Ageing Research Reviews 31 (2016) 36–54

Contents lists available at ScienceDirect

Ageing Research Reviews

journal homepage: www.elsevier.com/locate/arr

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, UT 84602, USA

a r t i c l e i n f o a b s t r a c t

Article history: Oxygen in biology is essential for life. It comes at a cost during normal cellular function, where reactive
Received 2 June 2016 oxygen species (ROS) are generated by oxidative metabolism. Human skin exposed to solar ultra-violet
Received in revised form 29 July 2016 radiation (UVR) dramatically increases ROS production/oxidative stress. It is important to understand
Accepted 4 August 2016
the characteristics of human skin and how chronological (intrinsic) aging and photo-aging (extrinsic
Available online 9 August 2016
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
Keywords:
one of the largest categories used in dermatology and cosmeceuticals to combat skin aging. An emerg-
Free radicals
Oxidative stress
ing botanical is equol, a polyphenolic/isoflavonoid molecule found in plants and food products and via
Antioxidant gastrointestinal metabolism from precursor compounds. Introductory sections cover oxygen, free radi-
Anti-aging cals (ROS), oxidative stress, antioxidants, human skin aging, cellular/molecular ROS events in skin, steroid
Equol enzymes/receptors/hormonal actions and genetic factors in aging skin. The main focus of this review cov-
Human skin ers the characteristics of equol (phytoestrogenic, antioxidant and enhancement of extracellular matrix
properties) to reduce skin aging along with its anti-aging skin influences via reducing oxidative stress
cascade events by a variety of biochemical/molecular actions and mechanisms to enhance human dermal
health.
© 2016 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
1.1. Oxygen in biology (Pros and cons) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
1.2. Free radicals (ROS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
1.3. Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
1.4. Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2. Human skin characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3. Aging of human skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.1. Intrinsic (chronological) and extrinsic (photo) aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.2. Cellular and molecular ROS events in human skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.3. Skin steroid enzymes, steroid hormones and steroid receptors in human aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.4. Genetics and steroid hormonal factors in skin aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4. Characteristics of equol: chemical structure, isomeric forms, metabolic and plant sources and biological actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.1. Antioxidant properties of equol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.2. Equol’s lipophilic nature and penetration into human skin via a reservoir delivery mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5. Equol’s anti-aging influence on human skin by reducing oxidative stress in cascade events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
5.1. Equol decreases ROS via sStimulation of Nrf2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

∗ Corresponding author at: Department of Physiology and Developmental Biol-

ogy and The Neuroscience Center, LS 4005, College of Life Sciences, Brigham Young
University, Provo, UT 84602, USA .
E-mail address: Edwin Lephart@byu.edu

http://dx.doi.org/10.1016/j.arr.2016.08.001
1568-1637/© 2016 Elsevier B.V. All rights reserved.
 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54 37

5.2. Equol acts as an antioxidant, stimulates antioxidant/detoxifying enzymes and inhibits skin aging biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.3. Equol protects DNA and enhances nerve and tissue repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.4. Equol inhibits AP-1 and neoplastic cell growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.5. Equol inhibits NFkappaB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.6. Equol inhibits the inflammatory response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.7. Equol inhibits MMPs and elastase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.8. Equol stimulates TIMP 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.9. Equol stimulates collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.10. Equol stimulates elastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
6. Equol: comparison to resveratrol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
7. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Conflict of interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

1. Introduction steroid enzymes/receptors and hormonal actions are covered along

with the influence of genetic factors in skin aging. Then, the main
Oxidative stress plays an important role in human skin aging sections of this review include the characteristics of equol and
and dermal damage (Gonzaga, 2009; Kammeyer and Luiten, 2015; its anti-aging influence on human skin via reducing oxidative
Natarajan et al., 2014; Rittie and Fisher, 2002; Zouboulis and stress cascade events by a variety of biochemical and molecular
Makrantonaki, 2011). The mechanisms of intrinsic (chronological) actions/mechanisms. Each section is self-contained with brief back-
and extrinsic (photo) aging include the generation of reactive oxy- ground information for each topic followed by examples/figures
gen species (ROS) via oxidative metabolism and exposure to sun showing human applications and/or analysis for the cited studies
ultra-violet (UV) light, respectively (Gonzaga, 2009; Kammeyer and presented.
Luiten, 2015; Natarajan et al., 2014). Photo-aging is dependent
upon the exposure to and intensity of solar UV radiation (UVR) 1.1. Oxygen in biology (Pros and cons)
(Gonzaga, 2009; Kammeyer and Luiten, 2015). Oxidant events and
molecular mechanisms of skin aging involve damage to DNA, the The respiration system in humans provides the breath of life that
inflammatory response, reduced production of antioxidants and is dependent upon oxygen. The oxygen from the atmosphere trans-
the generation of matrix metalloproteinases (MMPs) that degrade ferred into blood is utilized to metabolize dietary nutrients (e.g.,
collagen and elastin in the dermal skin layer (Gonzaga, 2009; fats, proteins & carbohydrates) in order to produce energy. In cellu-
Kammeyer and Luiten, 2015; Natarajan et al., 2014; Rittie and lar aerobic metabolic respiration, glucose is typically the molecule
Fisher, 2002; Zouboulis and Makrantonaki, 2011). All of these that is utilized to generate energy-rich ATP molecules within the
events lead to damaged skin and reflect the aging process: the mitochondria of the cell to maintain homeostasis (Lephart, 2009;
disruption of the extra cellular dermal matrix, the loss of tensile Martini, 2004).
strength and elasticity, impaired wound healing, the appearance of While biological systems are dependent upon atmospheric oxy-
wrinkles, age-spots and loss of skin tone. The goal is to delay aging gen, too little or too much oxygen can be damaging to cells and
onset and/or slow down the structural and visual appearance of tissues (Lephart, 2009; Martini, 2004). In this regard, oxygen is
skin aging with time. often referred to as a Janus gas that has both positive benefits and
Because phytochemicals are known to be constituents of animal potentially damaging side-effects in biological systems (Burton and
and human food sources, botanicals with polyphenolic structures Jauniaux, 2011). Oxygen participates in high-energy electron trans-
have received increased research study due to their potential sig- fers that supports the generation of abundant essential quantities
nificance and application in treating human cancers and other of ATP molecules through oxidative metabolism (Lephart, 2009;
age-related diseases including skin aging (Adlercreutz et al., Martini, 2004). However, biological systems and the numerous
2004; Evans and Johnson, 2010; Lephart et al., 2014; Mazur and chemical reactions within cells are not machines, and errors occur
Adlercreutz, 2000; Park and Pezzuto, 2015; Wang et al., 2014). resulting in the generation of very harmful molecules or particles
Isoflavonoid (plant-derived) molecules that fall under the polyphe- that are associated with oxygen such as free radicals.
nolic umbrella have been shown to decrease oxidative stress
(Bansal and Parle, 2010; Djuric et al., 2001; Yoon and Park, 2014). 1.2. Free radicals (ROS)
One of the important emerging isoflavonoid molecules is equol,
which can decrease oxidative stress (Ma et al., 2010; Zhang et al., While oxygen is essential for life, its benefits have a cost in
2013). In skin, equol has been shown to improve dermal health normal cellular function. As discussed by Halliwell and Gutteridge
by direct and downstream influences at several different steps (1999), unless electrons are transferred to oxygen during aerobic
of the oxidative stress cascade, while at the same time inhibit metabolism in the correct manner, potentially oxygen free radicals
MMP’s actions and simultaneously stimulate collagen and elastin can be formed easily. Occasionally electrons “escape,” and instead
(Gopaul et al., 2012; Lephart, 2013a). Finally, equol has two primary of completing the cellular respiration cycle, oxygen may become
properties: 1) an inherent antioxidative capacity resulting from its toxic and mutagenic (Halliwell and Gutteridge, 1999). For example,
polyphenolic molecular structure and 2) its well-known phytoe- a single oxygen atom is unstable and wants to bind a twin atom,
strogenic activity that enables it to reduce skin aging (Gopaul et al., forming molecular oxygen (O2 ). However, when this is not possible,
2012; Lephart, 2013a). the stability of this bond is compromised because only one pair of
In summary, the general concepts and principles of oxygen electrons is shared and two unpaired electrons remain (Fig. 1). This
in biology (pros and cons), free radicals (ROS), oxidative stress, complex represents a superoxide (O2 − ) and hydroxyl radical. These
antioxidants, human skin [intrinsic and extrinsic (photo)] aging are often referred to collectively as reactive oxygen species (ROS)
and, cellular and molecular ROS events are covered first. Also, due to the presence of an unpaired electron, which makes them
highly reactive (Buonocore et al., 2010; Halliwell and Gutteridge,
38 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54
Fig. 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 (H2 O2 ) 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., 2H2 O2 → 2H2 O + O2 ).
1999) and capable of chain reactions, which form another free rad-
ical 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 Fisher, 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
Fisher, 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, carcinogene-
sis, degeneration and other disorders and disease states involving
inflammation and aging. All major organ systems in humans includ-
ing skin, nervous, cardiovascular, respiratory, kidney, skeletal and
immune, etc., along with other multiple organs disorders such as
diabetes, sepsis, depression and trauma are impacted by oxida-
tive stress (Bar-Or et al., 2015; Kohen and Nyska, 2002; Maritim
et al., 2003; Maes et al., 2011). Thus, based upon numerous jour-
nal 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 Fisher, 2002; Valko et al., 2007; Zouboulis and
Makrantonaki, 2011). Oxidative stress may be defined as the imbal-
ance between the production of free radicals and the ability of the
body to counteract or detoxify their harmful effects through neu-
tralization by antioxidants. Since free radical (ROS) development
is unavoidable, the human body has adapted by establishing and
maintaining defense mechanisms that reduce their impact, partic-
ularly 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 detox-
ifying enzymes and antioxidant molecules (Krishnamurthy and
Wadhwani, 2012; Martini, 2004). In brief, the free radical detoxify-
ing 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 oxy-
gen (O2 ) or hydrogen peroxide (H2 O2 ) (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 perox-
idase is similar to catalase and represents a family of enzymes that
covert hydrogen peroxide into water and oxygen (Krishnamurthy
and Wadhwani, 2012). Notably, SOD, catalase and glutathione per-
oxidase are important antioxidant enzymes in human skin (Shindo
et al., 1994).
An antioxidant is any molecule that can block free rad-
icals 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 reac-
tions promoted by ROS. Antioxidants act at intra- and extra-cellular
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 glu-
tathione and uric acid are important antioxidants present in human
skin (Shindo et al., 1994).
Many different antioxidants are obtained from dietary or exoge-
nous 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 abun-
dance 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 antiox-
idants available that are ready to “neutralize” various free radicals
or ROS the body is either exposed to or that it produces. If an imbal-
ance occurs, then oxidative stress will result and homeostasis will
not be maintained.
2. Human skin characteristics
Human skin is the largest and most complex organ (represent-
ing one sixth of the total body weight) functioning as a physical
barrier to protect the body from water loss as well as environ-
mental 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, thermoregu-
lation, sensory input from mechanoreceptors and endocrine and
metabolic mechanisms to sustain optimal health (Lephart, 2009;
Martini, 2004; Qunshan and Nash, 2010). Finally, and most impor-
tantly, the antioxidant defense capacity of the skin would be
expected to be greater than that of internal organs due to its pro-
 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54
Fig. 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.
tective 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 stra-
tum corneum (10–30 ␮m) is the outermost layer of progressively
dying and flattened dead cells or corneocytes. The ‘bricks and mor-
tar’ cellular structure is composed of the live keratinocyte layers,
which form the majority of the epidermal layer (100–150 ␮m)
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 pigmen-
tation of the skin for photoprotection (Natarajan et al., 2014) (Fig. 2).
Conspicuously, the epidermis, composed mainly of ker-
atinocytes, 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 concentra-
tions 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 der-
mis bestows the scaffolding or structural fibers of the skin. In
general, the dermis is a dense and irregular layer of connective
tissue, 2–3 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 water-holding com-
ponents i.e., hyaluronic acid (responsible for normal turgor) and
39
Fig. 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).
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 tis-
sue 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), metallopro-
teinases (MMPs), elastase and many other molecules that maintain
skin health (Farage et al., 2010).
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 mil-
lion, 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 popu-
lation (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 fea-
tures 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 dele-
tions of mitochondrial DNA (Ashworth et al., 1999; Qin et al., 2014;
40 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54
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).
The first signs of skin aging occur around age 30 and especially
after menopause, where there is skin dryness, decreased skin firm-
ness and loss of elasticity (Burns and Breathnach, 2004; Escoffier
et al., 1989; Fazio et al., 1988; Freedberg et al., 2003; Hillebrand,
2010; Khol et al., 2011; Seite et al., 2006; Uitto, 2008). In fact, elas-
ticity 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 dis-
cussed 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 chrono-
logical 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 mech-
anisms (Gonzaga, 2009; Farage et al., 2010; Farage et al., 2008;
Kammeyer and Luiten, 2015; Khol et al., 2011; Zouboulis and
Makrantonaki, 2011). Intrinsic or chronological aging is an unavoid-
able 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 mito-
chondrial 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
photo-aging. 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 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 reach-
ing earth’s surface (Walker et al., 2003). While also damaging the
epidermis, UVA penetrates deeper into the dermis to degrade col-
lagen 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 Fisher, 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 Fisher, 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 min of mid-day sun every-other-day was sufficient to
maintain elevated levels of MMPs that are known to degrade der-
mal 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 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 com-
parisons can only be made on group means rather than on data
linked to individuals (Spiro and Buttriss, 2014; National Institutes
of Health, Vitamin D, 2016).
Thus, staying out of the sun is the best avoidable practice a per-
son can do to prevent ROS production and photo-aging. To illustrate
this point, Fig. 6 shows a 69-year-old man that drove trucks com-
mercially 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 chrono-
logical or intrinsic aging (Gonzaga, 2009; Kammeyer and Luiten,
2015; Natarajan et al., 2014; Rittie and Fisher, 2002; Zouboulis and
Makrantonaki, 2011). In intrinsic aging there is ROS production
via oxidative metabolism (Anderson et al., 2014; Gonzaga, 2009;
 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54 41

Fig. 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-makes-woman-age-50-years-in-just-a-few-days.html, October
14, 2011.

Fig. 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.

Meadows et al., 2014). All of the skin aging characteristics (covered important for optimal skin health, this will be a major focus for the
above) are associated with oxidative metabolism and subsequent cellular, molecular, enzymatic, antioxidant and other components
ROS generation that define this inevitable phenomenon. Since the that regulate these extracellular matrix proteins.
structural and functional aspects of collagen and elastin fibers are so
42 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54

Zouboulis and Makrantonaki, 2011). The activation of the inflam-

Fig. 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 (pro-
tected 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).
From molecular to cellular cascades, ROS production is known
to cause the activation of activator protein-1 (AP-1), which sup-
presses TGF␤ 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 Fisher, 2002;
Zouboulis and Makrantonaki, 2011). At the same time, AP-1 acti-
vation: a) stimulates MMPs that degrade collagen and b) activates
NF-kappaB, 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 Fisher, 2002;
matory response generates cytokines or interleukins (ILs), which
then in a positive feedback loop, stimulates the further produc-
tion of ROS (Gonzaga, 2009; Kammeyer and Luiten, 2015; Naylor
et al., 2011; Peng et al., 2015; Rittie and Fisher, 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 intrin-
sic aging (covered above), but the magnitude of the effects is
amplified, which explains why photo-aging accounts for approx-
imately 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 Fisher, 2002; Shindo et al., 1994; Thiele et al.,
1998; Warren et al., 1991; Zouboulis and Makrantonaki, 2011)
(Fig. 7). For instance, vitamin E sequestered in hydrophobic lipids,
can absorb the energy from UV light, and thus plays an impor-
tant role in photo-protection from ROS (Rhie et al., 2001; Weber
et al., 1997). However, exposure to UV light lowers vitamin E con-
tained 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 stimula-
tion of MMPs and a concept called “fibroblast collapse” (Gonzaga,
2009; Kammeyer and Luiten, 2015; Khol et al., 2011; Natarajan
et al., 2014; Rittie and Fisher, 2002; Zouboulis and Makrantonaki,
2011). In this situation fibroblasts increase the production of elas-
tase, 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

Fig. 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 (TGF␤).
 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54 43

Fig. 8. Steroid hormone enzymes, steroid hormones and steroid receptors in the epidermal and dermal layers during aging. Estrogen receptor beta (ER ␤) is the predominant
estrogen receptor subtype in human skin. The photomicrograph displays ER ␤ (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 5␣-reductase type I enzyme in fibroblasts converts
testosterone (T) into 5␣-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 ␤ are known to
decrease MMPs and enhance wound healing. Notably, ER ␣ expression in human skin is expressed at lower levels compared to ER ␤ expression (not shown). While ER ␣
expression does not change with menopause, ER ␤ expression declines by approximately 15–20%, 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, 2015; Makrantonaki and Zouboulis,
2009; Nitsch et al., 2004; Pelletier and Ren, 2004; Pomari et al., 2015; Thornton et al., 2003).

and Fisher, 2002; Zouboulis and Makrantonaki, 2011). In total, the
destructive actions associated with photo-aging are noticeable ini-
tially 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 Fisher, 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 der-
mal 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 production (Ramara et al.,
2007) and b) antioxidant enzyme expression is stimulated by estro-
gen via the ERK1 and ERK2[MAP]/NFkappaB cascade (Borras et al.,
2005). The ovary and skin fibroblasts are capable of synthesiz-
ing 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 sub-
types, estrogen receptor alpha (ER ␣) and estrogen receptor beta
(ER ␤; Fig. 8). The predominant subtype of ER in skin and scalp
hair is ER ␤, 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 ␤ in fibrob-
lasts in the dermis (Pelletier and Ren, 2004; Thornton et al., 2003).
The intracrine sex steroid synthesis and signaling in human epi-
dermal 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 ␤ and androgen receptors play important roles in
skin and hair health. In general, activation of ER ␤ enhances,
whereas, activation of ER ␣ or hormonal actions via andro-
gen 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 solar-stimulated UV radiation-induced immune suppres-
sion. Markiewicz et al. in (2013), using knock-out mice showed that
ER ␤ meditated skin dermal thickness/collagen deposition occur in
a unique manner. This 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 ␤ 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 ␤ activation and its beneficial influence in
human skin has been recently reviewed (Jackson et al., 2011). More-
over, estrogen related receptor (ERR) gamma (␥) has been identified
in keratinocytoes and fibroblasts in human skin that has positive
influences on dermal health. For instance, when ERR ␥ is activated
in skin, it is thought to protect against neoplastic growth (Krahn-
Bertil et al., 2008).
Also, the 5␣-reductase enzyme is present in fibroblasts. This
enzyme converts testosterone to the more potent androgen, 5␣-
dihydrotestosterone (5␣-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 pro-
duction, in general, androgens are known to increase free radical
production from data obtained in cardiovascular studies (Chignalia
et al., 2012; Tostes et al., 2016).
44 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54
Fig. 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).
3.4. Genetics and steroid hormonal factors in skin aging
Image analysis were performed examining monozygotic (“iden-
tical”) 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
(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 superim-
posed 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; Kuwazuru 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 hor-
monal 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 for-
mation 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 health. Evidence that equol is an emerging
botanical that addresses skin aging by decreasing ROS via hor-
monal 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). Phe-
nolics are a group of compounds having at least one hydroxyl
group attached to an aromatic ring. The most high-profile polyphe-
nolic 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
17␤-estradiol are steroids with a cyclo-hexane-phenantrene par-
ent chemical structure that is derived from cholesterol, equol is not
a steroid. However, equol and 17␤-estradiol have similar chemi-
cal structures/confirmations and molecular weights (C15 H14 O3 vs.
C18 H24 O2 ; 242.3 g/mol vs. 272.4 g/mol, respectively) (Fig. 10A 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 capa-
ble of producing higher levels of equol than others (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 10–20 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, 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 sig-
nificantly correlated with urinary equol levels in U.S. adults. Also,
there are some data that showed R-equol 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 differ-
ent 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
et al., 2010).
Of particular interest from a chemical messenger and molec-
ular 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,
 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54 45

Fig. 10. Chemical structures, formulas and molecular weights (MW) of Equol (panel A) and of 17␤-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 17␤-Estradiol (PubChem). While both Equol and 17␤-Estradiol
contain aromatic rings, 17␤-Estradiol 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.

that shows equol binding GPER1 in human skin. However, equol
has been shown to bind the orphan G-protein-coupled receptor
(GPR30) in 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 5␣-dihydrotestosterone (5␣-DHT) and
inhibit its potent negative actions in skin (Gopaul et al., 2012;
Lephart, 2013a). Equol has the ability to bind to ERR ␥, which
has important implications for anti-aging in human skin (Hirvonen
et al., 2011; Krahn-Bertil et al., 2008; Lephart, 2013a). Addition-
ally, when equol is bound to ERR ␥, it decreases inflammation
and has anti-proliferative effects on prostate and breast cancer
cells (Hirvonen et al., 2011). Finally, equol research has dramati-
cally 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, 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
age-related diseases (Lephart 2013a,b; Setchell and Clerici, 2010).
For example, in plants, the accumulation of polyphenolic com-
pounds such as flavonoids and phenolic molecules have been linked
to pathogen resistance (Hounsome et al., 2009, 2010; Lephart et al.,
2014). There are many polyphenolic molecules that act as antioxi-
dants including isoflavonoids (Bohn, 2010; Lephart et al., 2014; Li
et al., 2012; Hounsome et al., 2009, 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 activ-
ities, 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., 2010).
Additionally, equol protected against apoptosis and vascular injury
through oxidative stress in porcine pulmonary arteries and human
pulmonary artery endothelial cells (Chung et al., 2008; 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 antioxi-
dant 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, 2010). Of the
isoflavonoid compounds examined, equol was identified as being
present in cabbage at high levels similar to other polyphenolic com-
pounds that can serve as an antioxidant during the storage of white
cabbage to help prevent oxidative stress (Hounsome et al., 2009,
2010).
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 octanol-
water 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 high-
46 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54

Fig. 11. [3 H]-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: [3 H]-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 35–51, 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 (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 ␤) 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.

est absorption levels (80–85%) 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 compart-
ments (Lephart, 2013a). As displayed in Fig. 11A, the [3 H]-equol
penetrated into human skin with a profile showing an initial max-
imum peak flux occurring 6 h after dosing followed by a decline
in penetration with a secondary lower peak flux at approxi-
mately 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 ␤) in ker-
atinocytes for which S-equol has a high affinity (Gopaul et al., 2012;
Lephart, 2013a,b; Pelletier and Ren, 2004; Setchell et al., 2005;
Thornton et al., 2003). Thus, the epidermal “reservoir” delivery
mechanism of topically applied 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 17␤-estradiol (Chadha et al., 2011; Hung et al.,
2008; Xing et al., 2009; Zhong et al., 2009). This epidermal reser-
voir delivery mechanism of equol is depicted in Fig. 11B. Finally,
the delivery of approximately 14 nM racemic equol into the ker-
atinocytes (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 col-
lagen 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 mecha-
nism leading to skin aging (Gonzaga, 2009; Kammeyer and Luiten,
2015; Natarajan et al., 2014; Rittie and Fisher, 2002; Zouboulis and
Makrantonaki, 2011). Cell culture (primary or organotypic), molec-
ular, and gene expression/array methods have examined equol’s
influence on oxidative stress along with various biomarkers at dif-
ferent 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 infor-
mation is known. In a comprehensive investigation on equol as:
R-equol, racemic equol or S-equol from PCR/mRNA studies quan-
tifying 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 inhi-
bition) by R-equol and/or racemic equol, which suggest that, in
general, R-equol and/or racemic equol was more potent compared
 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54
Fig. 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), 5␣-dihydrotesterone (5␣-DHT), Pro-inflammatory Transcription Factor NF-kappB
(NFkappaB), Nuclear-factor-erythroid 2-related factor 2 (Nrf2), Estrogen receptor beta (ER ␤), AP-1 is a nuclear transcription element, Activator Protein − 1 (AP-1) and
Estrogen Related Receptor gamma (ERR ␥).
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 sStimulation 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 mas-
ter 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 capac-
ity to induce the expression of numerous genes, which encode
detoxifying enzymes and antioxidant proteins that provide pro-
tection 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, inflamma-
tion, 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 17␤-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 2-related 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 antiox-
idant response element (ARE) that is present in the promoter region
of genes for 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
47
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 ␤ 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 superox-
ide 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 oxida-
tive stress in equol-producing postmenopausal women using
vascular cell adhesion molecule-1 (VCAM-1) and nitric oxide (NO)
as markers of vascular endothelial function, and malonyldialde-
hyde (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-
48 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54
producing postmenopausal women with dietary soy isoflavone
supplementation.
One small human in vitro study reported that R-equol pro-
tected 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 com-
mon 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 antiox-
idant 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,
2013a,b). SOD 2 and TXNRD 1 are known to protect against free red-
icals, 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
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 stimu-
lated the gene expression of the SH-rich detoxifying proteins like
metallothionein-1H and metallothionein-2H that protect against
metal toxicity (Gopaul et al., 2012; Lephart, 2013a). Previous stud-
ies have shown that equol’s photo-protective 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 expres-
sion of type 1 5␣-reductase in human skin (Gopaul et al., 2012;
Lephart, 2013a). Recall, this enzyme converts testosterone to the
more potent androgen, 5␣-DHT in dermal fibroblasts that cause the
stimulation of MMPs and reduces tissue wound repair (Ma et al.,
2010; Makrantonaki and Zouboulis, 2009; Nitsch et al., 2004). In
cell cultures of human dermal fibroblasts 5␣-DHT was cytotoxic
at 10 nM (Gopaul et al., 2012). In this regard, clinical evidence has
demonstrated that the accumulation of 5␣-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 5␣-DHT 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).
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 protec-
tive 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 (2013a) 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 oxida-
tive stress cascade. It induces MMPs in human skin that in turn
activates collagen degradation (Huang et al., 1997). AP-1 also blocks
the positive pro-collagen actions of transforming growth factor-␤1
(Fisher et al., 1996). Kang et al. (2007a) reported that the anti-tumor
effects of racemic equol are due to the inhibition of cell transfor-
mation by the MEK signaling pathway by blocking AP-1. Racemic
equol dose-dependently 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 ␤ signaling has been
shown to protect against transplanted skin tumor growth in mice
(Cho et al., 2010), which 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 ␥ that in turn
enhanced the transcriptional activity of ERR ␥, which is known to
protect against neoplastic growth (Hirvonen et al., 2011). Equol’s
precursor molecule daidzein did not have this effect (Krahn-Bertil
et al., 2008). It is also known that ERR ␥ is present in keratinocytes
and fibroblasts in human skin, where its actions may show sim-
ilar favorable protection as that seen against breast and prostate
cancers (Hirvonen et al., 2011; Krahn-Bertil et al., 2008).
Finally, it has been demonstrated that ER ␤ signaling in the
prostate (and breast tissue) are associated with decreased inflam-
mation and neo-plastic growth (Lephart, 2014a). S-Equol is known
to have a high affinity for ER ␤, which is the predominate ER in ker-
atinocytes 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 radia-
tion exposure (Chang et al., 2010; Pomari et al., 2015; Widyarini
et al., 2006a). The high affinity for ER ␤ 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 excit-
ing and active area of investigation, where it is expressed in all cell
types and is evolutionarily conserved (Ghosh et al., 1998). NFkap-
paB is involved in the oxidative stress mechanism by the expression
of numerous genes such as the cytokines and plays a major role in
the pathology of inflammatory disease (Gilmore, 2006; Ghosh et al.,
1998; Hayden and Ghosh, 2012; Schreck et al., 1991).
Several investigators examined the inhibition of NFkappB by
equol. Kang et al. in (2005) showed that racemic equol inhibited
tumor necrosis factor-␣ gene expression by blocking NFkappB in
mouse macrophages that was independent of an estrogen recep-
tor mechanism. Tumor necrosis factor-␣ is a cell signaling protein
(cytokine) involved in systemic inflammation (Kang et al., 2005).
 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54
Furthermore, Kang et al. in (2007b) reported the dose-dependent
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 lipopolysac-
charide (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 dis-
ease (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 NF-kappaB 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 treatments (Evans and Johnson, 2010; Adlercreutz
et al., 2004; Park and Pezzuto, 2015). It is well documented that var-
ious 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 expres-
sion of several pro-inflammatory 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 and Athar, 2006; Natarajan
et al., 2014; Strickland et al., 1997).
It is also known that ER ␤ 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 bind-
ing to ER ␤ 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 stim-
ulates 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, 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 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, 17␤-estradiol decreased
the expression by 8.4% (Gopaul et al., 2012). Equol’s ability to inhibit
49
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 impor-
tant 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; Hornebeck, 2003). It is
known that increased TIMP expression and activation blocks the
negative influence of MMPs in causing skin damage. When the iso-
mers 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 pro-
tein that maintains optimal dermal health (Draelos and Puglises,
2011; Farage et al., 2010). While oxidative stress via a cascade
mechanism degrades collagen and reduces the deposition of col-
lagen (via MMPs) in the dermal layer, it is important not only to
block the negative insults of ROS production, but to enhance colla-
gen deposition. In both cell culture and gene array studies, racemic
equol stimulated pro-collagen type 1 and collagen levels (Gopaul
et al., 2012; Lephart, 2013a). When the equol isomers were exam-
ined 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 produc-
tion, but to enhance elastin deposition (Draelos and Puglises, 2011;
Farage et al., 2008; Mera et al., 1987; Tulah and Birch-Machin,
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 sag-
ging 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 hor-
mone, 17␤-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
When the above equol skin biomarker results are compared to
the botanical that has the highest recognition by the general pub-
lic or lay profile namely, resveratrol (Lephart et al., 2014; Park
50 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54
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 5␣-DHT
5. Inhibits 5␣-Reductase Enzyme
6. Activates Estrogen-Related Receptor Gamma (ERR␥)
7. Topical Reservoir Delivery
8. Stimulates ECM proteins at [nM]
9. Directly Stimulates Sirutins
10. Dietary Supplementation-Decreases Facial Wrinkles
CLogP = hydrophilicity index; 5␣-DHT = 5␣-dihydrotestosterone; [nM] = nanomolar concentration; [␮M] = micromolar concentration.
and Pezzuto, 2015), equol displayed better biochemical, molec-
ular, protective and delivery mechanisms of action to promote
and/or improve human skin health as a topical anti-aging ingredi-
ent/compound (Lephart, 2013a). This is interesting since equol and
resveratrol have similar molecular weights and octanol-water par-
titions (indicating lipid solubility). However, equol directly binds
the potent androgen 5␣-DHT, inhibits the 5␣-reductase enzyme
in fibroblasts, acts as a selective SERM modulator by bind ER␤
with higher affinity compared to ER␣ 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 dos-
ing (>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 effec-
tive 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 cellu-
lar processes that protect cells from stress and potentially treat
age-related conditions and lengthen a healthy life (Lephart et al.,
2014) (see Table 1). In a recent study, the combined administration
of equol with resveratrol enhanced SIRT1 expression in endothe-
lial cells demonstrating equol’s unique beneficial biological actions
(Davinelli et al., 2013).
Equol is currently used in skin treatments, and the positive influ-
ences 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 ␤ 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 expres-
sion 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
Equol Resveratrol
242.2 g/mol 228.2 g/mol
CLogP 3.2 CLogP 3.0
ER ␤ > ER ␣ mixed ER agonist
Yes No
Yes No
Yes No
Yes No
Yes No at [␮M]
No Yes
Yes Unknown
S-equol reduced facial wrinkles compared to placebo controls with-
out 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 oxida-
tive stress to ameliorate cellular dermal damage (Lobo et al., 2010;
Poljsak 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 cellu-
lar 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 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 patho-
logical 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 characteris-
tics of equol and evidence of its protective properties and biological
actions are discussed in reference to ROS generation and oxidative
 E.D. Lephart / Ageing Research Reviews 31 (2016) 36–54
stress events. Some of these topics include equol’s action of: stimu-
lation 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 appli-
cations but by dietary supplementation and/or functional foods.
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.
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