Skin aging and oxidative stress: Equol’s anti-aging effects via biochemical and molecular mechanisms

Accepted Manuscript Title: Skin Aging and Oxidative Stress: Equol’s Anti-Aging Effects via Biochemical and Molecular Mechanisms Author: Edwin D. Lephart PII: DOI: Reference:

S1568-1637(16)30109-X http://dx.doi.org/doi:10.1016/j.arr.2016.08.001 ARR 693

To appear in:

Ageing Research Reviews

Received date: Revised date: Accepted date:

2-6-2016 29-7-2016 4-8-2016

Please cite this article as: Lephart, Edwin D., Skin Aging and Oxidative Stress: Equol’s Anti-Aging Effects via Biochemical and Molecular Mechanisms.Ageing Research Reviews http://dx.doi.org/10.1016/j.arr.2016.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Skin Aging and Oxidative Stress: Equol‟s Anti-Aging Effects via Biochemical and Molecular Mechanisms

Edwin D. Lephart Department of Physiology and Developmental Biology and The Neuroscience Center Brigham Young University Provo, Utah, 84602, USA

Corresponding author: Edwin D. Lephart Department of Physiology and Developmental Biology and The Neuroscience Center LS 4005 College of Life Sciences Brigham Young University Provo, Utah USA 84602 Tel: 801 422-2006 FAX: 801 422 0700 Cell: 801 319 8173 Email: [email protected]

1

Highlights Reactive oxygen species (ROS) in skin are generated by exposure to solar UV radiation Skin‟s chronological & photo-aging occur via ROS by cascade signaling pathways Dermal aging is also influenced by steroid hormones (receptors) and genetic factors The botanical equol combats skin aging by reducing ROS events/biomarkers Equol acts through both its antioxidant and phytoestrogenic properties to reduce skin aging.

Abstract Oxygen in biology is essential for life. It comes at a cost during normal cellular function, where reactive oxygen species (ROS) are generated by oxidative metabolism. Human skin exposed to solar ultra-violet radiation (UVR) dramatically increases ROS production/oxidative stress. It is important to understand the characteristics of human skin and how chronological (intrinsic) aging and photo-aging (extrinsic aging) occur via the impact of ROS production by cascade signaling pathways. The goal is to oppose or neutralize ROS insults to maintain good dermal health. Botanicals, as active ingredients, represent one of the largest categories used in dermatology and cosmeceuticals to combat skin aging. An emerging botanical is equol, a polyphenolic/isoflavonoid molecule found in plants and food products and via gastrointestinal metabolism from precursor compounds. Introductory sections cover oxygen, free radicals (ROS), oxidative stress, antioxidants, human skin aging, cellular/molecular ROS events in skin, steroid enzymes/receptors/hormonal actions and genetic factors in aging skin. The main focus of this review covers the characteristics of equol (phytoestrogenic, antioxidant and enhancement of extracellular matrix properties) to reduce skin aging along with its anti-aging skin influences via

2

reducing oxidative stress cascade events by a variety of biochemical/molecular actions and mechanisms to enhance human dermal health. Keywords: Free Radicals; Oxidative Stress; Antioxidant; Anti-aging; Equol; Human Skin Highlights Reactive oxygen species (ROS) in skin are generated by exposure to solar UV radiation Skin‟s chronological & photo-aging occur via ROS by cascade signaling pathways Dermal aging is also influenced by steroid hormones (receptors) and genetic factors The botanical equol combats skin aging by reducing ROS events/biomarkers Equol acts through both its antioxidant and phytoestrogenic properties to reduce skin aging. 1. Introduction Oxidative stress plays an important role in human skin aging and dermal damage (Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). The mechanisms of intrinsic (chronological) and extrinsic (photo) aging include the generation of reactive oxygen species (ROS) via oxidative metabolism and exposure to sun ultra-violet (UV) light, respectively (Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014). Photo-aging is dependent upon the exposure to and intensity of solar UV radiation (UVR) (Gonzaga, 2009; Kammeyer and Luiten, 2015). Oxidant events and molecular mechanisms of skin aging involve damage to DNA, the inflammatory response, reduced production of antioxidants and the generation of matrix metalloproteinases (MMPs) that degrade collagen and elastin in the dermal skin layer (Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). All of these events lead to damaged skin and reflect the aging process: the disruption of the extra cellular dermal matrix, the loss of tensile strength and elasticity, impaired wound healing, the

3

appearance of wrinkles, age-spots and loss of skin tone. The goal is to delay aging onset and/or slow down the structural and visual appearance of skin aging with time. Because phytochemicals are known to be constituents of animal and human food sources, botanicals with polyphenolic structures have received increased research study due to their potential significance and application in treating human cancers and other age-related diseases including skin aging (Adlercreutz, et al., 2004; Evans and Johnson, 2010; Lephart et al., 2014; Mazur and Adlercreutz, 2000; Park and Pezzuto, 2015; Wang et al., 2014). Isoflavonoid (plantderived) molecules that fall under the polyphenolic umbrella have been shown to decrease oxidative stress (Bansal and Parle, 2010; Djuric et al., 2001; Yoon and Park, 2014). One of the important emerging isoflavonoid molecules is equol, which can decrease oxidative stress (Ma et al., 2010; Zhang et al., 2013). In skin, equol has been shown to improve dermal health by direct and downstream influences at several different steps of the oxidative stress cascade, while at the same time inhibit MMP‟s actions and simultaneously stimulate collagen and elastin (Gopaul et al., 2012; Lephart, 2013a). Finally, equol has two primary properties: 1) an inherent antioxidative capacity resulting from its polyphenolic molecular structure and 2) its well-known phytoestrogenic activity that enables it to reduce skin aging (Gopaul et al., 2012; Lephart, 2013a) In summary, the general concepts and principles of oxygen in biology (pros and cons), free radicals (ROS), oxidative stress, antioxidants, human skin [intrinsic and extrinsic (photo)] aging and, cellular and molecular ROS events are covered first. Also, steroid enzymes/receptors and hormonal actions are covered along with the influence of genetic factors in skin aging. Then, the main sections of this review include the characteristics of equol and its anti-aging influence on human skin via reducing oxidative stress cascade events by a variety of biochemical and molecular actions/mechanisms. Each section is self-contained with brief background

4

information for each topic followed by examples/figures showing human applications and/or analysis for the cited studies presented.

1.1 Oxygen in Biology (Pros and Cons) The respiration system in humans provides the breath of life that is dependent upon oxygen. The oxygen from the atmosphere transferred into blood is utilized to metabolize dietary nutrients (e.g., fats, proteins & carbohydrates) in order to produce energy. In cellular aerobic metabolic respiration, glucose is typically the molecule that is utilized to generate energy-rich ATP molecules within the mitochondria of the cell to maintain homeostasis (Lephart, 2009; Martini, 2004). While biological systems are dependent upon atmospheric oxygen, too little or too much oxygen can be damaging to cells and tissues (Lephart, 2009; Martini, 2004). In this regard, oxygen is often referred to as a Janus gas that has both positive benefits and potentially damaging side-effects in biological systems (Burton and Jauniaux, 2011). Oxygen participates in high-energy electron transfers that supports the generation of abundant essential quantities of ATP molecules through oxidative metabolism (Lephart, 2009; Martini, 2004). However, biological systems and the numerous chemical reactions within cells are not machines, and errors occur resulting in the generation of very harmful molecules or particles that are associated with oxygen such as free radicals. 1.2 Free Radicals (ROS) While oxygen is essential for life, its benefits have a cost in normal cellular function. As discussed by Halliwell and Gutteridge (1999), unless electrons are transferred to oxygen during aerobic metabolism in the correct manner, potentially oxygen free radicals can be formed easily. Occasionally electrons “escape,” and instead of completing the cellular respiration cycle, oxygen 5

may become toxic and mutagenic (Halliwell and Gutteridge, 1999). For example, a single oxygen atom is unstable and wants to bind a twin atom, forming molecular oxygen (O2). However, when this is not possible, the stability of this bond is compromised because only one pair of electrons is shared and two unpaired electrons remain (Fig. 1). This complex represents a superoxide (O2−) and hydroxyl radical. These are often referred to collectively as reactive oxygen species (ROS) due to the presence of an unpaired electron, which makes them highly reactive (Buonocore et al., 2010; Halliwell and Gutteridge, 1999) and capable of chain reactions, which form another free radical at each step (Buonocore et al., 2010; Halliwell and Gutteridge, 1999; Kohen and Nyska, 2002). The overproduction of ROS can have deleterious effects on cell structures, including damage to membranes, lipids, proteins, RNA and DNA (Buonocore et al., 2010, Gonzaga, 2009; Halliwell and Gutteridge, 1999; Kammeyer and Luiten, 2015; Kohen and Nyska, 2002; Natarajan et al., 2014; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). In this regard, ROS production leads to the mechanisms of oxidative stress in living systems that account for a majority of disorders, diseases and death worldwide. Finally, ROS are well known to be involved in human cutaneous aging, the dermal inflammatory response and skin cancers (Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). 1.3 Oxidative Stress Kohen and Nyska (2002) noted that ROS are involved in a variety of biological phenomena, such as mutation, carcinogenesis, degeneration and other disorders and disease states involving inflammation and aging. All major organ systems in humans including skin, nervous, cardiovascular, respiratory, kidney, skeletal and immune, etc., along with other multiple organs

6

disorders such as diabetes, sepsis, depression and trauma are impacted by oxidative stress (BarOr et al., 2015; Kohen and Nyska, 2002; Maritim et al., 2003; Maes et al., 2011). Thus, based upon numerous journal reports, ROS generation represents the root basis of oxidative stress that leads to oxidative damage in biological structures and molecules (Bar-Or et al., 2015; Gonzaga, 2009; Kammeyer and Luiten, 2015; Maes et al., 2011; Maritim et al., 2003; Natarajan et al., 2014; Rittie and Fischer, 2002; Valko et al., 2007; Zouboulis and Makrantonaki, 2011). Oxidative stress may be defined as the imbalance between the production of free radicals and the ability of the body to counteract or detoxify their harmful effects through neutralization by antioxidants. Since free radical (ROS) development is unavoidable, the human body has adapted by establishing and maintaining defense mechanisms that reduce their impact, particularly in human skin (Kammeyer and Luiten, 2015; Natarajan et al., 2014; Shindo et al., 1994; Zouboulis and Makrantonaki, 2011). 1.4 Antioxidants The body‟s two major defense systems are free radical detoxifying enzymes and antioxidant molecules (Krishnamurthy and Wadhwani, 2012; Martini, 2004). In brief, the free radical detoxifying enzyme systems include: superoxide dismutase (SOD), catalase and glutathione peroxidases. SOD is an enzyme that catalyzes the partitioning of the superoxide (●O2−) into either molecular oxygen (O2) or hydrogen peroxide (H2O2) (Fig. 1) (Krishnamurthy and Wadhwani, 2012). Catalase is another important detoxifying enzyme that converts hydrogen peroxide to water and molecular oxygen; it is also inducible and thus completes the action of SOD (Krishnamurthy and Wadhwani, 2012) (Fig. 1). Glutathione peroxidase is similar to catalase and represents a family of enzymes that covert hydrogen peroxide into water and oxygen

7

(Krishnamurthy and Wadhwani, 2012). Notably, SOD, catalase and glutathione peroxidase are important antioxidant enzymes in human skin (Shindo et al., 1994). An antioxidant is any molecule that can block free radicals and/or ROS from stealing electrons from other atoms (Krishnamurthy and Wadhwani, 2012; Martini, 2004). Or, in other words, an antioxidant is any substance that inhibits the damage due to oxygen (oxidation) that is caused by free radicals or reactions promoted by ROS. Antioxidants act at intra- and extracellular locations (Ames et al., 1981; Becker, 1993; Krishnamurthy and Wadhwani, 2012; Martini, 2004). Glutathione and uric acid are two molecules the body can synthesize that are endogenous sources of antioxidants (Krishnamurthy and Wadhwani, 2012). Both glutathione and uric acid are important antioxidants present in human skin (Shindo et al., 1994). Many different antioxidants are obtained from dietary or exogenous sources. The most commonly known antioxidants include, vitamins A, C and E (Krishnamurthy and Wadhwani, 2012; Martini, 2004). However, other dietary sources of antioxidants are carotenoids, lipoic acid and phenolic compounds found in abundance in many plant products (Krishnamurthy and Wadhwani, 2012; Shindo et al., 1994). Many of these dietary antioxidants are incorporated into the epidermal and dermal layers of human skin (Shindo et al., 1994). Finally, exposure to solar UVR, heavy metals or other toxic agents are known to increase free radical or ROS formation along with the inescapable generation of ROS by normal metabolic oxidation associated with mitochrondrial function (Tulah and Birch-Machin, 2013). Thus, a healthy life free of disease becomes a matter of balance; biological systems must have enough antioxidants available that are ready to “neutralize” various free radicals or ROS the body is either exposed to or that it produces. If an imbalance occurs, then oxidative stress will result and homeostasis will not be maintained.

8

2. Human Skin Characteristics Human skin is the largest and most complex organ (representing one sixth of the total body weight) functioning as a physical barrier to protect the body from water loss as well as environmental insults such as pathogens, chemicals, physical agents and solar UVR throughout life (Madison, 2003; Qunshan and Nash, 2010; Shindo et al., 1994). Moreover, the skin provides essential physiological functions including immune defense, thermoregulation, sensory input from mechanoreceptors and endocrine and metabolic mechanisms to sustain optimal health (Lephart, 2009; Martini, 2004; Qunshan and Nash, 2010). Finally, and most importantly, the antioxidant defense capacity of the skin would be expected to be greater than that of internal organs due to its protective structural and biological functioning of the dermal layers (Shindo et al., 1994). The skin is divided into three distinct layers: 1) the epidermis; 2) the dermis; and 3) the hypodermis or subcutaneous tissue (Lephart, 2009; Madison, 2003; Martini, 2004; Qunshan and Nash, 2010). See Fig. 2 for a basic graphic representation of human skin layers. However, in brief, only the epidermis and dermis will be covered due to their structural/functional components in reference to skin characteristics and aging. The epidermis is the major protective outer layer. The stratum corneum (10-30 microns) is the outermost layer of progressively dying and flattened dead cells or corneocytes. The „bricks and mortar‟ cellular structure is composed of the live keratinocyte layers, which form the majority of the epidermal layer (100-150 microns) and its active functional aspects (Farage et al., 2010; Madison, 2003; Menon, 2002; Qunshan and Nash, 2010; Shindo et al., 1994). Melanocytes near the epidermal-dermal junction provide pigmentation of the skin for photoprotection (Natarajan et al., 2014) (Fig. 2).

9

Conspicuously, the epidermis, composed mainly of keratinocytes, has a greater abundance of the antioxidant enzymes such as SOD (125 %), catalase (720 %) and glutathione peroxidase (60 %) compared to the dermal layer (Shindo et al., 1994). Also, seasonal variations (summer vs. winter) have been reported for catalase, while SOD enzyme activity remains stable, when human skin is exposed to UV light (Hellemans et al., 2003). Additionally, the biosynthesized antioxidants glutathione and uric acid, along with the dietary-derived antioxidants, vitamin D and vitamin E (tocopherols), are present at much higher concentrations in the epidermis compared to the dermal layer (Shindo et al., 1994). Thus, the epidermal layer contains the highest concentration of antioxidants and is the major line of antioxidant defense in skin (Natarajan et al., 2014; Seo and Chung, 2006; Shindo et al., 1994). While the epidermis provides the first line of defense, the dermis bestows the scaffolding or structural fibers of the skin. In general, the dermis is a dense and irregular layer of connective tissue, 2 to 3 mm thick, that comprises most of the skin thickness (Brincat et al., 2005. It contains mechanosensory receptors, sweat and oil (sebaceous) glands, and its primary waterholding components i.e., hyaluronic acid (responsible for normal turgor) and supportive glycosaminoglycans that maintain optimal skin health (Farage et al., 2010; Lephart, 2009; Martini, 2004). It is composed mainly of extracellular matrix proteins (such as collagen, elastin, etc.) that are secreted by fibroblasts and provide dermal strength and flexibility (Farage et al., 2010; Lephart, 2009; Martini, 2004; Qunshan and Nash, 2010). The elastic fiber network is responsible for recoil and elasticity of the skin, but it also plays a role in tissue repair (Lephart, 2009; Pierard et al., 2010; Mera et al., 1987) (Fig. 2). The dermal layer also contains other molecules such as tissue inhibitor of matrix metalloproteinases (TIMP), metalloproteinases (MMPs), elastase and many other molecules that maintain skin health (Farage et al., 2010).

10

3. Aging of Human Skin Aging is accompanied by the progressive loss of anatomical structure and physiological function in multiple organs. In 2050, the U. S. population aged 65 and over is projected to be 83.7 million, almost double its estimated population of 43.1 million in 2012 (Ortman et al., 2014). In Western countries it is estimated that women will spend more than one-third of their lifetimes in post menopause and more than 40 million postmenopausal women live in the U.S. today composing approximately 18 % of the total population (Brincat et al., 2005; Farage et al., 2008). As human beings age, the skin thins, dries, wrinkles, becomes pigmented unevenly with age or liver spots (solar lentigines), and wound healing is delayed (Friedman, 2005; Pierard et al., 2010). Specifically, the appearance of wrinkles around the eyes and mouth, and frown lines along the forehead are seen with uneven skin color and a general loss of skin tone (pale appearance). Sagging skin and thin skin are due to the loss of and definition/abundance of the underlying collagen and especially the elastin fibers in the dermal layer that provide full, robust and the elastic recoil properties of youthful skin (Duncan and Leffell, 1997). Skin collagen and elastin peaks around 30 years of age (Burns and Breathnach, 2004; Fazio et al., 1988; Freedberg et al., 2003; Khol et al., 2011; Seite et al., 2006), which corresponds with the peak of estrogen production around 25-30 years of age (Baumann, 2002; Jain et al., 2003; Schwartz and Mayaux, 1982) (Fig. 3). Reported features of aged human skin include fragmentation of collagen fibers by the action of the enzyme MMP-1 and increased mitochondrial ROS production and oxidative stress resulting in common deletions of mitochondrial DNA (Ashworth et al., 1999; Qin et al., 2014; Quan et al., 2015). Also, the importance of SOD in skin aging has been shown in SOD-deficient mice where epidermal thinning was induced by DNA breaks causing cellular senescence (Velarde et al., 2012).

11

The first signs of skin aging occur around age 30 and especially after menopause, where there is skin dryness, decreased skin firmness and loss of elasticity (Burns and Breathnach, 2004; Escoffier, 1989; Fazio et al., 1988; Freedberg et al., 2003; Hillebrand, 2010; Khol et al., 2011; Seite et al., 2006; Uitto, 2008). In fact, elasticity has been shown (via mechanical depression) to be highest in young (10-year-old) subjects with a continual gradual decline with chronological aging (Uitto, 2008) (Fig. 3). While skin aging first appears around 30 years of age, an important study has clearly demonstrated that the age-related loss in skin elasticity actually precedes the formation of visible wrinkles (Fujimura et al., 2007). Behind collagen, the importance of elastin is paramount, since loss of elastin can result in rapid skin aging (Farage et al., 2010; Qunshan and Nash, 2010). As discussed by Anderson (2012) when a 21-year-old women experienced an allergic food reaction, and all elastin was selectively destroyed that resulted in rapid skin damage and aging over a period of a few years (Fig. 4). Remarkably, as discussed by Anderson, there is not a clear line of distinction between medical procedures in dermatology and cosmetic applications to assist patients to feel better by having a positive perception about themselves (Anderson, 2012; Kligman and Koblenzer, 1997). For example, whether a person goes through dermal trauma from war or accidental means compared to individuals experiencing chronological skin aging, the impact on patients in regard to positive self-perception is paramount especially if the events/changes occur in highly visible body areas such as the face, neck, hands and arm regions (Anderson 2012; Kligman and Koblenzer, 1997). 3.1 Intrinsic (Chronological) and Extrinsic (Photo) Aging Skin aging can be classified by intrinsic and extrinsic mechanisms (Gonzaga, 2009; Farage et al., 2010; Farage et al., 2008; Kammeyer and Luiten, 2015; Khol et al., 2011; Zouboulis and

12

Makrantonaki, 2011). Intrinsic or chronological aging is an unavoidable phenomenon that includes several factors such as genetics, metabolism and the passage of time, where the “repair process” may become defective (Anderson et al., 2014; Meadows et al., 2014; Quan et al., 2015; Tulah and Birch-Machin, 2013; Velarde et al., 2012). The repair process is an important component with aging, since DNA and their gene products (i.e., proteins) play such an essential role in optimal dermal health. The generation of mitochondrial ROS by oxidative metabolism represents the inescapable production of free radicals by aerobic cellular chemical reactions in chronological aging leading to mitochondrial damage, altered cell function and, if the insult is too great, the affected cells may die (Anderson et al., 2014; Meadows et al., 2014; Quan et al., 2015; Tulah and Birch-Machin, 2013; Velarde et al., 2012). If damaged skin cells are not repaired, mutations occur that result in premature aging (Qin et al., 2014; Quan et al., 2015). On the other hand, extrinsic aging is a phenomenon that can be avoided to some extent. Extrinsic aging is caused by environmental exposure, primarily solar UV radiation (UVR) or ultra violet (UV) light from artificial tanning sources. This is more commonly termed photoaging. Photo-aging is the effect of long-term UV exposure and the resulting solar damage superimposed on intrinsically aged skin (Gonzaga, 2009; Kammeyer and Luiten, 2015; Zouboulis and Makrantonaki, 2014). It is thought that up to 80-to-90 % of skin aging is due to the deleterious effects of the sun or photo-aging (Gonzaga, 2009; Hillebrand, 2010; Kammeyer and Luiten, 2005; Khol et al., 2011; Natarajan et al., 2014). There are different types of solar UV light rays (UVC, UVB and UVA) pertaining to human skin photo-aging. UVC rays are blocked by the ozone layer and do not reach the surface of the earth (Gonzaga, 2009; Walker et al., 2003). UVA and UVB make up 95-98 % and 2-5 %, respectively, of the UV radiation reaching human skin (Walker et al., 2003). However, the

13

precise amount and type of UVR (especially UVB) exposure depends on a number of factors (solar zenith angle, latitude; stratospheric ozone levels, and pollution, weather-cloud cover, and altitude) (Walker et al., 2003) (Fig. 5). UVB (290-320 nm) represent only 2-5 % of the sun‟s emissions, it penetrates into epidermal cells, can damage DNA and activate a cascade of events leading to photo-aging (Walker et al., 2003). UVA (400 – 320 nm) represents 95-98 % of the total UV radiation reaching earth‟s surface (Walker et al., 2003). While also damaging the epidermis, UVA penetrates deeper into the dermis to degrade collagen and elastin fibers via oxidative stress and activating MMPs, thus, UVA is more cytotoxic than UVB (Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). The characteristics of light and UV light (UVC, UVB and UVA) penetration into human skin are summarized in Fig. 5. In sun-exposed skin areas, such as the face and neck, photo-aging pathological changes in cellular activities occur with the generation of ROS that lead to the gross disorganization of the dermal matrix and the decline of antioxidants (Gonzaga, 2009; Kammeyer and Luiten, 2015; Khol et al., 2011; Natarajan et al., 2014; Rittie and Fischer, 2002; Shindo et al., 1994; Zouboulis and Makrantonaki, 2011). How much sun exposure is required to cause photo-aging depends on a person‟s type of skin (fair vs. darker pigmentation; as classified by Fitzpatrick‟s skin types) and the exposure over time without skin protection to UVR. One study suggested that minimal repetitive exposure to UVR, equivalent to 5-15 minutes of mid-day sun every-other-day was sufficient to maintain elevated levels of MMPs that are known to degrade dermal collagen and elastin fibers (Rabe et al., 2006). Surprisingly, this is approximately the same amount of sun exposure internal medicine/endocrinologists suggest is needed to maintain healthy vitamin D

14

levels. (Gilchrest, 2008). In 1998, previous to the above data, the American Academy of Dermatology issued the “safe sun position” based upon the irrefutable facts that UV irradiation causes skin cancer, melanoma and photo-aging and that vitamin D can be obtained from the diet or from oral supplements (Robinson et al., 1998). However, controversy about dietary vitamin D increasing vitamin D status remains a controversy due to: 1) a suggested high prevalence of low vitamin D intakes and vitamin D deficiency or inadequate vitamin D status in Europe and 2) comparing vitamin D intakes estimated from foods and dietary supplements to serum 25-hydroxy vitamin D concentrations is problematic since comparisons can only be made on group means rather than on data linked to individuals (Spiro and Butriss, 2014; National Institutes of Health, Vitamin D, 2016). Thus, staying out of the sun is the best avoidable practice a person can do to prevent ROS production and photo-aging. To illustrate this point, Fig. 6 shows a 69-year-old man that drove trucks commercially for twenty-eight years. As Fig. 6 displays, the left side of this man‟s face (driver‟s side) shows dramatic photo-aging, while the right side of the face (protected from UVR), shows minimal photo-aging (Gordon and Brieva, 2012). This condition is unilateral dermatheliosis and demonstrates the dramatic impact photo-aging has on dermal health. 3.2 Cellular and Molecular ROS Events in Human Skin Recall the unavoidable component of skin aging is chronological or intrinsic aging (Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). In intrinsic aging there is ROS production via oxidative metabolism (Anderson et al., 2014; Gonzaga, 2009; Meadows et al., 2014). All of the skin aging characteristics (covered above) are associated with oxidative metabolism and subsequent ROS generation that define this inevitable phenomenon. Since the structural and functional aspects of

15

collagen and elastin fibers are so important for optimal skin health, this will be a major focus for the cellular, molecular, enzymatic, antioxidant and other components that regulate these extracellular matrix proteins. From molecular to cellular cascades, ROS production is known to cause the activation of activator protein-1 (AP-1), which suppresses TGFβ receptors that in turn blocks pro-collagen synthesis that reduce collagen levels (Gonzaga, 2009; Kammeyer and Luiten, 2015; Naylor et al., 2011; Peng et al., 2015; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). At the same time, AP-1 activation: a) stimulates MMPs that degrade collagen and b) activates NFkappaB, which is a major activator of the inflammatory response (Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014; Naylor et al., 2011; Peng et al., 2015; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). The activation of the inflammatory response generates cytokines or interleukins (ILs), which then in a positive feedback loop, stimulates the further production of ROS (Gonzaga, 2009; Kammeyer and Luiten, 2015; Naylor et al., 2011; Peng et al., 2015; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). These pathways are shown in Fig. 7. Now, turning to photo- or extrinsic aging, the exposure to UVR displays the same molecular to cellular responses as for intrinsic aging (covered above), but the magnitude of the effects is amplified, which explains why photo-aging accounts for approximately 80-to-90 % of skin aging. It is known that UVR exposure and ROS production in photo-aging cause damage to DNA, proteins, lipids and reduces the levels of antioxidants in the skin (Bickers and Athar, 2006; Gilchrest, 1989; Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014; Naylor et al., 2011; Peng et al., 2015; Rittie and Fischer, 2002; Shindo et al., 1994; Theile et al., 1998; Warren et al., 1991; Zouboulis and Makrantonaki, 2011) (Fig. 7). For instance, vitamin E

16

sequestered in hydrophobic lipids, can absorb the energy from UV light, and thus plays an important role in photo-protection from ROS (Rhie et al., 2001; Weber et al, 1997). However, exposure to UV light lowers vitamin E contained primarily in the stratum corneum (Shindo et al., 1994), which is an early and sensitive in vivo marker of UV induced photo-oxidation (Thiele et al., 1998). Also, vitamin E concentrations in the human epidermis decline with age (Rhie et al., 2001). However, at the same time, these events are also associated with stimulation of MMPs and a concept called “fibroblast collapse” (Gonzaga, 2009; Kammeyer and Luiten, 2015; Khol et al., 2011; Natarajan et al., 2014; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). In this situation fibroblasts increase the production of elastase, which degrades elastin that in turn reduce elastin levels (Akhtar et al., 2010; Gonzaga, 2009; Hillebrand, 2010; Kammeyer and Luiten, 2015; Khol et al., 2011; Natarajan et al., 2014; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). In total, the destructive actions associated with photo-aging are noticeable initially by wrinkles followed by skin damage that, if not treated, are irreversible. It is beyond the scope of this review to detail, especially the molecular factors, elements and pathways involved in the cascade with photo-aging and ROS production, assuredly, this information is discussed elsewhere (Bickers and Athar, 2006; Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014; Naylor et al., 2011; Peng et al., 2015; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). 3.3 Skin Steroid Enzymes, Steroid Hormones and Steroid Receptors in Human Aging It is well known that estrogen improves skin quality and dermal health, especially in postmenopausal women by increasing skin collagen, elastin deposition (elasticity) and hydration (Archer, 2012; Brincat et al., 2005; Freedberg et al., 2003; Friedman, 2005; Hillebrand, 2010; Khol et al., 2011; Wend et al., 2012). It is also known that: a) estrogen suppresses ROS

17

production (Ramara et al., 2007) and b) antioxidant enzyme expression is stimulated by estrogen via the ERK1 and ERK2[MAP]/NFkappaB cascade (Borras et al., 2005). The ovary and skin fibroblasts are capable of synthesizing estrogen from steroid precursors by the aromatase enzyme, but ovarian production dramatically decreases after 30 years of age and especially after menopause (Inoue et al., 2011; Jain et al., 2003; Schwartz and Mayaux, 1982). Topical moisturizers with science-based active ingredients and/or estrogen or oral hormone replacement therapy (ERT/HRT) have been shown to improve skin aging parameters and reverse the negative effects of skin aging compared to untreated skin (Archer, 2012; Brincat et al., 2005; Freedberg et al., 2003; Hillebrand, 2010; Wend et al., 2012). The most important steroid receptors in skin are estrogen and androgen receptors. Estrogen receptors (ER) are expressed as subtypes, estrogen receptor alpha (ER α) and estrogen receptor beta (ER β; Fig. 8). The predominant subtype of ER in skin and scalp hair is ER β, it is also in keratinocytes, fibroblasts and the hair bulb (Pelletier and Ren, 2004; Thornton et al., 2003). Androgen receptors (AR) are expressed at lower levels compared to ER β in fibroblasts in the dermis (Pelletier and Ren, 2004; Thornton et al., 2003). The intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts have been recently reported by Pomari et al. (2015) that includes the expression of the G-protein-coupled estrogen receptor (GPER1) that apparently is involved in estrogen signaling for both dermal cell types. The ER β and androgen receptors play important roles in skin and hair health. In general, activation of ER β enhances, whereas, activation of ER α or hormonal actions via androgen receptors decreases skin, hair and prostate health (Lephart, 2014a). For example, Widyarini et al. (2006a) demonstrated the mechanism by which estrogen receptor signaling protected against

18

solar-stimulated UV radiation-induced immune suppression. In (2013), Markiewicz et al. using knock-out mice showed that ER β meditated skin dermal thickness/collagen deposition in a unique manner that has implications for the protective effect of estrogen against exposure to UV light or photo-aging. Also, as discussed by Chang et al. in (2010) ER β activation blocked photo-aging (via inhibiting various inflammatory biomarkers such as IL- 1, IL-6 and NFkappaB) using selective estrogen receptor compounds. The importance of ER β activation and its beneficial influence in human skin has been recently reviewed (Jackson et al., 2011). Moreover, estrogen related receptor (ERR) gamma (γ) has been identified in keratinocytoes and fibroblasts in human skin that has positive influences on dermal health. For instance, when ERR γ is activated in skin, it is thought to protect against neoplastic growth (Krahn-Bertil et al., 2008). Also, the 5α-reductase enzyme is present in fibroblasts. This enzyme converts testosterone to the more potent androgen, 5α-dihydrotestosterone (5α-DHT) that decreases skin fibroblast cell viability and wound healing (Gopaul et al., 2012; Makrantonaki and Zouboulis, 2009; Nitsch et al., 2004) (Fig. 8). In reference to ROS production, in general, androgens are known to increase free radical production from data obtained in cardiovascular studies (Chignalia et al., 2012; Tostes et al., 2016). 3.4 Genetics and Steroid Hormonal Factors in Skin Aging Image analysis were performed examining monozygotic (“identical”) twins to determine how genetics influences or modulate photo-aging factors skin patterns and skin damage. Since each twin pair has an identical chromosomal DNA sequence and since most twins are raised in the same households and exposed to similar environments, similar severities of skin damage/wrinkling between the twins were expected. This is exactly what was found

19

(Hillebrand, 2010). The implications from these findings suggested that ROS generation and oxidative stress impact was similar in monozygotic twins. Next, to track the progression of photo-skin damage superimposed upon chronological aging an 8-year study was performed (Hillebrand, 2010). Each subject‟s pattern of wrinkling around the eyes and on the cheek at baseline was compared to the pattern observed after 8 years. The study found that subjects, who were in their forties at baseline, displayed a significantly faster rate of skin damage over the 8-year period compared to women in other age groups at baseline, confirming previous reports (Akazaki et al., 2002; Kuwazuruet et al., 2008). Finally, the relationship of menopausal status and rate of skin damage was examined. Women, who had entered menopause between baseline and 8 years, showed the highest rate of skin damage (> 95 % increase), suggesting that hormone replacement therapy (HRT) is most effective within 5 years of menopausal (Archer, 2012; Phillips et al., 2008). Thus aging can be described as a fast moving train without any stops along the way. Skin aging, along with prostate health changes (Lephart, 2014a) and male-pattern baldness (Lee et al., 2015), are associated with intrinsic or chronological alterations such as hormonal changes and for skin exposure to the sun or photo-aging. These aging processes are demonstrated in Fig. 9 showing a woman (in a split-face photograph) at 23-years-old vs. 63-years of age (Nkengne and Bertin, 2012). The split-face comparison displays decreased collagen and elastin deposition, increased wrinkle formation and dull skin tone. Therefore, the goal to slow down the fast moving train of skin aging needs to include lifestyle changes (decreased sun exposure) and treatments (decreased ROS production), in order to delay the onset/development of damaged cutaneous elements in order to support good dermal

20

health. Evidence that equol is an emerging botanical that addresses skin aging by decreasing ROS via hormonal and molecular mechanisms to provide good dermal health is presented below. 4. Characteristics of Equol: Chemical Structure, Isomeric Forms, Metabolic and Plant Sources and Biological Actions Traditionally, equol is classified as a polyphenolic compound that is a metabolite of daidzein (an isoflavone found in plant and food products) (Lephart, 2013b, Setchell and Clerici, 2010). Phenolics are a group of compounds having at least one hydroxyl group attached to an aromatic ring. The most high-profile polyphenolic molecule known to the general public is resveratrol that equol is structurally related (Lephart et al., 2014; Park and Pezzuto, 2015). Interestingly, genistein, another polyphenolic/isoflavonoid molecule, was highly studied in the 1980s to the mid-1990s until the equol hypothesis was proposed (Setchell and Clerici, 2010). Equol, an isoflavonoid, has two phenolic rings with hydroxyl groups on each ring that provide functional points for biological activity (Fig. 10A) (Lephart, 2013b; Lephart, 2015; Setchell and Clerici, 2010). While endogenous estrogenic hormones such as 17β-estradiol are steroids with a cyclo-hexane-phenantrene parent chemical structure that is derived from cholesterol, equol is not a steroid. However, equol and 17β-estradiol have similar chemical structures/confirmations and molecular weights (C15H14O3 vs. C18H24O2; 242.3 g/mol vs. 272.4 g/mol, respectively) (Fig. 10 A and B). Equol has a chiral center at carbon 3, and thus can exist in two mirror image forms known as enantiomers (S-equol and R-equol) (Lephart, 2013b; Setchell and Clerici, 2010). Racemic equol refers to exact equal portions of S-equol and R-equol (Lephart, 2013b). However, in man and animals only S-equol is produced by intestinal bacteria conversion from daidzein, and some individuals are capable of producing higher levels of equol than others

21

(Lephart, 2013b, Setchell and Clerici, 2010; Setchell et al., 2005). These individuals have been identified as “equol producer” (Lephart, 2013b, Setchell and Clerici, 2010). The term “equol producer” is a descriptive or arbitrary term for humans that maintain S-equol levels around or above 10 to 20 ng/ml after consumption of soy food products that infer protective health benefits (Lephart, 2013b). S-Equol has also been found in plant products such as beans, cabbage, lettuces, tofu and other food and animal products (Abiru et al., 2012; Common and Ainsworth, 1961; Hounsome et al., 2009; Hounsome et al., 2010; Jou et al., 2013). For example, S-equol has been reported in cow‟s milk (Bannwart et al., 1986; Hoikkälä et al., 2007; King et al., 1998 Lundh et al., 1990), and Frankenfeld (2011) showed that dairy consumption significantly correlated with urinary equol levels in U.S. adults. Also, there are some data that showed Requol levels in fermented soy products are higher compared to S-equol (Lephart, 2013b, Lephart, 2014b). Notably, the metabolism of R- and S-equol in humans appears to be similar (Setchell et al., 2009). Therefore, humans are exposed to this polyphenolic/isoflavonoid compound from different plant and food sources regardless of age, gender or geographical location with scientific data to support a consumption/exposure record that appears to be safe (Andres et al., 2012; Badger et al., 2009; Degen et al., 2011; Gilchrist et al., 2010; Hamilton-Reeves, 2010). Of particular interest from a chemical messenger and molecular perspective, equol has an affinity for estrogen receptor beta (ER ), which is abundant in keratinocytes of the epidermis and fibroblasts in the dermis (Pelletier and Ren, 2004; Setchell et al., 2005; Thornton et al., 2003). More recent data suggest that G-protein-coupled estrogen receptors (GPER1) are expressed in both keratinocytes and fibroblasts that mediate estrogenic signaling (Pomari et al., 2015). In this regard, there is no evidence, to date, that shows equol binding GPER1 in human skin. However, equol has been shown to bind the orphan G-protein-coupled receptor (GPR30) in

22

endothelial cells to activate endothelial NO synthase (eNOS) that has a positive influence on arterial blood pressure. Thus, equol may improve endothelial function and lower blood pressure and thus have a potential protective role in cardiovascular disease (Rowlands et al., 2011). Equol is also a selective androgen modulator (SAM), having the ability to specifically bind 5α-dihydrotestosterone (5α-DHT) and inhibit its potent negative actions in skin (Gopaul et al., 2012; Lephart, 2013a). Equol has the ability to bind to ERR γ, which has important implications for anti-aging in human skin (Hirvonen et al., 2010; Krahn-Bertil et al., 2008; Lephart, 2013a). Additionally, when equol is bound to ERR γ, it decreases inflammation and has anti-proliferative effects on prostate and breast cancer cells (Hirvonen et al., 2010). Finally, equol research has dramatically increased within the past two decades, and this polyphenolic molecule along with other botanical compounds is widely used in personal care products such as skin protectants, whitening, anti-wrinkle, and anti-aging ingredients as well as benefiting prostate health (Baumann, 2002; Draelos and Puglises, 2011; Evans and Johnson, 2010; Gopaul et al., 2012; Jackson et al., 2014; Lephart, 2013a; Lephart, 2014a; Lund et al., 2011; Weber et al., 1997). 4.1 Antioxidant Properties of Equol Equol has recently caught the interest of researchers because of its powerful antioxidant activity and its unique molecular and biochemical messenger properties with implications in treating agerelated diseases (Lephart 2013a; Lephart 2013b; Setchell and Clerici, 2010). For example, in plants, the accumulation of polyphenolic compounds such as flavonoids and phenolic molecules have been linked to pathogen resistance (Hounsome et al., 2009; Hounsome et al., 2010; Lephart et al., 2014). There are many polyphenolic molecules that act as

23

antioxidants including isoflavonoids (Bohn, 2010; Lephart et al., 2014; Li et al., 2012; Hounsome et al., 2009; Hounsome et al., 2010; Rice-Evans, 2001). Comparative studies examining polyphenolic compounds have demonstrated that equol is a superior antioxidant, having greater antioxidant capacity than vitamin C or vitamin E in several in vitro tests (Arora et al., 1998; Mitchell et al., 1998). In fact, in a more recent study, equol exhibited one of the highest antioxidant activities, when three different in vitro assays were used, and equol was more effective than the positive controls quercetin and ascorbic acid (Rufer and Kulling, 2006). Finally, equol has greater antioxidant activity (i.e., oxidative damage to lipid membranes, etc.) compared to genistein (Mitchell et al., 1998; Rufer and Kulling, 2006). In other tissue sites, equol was proven to have significant antioxidant effects in bovine aortic endothelial cells, where equol protected against peroxide-induced cell death (Cheng et al., 2008). Additionally, equol protected against apoptosis and vascular injury through oxidative stress in porcine pulmonary arteries and human pulmonary artery endothelial cells (Chung et al., 2010; Kamyama et al., 2009). Equol also significantly reduced oxidative stress and protected rats against focal cerebral ischemia (Ma et al., 2010). In food, a recent study examined the changes in antioxidant compounds in white cabbage during winter storage. The major groups of antioxidants identified in cabbage were vitamins, flavonoids and phenolic acids (Hounsome et al., 2009; Hounsome et al., 2010). Of the isoflavonoid compounds examined, equol was identified as being present in cabbage at high levels similar to other polyphenolic compounds that can serve as an antioxidant during the storage of white cabbage to help prevent oxidative stress (Hounsome et al., 2009; Hounsome et al., 2010).

24

4.2 Equol’s Lipophilic Nature and Penetration into Human Skin via a Reservoir Delivery Mechanism The lipophilic nature of equol is shown via its octanol-water partition coefficient of 3.2, which is higher than other polyphenolic molecules (Rothwell et al., 2005). For example, the octanolwater partition coefficients for resveratrol = 3.0; genistein = 3.0 and diadzein = 2.5. Additionally, intestinal absorption data supports this proposition, where equol (either as the R- or S-isomer) has the highest absorption levels (80 to 85%) compared with other isoflavonoids like genistein (at 15-20%) or daidzein (at 30-40%) (Setchell and Clerici, 2010; Setchell et al., 2009). Drug delivery studies have shown the more stable isomer is R-equol vs. S-equol (Alvira et al., 2008), and in vitro culture data in breast and prostate cancer cells suggested that the R-isomer accounts for the chemo-protective effects of equol rather than the S-isomer (Magee et al., 2006). Franz cell testing using tritiated racemic equol penetration into human skin has shown: 1) the percutaneous absorption and, 2) skin content distribution among the epidermal and dermal compartments (Lephart, 2013a). As displayed in Fig. 11A, the [3H]-equol penetrated into human skin with a profile showing an initial maximum peak flux occurring 6 h after dosing followed by a decline in penetration with a secondary lower peak flux at approximately 26-28 h after a single applied dose. Notably, when the actual epidermal/dermal content of the tritiated racemic equol was quantified, the epidermal content was higher than typically observed with most topical compounds or drugs representing an unusual delivery profile (Lephart, 2013a). Notably, racemic equol was sequestered into the epidermal compartment due, most likely, to the abundance of estrogen receptor subtypes (ER β) in keratinocytes for which S-equol has a high affinity (Gopaul et al., 2012; Lephart, 2013a; Lephart, 2013b; Pelletier and Ren, 2004; Setchell et al., 2005; Thornton et al., 2003). Thus, the epidermal “reservoir” delivery mechanism of topically applied

25

equol to the dermal layer over time has not been seen with other compounds such other polyphenolic molecules like resveratrol along with the isoflavonoid, genistein, or topical drugs like 17β-estradiol (Chadha et al., 2011; Hung et al., 2008; Xing et al., 2009; Zhong et al., 2009). This epidermal reservoir delivery mechanism of equol is depicted in Fig. 11B. Finally, the delivery of approximately 14 nM racemic equol into the keratinocytes (after a single dose) in the percutaneous absorption studies was similar to previous in vitro culture results, where the continual exposure of 10 nM equol significantly stimulated collagen and elastin, while at the same time significantly inhibited MMPs protein expression (Gopaul et al., 2012; Lephart, 2013a). 5. Equol’s Anti-Aging Influence on Human Skin by Reducing Oxidative Stress in Cascade Events The formation of ROS is a widely accepted pivotal mechanism leading to skin aging (Gonzaga, 2009; Kammeyer and Luiten, 2015; Natarajan et al., 2014; Rittie and Fischer, 2002; Zouboulis and Makrantonaki, 2011). Cell culture (primary or organotypic), molecular, and gene expression/array methods have examined equol‟s influence on oxidative stress along with various biomarkers at different cascade steps/events that lead to damaged skin. However, since equol can be expressed as isomers, the type of equol tested (racemic, S-equol or R-equol) is noted below, when this information is known. In a comprehensive investigation on equol as: Requol, racemic equol or S-equol from PCR/mRNA studies quantifying human skin gene expression, only three genes displayed the greatest significant expression by S-equol, whereas, 16 genes displayed the greatest significant levels (either stimulation or inhibition) by R-equol and/or racemic equol, which suggest that, in general, R-equol and/or racemic equol was more

26

potent compared to S-equol for optimal human skin health (Lephart, 2013a). The findings from various investigations are described below under specific headings and summarized in Fig. 12. 5.1 Equol Decreases ROS via Stimulation of Nrf2 Oxidative Stress via ROS production by UV-light-induced signal cascades is known to cause skin aging and the pathology of skin disease (Bickers and Athar, 2006; Khol et al., 2011; Naylor et al., 2011). Nuclear-factor-erythroid 2-related factor 2 (Nrf2) is a master regulator of the transcriptional response to oxidative stress, and it is structurally and functionally conserved from insects to humans (Lacher et al., 2015). Nrf2 plays a key role in the cellular defense against oxidative and xenobiotic stressors by its capacity to induce the expression of numerous genes, which encode detoxifying enzymes and antioxidant proteins that provide protection in endothelial cells, skin morphogenesis, wound repair and skin cancer (Beyer et al., 2007; Greenwald et al., 2015; Sekhar and Freeman, 2015; Zhang et al., 2013). Additionally, Nrf2 deficiency or Nrf2-knockdown is known to cause lipid oxidation, inflammation, and extra cellular matrix-protease expression [e.g., MMPs, cyclo-oxygenases (Cox)] in UVA-irradiated skin fibroblasts or heat shock-induced human dermal fibroblasts (Gruber et al., 2015; Park and Oh, 2015). Bottai et al. (2012) reported that 17β-estradiol protects human keratinocytes and fibroblasts against oxidative damage by counteracting hydrogen peroxide-mediated lipoperoxidation. Furthermore, equol has been shown to increase nuclear-factor-erythroid 2related factor 2 (Nrf2) (Zhang et al., 2013). As discussed by Jackson et al. (2014), the molecular mechanism involves the release of Nrf2 (transcription factor) from Keap 1, its cytoplasmic binding protein and subsequent binding to the antioxidant response element (ARE) that is present in the promoter region of genes for

27

antioxidant proteins and enzymes. Specifically, equol may increase Nrf2 levels and/or bind to the estrogen-responsive elements (EREs) in the promoter region the Nrf2 gene and/or increase gene expression of other antioxidant genes. In support of this concept, Zhang et al. (2013) showed that S-equol provided protection against peroxide-induced endothelial cell apoptosis by activation of estrogen receptor and Nrf2/ARE signaling pathways. Finally, Froyen and Steinberg (2011) reported that racemic equol increased the expression of the xenobiotic metabolizing enzyme quinone reductase (both mRNA and protein levels) via similar molecular mechanisms involving ER β and Nrf2. 5.2 Equol Acts as an Antioxidant, Stimulates Antioxidant/detoxifying Enzymes and Inhibits Skin Aging Biomarkers Few in vivo studies have been performed using equol as an antioxidant or protecting agent against disease. However, Ma et al. (2010) examined the hypothesis that genistein and equol were neuro-protective in transient focal cerebral ischemia in male and ovariectomized rats by inhibiting oxidative stress. In brief, the results of this study showed that equol supplementation via the diet was more potent than genistein, in general, in preventing transient focal cerebral ischemia in rats by decreased brain levels of superoxide production and NADPH oxidase (NOX) activity that are known to increase oxidative stress. As noted by Ma et al. (2010), equol is a better antioxidant than genistein or daidzein, and this may assist in the interpretation of the obtained results as supported by other studies (Arora et al., 1998; Mitchell et al., 1998; Rufer and Kulling, 2006). In a double-blind human investigation, Pusparini and Hidayat (2015) studied the effect of soy isoflavone supplementation (100 mg/day for 6 months) on endothelial dysfunction and oxidative stress in equol-producing postmenopausal women using vascular cell adhesion

28

molecule-1 (VCAM-1) and nitric oxide (NO) as markers of vascular endothelial function, and malonyldialdehyde (MDA) as an oxidative stress marker. The authors found that isoflavone supplementation in postmenopausal women with equol-producer status had a beneficial effect by decreased MDA concentrations, but without improvement of VCAM-1 and NO concentrations. Thus, the results of this study provided potential information that decreased oxidative stress exists in equol-producing postmenopausal women with dietary soy isoflavone supplementation. One small human in vitro study reported that R-equol protected gastric cells from oxidative stress and induced cell death (Asciutti et al., 2010). Another in vitro study reported that racemic equol inhibited LPS-induced oxidative stress and at the same time enhanced the immune response in chicken macrophages (Gou et al., 2015). ROS generation sits at the top of the pathway of the oxidative stress cascade, and oxidative stress can be considered the common denominator of both intrinsic and extrinsic aging by oxidative metabolism and exposure to UVR, respectively.

Previous studies have examined

the role of antioxidants in dermal aging by ROS production via gene expression analysis (Avantaggiato et al., 2014; Pandel et al., 2013). As pointed out above, equol is a strong antioxidant itself. Racemic equol has been shown to stimulate the gene expression of antioxidant enzymes in human skin such as SOD 2 and thioredoxin reductase 1 (TXNRD 1) (Gopaul et al., 2012; Lephart, 2013). SOD 2 and TXNRD 1 are known to protect against free redicals, oxidiative stress and UV-induced skin damage (Shindo et al., 1994; Velarde et al., 2012). In addition to dermal studies, equol had been shown to inhibit peroxide-induced bovine aortic endothelial cell death demonstrating equol has significant antioxidant effects to neutralize hydrogen peroxide (Chung et al., 2008). Furthermore, Widyarini et al. (2012) reported the positive biological activities of equol that include the UV-protective antioxidant effects via the

29

endogenous cutaneous antioxidant enzyme haem oxygenase (HO)-1. Haem oxygenase is a protector of lipid peroxidation and is part of the mitogen activated protein kinase (MAPK) pathway that results in the activation of antioxidant genes/enzyme via the Nrf2/KEAP 1/ARE pathway (Mann et al., 2009). Also, racemic equol stimulated the gene expression of the SH-rich detoxifying proteins like metallothionein-1H and metallothionein-2H that protect against metal toxicity (Gopaul et al., Lephart, 2013a). Previous studies have shown that equol‟s photoprotective effect in mouse and human skin is dependent on metallothionein (Widyarini et al., 2006b). Moreover, racemic equol was shown to inhibit the gene expression of human skin aging biomarkers: S100 calcium-binding protein (CBP) A8 and CBP A9 that are known to increase with skin aging (Gopaul et al., 2012). Also, racemic equol was reported to inhibit the gene expression of type 1 5α-reductase in human skin (Gopaul et al., 2012, Lephart, 2013a). Recall, this enzyme converts testosterone to the more potent androgen, 5α-DHT in dermal fibroblasts that cause the stimulation of MMPs and reduces tissue wound repair (Makrantonaki and Zouboulis, 2009; Nitsch et al., 2004). In cell cultures of human dermal fibroblasts 5α-DHT was cytotoxic at 10 nM (Gopaul et al., 2012). In this regard, clinical evidence has demonstrated that the accumulation of 5α-DHT in dermal papilla cells is implicated in androgenetic alopecia via the induction of ROS leading to cell senescence and cell death (Lee et al., 2015). Previous studies have demonstrated racemic equol‟s ability to specifically bind to 5αDHT and prevent its negative biological actions such as skin aging (Gopaul et al., 2012; Makrantonaki and Zouboulis, 2009; Nitsch et al., 2004). Finally, equol‟s positive effects not only have applications to human skin, but prostate health as well (Lephart, 2014a; Lund et al., 2011).

30

5.3 Equol Protects DNA and Enhances Nerve and Tissue Repair ROS production is known to damage DNA, proteins and lipids and other cellular components (Bickers and Athar, 2006; Gonzaga, 2009; Khol et al., 2011). In human skin an important aging biomarker is proliferating cell nuclear antigen (PCNA). It is involved in DNA repair, and it is known to decrease in aged skin (Goukassian et al., 2000; Takahashi et al., 2005). Racemic equol was shown to stimulate the gene expression of PCNA that is known for its protective skin effects (Gopaul et al., 2012; Lephart, 2013a). Several reports have shown that nerve growth factor (NGF) is important in cell survival, wound healing by stimulation of collagen and regeneration of cutaneous nerves (Marconi et al., 2003; Nithy et al., 2003; Yaar et al., 1991; Zhai et al., 1996). It is known that NGF expressed in keratinocytes is reduced by UVB exposure (by sun/photo-aging) (Marconi et al., 2003). Gopaul et al. (2012) and Lephart (2103a) showed that racemic equol stimulated NGF gene expression in human skin that suggested protective dermal effects by NGF from oxidative stress. 5.4 Equol Inhibits AP-1 and Neoplastic Cell Growth AP-1 is a nuclear transcription element that is part of the oxidative stress cascade. It induces MMPs in human skin that in turn activates collagen degradation (Hung et al., 1997). AP-1 also blocks the positive pro-collagen actions of transforming growth factor-β1 (Fischer et al., 1996). Kang et al (2007a) reported that the anti-tumor effects of racemic equol are due to the inhibition of cell transformation by the MEK signaling pathway by blocking AP-1. Racemic equol dosedependently attenuated TPA-induced activation of AP-1, whereas daidzein did not exert any effect when tested at the same concentrations (Kang et al., 2007a). Finally, ER β signaling has been shown to protect against transplanted skin tumor growth in mice (Cho et al., 2010), which

31

implicated a common mechanism of how the blocked AP-1 actions by equol reported by Kang et al. (2007a) may be mediated. Additionally, equol has been shown to bind to ERR γ that in turn enhanced the transcriptional activity of ERR γ, which is known to protect against neoplastic growth (Hirvonen et al., 2011). Equol‟s precursor molecule daidzein did not have this effect (Krah-Bertil et al., 2008). It is also known that ERR γ is present in keratinocytes and fibroblasts in human skin, where its actions may show similar favorable protection as that seen against breast and prostate cancers (Hirvonen et al., 2011; Krah-Bertil et al., 2008) Finally, it has been demonstrated that ER β signaling in the prostate (and breast tissue) are associated with decreased inflammation and neo-plastic growth (Lephart, 2014a). S-Equol is known to have a high affinity for ER β, which is the predominate ER in keratinocytes and fibroblasts in human skin (Pelletier and Ren, 2004; Setchell et al., 2005; Thornton et al., 2003) and this ER signaling mechanism protects against immune suppression by UV radiation exposure (Chang et al., 2010; Pomari et al., 2015; Widyarini et al., 2006a). The high affinity for ER β by S-equol may account for the unique topical dermal absorptive and „reservoir‟ penetration method into human skin that may account, in part, for its positive benefits (Lephart, 2013a). 5.5 Equol Inhibits NFkappaB The pro-inflammatory transcription factor NF-kappB has been studied for more than 30 years. It was first discovered by Sen and Baltimore in 1986 (Gupta et al., 2010). NF-kappaB remains an exciting and active area of investigation, where it is expressed in all cell types and is evolutionarily conserved (Ghosh et al., 1998). NFkappaB is involved in the oxidative stress mechanism by the expression of numerous genes such as the cytokines and plays a major role in

32

the pathology of inflammatory disease (Gilmore, 2006; Gosh et al., 1998; Hayden and Gosh, 2012; Schreck et al., 1991). Several investigators have examined the inhibition of NFkappB by equol. In (2005), Kang et al. showed that racemic equol inhibited tumor necrosis factor-α gene expression by blocking NFkappB in mouse macrophages that was independent of an estrogen receptor mechanism. Tumor necrosis factor-α is a cell signaling protein (cytokine) involved in systemic inflammation (Kang et al., 2005). Furthermore, Kang et al. in (2007b) reported the dosedependent inhibitory effects of racemic equol (by in vivo administration) on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in murine macrophages from lipopolysaccharide (LPS)-treated mice. The enzyme NO synthase (NOS) generates NO that is a free radical, and the overproduction of NO by iNOS is associated with the development of several diseases including atherosclerosis, stroke, septic shock and Alzheimer‟s disease (Kang et al., 2007b). The gene expression of iNOS is regulated mainly at the transcriptional level in macrophages and NF-kappB via the MAPK pathway and PI3K/Akt pathways. The MAPK pathway has been well known to be involved in the regulation of iNOS gene expression and NFkappaB activation (Kang et al., 2007b). In this study, LPS-induced activation of Akt was suppressed by racemic equol, and it also blocked LPS-induced NFkappaB activation. 5.6 Equol Inhibits the Inflammatory Response Extensive research during the last two decades has revealed the mechanism by which continued oxidative stress leads to chronic inflammation, which in turn, mediates most chronic diseases including cancer and skin damage (Bar-Or et al., 2015; Bickers and Athar, 2006; Droge, 2002; Maes et al., 2011; Maritim et al., 2003; Valko et al., 2007). Recent research on polyphenolic molecules from plants sources has expanded the horizon for potential therapeutic remedies and

33

treatments (Evans and Johnson, 2010; Adlercreutz et al., 2004; Park and Pezzuto, 2015). It is well documented that various pro-inflammatory markers in human skin are increased with UV exposure (Bickers and Athar, 2006; Khol et al., 2011; Strickland et al., 1997). It has been shown that racemic equol inhibited the gene expression of several proinflammatory biomarkers such as interleukin-1 alpha (IL-1A), IL-6, IL-8 and interleukin-1 receptor 2 as well as COX-1 and tumor necrosis factor receptor (Gopaul et al., 2012, Lephart, 2013a). These pro-inflammatory biomarkers are known to increase with UV exposure and aging (Bickers-Athar, 2006; Natarajan et al., 2014; Strickland et al., 1997). It is also known that ER β signaling protects epidermal cytokine expression and immune function by UVB exposure (Chang et al., 2010; Cho et al., 2008; Widyarini et al., 2012; Widyarini et al., 2006a). Since racemic equol has been utilized in most research investigations, the S-equol in racemic equol may act by binding to ER β receptors in keratinocytes. This may explain the obtained gene expression results, where equol inhibited several pro-inflammatory biomarkers (Gopaul et al., 2012). However, a comprehensive study that examined the equol isomers along with racemic equol in human skin via gene array analysis suggested that R-equol and/or racemic equol are better inhibitors of the pro-inflammatory biomarkers (Lephart, 2013a). This suggests that further research is warranted to determine the mechanism of how equol (isomers) inhibit pro-inflammatory markers. 5.7 Equol Inhibits MMPs and Elastase It is known that oxidative stress directly and/or indirectly stimulates the production of MMPs and elastase, that in turn breakdown collagen and elastin in the dermal matrix (Bickers and Athar, 2006; Farage et al., 2008; Farage et al., 2010; Mera et al., 1987; Tulah and Birch-Machin, 2013). Racemic equol was examined in two different studies for its influence on human skin gene

34

expression of these important biomarkers. Racemic equol inhibited the gene expression of MMP 1, MMP 3, and MMP 9 (Gopaul et al., 2012; Lephart, 2013a). In 8-week organotypic cell cultures of human dermal fibroblasts, racemic equol decreased the expression of the elastase enzyme by 35 %, while the potent natural steroid hormone, 17β-estradiol decreased the expression by 8.4 % (Gopaul et al., 2012). Equol‟s ability to inhibit MMPs and elastase has profound effects on maintaining the major extra cellular proteins (i.e., collagen and elastin) in opposition to the insult of oxidative stress. 5.8 Equol Stimulates TIMP 1 Since oxidative stress directly or indirectly activates MMPs, the tissue inhibitors of matrix metalloproteases (TIMPs) are important skin enzymes that inhibit the actions of MMPs (Draelos and Puglises, 2011; Fazio et al., 1988; Freedberg et al., 2003;). The TIMPs are known to decrease with exposure to UV light and with aging that contributes to dermal matrix degradation, impaired cell growth and survival (Farage et al., 2010; Horneback, 2003). It is known that increased TIMP expression and activation blocks the negative influence of MMPs in causing skin damage. When the isomers of equol along with racemic equol were examined in gene arrays using human epidermal/dermal skin equivalents, racemic equol showed the highest stimulation of TIMP 1 followed by S-equol, whereas, R-equol did not stimulate TIMP 1 at all (Lephart, 2013a). This characterization of the equol isomer‟s impact on TIMP 1 demonstrated the complex manner of extracellular dermal matrix regulation of collagen protection and maintenance. 5.9 Equol Stimulates Collagen Collagen is the most important extracellular dermal matrix protein that maintains optimal dermal health (Draelos and Puglises, 2011; Farage et al., 2010). While oxidative stress via a cascade

35

mechanism degrades collagen and reduces the deposition of collagen (via MMPs) in the dermal layer, it is important not only to block the negative insults of ROS production, but to enhance collagen deposition. In both cell culture and gene array studies, racemic equol stimulated procollagen type 1 and collagen levels (Gopaul et al., 2012; Lephart, 2013a). When the equol isomers were examined in human skin gene expression experiments, racemic equol stimulation of collagen was highest followed by R-equol and both were significantly higher than S-equol (Lephart, 2013a). 5.10 Equol Stimulates Elastin Just as the case with collagen, oxidative stress via a cascade mechanism degrades elastin and reduces the deposition of elastin (via MMPs, fibroblast collapse/elastase) in the dermal layer, it is important not only to block the negative insults of ROS production, but to enhance elastin deposition (Draelos and Puglises, 2011; Farage et al., 2008; Mera et al., 1987; Tulah and BirchMachin, 2013). While collagen fibers provide the structural framework of the skin, elastin provides the “bounce back” or the elasticity of the skin that prevents the rapid increase in wrinkle formation and sagging that is a hallmark of skin aging and dermal damage. In both cell culture and gene array studies, racemic equol stimulated elastin levels (Gopaul et al., 2012). Surprisingly, racemic equol stimulated elastin to a higher level compared to the endogenous steroid hormone, 17β-estradiol (Lephart, 2013a). In the examination of the equol isomers in human skin gene expression experiments racemic equol displayed the greatest stimulation of elastin followed by R-equol, whereas, S-equol did not stimulate elastin at all (Lephart, 2013a). A summary of equol‟s positive influences on human skin in reference to ROS and oxidative stress effects is shown in Fig. 12. 6. Equol: Comparison to Resveratrol

36

When the above equol skin biomarker results are compared to the botanical that has the highest recognition by the general public or lay profile namely, resveratrol (Lephart et al., 2014; Park and Pezzuto, 2015), equol displayed better biochemical, molecular, protective and delivery mechanisms of action to promote and/or improve human skin health as a topical anti-aging ingredient/compound (Lephart, 2013a). This is interesting since equol and resveratrol have similar molecular weights and octanol-water partitions (indicating lipid solubility). However, equol directly binds the potent androgen 5α-DHT, inhibits the 5α-reductase enzyme in fibroblasts, acts as a selective SERM modulator by bind ERβ with higher affinity compared to ERα and binds and activates estrogen related-receptor gamma, while resveratrol does not have these properties. This may account for equol‟s ability to stimulate collagen in human dermal fibroblast cell cultures at nano-molar concentrations, whereas, resveratrol requires micro-molar levels to increase collagen. Also, equol is highly absorbed after oral dosing (> 80 %), whereas resveratrol is rapidly metabolized which make this route of delivery challenging from a commercial perspective. In fact, dietary supplementation of equol appears to provide an effective route of delivery in the clinical application of decreasing facial wrinkles (see below). See Table 1 for a descriptive comparison of the chemical properties and actions of equol to that of resveratrol in human skin. In this regard, resveratrol is a known sirtuin 1 (SIRT1) or anti-aging activator, whereas, equol is not. The sirtuins are a class of NAD+-dependent protein deacetylase enzymes that regulate a wide variety of cellular activities, which promote cell survival and delay or attenuate many age-related changes such as preventing early mortality in obese animals (Lephart et al., 2014; Park and Pezzuto, 2015). Thus, activators of sirtuins may benefit fundamental cellular processes that protect cells from stress and potentially treat age-related conditions and lengthen a

37

healthy life (Lephart et al., 2014) (see Table 1). In a recent study, the combined administration of equol with resveratrol enhanced SIRT1 expression in endothelial cells demonstrating equol‟s unique beneficial biological actions (Davinelli et al., 2013). Equol is currently used in skin treatments, and the positive influences on gene and protein expression (anti-aging, anti-oxidant and anti-inflammatory properties) have been reported (Gopaul et al., 2012). Recently, it has been proposed that ER β agonists like S-equol may have better beneficial dermal applications (Chang et al., 2010), compared to R-equol or racemic equol, however, this proposition has not been confirmed by the available human skin gene expression data (Gopaul et al., 2012; Lephart, 2013a). A report by Oyama et al. (2012) suggested that isoflavone dietary supplementation (for 12 weeks) in postmenopausal Japanese women that contained S-equol reduced facial wrinkles compared to placebo controls without adverse hormonal or gynecological effects. While there is ample evidence that equol has the potential to decrease oxidative stress, further research is required to determine the exact mechanisms of how equol protects human skin from the damaging effects of ROS. Finally, it is interesting to consider how polyphenolic molecules via dietary supplementation or functional foods may prevent oxidative stress to ameliorate cellular dermal damage (Lobo et al., 2010; Poljask and Dehmane, 2012). 7. Summary and Conclusions Oxygen in biology is essential for life, but it comes at a cost, where ROS are generated by oxidative metabolism via normal cellular function. Human skin exposed to solar UV radiation dramatically increases ROS production and oxidative stress. It is important to understand the characteristics of human skin and how dermal damage occur with chronological (intrinsic) aging and photo-aging (extrinsic aging) via the impact of ROS production and

38

oxidative stress mechanisms. This includes a cascade of events that involve a variety of cell/molecular signaling pathways. However, it is well established that photo-aging accounts for a majority of the pathological changes in human skin and is dependent upon the exposure to and intensity of solar UV radiation. The oxidative stress effects on skin aging involve damage to DNA, the inflammatory response, reduced production of antioxidants and activation/inhibition of various signaling factors that ultimately lead to the production of matrix metalloproteinases (MMPs) that degrade collagen and elastin in the dermal skin layer. The outcome of this complex aging process is the disruption of the epidermal/dermal matrix, which has the highest antioxidant content and immune defense function in the body. The visually perceived appearance of wrinkles, age-spots and loss of skin tone reflect the aging process. The goal is to delay the onset of aging and/or slow down skin aging with time and maintain good dermal health. Botanicals, as active ingredients, represent one of the largest percentages of compounds in use in dermatology/cosmeceuticals today to combat aging. An emerging botanical is equol. Equol is a polyphenolic, isoflavonoid molecule found in plants/food products and is a metabolite of daidzein by intestinal bacteria in all humans and animals. Equol has the potential to decrease oxidative stress and increase skin cellular longevity in human skin. The characteristics of equol and evidence of its protective properties and biological actions are discussed in reference to ROS generation and oxidative stress events. Some of these topics include equol‟s action of: stimulation of Nrf2, antioxidant/detoxifying enzymes, and extra cellular matrix proteins along with DNA and tissue repair and, equol‟s inhibition of: NFkappaB, pro-inflammatory biomarkers and MMPs. Many phyto-chemical effects on the skin are still unknown, and further research is warranted to clarify how they protect against oxidative stress in skin aging and damage not only via topical applications but by dietary supplementation and/or functional foods.

39

Conflict of Interests The author declares no conflict of interest in the data/research presented in this review and regarding the publication of this manuscript. The author has equol patents (U.S. and worldwide).

Acknowledgement This study/review was supported, in part, by LS/TTO funding, 19-2215 at Brigham Young University.

References Abiru, Y., Kumenura, M., Ueno, T., Uchiyama, S., Masaki, K., 2012. Discovery of an S-equol rich food stinky tofu, a traditional fermented soy product in Taiwan. Intern. J. Food Sci. Nutr. 63, 964-970. Adlercreutz, H., Heinonen, S.M., Penalvo-Garcia, J., 2004. Phytoestrogens, cancer and coronary heart disease. Biofactors. 22, 229-236. Akazaki, S. Nakagawa, H., Kazama, H., 2002. Age-related changes in skin wrinkles assessed by a novel three-dimensional morphometric analysis. Br. J. Derm. 147, 689-695. Akhtar, K., Broekelmann, T.J., Miao, M., Keeley, F.W., Starcher, B.C., Pierce, R.A., Mecham, R.P., Adair-Kirk, T.L., 2010. Oxidative and nitrosative modifications of tropoelastin prevent elastic fiber assembly in vitro. J. Biol. Chem. 285, 37396-37404.

40

Alvira, E., Mayoral, J.A., J. I. Garica, J.I., 2008. Enantiodiscrimination of equol in βcyclodextrin: an experimental and computational study. J. Inclusion Phenomena. 60, 103-113. doi: 10.1007/s10847-007-9358-4 Ames, B.N., Cathcart, R., Schwiers, E., Hochstein, P., 1981. Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis. Proc. Natl. Acad. Sci (USA). 78, 6858-6862. Andres, A., Cleves, M.A., Bellando, J.B., Pivik, R.T., Casey, P.H., Badger, T.M., 2012. Developmental status of 1-year-old infants fed breast milk, cow‟s milk formula, or soy formula. Pediatrics. 129, 1134-1140. http://dx.doi.org/10.1542/peds.2011-3121 Anderson, A., Bowman, A., Boulton, S.J., Manning, P., 2014. A role for human mitochondrial complex II in the production of reactive oxygen species in human skin. Redox Biology. 2, 10161022. doi: 10.1016/j.redox.2014.08.005 Anderson, R.R., 2012. Cosmegizmoceuticals: The Physics and Chemistry of Looking Better. Society of Cosmetic Chemists Annual Meeting, December 6, NY, NY, vol. 43. Archer, D.F., 2012. Postmenopausal skin and estrogen. Gynecological Endocrinol. 28, 2-6. Arora, A., Nair, M.G., Strasburg, G.M., 1988. Antioxidant activities of isoflavones and their biological metabolites in a liposomal system. Arch. Biochem. & Biophys. 356, 133-141. Asciutti, S., Setchell, K.D.R., Gizzi, S., and C. Clerici, C., 2010. R-Equol protects human gastric cells from oxidative stress-induced cell death. Gastroenterology. 138, Suppl 1, pp. S-254 (1689. doi:org/10/1016/S0016-5085(10)61159-6 Ashworth, J.L., Murphy, G. Rock, M.J., Sherratt, M.J., Shapiro, S.D., Shuttleworth, C.A., Kieltry, C.M., 1999. Fibrillin degradation by matrix metalloproteinases: implications for connective tissue remodeling. Biochem. J. 340; 171-181.

41

Avantaggiato, A., Bertuzzi, G., Vitiello, U., Iannucci, G., Pasin, M., Cervelli, V., Carinci, F., 2014. Role of antioxidants in dermal aging: an in vitro study by q-RT-PCR. Aesthetic Plastic Surg. 38, 1011-1016. doi 10.1007/s00266-014-0380-9 Badger, T.M., Gilchrist, J.M., Pivik, R.T., Andres, A., Shandar, K., Chen, J-R., Ronis, M.J., 2009. The health implications of soy infant formula. Am. J. Nutr. 89, 1668S-1672S. http://dx.doi.org/10.3945/ajcn.2009.26736U Bannwart, C., Adlercreutz, H., Fotsis, T., Wähälä, K., Hare, T., Barrow, G., 1986. Identification of isoflavonic phytoestrogens and lignans in urine of human and in cow milk by GC/MS. Advances Mass Spec. 10, 661-622. Bansal, N., Parle, M. 2010. Soybean supplementation helps reverse age- and scopolamineinduced memory deficits in mice. J. Med. Food. 13, 1293-1300. doi:10.1089/jmf.2010.1132. Bar-Or, D., Bar-Or, R., Rael, L.T., Brody, E.N., 2015. Oxidative stress in severe acute illness. Redox Biology. 4, 340-345. doi.org/10/1016/j/redox.2015.01.006 Baumann, L., 2002. Cosmetic Dermatology: Principles and Practice, McGraw Hill, Hong Kong. Becker, B.F., 1993. Towards the physiological function of uric acid. Free Radical Biol. Med. 14, 615-631. Beyer, T.A., Keller, U., Braun, S., Schafer, M., Werner, S., 2007. Roles and mechanisms of action of the Nrf2 transcription factor in skin morphogenesis, wound repair and skin cancer. Cell Death Differentiation. 14, 1250-1554. Bickers D.R., Athar, M., 2006. Oxidative stress in the pathogenesis of skin disease. J. Invest. Derm. 126, 2565-2575. doi: 10.1038/sj.jid.5700340 Bohn, T., 2010. Isoflavone bioavailability from foods and supplements dietary factors impacting utilization. Argo Food Industry Hi-Technology. 21, 59-62.

42

Borras, C., Gambini, J., Gomez-Cabrera, M.C., Sastre, J., Pallardo, F.V., Mann, G.E., Vina, J., 2005. 17beta-oestradiol up-regulates longevity-related, antioxidant enzyme expression via the ERK1 and ERK2[MAPK]/NFkappaB cascade. Aging Cell. 4, 113-118. Bottai, G., Mancina, R., Muratori, M., Di Gennaro, P., Lotti, T., 2012. 17β-estradiol protects human skin fibroblasts and keratinocytes against oxidative damage. J. European Acad. Derm. Venereology. 27, 1236-1243. doi: 10.111/j.1468-3083.2012.0469.x Brincat, M.P., Baron, Y.M., and R. Galea, R., 2005. Estrogens and the skin. Climacteric. 8, 110113. Burns, D.A., S. M. Breathnach, S.M., 2004. Rook‟s Textbook of Dermatology, 7th ed, Blackwell Publishing Co, New York, Vol 1, 3.1 and 4.1. Buonocore, G., Perrone, S., M. L. Tataranno, M.L., 2010. Oxygen toxicity: chemistry and biology of reaction oxygen species. Seminars Fetal Neonatal Biology. 15, 186-190. Burton G.J., Jauniaux, E., 2011. Oxidative stress. Best Practice Res. Clin. Obstet. Gynaec. 25, 287-299. doi: 10.1016/j.bpobgyn.2010.10.016 Chadha G., Sathigari, S., Parsons, D.L., Jayachandra, B.R., 2011. In vitro percutaneous absorption of genistein from topical gels through human skin. Drug Dev. Industrial Pharmacy. 37, 498-505. Chang, K.C., Wang, Y., Oh, I.G., Freedman, L.P., Thompson, C.C., Chung, J.H., Nagpal, S., 2010. Estrogen receptor beta is a novel therapeutic target for photoaging. Molec. Pharm. 77, 744750. Cheng, C., Wang, X., Weakley, S.M., Kougias, P., Lin, P.H., Yao, Q., Chen, C., 2010. The soybean isoflavonoid equol blocks Ritonavir-induced endothelial dysfunction in porcine

43

pulmonary arties and human pulmonary artery endothelial cells. J. Nutr. 140, 12-17. doi: 10.3945/jn.109.110981 Chignalia, A.Z., Schuldt, E.Z., Camargo, L.L., Montezano, A.C., G. E. Callera, G.E., F. R. Laurindo, F.R., Lopes, L.R., Avellar, M.C.W., Carvalho, M.H.C., Fortes, Z.B., Touyz, R.M., Tostes, R.C., 2012. Testosterone Induces Vascular Smooth Muscle Cell Migration by NADPH Oxidase and c-Src–Dependent Pathways. Hypertension. 59, 1263-1271. Cho, J-L., Allanson, M., Domanski, D., Arun, S.J., V. E. Reeve, V.E., 2008. Estrogen receptorbeta signaling protects epidermal cytokine expression and immune function for UVB-induced impairment in mice. Photochem. Photobiol. Sci. 7, 120-125. Cho, J-L., Allanson, M., Domanski, D., Arun, S.J., Reeve, V.E., 2010. Oestrogen receptor-beta signaling protects against transplanted skin tumor growth in the mouse. Photochem. Photobiol. Sci. 9, 608-614. Chung, J.E., Kim, S.Y., Jo, H.H., Hwang, S.J., Chae, B., Kwon, D.J., Lew, Y.O., Lim, Y.T., Kim, J.H. Kim, E.J., Kim, M.R., 2008. Antioxidant effects of equol on bovine aortic endothelial cells. Biochem. Biophy. Res. Comm. 375, 420-424. doi: 10.1016/j.bbrc.2008.08.027 Common, R. H., Ainsworth, L., 1961. Identification of equol in the urine of the domestic fowl. Biochimica et Biophysica Acta. 53, 403-404. http://dx.doi.org/10.1016/0006-3002(61)90452-8 Davinelli, S., Nadia, S., Visentin, M., Zella, D., Scapagnini, G., 2013. Enhancement of mitochondrial biogenesis with polyphenols: combined effects of resveratrol and equol in human endothelial cells. Immunity Aging. 10, 29. doi:10.1186/1742-4933-10-28 Degen, G.H., Balaszkewicz, M., Shi, L.J., Buyken, A.E., Remer, T., 2011. Urinary isoflavone phytoestrogens in German children and adolescents- a longitudinal examination in the DONALD cohort. Molec. Nutr. Food Res. 55, 359-367. http://dx.doi.org/10.1002/mnfr.201000325

44

Droge, W., 2002. Free radicals in the physiologic control of cell function. Physiol. Rev. 82, 4795. Djuric, Z., Chen, G., Doerge, D.R., Heilburn, L.K. Kucuk, O., 2001. Effect of soy isoflavone supplements on markers of oxidative stress in men and women. Cancer Letters. 172, 1-6. Draelos Z.D., Puglises, P.T., 2011. Physiology of the Skin, 3rd ed., Allurebooks, Carol Stream, IL, USA. Duncan, K.O., Leffell, D.J., 1997. Preoperative assessment of the elderly patient. Derm. Clinic. 15, 583-593. Escoffier, C., de Rigal, J., Rachdfore, A., Vasselet, R., Lefeque, J.L., Agache, P.G., 1989. Agerelated mechanical properties of human skin: an in vivo study. J. Invest. Derm. 93, 353-357. Evans J.A. Johnson, E.J., 2010. The role of phytonutrients in skin health. Nutrients. 2, 903–928. Farage, M.A., Miller, K.W., Elsener, P., H. I. Maibach, H.I., 2008. Intrinsic and extrinsic factors in skin aging: a review. Internat. J. Cosmetic Sci. 30, 87-95. Farage, M.A., Miller, K.W., Maibach, H.I., 2010. “Degenerative Changes in Aging Skin,” In: (Eds.), Farage, M.A., Miller, K.W., Maibach, H.I., Textbook of Aging Skin. Springer-Verlag, Berlin Heidelberg, pp. 225-235. Fazio, M.J., Olsen, D.R., Kuizaniemi, H., Chu, M.L., Davidson, J.M., Rosenbloom, J., Uitto, J., 1988. Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts. Lab. Invest. 58, 270-277. Fisher, G.J., Datta, S.C., Taylor, H.S., 1996. Molecular basis of sun induced premature skin aging and retionoid antagonism. Nature. 379, 335-339.

45

Frankenfeld, C.L., 2011. Dairy consumption is a significant correlate of urinary equol concentration in representative sample of US adults. Am. J. Clin. Nutr. 93, 1109-1116. http://dx.doi.org/10.3945/ajcn.111.011825 Freedberg, I.M., Eisen, A.Z., Wolff, K., Austen, K.E., Goldsmith, L.A., Katz, S.I., 2003. Fritzpatrick‟s Dermatology in General Medicine, 6th ed, McGraw Hill, New York. Friedman, O., 2005. Changes associated with the aging face. Facial Plastic Surg. Clin. North Am. 13, 371-380. Froyen. E.B., Steinberg, F.M., 2011. Soy isoflavones increase quinone reductase in heap-1c1c7 cells via estrogen receptor beta and nuclear factor erythroid 2-related factor 2 binding to the antioxidant response element. J. Nutr.Biochem. 22, 843-848. Fujimura, T., Haketa, K., Hotta, M., T. Kitahara, T., 2007. Loss of skin elasticity precedes to rapid increase of wrinkle levels. J. Derm. Sci. 47, 233-239. Ghosh, S., May, M.J., Kopp, E.B., 1998. NF-kB and Rel Proteins: evolutionarily conserved mediators of immune responses. Ann. Rev. Immunology.16, 225-260. doi: 10.1146/annurev.immunol.16.1.225 Gilchrest, B.A., 1989. Skin aging and photoaging: an overview. J. Am. Acad. Derm. 21, 610613. Gilchrest B.A., 2008. Sun exposure and vitamin D sufficiency. Am. J. Clin. Nutr. 88, 570S577S. Gilchrist, J.M., Moore, M.B., Andres, A., Estroff, J.A., Badger, T.M., 2010. Ultrasonographic patterns of reproductive organs in infants fed soy formula: comparisons to infants fed breast milk and milk formula. J. Pediatrics. 156, 215-220. http://dx.doi.org/10.1016/j.jpeds.2009.08.043

46

Gilmore, T.D., 2006. Introduction to NF-kB: players, pathways, perspectives. Oncogene. 25, 6680-6684. doi:10.1038/sj.onc.1209954 Gonzaga, E.R., 2009. Role of UV light in photoaging, skin aging, and skin cancer,” Am. J. Clin. Derm.10, Suppl. 1, pp. 19-24. Gopaul, R., Knaggs, H., Lephart, E.D., 2012. Biochemical investigation and gene analysis of equol: A plant and soy-derived isoflavonoid with anti-aging and anti-oxidant properties with potential human skin applications. Biofactors. 38, 44-52. Gordon, J.R.S., Brieva, J.C., 2012. Unilateral dermatheliosis. New England J. Med. 366, e25. doi: 10.1056/NEJMicm1104059 Gou, Z., Jiang, S., Zheng, C., Tian, Z., X. Lin, X., 2015. Equol inhibits LPS-induced oxidative stress and enhances the immune response in chicken HD11 macrophages. Cell. Physio. Biochem. 36, 611-621. doi:10.1159/000430124 Goukassian, D., Gad, F., Yaar, M., Eller, M.S., Nahal, U.S., Gilchrest, B.A., 2000. Mechanisms and implications of the age-associated decrease in DNA repair capacity. FASEB J. 14, 13251334. Greenwald, M.B.Y., Frusic-Zlotkin, M., Soroka, Y., Sasson, S.B., Bianco-Peled, H., Bitton, R., Kohen, R., 2015. Nitroxide delivery system for Nrf2 activation and skin protection,” Euro. J. Pharm. Biopharmaceutics. 94, 123-134. doi.org/10.1016/j.ejpb.2015.05.008 Gruber, F., Ornelas, C.M., Karner, S., Narzt, M.S., Nagelreiter, M.I., Gschwandtner, M., Bochkov, V., Tschachler, E., 2015. Nrf2 deficiency causes lipid oxidation, inflammation, and matrix-protease expression in DHA-supplemented and UVA-irradiated skin fibroblasts. Free Radical Biol. Med. 88, 439-451. doi.org/10.1016/j/freeredbiomed.2015.05.006

47

Gupta, S.C., Sundaram, C., Reuter, S., Aggarwal, B.B., 2010. Inhibiting NFkappaB activation by small molecules as a therapeutic strategy.” Biochim et Biophysics Acta. 1799, 775-787. doi: 10.1016/k.bbagrm.2010.05.004 Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine, 3rd edition, Oxford University Press, New York, pp. 617-783. Hamilton-Reeves, J.M., Vazque, G., Duval, S.J., Phipps, W.R., Kurzer, M.S., Messina, M.J., 2010. Clinical studies show no effects of soy protein or isoflavones on reproductive hormones in men: results of a meta-analysis. Fertil. Steril. 94, 997-1007. http://dx.doi.org/10.1016/j.fertnstert.2009.04.038 Hayden, M.S., Ghosh, S., 2012. NF-kB, the first quarter-century: remarkable progress and outstanding questions. Genes Dev. 26, 203-234. doi: 10.1101/gad.183434.111 Hellemans, L., Cortstjens, H., Neven, A., Declercq, L., Maes, D., 2003. Antioxidant enzyme activity in human stratum corneum shows seasonal variations with an age-dependent recovery. J. Invest. Derm. 120, 434-439. Hillebrand, G.G., 2010. Facial Wrinkling: The Marquee Clinical Sign of Aging Skin. In: Farage, M.A., Miller, H.I., Maibach, H.I. (Eds.), Textbook of Aging Skin. Springer-Verlag, Berlin, Heidelberg, pp. 911-918. Hirvonen, J., Rajalin, A.M., Wohlfart, G., Adlercreutz, H., Wähälä, K., Aarnisalo, P., 2010. Transcriptional activation of estrogen-related receptor gamma (ERRgamma) is stimulated by the phytoestrogen equol. J. Steroid Biochem. Molec. Biol. 123, 46-57. doi: 10.1016/j/jsbmb.2010.11.001

48

Hoikkälä, A., Mustonen, Saastamoinen, S.I., Jokela, T., Taponen, J., Saloniemi, H., Wähälä, K., 2007. High levels of equol in organic skimmed Finnish cow milk. Molec. Nutr. Food Res. 51, 782-786. http://dx.doi.org/10.1002/mnfr.200600222 Hornebeck, W., 2003. Down-regulation of tissue inhibitor of matrix metalloprotease-1 (TIMP-1) in aged human skin contributes to matrix degradation and impaired cell growth and survival. Pathologie Biologie. 51, 569-573. Hounsome, N., Hounsome, B., Deri Tomos, A., Edwards-Jones, G., 2009. Changes in antioxidant compounds in white cabbage during winter storage. Postharvest Biol. Tech. 52, 173-179. Hounsome, H., Grail, B., Deri Tomos, A., Hounsome, B., Edwards-Jones, G., 2010. Highthroughput anti-oxidant profiling in vegetables by Fourier-transform ion cyclotron resonance mass spectrometry. Functional Plant Sci. Biotech. 4, 1-10. Huang, C., Schmid, P.C., Ma, W.Y., Schmidt, H.H., Dong, Z., 1997. Phosphatidylinositol-3 kinase is necessary for 12-O-tetradecanoylphorbol 13-acetate-induced cell transformation and activated protein 1 activation. J. Biol. Chem. 272, 4187-4194. Hung, C-F., Lin, Y-K., Huang, Z-R., Fang, J.Y., 2008. Delivery of resveratrol, a red wine polyphenol, for solutions and hydrogels via the skin. Biol. Pharm. Bulletin. 31, 955-962. Inoue, T., Miki, Y., Abe, K., Haton, M., Hosaka, M., Kariya, Y., Kakuo, S., Fujimura, T., Hachiya, A., Aiba, S., Sasano, H., 2011. The role of estrogen-metabolizing enzymes and estrogen receptors in human epidermis. Molec. Cell. Endocrinol. 344, 35-40. doi: 10.1016/j.mce.2011.06.015. Jackson, R.L., Greiwe, G.S., Schwen, R.J., 2011. Ageing skin: oestrogen receptor β agonists offer an approach to change the outcome. Exp. Derm. 20, 879-882.

49

Jackson, R.L., Greiwe, J.S. Schwen, R.J., 2014. S-equol, an antioxidant metabolite of soy dadizein, and oxidative stress in aging: a focus on skin and the cardiovascular system. In: Aging: Oxidative Stress and Dietary Antioxidants, UK, Elsevier Inc., London, pp. 145-155. Jain, T., Klein, N.A., Lee, D.M., Sluss, P.M., Soules, N.R., 2003. Endocrine assessment of relative reproductive age in normal eumenorrheic younger and older women across multiple cycles. Am. J. Obste. Gynec. 189, 1080-1084. Jou, H-J., Tsai, P.-J., Tu, J-H., Wu, W-H., 2013. Stinky tofu as a rich source of biavailable Sequol in Asian diets. J. Funct. Foods, 5, 651-659. Kammeyer, A., Luiten, R.M., 2015. Oxidative events and skin aging. Ageing Res. Rev. 21, 1629. doi.org/10.1016/j.arr.2015.01.001 Kamyama, M., Kishimoto, Y., Tani, M., Utsunomiya, K., Kondo, K., 2009. Effects of equol on oxidized low-density lipoprotein-induced apoptosis in endothelial cells. J. Atherosclerosis Thrombosis.16, 239-249. Kang, J.S., Yoon, Y.D., Han, M.H., Han, S.B., Lee, K., Kang, M.R., Moon, E.Y., Jeon, Y.J., Park, S.K., Kim, H.M., 2005. Estrogen receptor-independent inhibition of tumor necrosis factoralpha gene expression by phytoestrogen equol is mediated by blocking nuclear factor-kappa B activation in mouse macrophages. Biochem. Pharm. 71, 136-143. doi: 10.1016/j/bcp.2005.10.009 Kang, N.J., Lee, K.W., Rogozin, E.A., Cho, Y-Y., Heo, Y-S., Bode, A.M., Lee, H.J., Dong, Z., 2007a. Equol inhibits neoplastic cell transformation by targeting the MEK/ERK/p90RSK/ activator protein – 1 pathway. J. Biol. Chem. 45, 32856-32866.

50

Kang, J.S., Yoon, Y.D., Han, M.H., Han, S.D., Lee, K., Park, S.K., Kim, H.M., 2007b. Equol inhibits nitric oxide production and inducible nitric oxide synthase gene expression through down-regulating the activation of Akt. Internat. Immunopharmacology. 7, 491-499. doi: 10.1016/j.intimp.2006.12.004 Khol, E., Steinbauer, J., Landthaler, M., Szeimies, R.M., 2011. Skin Aging. J. Euro. Acad. Derm. Venereology. 25, 873-884. King, R.A., Mano, M.M., Head, R.J., 1998. Assessment of isoflavonoid concentrations in Australian bovine milk samples. J. Dairy Res. 65, 479-489. http://dx.doi.org/10.1017/S0022029998002891 Kligman, A.M., Koblenzer, C., 1997. Demographics and psychological implications for the aging population. Derm. Clin. 15, 549-553. Kohen, R. Nyska, A., 2002. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Tox. Pathology. 30, 620-650. doi: 10.1080/01926230290166724 Krahn-Bertil, E., Bolzinger, M.A., Andre, V., Orly, I., Kanitakis, J., Rousselle, P., Damour, O., 2008. Expression of estrogen-related receptor gamma (ERRgamma) in human skin. Euro. J. Derm. 18, 427-432. Krishnamurthy, P., Wadhwani, A., 2012. “Antioxidant Enzymes and Human Health, chapter 1,” In: El-Missiry, M.A. (Ed.), Antioxidant Enzyme- InTech Science, Technology & Medicine, Vienna, Austria, pp. 4-18. doi: 10.5772/48109. Kuwazuru, O., Saothong, J., and Y. Yoshikawa, Y.Y., 2008. Mechanical approach to aging and wrinkling of human facial skin based on the multistage buckling theory. Med. Eng. Physics. 30, 516-522.

51

Lacher, S.E., Lee, J.S., Wang, X., Campbell, M.R., Bell, D.A., 2015. Beyond antioxidant genes in the ancient Nrf2 regulatory network. Free Radical Biol. Med. 88, 452-465. doi.org/101016/j.freeradbiomed.2015.06.044 Lee, M.J., Cha, J.H., Lim, K.M., Lee, O-K., Bae, S., Kim, C-H., Lee, K-H., Lee, Y.N., Ahn, K.J., An, S., 2015. Analysis of the microRNA expression profile of normal human dermal papilla cells treated with 5α-dihydrotestosterone. Molec. Med. Reports. 12, 1205-1212. doi: 10.3892/mmr.2015.3478 Lephart, E.D., 2009. Review Human Biology. Kendell/Hunt, Dubuque, Iowa., pp. 68-94. Lephart, E.D., 2013a. Protective effects of equol and their polyphenolic isomers against dermal aging: Microarray/protein evidence with clinical implications and unique delivery into human skin. Pharm. Biol. 51, 1391-1400. Lephart, E.D., 2013b. Isoflavones and prenatal exposure to equol. In: Preedy, V.R. (Ed.), Isoflavones: Chemistry, Analysis, Function. The Royal Society of Chemistry, Thomas Graham House, Science Park, Cambridge, England, UK, pp. 480-499. Lephart, E.D., 2014a. Review: Antioxidant and anti-aging properties of equol in prostate health (BPH). Open J. Endo. Metab. Disorders. 4. doi: 10.4236/ojemd.2014.41001. Lephart, E.D., 2014b. Safety assessment of R,S-equol as a dietary supplement for benign prostatic hyperplasia. Proc. Soc. Tox. pbn. 1585, p. 244. Lephart, E.D., Summerfeldt, J.M., Andrus, M.B., 2014. Resveratrol: Influences on gene expression in human skin. J. Funct. Foods. 10, 377-384. Lephart, E.D., 2015. Modulation of aromatase by phytoestrogens. Enzyme Res. article ID 594656, 2015, doi: 10.1155/2015/594656

52

Li, H., Tsao, R., Deng, A., 2012. Factors affecting the antioxidant potential and health benefits of plant foods. Canadian J. Plant Sci. 92, 1101-1111. doi:10.4141/CJPS2011-239 Lobo, V., Patil, A., Phatak, A., Chandra, N., 2010. Free radicals, antioxidants and functional foods: impact on human health. Pharmacognosy Review. 4, 118-126. Lund, T.D., Blake, C., Bu, L., Hamaker, A.N., Lephart, E.D., 2011. Equol an isoflavonoid: potential for improved prostate health, in vitro and in vivo evidence. Reprod. Biol. Endocrinol. 9, 4. doi:10.1186/1477-7827-9-4 Lundh, T.J.O., Petersson, H.I., Martinsson, K.A., 1990. Comparative levels of free and conjugated plant estrogens in blood plasma of sheep and cattle fed oestrogenic silage. J. Agr. Food Chem. 38, 1530-1534. http://dx.doi.org/10.1021/jf00097a022 Ma, Y., Sullivan, C., Schreihofer, D.A., 2010. Dietary genistein and equol (4‟,7 isoflavandiol) reduce oxidative stress and protect rats against focal cerebral ischemia. Am. J. Physiol.- Reg. Integrative Comp. Physiol. 299, R871-R877. doi:10.1152/ajpregu.00031.2010 Madison, K.C., 2003. Barrier function of the skin: „la raison d‟etre‟ of the epidermis. J. Invest. Derm.121, 231-241. Maes, M., Galecki, P., Chang, Y.S., Berk, M., 2011. A review on the oxidative and nitrosative (O&NS) pathways in major depression and their possible contribution to the (neuro)degenerative process in that illness. Prog. Neuro-Psychpharm. Biol. Psychia. 35, 676-692. doi:10.1016/j.pnpbp.2010.05.004 Magee, P.J., Raschke, M., Steiner, C., Duffin, J.G., Pool-Zobel, B.L., Jokela, T., Wähälä, K., Rowland, R.I., 2006. Equol: a comparison of the effects of the racemic compound with that of the purified S-enantiomer on the growth, invasion, and DNA integrity of breast and prostate cells in vitro. Nutr. Cancer. 54, 232-242.

53

Makrantonaki, E., Zouboulis, C.C., 2009. Androgens and ageing of the skin. Curr. Opinion Endocrinol. Diabetes Obesity. 16, 240-245. doi: 10.1097/MED.0b013e32832b71dc Mann, G.E., Bonacasa, B., Ishii, T., Siow, R.C.M., 2009. Targeting the redox sensitive Nrf2Keep1 defense pathway in cardiovascular disease: protection afforded by dietary isoflavones. Curr. Opinion Pharmacology. 9, 139-145. Marconi, A., Terracina, M., Fila, C., Franchi, J., Bonte, F., Romagnoli, G., Maurelli, R., Failla, C.M., Dumas, M., Pincelli, C., 2003. Expression and function of neurotrophins and their receptors in cultured human keratinocytes. J. Invest. Derm. 121, 1515-1521. doi: 10.1111/j.1523-1747.2003.12624.x Maritim, A.C., Sanders, R.A., Watkins III, J.B., 2003. Diabetes, oxidative stress, and antioxidants: A review. J. Biochem. Molec. Tox. 17, 24-38.

doi: 10.1002/jbt.10058

Markiewicz, M. Znoyko, S., Stawski, L., Ghatnekar, A., Gilkeson, G., Trojanowska, M., 2013. A role for estrogen receptor-α and estrogen receptor-β in collagen biosynthesis in mouse skin. J. Invest. Derm. 133, 120-127. Martini, F., 2004. Fundamentals of Anatomy and Physiology, San Francisco, CA, BenjaminCummings. Mazur, W., Aldercreutz, H., 2000. Overview of naturally occurring endocrine-active substances in the human diet in relation to human health. Nutrition. 16, 654-658. Meadows, C., Moore, D.J., Moore, D.M., Draelos, Z.D., Kern, D., 2014. Age-related NADH oxidase (arNOX)-catalyzed oxidative damage to skin protein. Arch. Derm. Res.306, 645-652. doi: 10.1007/s00403-014-1472-8 Menon, G.K., 2002. New insights into skin structure: scratching the surface. Adv.Drug Delivery Review. 54, S3-S17.

54

Mera, S.L., Lovell, C.R., Russell Jones, R., Davies, J.D., 1987. Elastic fibers in normal and sundamaged skin: an immunohistochemical study. Br. J. Derm. 117, 21-27. Mitchell, J.H., Gardner, P.T., McPhail, D.B., Morrice, P.C., Collins, A.R., Duthie, G.C., 1998. Antioxidant efficacy of phytoestrogens in chemical and biological model systems. Arch. Biochem. Biophysics. 360, 142-148. Natarajan, V.T., Ganju, P., Ramkumar, A., Grover, R., Gokhale, R.S., 2014. Multifaceted pathways protect human skin from UV radiation. Nature Chem. Biol. 10, 542-551. doi:10.1038/nchembio.1548 National Institutes of Health, Office of Dietary Supplements, Vitamin D, 2016. https/ods.od.nih.gov/factsheets/vitamin-healthprofessional/#h4. Naylor, E.C., Watson, R.E.B., Sherratt, M., 2011. Molecular aspects of skin aging. Maturitas. 69, 249-256. doi: 10.1016/j.maturitas.2011.04.011 Nithy, M., Suguna, S., Rose, C., 2003. The effect of nerve growth factor on the early responses during the process of wound healing. Biochimica et Biophysica Acta. 1620, 25-31. Nitsch, S.M., Wittmann, F., Angele, P., Wichmann, M.W., Hatz, R., Hernandez-Richter, T., Chaudry, I.H., Jauch, K.W., Angele, M.R., 2004. Physiological levels of 5 alphadihydrotesterone depress wound immune function and impair wound healing following traumahemorrhage. Arch. Surgery, 139, 157-163. Nkengne. A., Bertin, C., 2012. Aging and facial changes-documenting clinical signs. Part 1: clinical changes of the aging face. SkinMed. 10, 284-289. Ortman, J.M., Velkoff, V.A., Hogan, H., 2014. An Aging Nation: The Older Population in the United States. US Bureau of the Census, May.

55

Oyama, A., Ueno, T., Uchiyama, S., Aihara, T., Miyake, A., Kondo, S., Matsunage, K., 2012. The effects of natural S-equol supplementation on skin aging in postmenopausal women: a pilot randomized placebo-controlled trail. Menopause. 19, 202-210. doi: 10.1097/gme.0b013e318227427b. Pandel, R., Poljsak, B., Godic, A., Dahmane, R., 2013. Skin photoaging and the role of antioxidants in its prevention. ISRN Dermatology. article ID 930164. http://dx.doi.org/10.1155/2013/930164 Park, E-J., Pezzuto, J.M., 2015. The pharmacology of resveratrol in animals and humans. Biochimica et Biophysica Acta. 1852, 1071-1113. Park, G., Oh, M.S., 2015. Acceleration of heat shock-induced collagen breakdown in human dermal fibroblasts with knockdown of NF-E2-related factor 2. BMB Reports. 48, 467-472. dio.org/10.5483/BMBRep.2015.48.8.215 Pelletier, G., Ren, L., 2004. Localization of sex steroid receptors in human skin. Histo. Histopathology. 19, 629-636. Peng, Y., Xuan, M., Leung, V.Y. L., Cheng, B., 2015. Stem cells and aberrant signaling of molecular systems in skin aging. Ageing Res. Rev. 19, 8-21. doi: 10.1016/j.arr.2014.10.006 Phillips, T.J., Symons, J., Menon, S., 2008. HT Study Group. Does hormone therapy improve age-related skin changes in postmenopausal women? J. Am. Acad. Derm. 59, 397-404. Pierard, G.E., Paquet, P., Xhauflaire-Uhoda, E., Quatresooz, P., 2010. Physiological Variations During Aging. In: Farage, M.A., Miller, K.W., Maibach, H.I. (Eds.), Textbook of Aging Skin. Springer-Verlag, Berlin Heidelberg, pp. 45-54. Poljsak, B., Dehmane, R., 2012. Free radicals and extrinsic skin aging. Derm. Res. Practice. article ID: 135206.

doi: 10.1155/2012/135206

56

Pomari, E., Valle, L.D., Pertile, P, Colombo, L. Thorton, M.J., 2015. Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts. FASEB J. 29, 508-524. doi: 10.1096/fj.14-251363 Pusparini, Y., Hidayat, A., 2015. Effect of soy isoflavone supplementation on endothelia dysfunction and oxidative stress in equol-producing postmenopausal women. Endocrine Metabolic Immune Disorders – Drug Targets. 15, 71-79. doi:10.2174/1871530314666141202123309 Qin, Z., Voorhees, J.J., Fisher, G.J., Quan, T., 2014. Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts. Aging Cell. 13, 1028-1037. doi: 10.111.acel.12265 Quan, C., Cho, M.K., Perry, D., Quan, T., 2015. Age-associated reduction of cell spreading induces mitochondrial DNA common deletion by oxidative stress in human skin dermal fibroblasts: implication for human skin connective tissue aging. J. Biomed. Sci. 22, 62. doi: 10.1186/s 12929-105-0167-6 Qunshan, J., Nash, J.F., 2010. Pathology of aging skin. In: Farage, M.A., Miller, K.W., Maibach, H.I. (Eds.), Textbook of Aging Skin, Springler-Verlag, Berlin Heidelberg, pp. 277291. Rabe, J.H., Mamelak, A.J., McElgunn, P.J., Morison, W.L., Sauder, D.N., 2006. Photoaging: mechanisms and repair. J. Am. Acad. Derm. 55, 1-19. Ramara, A., Duckles, S.P., Krause, D.N., Proaccio, V., 2007. Estrogen suppresses brain mitochondrial oxidative stress in female and male rats. Brain Res. 1176, 71-81.

57

Rhie, G., M. Shin, M.H., Seo, J.Y., Choi, W.W., Kim, K.H., Park, K.C., Eun, H.C., Chung, J.H., 2001. Aging- and photoaging-dependent changes of enzymic and nonenzymic antioxidants in the epidermis and dermis of human skin in vitro. J. Invest. Derm.117, 1212-1217. Rice-Evans, C., 2001. Flavonoid antioxidants. Curr. Med. Chem. 8, 797-807. Rittie, L., Fisher, G.J., 2002. UV-light-induced signal cascades and skin aging. Ageing Res. Rev. 1, 705-720. Robinson, J.K., Amonette, R., Wyatt, S.W., Bewerse, B.A., Bergfeld, W.F., Farris, P.K., 1998. Executive summary of the national „Sun Safety: Protecting Our Future‟ conference. American Academy of Dermatology and Centers for Disease Control and Prevention. J. Am. Acad. Derm. 38, 774-780. Rothwell, J.A., Day, A.J., Morgan, M.R., 2005. Experimental determination of octanol-water partition coefficients of quercetin and related flavonoids. J. Agr. Food Chem. 53, 4355-4360. Rowlands, D.J., Chapple, S., Siow, R.C.M., Mann, G.E., 2011. Equol-stimulated mitochrondrial reactive oxygen species activate endothelial nitric oxide synthase and redox signaling in endothelial cells, roles for F-actin and GPR30. Hypertension 57, 833-840. Rufer, C.E., Kulling, S.E., 2006. Antioxidant activity of isoflavones and their major metabolites using different in vitro assays. J. Agr. Food Chem. 54, 2926-2931. Schreck, R., Rieber, R., Baeuerle, P.A., 1991. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kB transcription factor and HIV-1. EMBO J. 10, 2247-2258. Schwartz, D., Mayaux, M.J., 1982, Female fecundity as a function of age: results of artificial insemination in 2193 nulliparous women with azoospermic husbands. New England J. Med. 306, 404-406.

58

Seite, S., Zucchi, H., Sepiter, D., Igondjo-Tchen, S., Senni, K., Godeau, G., 2006. Elastin changes during chronological and photo-ageing: the important role of lysozyme. J. Euro. Acad. Derm. Venereology. 20, 980-987. doi: 10.1111/j.1468-3083.2006.01706.x Sekhar. K.R., Freeman, M.L., 2015. Nrf2 promotes survival following exposure to ionizing radiation. Free Radical Biol. Med. 88. 268-274. doi.org/10.1016/j/freeredbiomed.2015.04.035 Seo, J.Y., Chung, J.H., 2006. Thermal aging: a new concept of skin aging. J. Derm. Sci. Suppl. 2, S13-S22. Setchell, K.D.R., Clerici, C., Lephart, E.D., Cole, S.J., Heenan, C., Castellani, D., Wolfe, B.E., Nechemias-Zimmer, L., Brown, N.M., Lund, T.D., Handa, R.J., 2005. S-Equol, a potent ligand for estrogen receptor {beta}, is the exclusive enantiomeric form of the soy isoflavone metabolite produced by human intestinal bacterial flora. Am. J. Clin.Nutr. 81, 1072-1079. Setchell, K.D.R., Zhao, X., Jah, P., Heubi J.E., Brown, N.M., 2009. The pharmacokinetic behavior of the soy isoflavone metabolite S-(-) equol and its diastereoisomer R-(+)equol in healthy adults determined by using stable-isotope-labeled tracers. Am. J. Clin. Nutr. 90, 10291037. Setchell, K.D.R., Clerici, C., 2010. Equol: history, chemistry, and formation. J. Nutr.140, pp. 1355S-1362S. Shindo, Y., Witt, E., Han, D., Epstein, W., Packer, L., 1994. Enzymic and non-enzymic antioxidants in epidermis and dermis in human skin. J. Invest. Derm. 102, 122-124. Spiro, A., Buttriss, J.L., 2014. Vitamin D: An overview of vitamin D status and intake in Europe. Nutr. Bull. 39, 322-350.

59

Strickland, I., Rhodes, L.E., Flanagan, B.F., Friedmann, P.S., 1997. TNFα and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neurtrophil accumulation and E-selection expression. J. Invest. Derm. 108, 763-768. Takahashi, Y., Moriwaki, S., Sugiyama, Y., Yamazaki Y.E-K., Mori, T., Takigawa, M., 2005. Decreased gene expression responsible for post-ultraviolet DNA repair synthesis is aging: a possible mechanism of age-related reduction in DNA repair capacity. J. Invest. Dermat. 124, 435-442. Thiele, J.J., Traber, M.G., Packer, L., 1998. Depletion of human stratum corneum vitamin E: an early and sensitive in vivo marker of UV induced photo-oxidation. J. Invest. Derm.110, 756-761. Thornton, M.J., Taylor, A.H., Mulligan, K., Al-Azzawi, F., Lyon, C.C., O‟Driscoll, J., Messenger, A.G., 2003. Oestrogen receptor beta is the predominant oestrogen receptor in human scalp skin. Exp. Derm. 12, 181-190. Tostes, R.C., Carneiro, F.S., Carvalho, M.H.C., Reckelhoff, J.F., 2016. Reactive oxygen species: players in the cardiovascular effects of testosterone. Am. J. Physiology-Regulatory Integrative Comp. Physiology. 310, R1-R14 Tulah, A.S., Birch-Machin, M.A., 2013. Stressed out mitochondria: The role of mitochondria in ageing and cancer focusing on strategies and opportunities in human skin. Mitochondrion. 13, 444-453. doi:10.1016/j.mito.2012.11.007. Uitto, J., 2008. The role of elastin and collagen in cutaneous aging: intrinsic aging versus photoexposure. J. Drugs Derm. 7, S12-S17. Valko, M., Leibfritz, D., Moncol, J., Mazur, M.T., J. Telser, J., 2007. Free radicals and antioxidants in normal physiological functions and human disease. Internat. J. Biochem. Cell Biol. 39, 44-84.

60

Velarde, M.C., Flynn, J.M., Day, N.U., Melvo, S., Campisi, J., 2012. Mitochondrial oxidative stress caused by SOD deficiency promotes cellular senescene and aging phenotypes in the skin. Aging. 4, 3-12. Walker, S.L., Hawk, J.L.M., Young, A.R., 2003. Acute and chronic effects of ultraviolet radiation on the skin. In: Freedberg, IM., Eisen, A.Z., Wolff, K.F., Austen, L.A., Goldsmith, L.A, Katz, S.I. (Eds.), Fitzpatrick‟s Dermatology in General Medicine, 6th edition, McGraw-Hill New York, pp. 1275-1282. Wang, S., Moustaid-Moussa, N., Chen, L., Mo, H., Shastri, A., Su, R., Bapat, P., Kwun, I., Shen, C-L., 2014. Novel insights of dietary polyphenols and obesity. J. Nutr. Biochem. 25, 1–18. Warren, R., Gartstein, V., Kligman, A.M., Montagna, W., Allendorf, R.A., Riddler, G.M., 1991. Age, sunlight, and facial skin: a histologic and quantitative study. J. Am. Acad. Derm. 25, 751760. Weber, C., Podda, M., Rallis, M., Thiele, J.J., Traber, M.G., Packer, L., 1997. Efficacy of topically applied tocophenols and tocotrenols in protection of murine skin from oxidative damage induced by UV-irradiation. Free Radical Biol. Med. 22, 761-769. Wend, K., Wend, P., Krum, S.A., 2012. Tissue-specific effects of loss of estrogen during menopause and aging. Front. Endocrinol. (Lausanne). 3, 1-19. Widyarini, S., Domanski, D., Painter, N., Reeve, V.E., 2006a. Estrogen receptor signaling protects against immune suppression by UV radiation exposure. Proc. National Acad. Sci. (USA). 103, 12837-12842. doi/10.1073/pnas.0603642103 Widyarini, S., Allanson, M., Gallagher, N.L., Pedley, J., Boyle, G.M., Parsons, P.G., Whiteman, D.C., Walker, C., Reeve, V.E., 2006b. Isoflavonoid photoprotection in mouse and human skin is dependent on metallothionein. J. Invest. Derm.126, 198-204. doi: 10.1038/sj.jid. 5700013

61

Widyarini, S., Domanski, D., Painter, N., Reeve, V.E., 2012. Photoimmune protective effect of the phytoestrogenic isoflavonoid equol is partially due to its antioxidant activities. Photochem. Photobiol. Sci. 11, 1186-1192. doi: 10.1039/c2pp25022e Xing, M.H., Hui, X., Zhong, W., 2009. In vitro human topical bioactive drug transdermal absorption: Estradiol. Cutaneous Ocular Tox. 28, 171-175. Yaar, M., Grossman, K., Eller, M., Gilchrest, B.A., 1991. Evidence for nerve growth factormediated paracrine effects in human epidermis. J. Cell Biol. 115, 821-828. Yoon, G-A., Park, S., 2014. Antioxidant action of soy isoflavones on oxidative stress and antioxidant enzyme activities in exercised rats. Nutr. Res. Prac. 8, 618-624. Zhai, S., Yarr, M., Doyle, S.M., Gilchrest, B.A., 1996. Nerve growth factor rescues pigment cells from ultraviolet-induced apoptosis by upregulating BCL-2 levels. Exp. Cell Res. 224, 335-343. Zhang, T., Liang, X.Y., Shi, L.Y., Wang, L., Chen, J.L., Kang, C., Zhu, J.D., Mi, M.T., 2013. Estrogen receptor and PI3K/Akt signaling pathway involvement in S-equol-induced activation of Nrf2/ARE in endothelia cells. PLOS One. 8, 2013, doi: 10.1371/journal.pone.0079075 Zhong, W., Xing, M.M.Q., Hui, X., Maibach, H.I., 2009. A stochastic model for transepidermal drug delivery. Skin Res. Tech. 15, 407-411. Zouboulis, C.C., Makrantonaki, E., 2011. Clinical aspects and molecular diagnostics of skin aging. Clin. Derm. 29, 3-14. doi:10.1016/j.clindermatol.2010.07.001

62

Figure legends

Figure 1. Electron structures of some common reactive oxygen species (ROS). Each structure is provided with its name and chemical formula. The red ● indicates an unpaired electron. Molecular oxygen (O2) becomes Superoxide (●O2-) with the loss of an electron. Hydrogen Peroxide (H2O2) becomes Peroxide (●O2-2) with the loss of two electrons. Superoxide Dismutase (SOD) converts Superoxide into either Molecular Oxygen or Hydrogen Peroxide and then Catalase (CAT) converts Hydrogen Peroxide into Water and Molecular Oxygen (e.g., 2H2O2 → 2H2O + O2).

63

Figure 2. A cartoon graphic displaying the three skin layers in humans. Keratinocytes are the major cellular type in the epidermis. Melanocytes near the epidermal-dermal junction provide pigmentation of the skin for photo-protection. In the dermis, the blue horizontal bars represent collagen fibers while the green vertical bars indicate elastin fibers.

64

Figure 3. Production of estrogen, collagen/elastin and elasticity profiles/patterns in women with age. Estrogen levels peak in the late twenties, while collagen and elastin peak around age 30. Elasticity (mechanical “bounce back or recoil”) is greatest in very young individuals (e.g., at 10 years of age), which continually decline with age. Profiles were generated via composite data from references (Escoffier et al., 1989; Hillebrand, 2010; Jain et al., 2003; Schwartz and Mayaux, 1982; Seite et al., 2006; Uitto, 2008).

65

Figure 4. A 21 year-old women (left panel) experienced an allergic food reaction, and all elastin was selectively destroyed resulting in rapid skin damage and aging over a period of a few years (right panel at age 26) as reported by Anderson (2012). Photo source: http://www.telegraph.co.uk/news/newstopics/howaboutthat/8826277/Mystery-condition-makeswoman-age-50-years-in-just-a-few-days.html, October 14, 2011.

66

Figure 5. The solar radiation spectrum and the influences of ultra violet (UV) light on skin aging. Brief descriptions of UVC, UVB and UVA characteristics are shown especially in reference to specific amounts and patterns of UV penetration into the skin and the potential damage each can cause with exposure. For instance, UVC rays do not reach the surface of the earth. They are blocked by the ozone layer of the earth‟s atmosphere (Walker et al., 2003). UVA and UVB make up 95-98 % and 2-5 %, respectively, of the UV radiation reaching human skin (Walker et al., 2003). UVB rays penetrated into the epidermal cells can damage DNA and activate the ROS cascade of events leading to photo-aging. UVA rays while also damaging the epidermis penetrate deeper into the dermis to degrade collagen and elastin fibers via oxidative stress and activating MMPs. UVA is more cytotoxic in skin than UVB rays.

67

Figure 6. Photograph of a 69-year-old man that drove commercial trucks for 28 years that displays the effects of extrinsic or photo-aging. The left-side of this man‟s face (driver‟s side) shows dramatic photo-aging, while the right side of his face (protected from UVR), shows minimal photo-aging. This is a clear example of unilateral dermatheliosis from photo-aging. Reproduced with permission from Massachusetts Medical Society License to E.D. Lephart (Gordon and Brieva, 2012).

68

Figure 7. Chronological aging via oxidative metabolism and photo-aging by exposure to UV light (extrinsic aging) through cellular/molecular signaling mechanisms is shown. The cascade events including the major impact of oxidative stress via the generation of reactive oxygen species (ROS) is displayed in reference to the appearance of damaged skin and wrinkles due to changes in human dermal structural proteins (collagen and elastin). Pro-inflammatory Transcription Factor NF-kappB (NFkappaB), AP-1 is a nuclear transcription element, Activator Protein – 1 (AP-1) and Transforming Growth Factor beta (TGFβ)

69

Figure 8. Steroid hormone enzymes, steroid hormones and steroid receptors in the epidermal and dermal layers during aging. Estrogen receptor beta (ER β) is the predominant estrogen receptor subtype in human skin. The photomicrograph displays ER β (rust-colored) staining in the keratinocytes in the epidermis and fibroblasts in the dermis. The aromatase enzyme is also present in dermal fibroblasts that convert androgens and estrogens (not shown). Also, the 5αreductase type I enzyme in fibroblasts converts testosterone (T) into 5α-dihydrotestosterone (DHT), which in turn binds to the androgen receptor (AR) that has negative effects on skin. For example, androgen hormonal actions via the AR are known to stimulate matrix metalloproteinases (MMPs) and decrease wound healing. Whereas, estrogenic hormonal actions via ER β are known to decrease MMPs and enhance wound healing. Notably, ER α expression in human skin is expressed at lower levels compared to ER β expression (not shown). While ER α expression does not change with menopause, ER β expression declines by approximately 15 to

70

20 %, thus decreasing the positive action of this estrogen receptor subtype for skin health. Information via composite data from references (Brincat et al., 2005; Gopaul et al., 2012; Inoue et al., 2011; Lephart 2013a; Lephart 2015; Makrantonaki and Zouboulis, 2009; Nitsch et al., 2004; Pelletier and Ren, 2004; Pomari et al., 2015; Thorton et al., 2003).

71

Figure 9. Chronological and photo-aging is shown by split-face photographs of a 23-year-old Caucasian women (left panel) compared to the same women at 61 years old (right panel). The right panel photograph shows: sagging jaw line, changes in lip thickness and cheek fullness, skin texture, the dermal expression displays solar elastosis and wrinkles with dull skin color and uneven skin tone. Reproduced with permission from MedSkin (Nkengne and Bertin, 2012).

72

Figure 10. Chemical structures, formulas and molecular weights (MW) of Equol (panel A) and of 17β-Estradiol (panel B). The 2-dimensional chemical structures are shown in the top half of the figure, while the bottom half displays the 3-dimensional chemical structures of Equol and 17β-Estradiol (PubChem). While both Equol and 17β-Estradiol contain aromatic rings, 17βEstradiol is a steroid hormone while Equol is not. Also, Equol has a chiral center at carbon 3 (see blue circle, panel A), and thus can exist in two mirror image forms known as enantiomers (i.e., S-equol and R-equol). Racemic equol refers to exact equal portions of S-equol and R-equol (Lephart, 2013b). The red spheres indicate oxygen atoms, blue spheres designate carbon atoms and, the small green spheres are hydrogen atoms.

73

Figure 11. [3H]-Equol percutaneous absorption profile into human skin (panel A) and Cartoon of Equol penetration into keratinocytes and pooling via a “reservoir” mechanism in the epidermis with delayed release into the dermis over time (panel B). Panel A: [3H]-equol was used to determine percutaneous absorption into human skin with a profile showing an initial maximum peak flux occurring 6 h after dosing followed by a decline with a secondary lower peak at approximately 26-28 h after a single applied dose. Human trunk skin obtained from 4 individuals (2 males and 2 females of Caucasian and Hispanic decent, ages 35 to 51, n = 3 per subject) were tested by Franz cell techniques (Lephart, 2013a). The standard error of the mean (SEM) among the time point collections ranged from 0.005 to 0.024 (not shown graphically) (Lephart, 2013a). Panel B: Cartoon display of typical active ingredients versus equol in penetrating human skin layers. Some topical dermal formulations penetrate directly through the human skin layers 74

(shown by dashed gray arrows), while other topical dermal formulations do not penetrate through the skin layers (shown by the blue dashed arrow). Based upon the percutaneous absorption profile of equol through human skin it is sequestered into the epidermal compartment due to its affinity to and the abundance of estrogen receptor beta (ER β) in keratinocytes (green dashed arrows) and forms an equol “reservoir” that has a time-release prolife into the dermis to act upon fibroblasts and promote anti-aging effects in human skin.

75

Figure 12. Summary of equol‟s positive influences on human skin via inhibition and/or decrease of reactive oxygen species (ROS) and/or protection against oxidative stress (OS). Tissue Inhibitor of Matrix Metalloproteinases (TIMP), Matrix Metalloproteinases (MPPs), 5αdihydrotesterone (5α-DHT), Pro-inflammatory Transcription Factor NF-kappB (NFkappaB), Nuclear-factor-erythroid 2-related factor 2 (Nrf2), Estrogen receptor beta (ER β), AP-1 is a nuclear transcription element, Activator Protein – 1 (AP-1) and Estrogen Related Receptor gamma (ERR γ)

76

Tables Table 1. Comparison of the chemical properties and actions of equol to resveratrol in human skin

1. Molecular Weight 2. Octanol-water partition 3. Estrogen Receptor (ER) Binding 4. Directly Binds 5α-DHT 5. Inhibits 5αReductase Enzyme 6. Activates Estrogen-Related Receptor Gamma (ERRγ) 7. Topical Reservoir Delivery 8. Stimulates ECM proteins at [nM] 9. Directly

Equol

Resveratrol

242.2 g/mol

228.2 g/mol

CLogP 3.2

CLogP 3.0

ER β > ER α

mixed ER agonist

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No at [µM]

No

Yes 77

Stimulates Sirutins 10. Dietary Yes SupplementationDecreases Facial Wrinkles

Unknown

CLogP = hydrophilicity index; 5α-DHT = 5α-dihydrotestosterone; [nM] = nanomolar concentration; [µM] = micromolar concentration

78