L-Hydroxyproline & Low-Molecular Weight Hyaluronic Acid

The continued robust growth of the market for functional cosmetic products has formulators searching for the newest, most effective ingredients that promise not only to protect against the environment and the ravages of time, but also to repair skin damage. Trends in the cosmetic industry have evolved from superficial moisturizers, UV filters and free radical quenchers to today’s interest in amino acids, peptides and other bio-actives that can deliver anti-aging benefits at the cellular level. Amino acids, the “building blocks” of protein, are naturally occurring nutrients essential for the growth and maintenance of the connective tissues, skin, hair and nails. They are the major components of natural moisturizing factor (NMF) in the outermost layer of the epidermis (stratum corneum), which maintains healthy moisture levels in skin. Topical application of amino acids provides effective moisturization to this layer. In addition, certain amino acids are essential for the synthesis of collagen and elastin, which give skin suppleness and strength from within.

Collagen is the major protein of connective tissues and is responsible for providing structure and strength to these tissues. Elastin refers to the bundles of protein fibers that give skin and other tissues the ability to stretch and snap back to their original length. Of particular interest to the cosmetic industry is the amino acid, L-hydroxyproline, a major constituent of collagen. Recent research shows L-hydroxyproline is not just a building block of collagen, but an essential bio-active factor needed for collagen and elastin formation.

In the skin, collagen and elastin are produced by fibroblast cells, which are present in the dermis, the deeper layer beneath the epidermis. To have a sound physiological effect, active ingredients that support fibroblast cell health must be able to penetrate the skin to that dermal layer. In the past, this has been a technological challenge, since penetration of many actives into the skin is relatively low when applied alone. Ideally, actives are partnered with penetration-enhancing ingredients or incorporated into alternative delivery systems in order to impart their functional benefits. Another possible route to the skin is systemic, raising the question of whether oral supplementation with amino acids can sufficiently enhance the activity of fibroblasts to produce measurable benefits in skin.

Researchers at Kyowa Hakko Kogyo Co., a global supplier of amino acids and other bulk ingredients used in pharmaceuticals, dietary supplements, cosmetics and other products, conducted a series of experiments designed to test the effects of the company’s patented form of L-hydroxyproline (Lumistor™) when applied topically, either alone or in combination with other ingredients, and when consumed orally as a supplement.

First, the effect of L-hydroxyproline (2-percent Lumistor in a cream base) was tested on the skin of female volunteers (age 45+) with moderate signs of superficial fine lines and wrinkles. Participants applied the cream twice a day and visited the testing center after two, five and seven weeks. At week five, Lumistor reduced fine lines and wrinkles by 25.3 percent compared to the placebo group. At week seven, the reduction was 32.7 percent (Table 1). This clearly showed L-hydroxyproline reduced the appearance of fine lines and wrinkles after five weeks. Previous in vitro studies showed the effect of L-hydroxyproline is seen via skin epidermal cell proliferation.

In the next phase of this study, researchers sought to further enhance the skin penetration of L-hydroxyproline by combining it with low molecular weight hyaluronic acid (LMW-HA). Kyowa’s LMW-HA, Resilen™-200, has a molecular weight of 150,000 to 200,000 and has been shown to penetrate the stratum corneum, helping to increase skin moisture and smoothness, and to reduce fine lines and wrinkles. In addition, HA enhances skin penetration of other bio-active compounds. Using the same protocol, at week five, L-hydroxyproline plus HA (2-percent Lumistor plus 1- percent Resilen-200 in a cream base) decreased fine lines and wrinkles by 35.8 percent; at week seven, the reduction was 45 percent (Table 1). The study showed the combination formula enhanced L-hydroxyproline’s ability to reduce the appearance of fine lines and wrinkles.

Earlier studies have shown continuous topical application of vitamin C can increase collagen formation; but, since most of the available vitamin C forms are unstable to oxygen and moisture, cosmetic products containing them have a short shelf life. To address this issue, Kyowa Hakko used a vitamin C derivative—magnesium ascorbyl phosphate—that is highly stable in aqueous systems. This ingredient was tested in combination with L-hydroxyproline and HA to assess the additional benefits in reduction of wrinkles.

The effects of L-hydroxyproline plus HA (Product A, 2- percent Lumistor plus 1-percent Resilen-200 in a cream base) were compared to those of L-hydroxyproline, HA plus vitamin C (Product B, 2-percent Lumistor plus 1-percent Resilen-200 + 5-percent magnesium ascorbyl phosphate in a cream base). Two groups of 15 women (age 45+) with moderate signs of superficial fine lines and wrinkles were tested at baseline; they then applied either product A or product B twice daily and were tested after two, four and six weeks.

Superficial fine lines and wrinkles were assessed using the method as described by Packman and Gans (J Soc Cos Chem. 1976;29:70-90).

As described in Table 2, product A decreased superficial fine lines and wrinkles by 30.4 percent and 47.6 percent after four and six weeks, respectively, as compared to baseline. Product B decreased superficial fine lines and wrinkles by 38.3 percent and 58.8 percent after four and six weeks of treatment, respectively, as compared to baseline, suggesting vitamin C, as magnesium ascorbyl phosphate, enhanced the functional benefits of Lumistor and Resilen in the skin.

In conclusion, L-hydroxyproline is a viable bioactive compound that, when used topically, could reduce the appearance of fine lines and wrinkles. In addition, L-hydroxyproline, when combined with LMW-HA, could achieve its maximum benefits as a natural skin penetration enhancerand stabilized form of vitamin C.

A third experiment was conducted to determine the potential benefit of dietary supplementation with L-hydroxyproline on dry and rough skin. Twenty-seven female volunteers (ages 48 to 66) with chronically dry, rough skin participated in this study. One group of women took a placebo made with cornstarch for eight weeks; the other group took 2 g/d of Lumistor capsules for eight weeks. Facial skin moisture content was examined by trained technicians at baseline and again at weeks four and eight during both protocols, using the Corneometer methodology. Participants were also asked to give a subjective assessment of the condition of their skin, using a scale of one to 10.

After eight weeks of dietary supplementation with Lumistor, skin moisture content improved significantly as compared to placebo (Table 3). The volunteers’ perception of facial skin elasticity and other skin parameters, including dryness and wrinkles, were also significantly improved (Table 4).

This is the first study to show oral intake of L-hydroxyproline can reduce fine lines and wrinkles by increasing facial water content. Previous studies have shown the effectiveness of oral HA (J New Remedies Clinics. 2001;50(5):541-560); hence, the combination of the two might result in an effective solution to wrinkle reduction.

Based on these studies, Kyowa Hakko developed a new, stable, non-irritating and highly active combination of Lumistor and Resilen-200 for use in products designed to combat premature wrinkling and aging of skin. The measurable improvements in skin found in these studies are attributed not only to superior moisture retention, but also to the physiological activities of L-hydroxyproline, including stimulation of collagen synthesis by fibroblast cells; stabilization of collagen molecules; and normalization of epidermal cells by improving the health of the basal layer.

Combining these ingredients with vitamins A, B5, C and E, and other antioxidants, in conjunction with topical use of UV filters, can create synergy and further increase the efficacy of cosmetics and skin care products. When an effective topical skin care regimen is supported by dietary supplementation, even greater improvements in skin moisture content and a decrease in fine lines and wrinkles can be achieved. In light of these new studies, a combination of topical application of Lumistor and Resilen-200, plus oral supplementation of Lumistor, is recommended for optimal efficacy. 

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