Sirtuin is a class of protein enzymes with potential antiaging effects in humans. The name comes from silent information regulator-2, or Sir2, the gene responsible for cellular regulation in yeast.1 Sirtuins are known to affect cellular metabolism in plants and animals, and therefore may play a role in manifestations of aging. But there are questions: Which particular sirtuin? What is the mechanism? How can humans intervene to regulate that mechanism externally? The answers would have great appeal to cosmetics manufacturers and users.
One source1 describes sirtuin as NAD-dependent histone deacetylases, which means they remove acetyl groups from histone tails, causing the histones to wrap more tightly around the DNA and interfere with the transcription of genes by blocking access by transcription factors. The overall result of histone deacetylation is a global (nonspecific) reduction in gene expression, the process by which the DNA sequence and other inheritable information in a gene is made into a protein or other functional gene product. A sirtuin is one of those proteins.
Normally, sirtuin activity in humans is inhibited when nicotinamide binds to a specific receptor site—thus the dependency of sirtuins on NAD or nicotinamide adenine dinucleotide.2 Drugs that interfere with this binding should increase sirtuin activity; also, the resveratrol found in red wine has been shown to inhibit this binding activity. Nicotinamide, the amide of niacin or vitamin B3, is used to treat arthritis. It also has demonstrated anti-inflammatory actions that may be of benefit in patients with inflammatory acne vulgaris. Nicotinamide, therefore, would appear to play a positive role in inhibiting inflammation but a negative role in aging. Are there other ways to stimulate sirtuins?
Research is ongoing in this area, but a few approaches have been documented in the search for methods to stimulate sirtuins.
Caloric restriction: Starting in the 1990s, MIT’s Leonard Guarente and Harvard’s David Sinclair have investigated the ways in which a healthful diet low in calories may prolong both health and lifespan in yeast. Both agree on the importance of the central role played by Sir2, the gene responsible for cellular regulation in yeast, but they disagreed about the mechanism, and now agree that both mechanisms may play a role.
Guarente and Sinclair each started separate companies to develop life- and health-extending drugs for humans using the sirtuin enzymes made by the seven SIRT genes that are the human counterparts of Sir2. In November 2007, Guarente completed a break with his company, Elixir Pharmaceuticals, and joined Sinclair’s company, Sirtris Pharmaceuticals, as co-chair of its scientific advisory board.
Also in November, Sirtris announced results of tests on some 500,000 small molecules that have the same sirtuin-stimulating effect as resveratrol. The tests identified a handful that are 1,000 times more potent than resveratrol. These drug compounds helped reverse diabetes symptoms and reduce insulin sensitivity in two different studies in diabetic mice and one in rats. Human tests are planned.
Activating compounds: US patent application 20060084135 by Howitz and Zipkin3 discloses compositions for manipulating the lifespan and stress response of cells and organisms by contacting the cell or organism with an “activating compound” that modulates the activity of the human SIRT1 protein or the yeast Sir2 protein. The activating compound may be a polyphenol compound, such as a plant polyphenol or an analog or derivative thereof. Exemplary compounds are selected from the group consisting of flavones, stilbenes, flavanones, isoflavones, catechins, chalcones, tannins and anthocyanidins. For example, compounds are selected from the group of resveratrol, butein, piceatannol, isoliquiritgenin, fisetin, luteolin, 3,6,3’,4’-tetrahydroxyflavone and quercetin.
Some of the activating compounds are effective at extending the lifespan of eukaryotic cells, i.e., cells with nuclei, such as all cells in humans and other mammals. By increasing the cell’s resistance to stress, these compounds—called “lifespan extending compounds”—extend the cell’s lifespan. The compound may be selected from the group consisting of resveratrol, butein and fisetin and analogs and derivatives thereof. The method calls for contacting the cell with one of those compounds at a concentration of approximately 10–100 μM. The cell may be in vitro or in vivo. If the cell is in a subject, the method may require administering the compound to the subject.
Exemplary lifespan extending compounds include polyphenol compounds and compounds of the stilbene, flavone and chalcone families, with which cells can be treated in vivo to increase their lifespan or prevent apoptosis. For example, skin can be protected from aging, e.g., developing wrinkles, by treating skin with a pharmaceutical or cosmetic composition comprising one of the activating compounds. Applicable skin afflictions or skin conditions include disorders or diseases associated with or caused by inflammation, sun damage or natural aging. The formulations may be administered topically, to the skin or mucosal tissue, as an ointment, lotion, cream, microemulsion, gel, solution or the like, within the context of a dosing regimen effective to bring about the desired result. A dose of active agent would typically be in the range of 0.075–0.5 μM per kg per day.
Curiously, Sinclair published a remarkably similar patent application4 one year earlier.
Resveratrol and stilbenes: Of current research interest is substances that mimic the conditions of calorie restriction (CR) at normal levels of nutritional intake, but which trigger the same molecular biological mechanisms. One of the most promising CR mimetics is resveratrol, a secondary plant substance found primarily in red wine. The group led by David Sinclair has been able to demonstrate that the same longevity promoting effect is found in lower organisms with resveratol as with calorie restriction. In animal studies, therefore, resveratrol is the only substance so far shown to achieve lengthening of the lifespan.5
The polyphenol resveratrol (3,5, 4’-trihydroxy-trans-stilbene) is already well-known to the cosmetics industry as an antioxidant and tyrosinase inhibitor. As a stilbene phytoalexin, it continues to receive increasing attention for its role in mitigation of numerous and diverse human pathological processes including inflammation, atherosclerosis and carcinogenesis.
Now, in WO/2005/069998, Merritt Andrus and Jing Liu identify resveratrol as the most potent activator of sirtuins.6 Potent, but not optimal, according to these inventors, because it is difficult to isolate stereo-selective extracts from plant sources. More significantly, resveratrol is highly unstable due to its potential for oxidation, resulting in the formation of unstable radicals and quinones, and requiring that the final isolation of the product be carried out from impure mixtures containing multiple components. Therefore, Andrus and Liu have devised a novel and efficient route to synthetic resveratrol and novel derivative compounds of resveratrol having significantly improved activity and stability. Furthermore, their invention provides a novel and efficient route to synthetic stilbene compounds. These compounds have significantly improved biological activity and can be stored for a long period without alteration. Applications in the food, cosmetic and pharmaceutical industries are described.
High-density grapes: In US patent application 20060078530, Kay Miyakawa Liu reports the use of resveratrol-containing extracts from grape botanicals to stimulate sirtuins.7
The extracts are obtained from the normal steps of producing red wine. The skins and juices from the grape botanicals are stored several weeks for fermentation and then sanitized in an extraction process that dilutes the water and alcohol from the juices. The remaining portion from the sanitation process, which is mostly sediment, is then mixed with isoprene glycol and nonionic water is re-inserted. Water and any remaining alcohol are again diluted from the solution, and the remaining portion is allowed to sit until dry. Once dry, the mixture is granulated several times into a fine powder called resveratrol amalgam.
The resveratrol amalgam is further combined with various vitamins and minerals known in the art of cosmetology to rejuvenate and protect the skin. Examples include soy and vitamins C and K. This chemical system is delivered to the layers of the skin by use of an ultrasonic device or any other application method known in the art. Upon delivery of the chemical system past the dermal layers, the system works with the metabolism to regenerate the skin.
According to Liu, combining the resveratrol amalgam with the various specified ingredients achieves significant effects such as the following: inhibits fatty acid oxidation and helps to keep cell membranes in good condition; restores venous tone and helps palliate “heaviness of the legs;” stimulates collagen and elastin synthesis in the skin thereby keeping it firm and elastic; inhibits the activity of various enzymes involved in inflammation, thereby soothing the skin and combating the aging process; reduces melanin synthesis and inhibits tyrosinase activity, thereby lightening the skin, restoring the brightness of the complexion and preventing the appearance of pigmented spots; and inhibits glycosylation processes, thereby cutting down the cross-linking of collagen fibers that is responsible for so many of the phenomena associated with the aging of the skin.
Yeast Kluyveromyces biopetides: Researchers at LVMH Recherche–Parfums Christian Dior in France investigated the role of yeast Kluyveromyces biopetides in stimulating the expression of sirtuin in human cutaneous cells and the benefit for the skin of an active skin care product containing those biopetides,8 as reported in this abstract.
SIRT1 was investigated by immunostaining, Western blotting, and cytometry on normal human skin cells in culture and on healthy skin samples ex vivo. Cellular integrity and aging was followed by comet assays measuring DNA fragmentation and β-galactosidase activity (a marker of senescence). The test product was yeast Kluyveromyces biopeptides. Thirty-three female subjects aged 37 to 64 years (mean 51.6 years)
enrolled in the study. Subjects applied a formulation enriched with 1% of the yeast biopeptides SIRT1 activator once daily to the face and neck for four weeks. Dermatologists used a graded scale (1–9) to score fine lines and wrinkles, hydration, pigment color intensity, complexion radiance, skin density, firmness, complexion homogeneity, and texture of the skin before and after the first application and again after four weeks of use. A Pixel Skin method, based on an analysis of the gray-level variance and surface of imperfections (age-related parameters) from numerical pictures of the faces, was used to objectively measure the skin care efficacy.
The yeast Kluyveromyces biopeptides significantly increased SIRT1 expression in normal human dermal skin fibroblasts in vitro (+172%) and in epidermal cells of healthy human skin ex vivo. The biopeptides also decreased cell senescence and DNA fragmentation induced by UVB stress. At the end of the study, facial improvements could be seen on fine lines and wrinkles, hydration, pigmented spot color intensity, complexion radiance, firmness, complexion homogeneity and texture. Improvement in hydration was significant immediately after the first application. Skin-pixel measurement and analysis showed a significant reduction of the gray variance linked to pixel heterogeneity (–4.2%) and a significant reduction of the surface of skin imperfections (–30.4%). All the indicators from clinical evaluation to the objective measurements of the skin showed a significant improvement of the aged skin, the researchers reported.
These results demonstrate the efficacy of the yeast Kluyveromyces biopeptides in activating SIRT1 of human skin cells, improving their DNA resistance and senescence, and of a formulation enriched in this ingredient in treating multiple skin aging signs, according to these researchers.
Sirtuin Activators in Cosmetics
In December 2007, when this article was written, a search at the US Patent Office Web site did not locate any patents addressing sirtuins in cosmetics. Among patent applications, there were fewer than two dozen with the word sirtuin in the abstract, and they are aimed principally at pharmacologic uses.
Elsewhere in this magazine, Isabelle Imbert and colleagues at Vincience review the scientific evidence confirming the presence of sirtuins in the skin, though the article provides few details on sirtuin stimulators in cosmetic products.
In the market place, the concept of sirtuins has apparently not yet appealed to consumers. However, product developers and marketers are working on this. Already a year ago, Actichem and Jan Dekker were promoting Resveratroxa, their sirtuins activator for a deep wrinkles treatment. Resveratrox is obtained from Vitis vinifera cultivated in the surroundings of Bordeaux, France. The extract is concentrated in resveratrol monomers and oligomers, described in company literature as “unique factors of DNA integrity protection.” SIRT1 activation was demonstrated in aged fibroblasts in culture and in keratinocytes in reconstructed epidermis. In addition, clinical measurements showed a reduction of deep wrinkles for 80% of the panelists.
Avon and Estee Lauder are among the finished goods manufacturers with recently announced sirtuin-stimulating products.
Pro-Sirtuin TX: By March of this year, Avon will have launched the first three products in its Anew Ultimate Age Repair line of formulations (Figure 1)
designed with patent-pending Pro-Sirtuin TX to boost these critical skin proteins.
This antiaging breakthrough reportedly was seven years in the making. Other attempts at skin rejuvenation and antiaging formulations were also reported during those years at Avon, though none of these US patent applications claims sirtuin stimulation as its method of action:
• 20030091665 discloses topical compositions for skin rejuvenation. One composition is a blend of neem seed cell broth and one or more additional botanical ingredients. Another composition has pomegranate fruit extract and, optionally, one or more additional botanical ingredients.
• 20030118617 relates to reducing, preventing, ameliorating, and/or inhibiting the cosmetic signs of dermatological aging via topically applied compositions containing at least one resveratrol analogue.
• 20040091509 discloses skin treatment compositions containing cell signaling compounds, which induce and promote the biosynthesis and/or bioactivity of endogenous chemicals that mediate cell-to-cell communication in the skin between keratinocytes, fibroblasts and other cell types present in the skin. The cell signaling compound is selected from the group consisting of: andrographolide and its derivatives; adenosine cyclic phosphate and its derivatives; hydrolyzed milk proteins; sunflower seed extract; plankton extract; phytol and its derivatives; and mixtures thereof.
• 20050048140 discloses topical compositions for alleviation of skin irritation symptoms or conditions having at least one plant extract effective to inhibit COX-2 enzyme, NGF protein, and/or TNF-alpha protein activity. Preferably, the compositions have a cosmetically, dermatologically, or pharmaceutically acceptable vehicle. In addition to the enzyme inhibitory plant material and acceptable vehicle, compositions may also have at least one active ingredient known to produce skin irritation. There is also a method for treatment of skin irritation symptoms or conditions involving topically applying compositions of the invention. Also disclosed are compositions and methods for topical administration of compositions to the skin that improve the aesthetic appearance of skin and/or provide an antiaging benefit to the skin.
• 20050153003 describes topical applications of crape myrtle extracts to prevent and/or ameliorate the effects of aging on skin or hair.
The exclusive Pro-Sirtuin TX technology was developed by Avon’s global research and development team. As part of its strategic alliance program, Avon’s scientists collaborated with leading European biotechnology companies to create three proprietary “designer” peptides, each with a unique structural sequence, to actively target the production of the skin’s sirtuin protein. Patent applications related to the Pro-Sirtuin TX technology have been filed globally.
A night cream and an elixir using this Pro-Sirtuin TX technology are already on the market. A day cream with SPF is scheduled for launch in March along with an eye cream/elixir, called Anew Ultimate Contouring Eye System.
Resveratrate: Billed as a new “youth molecule,” Resveratrate is the key ingredient in Re-Nutriv Ultimate Youth Creme, which launches this month as the latest addition to Estée Lauder’s Re-Nutriv line.
Resveratrol has been used in earlier Re-Nutriv products, such as the Ultimate Lifting Eye Creme. But the new Resveratrate molecule is able to penetrate into the skin more deeply than resveratrol, creating a “reservoir” for the skin to draw upon throughout the day, according to the company. Available documents do not explain how the difference is achieved, except to cite Resveratrate’s biomimetic (skin-friendly) quality.
The use of sirtuin activators in cosmetic products would seem to face the same challenges encountered by many of the new bioactive ingredients of interest.
• Their research and development is primarily in the pharmaceutical industry. Most of the patent filings are in that area. The two competing companies in this field are drug companies. Cosmetic claims would have to be carefully worded.
• The human system is complicated. Whereas Guarente and Sinclair have established that they can stimulate the gene that controls longevity in yeast, researchers are having a more difficult time with the human system, in which at least seven genes affect aging. In fact, Guarente says that both activation and inhibition of SIRT1 could be useful, if during caloric restriction the gene’s activity goes up in some tissues and down in others.9
• Cosmetic formulations are complicated, and they may have unexpected effects in the presence of sirtuin activators. For example, researchers have learned that dihydrocoumarin, a flavoring compound commonly added to food and cosmetics, inhibits the activity of both the yeast sirtuin and the human SIRT1.10 What other unexpected effects will formulators have to deal with?
The antiaging appeal of sirtuin activators is strong. In the world of the sirtuin, that much is certain..
1. http://en.wikipedia.org/wiki/Sirtuin (Accessed Nov 19, 2007)
2. http://en.wikipedia.org/wiki/Nicotinamide (Accessed Nov 19, 2007)
3. US Pat Applic 20060084135, Compositions for manipulating the lifespan and stress response of cells and organisms, KT Howitz and RE Zipkin (Apr 20, 2006)
4. US Pat Applic 20050096256, Compositions for manipulating the lifespan and stress response of cells and organisms, DA Sinclair (May 5, 2005)
5. B Kleine-Gunk, Anti-aging medicine—Hope or nonsense?, Dtsch Arztebl 104(28–29) A 2054–2060 (2007)
6. WO/2005/069998, Novel sirtuin activating compounds and methods for making the same, MB Andrus and J Liu (Aug 4, 2005)
7. US Pat Applic 20060078530, Using high-density grapes to rejuvenate human skin, KM Liu (Apr 13, 2006)
8. M Moreau et al, Enhancing cell longevity for cosmetic application: a complementary approach, J Drugs Dermatol 6(6 Suppl) s14–19 (2007)
9. N Wade, The quest for a way around aging, International Herald Tribune (Nov 8, 2006)
10. AJ Olaharski et al, The flavoring agent dihydrocoumarin reverses epigenetic silencing and inhibits sirtuin deacetylases, PLoS Genet 1(6) e77 (2005)