 1 hour after the final treatment. Samples B-L will be taken in the same manner, but at different time intervals according to the supplementary chart.

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. 

 According to the published protocol (Rossetti et al 2010), an elastin antibody staining protocol will be followed to measure elastin production at the various time points (Fig 2) over the course of 12 days. The skin samples will be isolated, dehydrated, and embedded in paraffin. Sections will be cut at a thickness of 5mm at intervals of 150 mm. The sections will then be stained with Luna staining (fig 4), 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 mL qPCR Mastermix (Sabio Sciences, CAS# 330520), 11mL cDNA, and 1.5 mL of water.

Predicted Results:

I predict that elastin production will significantly increase over the 12 days of this experiment (Fig 3). I predict that elastin production will be highest at the 12th day mark. Previous research has shown that elastin synthesis takes time, and that prolonged exposure to topical treatments has an increased effect the more they are used (Rossetti). In this study, it was found that after 12 days, the amount of elastin was highest in the retinol sample compared to the control. 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.

I cannot make a prediction as to the ability of beta glucan to promote greater elastin production in comparison to retinol. There have not been any prior studies comparing the two in any regard, therefore any prediction would be purely speculative, and not have any scientific backing.

Pitfalls: It is possible that no elastin increase will be observed when the beta glucan samples are compared to the control. If this occurs, I would suggest extending the length of the experiment from 12 days to 30 days.

Bibliography

Abraham, P.A., Perejda, A.J., Carnes, W.H., and Uitto, J. (1982). Marfan syndrome. Demonstration of abnormal elastin in aorta. J Clin Invest 70, 1245-1252.

Antille, C., Tran, C., Sorg, O., and Saurat, J.H. (2004). Topical beta-carotene is converted to retinyl esters in human skin ex vivo and mouse skin in vivo. Exp Dermatol 13, 558-561.

Bernstein, E.F., Chen, Y.Q., Kopp, J.B., Fisher, L., Brown, D.B., Hahn, P.J., Robey, F.A., Lakkakorpi, J., and Uitto, J. (1996). Long-term sun exposure alters the collagen of the papillary dermis. Comparison of sun-protected and photoaged skin by northern analysis, immunohistochemical staining, and confocal laser scanning microscopy. J Am Acad Dermatol 34, 209-218.

Brown, G.D., and Gordon, S. (2001). Immune recognition. A new receptor for beta-glucans. Nature 413, 36-37.

Chen, V.L., Fleischmajer, R., Schwartz, E., Palaia, M., and Timpl, R. (1986). Immunochemistry of elastotic material in sun-damaged skin. J Invest Dermatol 87, 334-337.

Davis, B.H., Kramer, R.T., and Davidson, N.O. (1990). Retinoic acid modulates rat Ito cell proliferation, collagen, and transforming growth factor beta production. J Clin Invest 86, 2062-2070.

Di Lullo, G.A., Sweeney, S.M., Korkko, J., Ala-Kokko, L., and San Antonio, J.D. (2002). Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem 277, 4223-4231.

Fisher, G.J., Kang, S., Varani, J., Bata-Csorgo, Z., Wan, Y., Datta, S., and Voorhees, J.J. (2002). Mechanisms of photoaging and chronological skin aging. Arch Dermatol 138, 1462-1470.

Fujii M, Yamanouchi S, Hori N, Iwanaga N, Kawaguchi N, Matsumoto M. (1997). Evaluation of Yucatan micropig skin for use as an in vitro model for skin permeation study. Biological & Pharmaceutical Bulletin. 20(3): 249-254.

Hamel, B.C., Pals, G., Engels, C.H., van den Akker, E., Boers, G.H., van Dongen, P.W., and Steijlen, P.M. (1998a). Ehlers-Danlos syndrome and type III collagen abnormalities: a variable clinical spectrum. Clin Genet 53, 440-446.

Gysler, A., Burkhard, K., Sippl, W., Lange, K., Korting, C.H., Holtje, H.D., Schafer-Kortin, M. (1999) Skin penetration and metabolism of topical glucocorticoids in reconstructed epidermis and in excised human skin. Pharmaceutical Research 16(9), 1386-1391. 

Lavker, R.M., Dong, G., Zheng, P.S., and Murphy, G.F. (1991). Hairless micropig skin. A novel model for studies of cutaneous biology. Am J Pathol 138, 687-697.

McGowan, S.E., Doro, M.M., and Jackson, S.K. (1997). Endogenous retinoids increase perinatal elastin gene expression in rat lung fibroblasts and fetal explants. Am J Physiol 273, L410-416.

Mercer, R.R., and Crapo, J.D. (1990). Spatial distribution of collagen and elastin fibers in the lungs. J Appl Physiol (1985) 69, 756-765.

Miura, N.N., Ohno, N., Aketagawa, J., Tamura, H., Tanaka, S., and Yadomae, T. (1996). Blood clearance of (1-->3)-beta-D-glucan in MRL lpr/lpr mice. FEMS Immunol Med Microbiol 13, 51-57.

Mukherjee, S., Date, A., Patravale, V., Korting, H.C., Roeder, A., and Weindl, G. (2006). Retinoids in the treatment of skin aging: an overview of clinical efficacy and safety. Clin Interv Aging 1, 327-348.

Nevins, C.P., Vierck, J.L., Bogachus, L.D., Velotta, N.S., Castro-Munozledo, F., and Dodson, M.V. (2005). An Inexpensive Method for Applying Nitrogen Evaporation to Hexane-containing 24- or 96-well Plates. Cytotechnology 49, 71-75.

Oh, S.Y., Fujii, M., Takeda, Y., Yoda, K., Utoguchi, N., Matsumoto, M., Watanabe, Y. (2002) The effect of ethanol on the simultaneous transport and metabolism of methyl p-hydroxybenzoate in excised skin of Yucatan micropig. International Journal of Pharmaceuticals, 240(1-2):115.

Oh, Y.K., Kim, M.Y., Shin, J.Y., Kim, T.W., Yun, M.O., Yang, S.J., Choi, S.S., Jung, W.W., Kim, J.A., and Choi, H.G. (2006). Skin permeation of retinol in Tween 20-based deformable liposomes: in-vitro evaluation in human skin and keratinocyte models. J Pharm Pharmacol 58, 161-166.

Overbeek, S.A., Braber, S., Koelink, P.J., Henricks, P.A., Mortaz, E., LoTam Loi, A.T., Jackson, P.L., Garssen, J., Wagenaar, G.T., Timens, W., et al. (2013). Cigarette smoke-induced collagen destruction; key to chronic neutrophilic airway inflammation? PLoS One 8, e55612.

Pillai, R., Redmond, M., Roding, J. (2005). Anti-wrinkle therapy: significant new findings in the non-invasive cosmetic treatment of skin wrinkles with beta-glucan. International journal of cosmetic science. 27(5): 292.

Rossetti, D. Kielmanowicz, M.G., Vlgodman, S., Hu, Y.P., Chen, N., Nkengne, A., Oddos, T., Fischer, D., Seiberg, M., Lin, C.B. (2010). A novel anti-ageing mechanism for retinol: induction of dermal elastin synthesis and elastin fibre formation. International Journal of Cosmetic Science. 33(1):62-69.

Schwartz, E., and Kligman, L.H. (1995). Topical tretinoin increases the tropoelastin and fibronectin content of photoaged hairless mouse skin. J Invest Dermatol 104, 518-522.

Wei, D., Zhang, L., Williams, D.L., and Browder, I.W. (2002). Glucan stimulates human dermal fibroblast collagen biosynthesis through a nuclear factor-1 dependent mechanism. Wound Repair Regen 10, 161-168.