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CHAPTER 1 PART II 5.1. INTRODUCTION
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There are many fairness products that inhibit melanogenesis and are in great demand. But
there are some facts that are often ignored; fairness is not a measure of skin health.
Complexion that is clear, bright, and glows with health is the hallmark of beautiful skin.
What skin needs is just “adequate protection, cleansing and nourishment” by topical and
internal care (Fig. 5.1.1). That is all the pampering that skin needs for attaining “Bright &
Glowing” sheen.
Figure 5.1.1: Skin care topically and internally
Healthy Skin
Topical Care Internal Care
Protection by Sunscreen
Cleansing by Astringent
Nourishment by Antioxidant rich
Moisturizers, conditioners &
cell rejuvenators
Protection by Antioxidants
Cleansing by Antioxidants that
detoxify blood
Nourishment by nutrition rich diet
& supplements (Nutricosmetics)
PART II
SCREENING OF ACTIVES THROUGH VARIOUS
MECHANISMS OF MELANOGENESIS AND POSITIONING
THEM IN ACCORDANCE TO THEIR SPECIFIC MODE OF
ACTION IN RECTIFYING PIGMENTATION DISORDERS
5.1. INTRODUCTION
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5.1.1. Skin color and melanin:
All organisms, from simple invertebrates to complex human beings, exist in different
colors and patterns, which arise from the unique distribution of pigments throughout the
body. Human skin exists in a wide range of colors and gradations, ranging from white to
brown to black. Pigmentation is highly heritable, being regulated by genetic,
environmental, and endocrine factors that modulate the amount, type, and distribution of
melanins in the skin, hair, and eyes.
Melanin is a chemically inert and stable pigment, which is produced deep inside
the skin but is displayed as a mosaic at the surface of the body. Melanin is therefore
responsible for the most striking polymorphic traits of humans and for the most obvious
and thoroughly discussed aspect of human geographical variability: skin color. In
addition to its roles in heat regulation and color variation, melanin protects against
Ultraviolet radiation (UV), environmental factors etc., and thus is an important defense
system in human skin. Melanin plays a major photoprotective role in human skin by
absorbing, scattering, photo-oxidizing, and scavenging free radicals and acting as a
pseudo-dismutase to minimize the toxic effects of ROS and to prevent damage to DNA,
proteins, and cell membrane lipids (Pathak M A and Fitzpatrick T B, 1993). It is known
that UV-A produces harmful oxygen species such as O.2-, .OH, and 1O2 and that melanin
interacts with them, thus protecting the skin against the damage that could occur (Pathak
M A and Fitzpatrick T B, 1993; Pathak, M A and Stratton K, 1968).
5.1.2. Skin and stress:
Being the largest organ of the body that is always under the influence of internal and
external factors, the skin often reacts to those agents by modifying the constitutive
pigmentation pattern. Minor changes in the physiological status of the human body or
exposure to harmful external factors can affect pigmentation patterns either in transitory
or permanent manners. Understanding the mechanisms by which different factors and
compounds affect melanogenesis is of great interest pharmaceutically (as therapy for
pigmentary diseases like vitiligo) and cosmeceutically (e.g., to design depigmentation
products with potential to reduce skin darkening). Other than genetic factors, many
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factors like endocrine factors that induce temporary (e.g., during pregnancy) or
permanent (e.g., during ageing) changes in skin color, environmental factors (e.g., UV,
pollution), certain drugs, and chemical compounds, etc. play an important role in skin
pigmentation.
Undesirable excess pigmentation can be prevented before it manifests in
permanent manner. Skin pigmentation is the result of the intricate cellular and molecular
interactions between melanocytes and keratinocytes, which together compose the
epidermal melanin unit. All of the other types of cells distributed within different layers
of the skin and the intracellular signaling pathways often overlapping and involving cross-
talking also play a role in skin pigmentation. The skin reacts to stress through all its
cellular and molecular components, which form a complicated, sophisticated, and highly
sensitive signaling network.
5.1.3. Skin structure and functions:
An understanding of skin structure is prerequisite to understand pigmentation
mechanisms. The skin plays an extremely important role, providing a vast physical
barrier against mechanical, chemical, and microbial factors that may affect the
physiological status of the body (Haake A and Holbrook K, 1999). In addition to those
functions, the skin also acts as an immune network and, through its pigments, provides a
unique defense system against UV radiation (UV) (Pathak M A, 1995). Thus,
melanocytes transfer melanosomes through their dendrites to keratinocytes, where they
form the melanin caps that reduce UV-induced DNA damage in human epidermis. The
skin’s layers are represented by the epidermis, the dermis, and the hypodermis, the latter
consisting of fatty tissue that connects the dermis to underlying skeletal components. The
structure of skin in illustrated and discussed in detail (Fig. 5.1.2).
5.1.3.1. Epidermis: The epidermis is an external, stratified epithelium devoid of blood or
nerve supplies of 5–100 µm thickness (which can reach 600 µm on palms and soles)
(Tobin D J, 2006). It is composed of several distinct cell populations; keratinocytes and
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melanocytes are the main constituents, of which the first comprise 95% of the epidermis
and are arranged in four layers, as shown in Fig. 5.1.2.
A) Different layers and components of skin B) Layers of the epidermis Figure 5.1.2: Structure of skin
Stratum basale (also known as the stratum germinativum) is a single layer of cells
attached to a noncellular basement membrane that separates the epidermis from the
dermis. The stratum basale consists mostly of basal keratinocytes, which have stem cell-
like properties, and at least two different types of neural crest-derived cells: Merkel cells
(neuroendocrine cells responsible for the transmission of touch sensation through the
cutaneous nerves) and melanocytes.
Stratum spinosum contains irregular polyhedral keratinocytes with some limited
capacity for cell division. Also found here are the bone marrow-derived sentinel cells of
the immune system called Langerhans’ cells, which represent the antigen-presenting cells
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of the skin and play a vital role in immunological reactions such as allergic contact
dermatitis.
Stratum granulosum contains flattened, polyhedral nondividing keratinocytes
producing granules of a protein called keratinohyalin. These granules increase in size and
number as the cell nuclei gradually degenerate and the cells die. These cells flatten as
dividing cells underneath them progressively push them toward the skin surface.
Stratum corneum contains nonviable, but biochemically active cells called
corneocytes. The keratinocytes continue to differentiate as they move from the basal layer
to the stratum corneum, the result being cornified cells that contain abundant keratin and
lack cytoplasmic organelles. It is these cornified cells that provide a barrier against the
physical and chemical agents in the environment that may adversely affect the body.
More specifically, this epidermal barrier functions to reduce transepidermal water loss
from within and to prevent invasion by infectious agents and noxious substances from
without (Elias P M, 2005).
5.1.3.2. Dermis: The dermis is a 2 to 4 mm-thick layer of connective tissue and
fibroblasts that houses the neural, vascular, lymphatic, and secretory apparatus of the skin.
The main cell type, fibroblasts, is required for synthesis and degradation of the
extracellular matrix (ECM) (Haake A and Holbrook K, 1999). This matrix is a complex
structure composed of highly organized collagen, elastic, and reticular fibers. The dermis
also hosts multifunctional cells of the immune system such as macrophages and mast
cells, the latter being able to trigger allergic reactions by secreting bioactive mediators
such as histamine. Structures within the dermis include: 1) Excretory and secretory glands
(sebaceous, eccrine, and apocrine). Sebaceous glands secrete triglyceride and cholesterol-
rich sebum that lubricate the skin and keep it supple and waterproof. They are often
associated with hair shafts. 2) Hair follicles and nails: in addition to generating the hair
shaft, the hair follicle provides a protective niche to several stem cell populations in the
skin, including keratinocyte stem cells, melanocyte stem cells, a population of epidermal
neural crest stem cells, and the dermal stem cell compartment, known as the dermal
papilla (Cotsarelis G et al., 1990 and Ito M et al., 2005). These stem cells are required
most visibly during wound healing. 3) Sensory nerve receptors of Merkel and Meissner’s
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corpuscles (for touch), Pacinian corpuscles (for pressure), and Ruffini corpuscles
(mechano-receptors). As illustrated in Fig. 5.1.3, dermis contains various skin structural
proteins that confer integrity to the skin. In the dermis, collagen provides the skin with
tensile strength and tissue integrity whereas elastin provides elasticity and resiliency.
Besides collagen and elastic fibers, the dermis contains the extrafibrillar matrix, which is
extracellular and composed of a complex mixture of proteoglycans, glycoproteins,
glycosaminoglycans, water, and hyaluronic acid. The most significant
glycosaminoglycans, which bind to proteins to form the proteoglycans of the skin, are
chondroitin sulfate, dermatan sulfate, keratin sulfate, heparan sulfate, and heparin. The
most important proteoglycans of the skin are versicans, which are involved in assuring
the tightness of the skin, and perlecan, found in basement membranes. Glycoproteins,
such as laminins, matrilins, fibronectin, tenascins, etc., are involved in cell adhesion, cell
migration, and cell-cell communication, which are extremely important processes taking
place in the skin.
A B A) Collagen and elastic fibers in dermis B) Stratified epidermis and vascular dermis Figure 5.1.3: Skin structural proteins
Epidermis
Skin structural proteins
in dermis
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5.1.4. Synthesis and distribution of melanin in skin under normal conditions:
Melanin biosynthesis is a complex pathway that appears in highly specialized cells, called
melanocytes, within membrane-bound organelles referred to as melanosomes (Hearing V
J, 1997). Melanosomes are transferred via dendrites to surrounding keratinocytes, where
they play a critical role in photoprotection. The anatomical relationship between
keratinocytes and melanocytes is known as "the epidermal melanin unit" and it has been
estimated that each melanocyte is in contact with 40 keratinocytes in the basal and
suprabasal layers (Fitzpatrick T B and Breathnach A S, 1963). Several important steps
must occur for the proper synthesis and distribution of melanin, as described briefly
below (Boissy R E and Nordlund J J, 1997).
5.1.4.1. The development of melanocyte precursor cells (melanoblasts) and their
migration from the neural crest to peripheral sites:
Prospective melanocytes, known as melanoblasts, derive from the neural crest beginning
in the second month of human embryonic life and migrate throughout the mesenchyme of
the developing embryo. They reach specific target sites, mainly the dermis, epidermis,
and hair follicles, the uveal tract of the eye, the stria vasculare, the vestibular organ and
the endolymphatic sac of the ear, and leptomeninges of the brain. In humans, this
migration process takes place between the 10th and the 12th wk of development for the
dermis and 2 wk later for the epidermis (Haake A and Holbrook K, 1999).
5.1.4.2. Differentiation of melanoblasts into melanocytes:
Once melanoblasts have reached their final destinations, they differentiate into
melanocytes, which at about the sixth month of fetal life are already established at
epidermal-dermal junction sites (Haake A and Holbrook K, 1999).
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5.1.4.3. Survival and proliferation of melanocytes:
Melanocytes have been identified within fetal epidermis as early as 50 days of gestation.
Dermal melanocytes decrease in number during gestation and virtually disappear by birth,
whereas epidermal melanocytes established at the epidermal-dermal junction continue to
proliferate and start to produce melanin. 5.1.4.4. Formation of melanosomes and production of melanins:
Once established in situ, melanocytes start producing melanosomes, highly organized
elliptic membrane-bound organelles in which melanin synthesis takes place.
Melanosomes are typically divided into four maturation stages (I–IV) determined by their
structure and the quantity, quality, and arrangement of the melanin produced (Seiji M et
al., 1963 and Kushimoto T et al., 2001). Nascent melanosomes are assembled in the
perinuclear region near the Golgi stacks, receiving all enzymatic and structural proteins
required for melanogenesis. Stage I melanosomes are spherical vacuoles lacking
tyrosinase (TYR) activity (the main enzyme involved in melanogenesis) and have no
internal structural components. However, TYR can be detected in the Golgi vesicles, and
it has been shown that it is subsequently trafficked to stage II melanosomes. At this point,
the presence and correct processing of Pmel17, an important melanosomal structural
protein, determine the transformation of stage I melanosomes to elongated, fibrillar
organelles known as stage II melanosomes (Kushimoto T et al., 2001 and Berson J F et
al., 2001); they contain tyrosinase and exhibit minimal deposition of melanin. After this,
melanin synthesis starts and the pigment is uniformly deposited on the internal fibrils, at
which time the melanosomes are termed as stage III. Their last developmental stage (IV)
is detected in highly pigmented melanocytes; these melanosomes are either elliptical or
ellipsoidal, electron-opaque due to complete melanization, and have minimal TYR
activity. The developmental stages detailed above refer mainly to eu-melanosomes
(containing black-brown pigments); however, they are quite similar to pheo-melanosomes
(containing yellow-reddish melanin), the only difference being that the latter remain
round and are not fibrillar during maturation.
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Within melanosomes, at least three enzymes are absolutely required to synthesize
different types of melanin. While tyrosinase is responsible for the critical steps of
melanogenesis, tyrosinase-related protein 1 (TYRP1) and DOPAchrome tautomerase
(DCT) are further involved in modifying the melanin into different types.
TYR (monophenol, 3,4-ß-dihydroxyphenylalanine oxygen oxidoreductase, EC
1.14.18.1) is a single chain type I membrane glycoprotein catalyzing the hydroxylation of
tyrosine to β-3,4-dihydroxyphenylalanine (DOPA) (which is the initial rate-limiting step
in melanogenesis) and the subsequent oxidation of DOPA to DOPAquinone. TYR,
TYRP1, and DCT share numerous structural similarities and follow quite similar
biosynthetic, processing, and trafficking pathways (Hearing V J and Tsukamoto K, 1991).
Their maturation is assisted by chaperones, calnexin being the most important one due to
its involvement in the correct folding of tyrosinase (Halaban R et al., 1997; Branza-
Nichita N et al., 1999 and Branza-Nichita N et al., 2000). The subsequent metabolism of
DOPA and its derivatives by various melanocyte-specific enzymes, including TYRP1 and
DCT, results in the synthesis of eumelanin, a black-brown pigment. The synthesis of
pheomelanin involves the production of cysteinyldopa conjugates from DOPAquinone
after the production of DOPA from tyrosine. TYRP1 is important for the correct
trafficking of tyrosinase to melanosomes (Toyofuku K et al., 2001), and DCT also seems
to be involved in the detoxification processes (Urabe K et al., 1994) taking place within
melanosomes.
Melanins are polymorphous and multifunctional biopolymers that include
eumelanin, pheomelanin, mixed melanins (a combination of the two), and neuromelanin.
Mammalian melanocytes produce two chemically distinct types of melanin pigments:
black-brown eumelanin and yellow-reddish pheomelanin (Prota G, 1992). Although they
contain a common arrangement of repeating units linked by carbon-carbon bonds,
melanin pigments differ from each other with respect to their chemical, structural, and
physical properties. Eumelanin is a highly heterogeneous polymer consisting of 5,6-
dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units in
reduced or oxidized states, as detailed above; pheomelanin consists mainly of sulfur-
containing benzothiazine derivatives (Ito S et al., 2000). Due to their chemical structure,
both eumelanin and pheomelanin are involved in binding to cations, anions, drugs, and
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chemicals, etc., and therefore play an important protective role within melanocytes
(Nordlund J. J, 1985). Neuromelanin, which is produced in dopaminergic neurons of the
human substantia nigra, can also chelate redox active metals (Cu, Mn, Cr) and toxic
metals (Cd, Hg, Pb), and thus protects against their ability to promote neurodegeneration
(Zecca L et al., 1996).
Given their complexity, melanosomes can be used as a model to study organelle
biogenesis, protein trafficking and processing, organelle movement, and cell-cell
interactions (like those occurring during melanin transfer between melanocytes and
keratinocytes) (Hearing V J, 2000). Therefore, even minor changes in the cellular
environment affect melanosomes and pigmentation. Numerous intrinsic and extrinsic
factors, including body distribution, ethnicity/gender differences, variable hormone-
responsiveness, genetic defects, hair cycle-dependent changes, age, UV-R, climate/season,
toxin, pollutants, chemical exposure and infestations, are responsible for a whole range of
responses in melanosome structure and distribution under different types of stress.
Cutaneous pigmentation is the outcome of two important events: the synthesis of
melanin by melanocytes and the transfer of melanosomes to surrounding keratinocytes
(Fitzpatrick T B and Szabo G, 1959). Although the number of melanocytes in human skin
of all types is essentially constant, the number, size, and manner in which melanosomes
are distributed within keratinocytes vary. The melanin content of human melanocytes is
heterogeneous not only between different skin types but also between different sites of
the skin from the same individual. This heterogeneity is highly regulated by gene
expression, which controls the overall activity and expression of melanosomal proteins
within individual melanocytes (Sturm R A et al., 1998). It has been shown that
melanocytes with low melanin content synthesize TYR more slowly and degrade it more
quickly than melanocytes with a higher melanin content and TYR activity (Halaban R et
al., 1983). In general, highly pigmented skin contains numerous single large melanosomal
particles (0.5–0.8 mm in diameter), which are ellipsoidal and intensely melanotic (stage
IV). Lighter pigmentation is associated with smaller (0.3–0.5 mm in diameter) and less
dense melanosomes (stages II and III), which are clustered in membrane-bound groups
(Toda K et al., 1972). These distinct patterns of melanosome type and distribution are
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present at birth and are not determined by external factors (such as sun exposure). They
are responsible for the wide variety of skin complexions.
5.1.4.5. Epidermal melanin unit and the involvement of keratinocytes in melanin
production:
Epidermal melanin unit is a functional and structural complex within the epidermis
consisting of two cell types: melanocytes and keratinocytes. The variation in skin color
among various races is determined mainly by the number, melanin content, and
distribution of melanosomes produced and transferred by each melanocyte to a cluster of
keratinocytes surrounding it (Jimbow K et al., 1976). Once in keratinocytes, the melanin
granules accumulate above the nuclei and absorb harmful UV before it can reach the
nucleus and damage the DNA. When melanin is produced and distributed properly in the
skin, dividing cells are protected at least in part from mutations that might otherwise be
caused by harmful UV (Kobayashi N et al., 1998). The melanocyte-keratinocyte complex
responds quickly to a wide range of environmental stimuli, often in paracrine and/or
autocrine manners and further triggers various molecular responses as illustrated in Fig. 5.
Thus, melanocytes respond to UV, melanocyte-stimulating hormone (MSH), endothelins,
growth factors, cytokines, etc. After UV-R exposure, melanocytes increase their
expression of proopiomelanocortin (POMC, the precursor of MSH) and its receptor
melanocortin 1 receptor (MC1-R), TYR and TYRP1, protein kinase C (PKC), and other
signaling factors (Chakraborty A K et al., 1996 and Funasaka Y et al., 1998). On the
other hand, it is known that UV stimulates the production of endothelin-1 (ET-1) by
keratinocytes and that those factors can then act in a paracrine manner to stimulate
melanocyte function (Tada A et al., 1998 and Abdel-Malek Z et al., 2000). ET-1 is a 2l
amino acid peptide with vasoactive properties first isolated from endothelial cells and
later found to be synthesized and secreted by keratinocytes as well (Imokawa G et al.,
1992; Yohn J J et al., 1993 and Hara M et al., 1995), particularly after exposure to UV-R
(Imokawa G et al., 1992; Yohn J J et al., 1993 and Hara M et al., 1995). The overall
effect of ET-1 is the increase of melanocyte dendricity and the enhancement of
melanocyte migration and melanization (Hara M et al., 1995). Binding of ET-1 to its G
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protein-coupled receptor (ETBR) on melanocytes activates a cascade of signaling
pathways, resulting in mobilization of intracellular calcium, activation of PKC, elevation
of cAMP levels, and activation of mitogen-activated protein kinase (MAPK) (Swope V B
et al., 1995 and Imokawa G et al., 1996). UV stimulates keratinocytes to produce ET-1
and also induces interleukin-1 (IL-1) production in these cells. IL-l is known to induce
ET-1 in keratinocytes in an autocrine manner. Therefore, it has been suggested that these
intracellular events in keratinocytes lead to increased TYR mRNA, protein, and enzymatic
activity in neighboring melanocytes as well as to an increase in melanocyte number
(Imokawa G et al., 1995).
In addition to keratinocytes, fibroblasts, and possibly other cells in the skin
produce cytokines, growth factors, and inflammatory mediators that can increase melanin
production and/or stimulate melanin transfer to keratinocytes by melanocytes. Melanocyte
growth factors affect not only the growth and pigmentation of melanocytes but also their
shape, dendricity, adhesion to matrix proteins, and mobility.
α-MSH, Adenocorticotropic hormone (ACTH), basic fibroblast growth factor
(bFGF), nerve growth factor (NGF), endothelins, granulocyte-macrophage colony-
stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and hepatocyte growth
factor (HGF) are keratinocyte-derived factors that are thought to be involved in the
regulation of the proliferation and/or differentiation of melanocytes (Hirobe T, 2005),
some acting through receptor-mediated signaling pathways (Fig. 5). It has been shown
that in human epidermis, -MSH (Chakraborty A K et al., 1996 and Slominski A et al.,
2000) and ACTH (Chakraborty A K et al., 1996; Slominski A et al., 2000 and
Wakamatsu K et al., 1997) are produced in and released by keratinocytes and are
involved in regulating melanogenesis and/or melanocyte dendrite formation. -MSH and
ACTH bind to a melanocyte-specific receptor, MC1-R (Cone R D et al., 1996), which
activates adenylate cyclase through G-protein, which then elevates cAMP from adenosine
triphosphate (Im S et al., 1998). Cyclic AMP exerts its effect in part through protein
kinase A (PKA) (Insel P A et al., 1975), which phosphorylates and activates the cAMP
response element binding protein (CREB) that binds to the cAMP response element
(CRE) present in the M promoter of the microphthalmia-associated transcription factor
(MITF) gene (Busca R and Ballotti R, 2000 and Tachibana M, 2000). The increase in
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MITF-M expression induces the up-regulation of TYR, TYRP1, and DCT (Busca R and
Ballotti R, 2000 and Tachibana M, 2000), which leads to melanin synthesis.
Figure 5.1.4: Scheme of signaling pathways within the epidermal melanin unit and mechanisms by which keratinocyte-derived factors act on human melanocyte proliferation and differentiation
Prostaglandin (PG) E2 and PGF2 are known to be produced and released from
human keratinocytes by the stimulation of proteinase-activated receptor 2 (PAR-2). PGE2
and PGF2 stimulate the dendritogenesis of human epidermal melanocytes in culture
(Scott G et al., 2004) through Prostaglandin E receptor 1 (EP1), Prostaglandin E receptor
3 (EP3) and Prostaglandin F receptor (FP). Their influence on melanocyte dendricity has
been suggested to be cAMP-independent and might be mediated through phospholipase C
(PLC) (Scott G et al., 2004). Hence, melanin formation is a complex mechanism which is
summarized briefly in Fig. 5.1.4.
The epidermis has a complex network that secretes as well as responds to
autocrine and paracrine cytokines produced by keratinocytes and melanocytes,
respectively. Human melanocyte proliferation requires the cross-talking of several
CHAPTER 5 PART II 5.1. INTRODUCTION
195
signaling pathways including the cAMP/PKA, PKC, and tyrosine kinase pathways
(Costin G E and Hearing VJ, 2007). Therefore, the mechanisms by which various factors
increase skin pigmentation are closely inter-related and briefly illustrated in Fig. 5.1.5.
Figure 5.1.5: Summarized mechanism of Skin pigmentation
5.1.5. Melanogenesis and its importance for cosmetic purposes:
• Melanogenesis is a normal biological mechanism of skin defense from UV,
pollution and other forms of stress that skin undergoes.
• Hence, a safe & effective fairness product is one that does not alter any normal
biological mechanism.
• The cause and mechanism of melanognesis & skin darkening has to be understood
before developing any fairness product.
Activation of MITF cAMP / α-MSH
Activation of Tyrosinase genes
Activation of Tyrosinase
Melanogenesis
Melanin transfer from MELANOCYTES to
peripheral KERATINOCYTES
Pigmentation
Serine protease & PAR 2 activation
CHAPTER 5 PART II 5.1. INTRODUCTION
196
• Pigmentation disorders like excess pigmentation and uneven pigmentation should
only be rectified by fairness products. Fairness products are expected to bring
back the skin color to normal as per the genetic predisposition of the person.
• Fairness products should only work on pigmentation disorders and not on normal
pigmentation mechanism. They should normalize pigmentation disorders like:
Excess or uneven pigmentation due to skin damage by UV & pollution, Acne
marks and under eye dark circles.
• Similarly tanning products are also in demand for cosmetic purposes. Skin
tanning is also a way of protecting fair-skinned people from skin cancer caused by
exposure to sunlight.
5.1.6. Hyperpigmentation:
There are numerous internal and external stresses that affect human skin pigmentation.
The list is fairly long, so the present study focuses on the common stress conditions
whose mechanisms of action are known to some extent or are currently under
investigation and whose use may affect the discovery of new approaches to reduce
hyperpigmentation. The common external factors are UV radiation that causes tanning
and photoageing; drugs, chemicals, etc. and internal factors are hormonal influences and
inflammation that cause postinflammatory hyperpigmentation.
5.1.6.1. Hyperpigmentation induced by external factors:
5.1.6.1.1. UV influence on human pigmentation:
The skin responds to UV exposure by developing two defensive barriers: thickening of
the stratum corneum and the elaboration of a melanin filter in cells of the epidermis. The
palms and soles are the regions with the thickest stratum corneum, and they are
exceptionally resistant to UV damage. UV triggers various mechanisms in the skin
keratinocytes and melanocytes (Fig. 5.1.6). The keratins and proteins within the stratum
corneum act mainly by scattering and absorbing the UV. UV sets in action an integrated
mechanism for increase in the number of melanocytes as well as stimulation of melanin
CHAPTER 5 PART II 5.1. INTRODUCTION
197
synthesis and melanocyte dendricity, a crucial morphological feature required for melanin
transfer from melanocytes to keratinocytes within the melanosomes. In humans, apart
from DNA damage and cancer, an increase of skin pigmentation over the basal
constitutive level called tanning, is mainly stimulated by UV.
The tanning response is determined by a complex set of regulatory processes involving direct effects of UV on melanocytes and indirect effects through the release of keratinocyte-derived factors Figure 5.1.6: Mechanisms involved in the hyperpigmentation induced by UV
This mechanism is probably triggered by keratinocytes, which respond to UV-R with
bursts of mitoses and with increased production of ET-1 and POMC, thus creating a new
demand for melanosomes. After UV, the epidermal melanin unit responds with increased
levels of TYR activity, increased synthesis of melanosomes, and higher rates of
melanosome transfer to keratinocytes to meet the new demand for melanosomes created
by the proliferation of keratinocytes (Robins A H, 1991). UV-A also penetrates deep into
the dermis; it is estimated that 19–50% of the solar UV-A can reach the depth of
melanocytes, whereas only 9–14% of solar UV-B reaches these cells. Therefore, UV-A
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198
stimulates melanin pigmentation, but the resultant tan appears to be transient and less
protective against UV-induced injury than tans generated after UV-B exposure. UV-B is
responsible for causing the sunburn reaction within the skin and is absorbed mainly by
the epidermis and upper dermis. Like UV-A, UV-B stimulates the production of melanin,
which constitutes the basis for tanning. UV-B has great potential to induce erythema, and
therefore its influence on the skin has been thoroughly investigated in vitro and in vivo
(Robins A H, 1991). One role of melanin in the skin is to neutralize the ROS generated by
a variety of factors, including UV-B (Nordlund J J, 1985), therefore functioning like a
natural sunscreen. The influence of UV on human pigmentation from the perspective of
tanning as well as photoageing is a perfect example of factors sharing intracellular
pathways with slightly different end results on the skin.
5.1.6.1.1.1. Tanning response to UV: The tanning response has been shown to have two
distinct phases, termed immediate pigment darkening and delayed tanning. Both have
strong genetic determinants and are generally more pronounced in individuals with dark
baseline (constitutive) pigmentation (Gilchrest B A et al., 1996).
Immediate tanning is a quick but transient brownish tan that follows the exposure
of skin to UV-A or visible light. It begins immediately after exposure, reaches a
maximum within 1–2 h, then fades between 3 and 24 h after exposure (Gilchrest B A et
al., 1996). Immediate tanning reaction is based on the photoxidation of preexisting
melanin, melanin precursors, or even of other epidermal constituents and/or their
redistribution in the epidermis.
Delayed tanning gives rise to a durable tan induced by repeated exposure mainly
to UV-B. It is a gradual process in which the skin starts darkening 48–72 h after
irradiation, reaches a maximum 3 wk after exposure, and the skin does not return to its
original melanin content until 8–10 months later (Gilchrest B A et al., 1996). Delayed
tanning is dependent on both qualitative and quantitative changes within melanocytes,
which enlarge in size, increase their dendricity, and develop a diffuse distribution of thick
filaments in their cell bodies. Therefore, delayed tanning is due to an increase in
melanocyte numbers and melanogenesis.
CHAPTER 5 PART II 5.1. INTRODUCTION
199
5.1.6.1.1.2. UV induced ROS, inflammation and effect on melanogenesis: UV also
causes peroxidation of lipids in cellular membranes, leading to generation of ROS, which
may stimulate melanocytes to produce excess melanin (Sies H and Stahl W, 2004).
Usually, only lipids containing two or more conjugated double bonds in their structure
absorb UV-B and thus liberate arachidonic acid, which is subsequently metabolized to
various species of PGs and leukotrienes, generates previtamin D3 from 7-
dehydrocholesterol with subsequent processing to various photoproducts and the
biologically active 1 , 25-dihydroxy-vitamin D3, and releases diacylglycerol (DAG),
which in turn activates PKC, among other possible roles in signal transduction (Nishizuka
Y, 1986; Fig. 5). It was observed that addition of DAG to cultured human melanocytes
increases their melanin content several fold within 24 h (Gordon P R and Gilchrest B A,
1989), and subsequent work demonstrated that UV-R acts synergistically with DAG to
enhance melanogenesis (Friedmann P S et al., 1990). Direct melanogenic effects of UV
on melanocytes might also involve the production of Nitric oxide (NO), which is
considered a major intra- and intercellular messenger molecule. NO elicits its effects
through the activation of a soluble guanylate cyclase, leading to an increase in
intracellular cyclic guanosine monophosphate (cGMP) content and the activation of
cGMP-dependent protein kinase. Furthermore, it has been shown that UV-R increases
both NO and cGMP production, suggesting they are both required for UV-B-induced
melanogenesis (Fig. 5.1.6).
5.1.6.1.2. The action of drugs, chemicals, etc., on human skin pigmentation:
Numerous common drugs can stimulate human skin hyperpigmentation such as certain
antibiotics (sulfonamides and tetracyclines), diuretics, nonsteroidal antiinflammatory
drugs, pain relievers, and some psychoactive medications. The use of oral contraceptives
has been associated with the development of discoloration of the cheeks, forehead, and
nose (Goh C L and Dlova C N, 1999) similar to chloasma with increased melanogenesis
and enlarged melanocytes. Certain antiepileptic agents (mainly hydantoins) may also
cause skin hyperpigmentation (Levantine A and Almeyda J, 1973). Their long-term use
induces a brownish coloration of the face and neck, similar to chloasma of pregnancy. It
CHAPTER 5 PART II 5.1. INTRODUCTION
200
is already known that chloroquine has an affinity for melanin and causes skin
hyperpigmentation. Different studies have detected melanin in the dermis of patients
undergoing chloroquine treatment (Levy H, 1982).
Levodopa, often used to treat Parkinson’s disease, also induces hyperpigmentation
of the skin (Robins A H, 1991). DOPA is normally transformed into melanin within
melanosomes; therefore, DOPA therapy (applied as levodopa treatment) may possibly
enhance melanin biosynthesis. Heavy metals can also elicit hyperpigmentation, which can
arise after the extensive use of drugs containing arsenic, bismuth, gold, or silver
(Molokhia M M and Portnoy B, 1973). The metals are believed to act by binding, and
thereby inactivating, sulfhydryl compounds in the skin that normally inhibit TYR activity.
Removal of this inhibition stimulates melanogenesis. Mercury products inactivate TYR
probably by replacing the essential copper in the enzymatic site of that protein. Some
chemotherapy agents also can cause hyperpigmentation, the most common ones being
cyclophosphamide, 5-fluorouracil, doxorubicin, daunorubicin, and bleomycin. Their
mechanisms of action are currently unknown but may involve direct toxicity, stimulation
of melanocytes, and/or inflammation.
5.1.6.2. Hyperpigmentation induced by internal factors:
5.1.6.2.1. Hormonal influence on human skin pigmentation:
Hyperpigmentation is sometimes seen during pregnancy and this condition is called
melasma, chloasma, or mask of pregnancy; it occurs mainly on the cheeks, upper lip, chin,
and forehead. It is characterized by a symmetrical hypermelanosis with an irregular
coloration, ranging from light brown to gray and dark brown. Although melasma is
usually associated with pregnancy, multiple other factors can contribute to its
development including UV exposure, hormone therapy, estrogen-containing oral
contraceptives, genetic influences, certain cosmetics, endocrine or hepatic dysfunction,
and selected antiepileptic drugs (Table 5.1.1). Of the environmental sources, UV is the
most influential (Ortonne J P et al., 2003 and Barankin B et al., 2002).
The areas of hyperpigmentation seen in melasma exhibit increased deposition of
melanin in the epidermis and dermis (Kang W H et al., 2002 and Grimes P E et al., 2005).
CHAPTER 5 PART II 5.1. INTRODUCTION
201
No increase in the number of melanocytes in those areas is observed, but the melanocytes
are larger, more dendritic, and show increased melanogenesis, producing especially
eumelanin (Grimes P E et al., 2005). Studies confirmed an increased number of
melanosomes in keratinocytes, melanocytes, and dendrites in lesional skin compared with
nonlesional skin. During pregnancy (especially in the third trimester), elevated levels of
estrogen, progesterone, and MSH have often been found in association with melasma
(Smith A G et al., 1977 and Parker F, 1981). TYR activity increases and cellular
proliferation is reduced after treatment of melanocytes in culture with ß-estradiol (Ranson
M et al., 1988). Sex steroids increase transcription of genes encoding melanogenic
enzymes in normal human melanocytes, especially those for DCT and TYR
(Kippenberger S et al., 1998). These results are consistent with the significant increases in
melanin synthesis and TYR activity reported for normal human melanocytes under
similar conditions in culture (McLeod S D et al., 1994). It is known that estrogens
improve skin moisture and also increase its thickness and collagen content. Therefore,
estrogen plays a key role in skin ageing homeostasis given the fact that skin appearance
declines quickly in the postmenopausal years. Despite the knowledge that estrogens have
such important effects on skin, their cellular and molecular mechanisms of action are still
poorly understood and their influence on pigmentation is still far from clear.
Examination of the effects of estrogen treatment on TYR activity has revealed a
stimulation of this melanogenic enzyme (Ranson M et al., 1988 and Kippenberger S et al.,
1998). It was recently demonstrated that androgens modulate TYR activity via regulation
of cAMP, a key regulator of skin pigmentation (Tadokoro T et al., 2003). The sum of
these studies emphasizes the importance of both sex hormones in regulating skin
pigmentation.
5.1.6.2.2. Postinflammatory hyperpigmentation of the skin:
Postinflammatory hyperpigmentation is manifested by discrete, hyperpigmented macules
with hazy, feathered margins, which may involve the epidermis and/or dermis. This
usually develops after resolution of inflammatory skin eruptions like acne, contact
dermatitis, or atopic dermatitis. Postinflammatory hyperpigmentation is more common in
CHAPTER 5 PART II 5.1. INTRODUCTION
202
patients with darker skin and, at the cellular level, is characterized by a normal number of
melanocytes that have increased melanin production (Table 5.1.1).
Arachidonate-derived chemical mediators, especially leukotrienes and
thromboxanes may be responsible for the induction of post inflammatory
hyperpigmentation of the skin because they can stimulate normal human melanocytes in
vitro. These cells become swollen and more dendritic with increased amounts of
immunoreactive TYR. Such morphological changes are thought to be required for the
transfer of melanosomes to surrounding keratinocytes. Those effects were stronger than
that elicited by PGE2, which, together with PGE1 and PGD2, are known to be important
endogenous regulators of inflammatory diseases in the skin and to stimulate mammalian
pigment cells in vitro (Tomita Y et al., 1987) and in vivo (Nordlund J J et al., 1986).
Despite the common frequency of skin hyperpigmentation following inflammation, the
mechanisms responsible for melanin synthesis have not yet been completely clarified, but
some data have became available recently, as follows.
In the skin, PGs (especially PGE2, PGF2 , and small quantities of prostacyclin)
are produced (Pentland A P and Mahoney M G, 1990) and rapidly released by
keratinocytes after UV-R (Hanson D and DeLeo V, 1990 and Pentland A P et al., 1990).
They are chronically present in inflammatory skin lesions and are involved in wound
healing (Pentland A P et al., 1987). UV-R stimulates production of PGF2 by
melanocytes, which in turn stimulates the activity and expression of TYR, suggesting that
PGF2 could act as an autocrine factor for melanocyte differentiation (Scott G et al.,
2005).
On the other hand, PAR-2 is an important factor regulating skin pigmentation
because its activation in keratinocytes stimulates their uptake of melanosomes through
phagocytosis. It has been reported that activation of PAR-2 in keratinocytes stimulates the
release of PGE2 and PGF2 , which act as paracrine factors that stimulate melanocyte
dendricity (Scott G et al., 2004). Melanocyte dendrite formation has been linked to the
cAMP-dependent activation of Rac and the inhibition of Rho (Busca R et al., 1998; Scott
G, 2002 and Scott G and Leopardi S, 2003). However, recent studies demonstrated that
neither PGE2 nor PGF2 stimulates cAMP in melanocytes, thus demonstrating that these
PGs stimulate dendrite formation in a cAMP-independent manner (Scott G et al., 2004).
CHAPTER 5 PART II 5.1. INTRODUCTION
203
These data suggest that PAR-2 mediates cutaneous pigmentation through regulation of
melanosome uptake and production of PGs, which act as paracrine factors to stimulate
melanocyte dendricity.
All the inflammatory factors and pathways described above interact within the
skin; the final result is an increase of TYR activity and melanocyte dendricity, which
promotes the production of melanin and its distribution to keratinocytes. Therefore,
different factors are responsible for increasing human skin pigmentation via various
intracellular pathways. Table 5.1.1 summarizes the various conditions of
hyperpigmentation, their characteristics and causative factors. Table 5.1.2 summarizes
some of the internal or external stresses and the secondary messengers and effectors that
are involved.
CHAPTER 1 PART II 5.1. INTRODUCTION
204
Tabl
e 5.
1.1:
Sum
mar
y of
hyp
erpi
gmen
tatio
n co
nditi
ons,
caus
ativ
e fa
ctor
s, cl
inic
al fe
atur
es a
nd c
ellu
lar c
hara
cter
istic
s
Mol
ecul
ar m
arke
rs a
ffec
ted
1. In
crea
sed
TYR
-pos
itive
cel
ls p
er le
ngth
of t
he
derm
al/e
pide
rmal
inte
rfac
e co
mpa
red
with
un
affe
cted
skin
. 2.
Ker
atin
ocyt
es’ p
oten
tial t
o pr
oduc
e ET
-1 is
si
gnifi
cant
ly h
ighe
r com
pare
d w
ith u
naff
ecte
d sk
in.
3. T
NF-
α is
up-
regu
late
d in
the
SL le
sion
al
epid
erm
is.
1. H
igh
leve
ls o
f pro
gest
eron
e, e
stro
gen,
and
M
SH.
2. In
crea
sed
trans
crip
tion
of g
enes
enc
odin
g D
CT,
TY
R.
3. M
elan
ocyt
es a
re la
rger
and
mor
e de
ndrit
ic.
1. P
GE2
and
PG
F2 sy
nthe
sis i
s up-
regu
late
d;
they
act
as p
arac
rine
fact
ors w
hich
stim
ulat
e m
elan
ocyt
e de
ndric
ity.
2. L
euko
trien
es, T
NF-
α an
d th
rom
boxa
nes m
ay
be re
spon
sibl
e fo
r the
indu
ctio
n of
pos
t-in
flam
mat
ory
hype
rpig
men
tatio
n.
Incr
ease
d R
OS
gene
ratio
n an
d su
bseq
uent
cel
l da
mag
e re
sulti
ng in
ove
r exp
ress
ion
of
Tyro
sina
se, M
SH &
cA
MP
as a
stre
ss re
spon
se.
Cel
lula
r ch
arac
teri
stic
s
1. In
crea
sed
mel
anin
pr
oduc
tion.
2.
Slig
ht
incr
ease
in
num
ber o
f m
elan
ocyt
es.
1. In
crea
sed
mel
anin
pr
oduc
tion.
2.
Nor
mal
nu
mbe
r of
mel
anoc
ytes
1.
Incr
ease
d m
elan
in
prod
uctio
n.
2. N
orm
al
num
ber o
f m
elan
ocyt
es.
Tem
pora
ry
incr
ease
in
mel
anin
pr
oduc
tion
Clin
ical
feat
ures
1. C
ircum
scrib
ed, b
row
n to
bl
ack
mac
ules
. 2.
Ran
ge fr
om <
1 m
m to
se
vera
l cm
. 3.
Occ
ur in
epi
derm
is.
4. F
ound
on
UV
-exp
osed
are
as
of th
e bo
dy su
ch a
s the
face
, do
rsum
of t
he h
and,
ext
enso
r fo
rear
m a
nd u
pper
bac
k.
1. S
ymm
etric
faci
al
hype
rpig
men
tatio
n.
2. M
ay in
volv
e ep
ider
mis
, de
rmis
or b
oth.
1.
Dis
cret
e hy
perp
igm
ente
d m
acul
es w
ith h
azy
mar
gins
. 2.
May
invo
lve
epid
erm
is,
derm
is o
r bot
h.
Loss
in sk
in g
low
& ta
nnin
g on
U
V-e
xpos
ed a
reas
of t
he b
ody
such
as t
he fa
ce, d
orsu
m o
f the
ha
nd, e
xten
sor f
orea
rm a
nd
uppe
r bac
k.
Cau
sativ
e fa
ctor
(s)
Indu
ced
by U
V
Sun
expo
sure
, pr
egna
ncy,
or
al
cont
race
ptiv
es,
anti-
epile
ptic
s et
c.
Dev
elop
s aft
er
reso
lutio
n of
ac
ne, c
onta
ct
derm
atiti
s, et
c.
Exp
osur
e to
U
V &
pol
lutio
n
Hyp
erpi
gmen
tatio
n di
sord
er
Sola
r le
ntig
ines
(S
L)
Mel
asm
a
Post
-in
flam
mat
ory
hype
r pi
gmen
tatio
n
Skin
tann
ing
CHAPTER 1 PART II 5.1. INTRODUCTION
205
Table 5.1.2: Summary of external and internal stress increasing human skin pigmentation and their intracellular secondary messengers and effectors
Stress Secondary messenger Secondary effector UV induces the production of: NO cGMP Protein kinase G (PKG) ET-1 DAG Protein kinase C (PKC) -MSH, ACTH, PGE2 cAMP Protein kinase A (PKA)
Hormones (non classical pathway)
cAMP Mitogen activated protein kinase (MAPK)
Inflammation Inositol 1,4,5 triphosphate MAPK/PKC
5.1.7. Skin lightening:
Skin lightening effect is brought about effectively by a synchronized combination of
various biological mechanisms in skin cells. Main targets like Tyrosinase inhibition and
Melanogenesis inhibition when supported by Antioxidant and Anti inflammatory
mechanisms exert a positive effect on skin cells, and that is a crucial step in creating or
maintaining light pigmented healthy and conditioned skin.
5.1.7.1. Major targets for skin lightening:
Given the complexity of skin and the pathways involved in regulating melanogenesis, one
can assume that stimulating or inhibiting more than one pathway affected by stress would
lead to synergistic effects in increasing or decreasing pigmentation. Shedding light on the
molecular mechanisms underlying hyperpigmentation induced by internal or external
factors, research can be applied to various ends like finding new technologies or
compounds that could decrease pigmentation. Fairness products are expected to rectify
stress related abnormalities in pigmentation mechanism as describe in Table 5.1.1.
Therefore, understanding the mechanisms by which different compounds affect
melanogenesis is of great interest pharmaceutically and cosmeceutically. Table 5.1.3
summarizes the major targets for skin lightening. Table 5.1.4 summarizes the melanin
inhibitory pathways that result in adverse effects.
CHAPTER 5 PART II 5.1. INTRODUCTION
206
5.1.7.1.1. Peptide hormone (Melanocyte stimulating hormone - MSH) inhibition for
skin lightening:
The Melanocyte-stimulating hormones are a class of peptide hormones that in nature are
produced by cells in the intermediate lobe of the pituitary gland. They stimulate the
production and release of melanin (melanogenesis) by melanocytes in skin and hair.
An increase in MSH will cause a darkening in humans. Melanocyte-stimulating hormone
increases in humans during pregnancy. This, along with increased estrogens, causes
increased pigmentation in pregnant women. Melanocyte-stimulating hormone belongs to
a group called the melanocortins. This group includes ACTH, α-MSH, β-MSH and γ-
MSH; these peptides are all cleavage products of a large precursor peptide called pro-
opiomelanocortin (POMC). α-MSH is the most important melanocortin for pigmentation.
Hence, inhibition of α-MSH is one of the major targets for skin lightening.
CHAPTER 1 PART II 5.1. INTRODUCTION
207
Table 5.1.3: Major Targets for skin lightening
Target Mechanism Remarks
Peptide hormone (MSH) inhibition
MSH induces melanogenesis in melanocytes.
Inhibitors of MSH induced melanogenesis are potential skin lighteners.
Tyrosinase inhibition
Tyrosinase expression results in melanin formation.
Inhibitors of Tyrosinase are potential skin lighteners.
Protease inhibition Serine proteases induce pigmentation through inflammation.
Inhibitors of serine proteases (elastase, collagenase and hyaluronidase) have potential for skin lightening.
Anti inflammatory potential
Overstay of Inflammatory response by markers like TNF α etc. induces pigmentation by dermal matrix damage and induction of pigmentation by affected melanocytes.
Inhibitors of inflammatory markers like TNF α etc. have potential for skin lightening.
Antioxidant potential
Free radical damage induces pigmentation.
Antioxidants have skin lightening potential.
UV protection UV induces pigmentation through NO and free radical induction.
UV protectants inhibit NO and ROS induced melanognesis.
cAMP induced melanogenesis
cAMP upregulates melanin production by PKC pathway.
Inhibitors of cAMP induced melanogenesis have potential for skin lightening
Melanin transfer in melanocyte-keratinocyte coculture
Endothelin induces transfer of melanin from melanocytes to keratinocytes
Endothelin antagonists inhibit melanin migration to upper keratinocyte layers. Hence inhibitors of melanin migration in cocultures are good skin lighteners.
MITF activation. Upregulation of MITF expression mediates melanogenesis stimulated by cAMP.
Inhibitors of MITF have good skin lightening potential.
CHAPTER 5 PART II 5.1. INTRODUCTION
208
Table 5.1.4: Melanin inhibitory targets that result in adverse effects
Target Mechanism Remarks
Phenylalanine hydroxylase inhibition
Phenylalanine hydroxylase catalyses the formation of tyrosine which is the precursor of melanin.
Inhibitors of Phenylalanine hydroxylase inhibit the melanin formation pathway. Tyrosine is required for other metabolic pathways. Inactive phenylalanine hydroxylase results in disorders like phenylketonuria etc.
Inhibition of tyrosinase glycosylation
Tyrosinase gets glycosylated in the endoplasmic reticulum and gets into its active form.
Inhibition of glycosylation of tyrosinase results in the formation of inactive tyrosinase that cannot catalyze the formation of melanin. Inhibition of tyrosinase glycosylation results in albinism.
5.1.7.1.2. Tyrosinase inhibition for skin lightening:
Tyrosinase (monophenol, l-dopa:oxygen oxidoreductase, EC 1.14.18.1) is a copper-
containing enzyme present in plant and animal tissues that catalyzes the production of
melanin and other pigments by oxidation of phenols such as tyrosine. Tyrosinases from
different species are diverse in terms of their structural properties, tissue distribution and
cellular location. Human tyrosinase is a transmembrane protein. In humans, tyrosinase is
sorted into melanosomes and the catalytically active domain of the protein resides within
melanosomes. Only a small enzymatically non-essential part of the protein extends into
the cytoplasm. As described earlier and represented in fig, the gene for Tyrosinase is
regulated by the Microphthalmia-associated transcription factor (MITF). Preventing
the maturation or intracellular trafficking of tyrosinase is an alternative way to reduce the
effect of the enzyme on pigmentation (Halaban R et al., 1983; Petrescu S M et al., 1997
and Francis E et al., 2003). Various natural extracts can also influence tyrosinase mRNA
at the transcription level; also mRNA of the other tyrosinase-related proteins or MITF
can be affected (Lee M H et al., 2006; Kim J H et al., 2008 and Zi S X et al., 2009).
Hence, Inhibition of Tyrosinase and/or MITF is one of the major targets for skin
lightening.
CHAPTER 5 PART II 5.1. INTRODUCTION
209
Figure 5.1.7: Metabolic pathway of Tyrosine conversion to Melanin
Pigmentation is a multistep process critically dependent on the functional integrity of
tyrosinase, the rate-limiting enzyme in melanin synthesis. As illustrated in Fig. 5.1.7,
biosynthesis of melanin is initiated by the catalytic oxidation of tyrosine to 3,4 dihydroxy
phenylalanine (dopa) by tyrosinase. Subsequent reactions happen spontaneously where
tyrosine catalyzes the dehydrogenation of dopa to dopaquinone and 5,6-dihydroxyindole
to indole-5,6-quinone, key reactions in melanin biosynthesis (Fitzpatrick T B et al., 1949;
Hearing V J and Ekel T M, 1976; Korner A and Pawelek J, 1982 and Tripathi R K et al.,
CHAPTER 1 PART II 5.1. INTRODUCTION
210
1992), eventually resulting in the synthesis of melanin. The amino acid sequences
deduced from human and mouse tyrosinase (TYR and Tyr, respectively) cDNAs predict a
type I membrane glycoprotein with an N-terminal signal sequence and catalytic copper
binding regions with conserved positions of histidine and cysteine residues (Kwon B S et
al., 1987; Kwon B S et al., 1989; Muller G et al., 1988; Yamamoto H et al., 1989 and
Bouchard B et al., 1989). The 60-kDa tyrosinase core polypeptide is modified in the
endoplasmic reticulum (ER) by cotranslational addition of multiple N-linked glycans,
producing the 70-kDa species (Halaban R et al., 1983 and Halaban R et al., 1984).
Complex sugar modifications in the Golgi apparatus further increases tyrosinase's
molecular mass to 80 kDa, the size of the mature wild-type (WT) isoform (Halaban R et
al., 1983; Halaban R et al., 1984 and Halaban R et al., 1997). In normal melanocytes the
70-kDa protein eventually is released from this complex and proceeds to the Golgi
apparatus en route to the melanosomes, the site of melanin synthesis. Tyrosinase is a
melanocyte-specific enzyme critical for the synthesis of melanin, a process normally
restricted to a post-Golgi compartment termed the melanosome. Therefore, inhibition of
tyrosinase activity but not the inhibition of tyrosinase formation at the molecular level is
a major target for skin lightening.
Loss-of-function mutations in tyrosinase are the cause of albinism, demonstrating
the importance of the enzyme in pigmentation. Mutations in tyrosinase are the cause of
classic type I oculocutaneous albinism, an autosomal recessive genetic disorder
characterized by the absence of melanin in melanocytes (Oetting W S and King R A,
1999). Trafficking of albino tyrosinase from the endoplasmic reticulum (ER) to the Golgi
apparatus and beyond is disrupted. Albinism, at least in part, is an ER retention disease.
Mutant proteins, representatives of the albino phenotype, are retained in the ER bound to
calnexin and calreticulin and are not released to the targeted organelle, the melanosome.
Albinism is a disease associated with retention of malfolded protein in the ER that
includes cystic fibrosis and emphysema (Callea F et al., 1992; Sifers R N, 1995;
Kuznetsov G and Nigam S K, 1998 and Kopito R R, 1999). TYR(R402Q)/Tyr(H402A)
gene mutations behaved like the much-studied CFTR(ΔF508) mutation that is responsible
for the large majority of cases of cystic fibrosis (Kopito R R, 1999), and the model
trafficking thermosensitive protein vesicular stomatitis virus G protein (tsO45 strain)
CHAPTER 1 PART II 5.1. INTRODUCTION
211
(Presley J F et al., 1997). Curcumin and its derivative (tetrahydrocurcumin) are reported
to have a very significant tyrosinase inhibitory activity. Curcumin and its derivatives are
also reported to rescue for CFTR (ΔF508) mutation for the treatment of cystic fibrosis
(Lipecka J et al., 2006 and Patent No: 7521580). This indicates that although cucumin
and its derivatives do inhibit tyrosinase, they do not have any effect on the gene and
protein mechanisms responsible for normal tyrosinase formation and in fact they could
probable have a positive effect on the molecular events resulting in albinism.
5.1.7.1.3. Serine protease inhibition for skin lightening:
Serine proteases or serine endopeptidases are proteases (enzymes that cut peptide bonds
in proteins) in which one of the amino acids at the active site is serine. Serine protease
activated receptor, PAR-2 regulates pigmentation by affecting keratinocyte phagocytosis.
PAR-2 activation increases the ability of keratinocytes to ingest melanosomes, resulting
in skin darkening. Inhibition of PAR-2 activation by serine protease inhibitors reduces
pigment transfer and leads to depigmentation. Inhibition of PAR-2 activation also
prevents UVB induced pigmentation and reduces tanning. Protease activated receptor 2
(PAR-2) is important for melanosomal transfer from melanocytes to keratinocytes and
this transfer can be used as a target for skin lightening (Sharlow E R et al., 2000; Seiberg
M et al., 2000 and Seiberg M, 2001). Hence, Serine protease inhibition is also one of the
targets for skin lightening.
5.1.7.1.4. Inhibition of free radicals and inflammation for skin lightening:
Free radical damage can also induce pigmentation. Free radicals generated in the body
due to stress conditions like UV exposure, pollution, unhealthy food habits and ageing,
primarily damage the skin. As described in detail earlier, free radicals trigger
inflammatory markers that eventually cause skin damage. As a result, excess melanin is
produced in a defense mechanism, resulting in pigmentation. Hence, antioxidant and anti
inflammatory properties are desirable for effective skin lightening. Topically-applied
antioxidants do have merit for all skin types to keep skin healthy and help prevent sun
damage and improve cell function. Antioxidants have been conclusively shown to exert a
CHAPTER 5 PART II 5.1. INTRODUCTION
212
positive effect on reducing skin irritation and inflammation, and that is a crucial step in
creating or maintaining healthy, vibrant skin and, therefore potentially reducing wrinkles. Hence, Anti oxidant and anti inflammatory actives play a significant role in healthy skin
(Rasik A M and Shukla A, 2000 and Kalka K et al., 2000). Although all antioxidant and
anti inflammatory actives do not necessarily inhibit melanin synthesis directly, they do
have a positive synergistic effect for skin lightening. For example, Glutathione is a
significant antioxidant and not a direct inhibitor of melanin synthesis. However, when
taken internally as a nutricosmetic, it helps in skin lightening.
5.1.7.1.5. UV protection to reduce skin darkening:
As described in detail earlier and in the illustrations in Fig. 5.1.8, UV exposure leads to
free radical damage and excessive pigmentation due to the migration of mature
melanosomes from melanocytes to keratinocytes, as a defense mechanism. Hence, UV
protection is important to prevent skin darkening.
A B A) Transfer of mature melanosomes to keratinocytes B) Melanocyte surrounded by keratinocytes, melanin synthesis and release of melanin granules in keratinocytes Figure 5.1.8: Melanin synthesis in melanocytes and transfer to keratinocytes
CHAPTER 1 PART II 5.1. INTRODUCTION
213
5.1.7.1.6. Inhibition of cAMP induced pigmentation for skin lightening:
As described earlier and illustrated in Fig. 5.1.9, cAMP up regulates melanin production
by Protein kinase pathway. MITF is stimulated via cAMP and PKA pathway. Through a
series of steps, tyrosinase is activated which results in pigmentation. Hence, inhibitors of
cAMP induced melanogenesis are potential skin lighteners.
Figure 5.1.9: Effect of cAMP on Tyrosinase activity
5.1.7.1.7. Inhibition of melanin transfer from melanocyte to keratinocyte for skin
lightening:
Fig. 5.1.10 illustrates the transfer of melanin and
melanoma formation in the epidermis of skin. Hence, the
transfer of melanin from melanocytes to keratinocytes is
the ultimate step that results in darkening of the peripheral
layers of the skin. Antagonists of melanin migration to
peripheral keratinocyte layers prevent pigmentation.
Hence inhibitors of melanin migration in cocultures are
good skin lighteners.
Figure 5.1.10: Melanin migration and skin darkening
CHAPTER 5 PART II 5.1. INTRODUCTION
214
5.1.7.2. Cell proliferation enhancement and its role in skin lightening:
Some of the actives with no significant antioxidant, anti inflammatory, UV protection or
skin lightening potential but yet with a significant cell proliferation potential will have a
very good effect in skin lightening as well, not by any direct activity but by enhancing the
cell rejuvenation. For example, when the cell proliferation enhancer is taken in
combination with significant skin lightening actives, while the actives lighten the skin
cells, the cell proliferation enhancer helps in rejuvenation of the cells, with an effect that
the darker skin is continuously replenished by fresh lightened skin cells. In the process
the skin lightening process is fastened as the dry dead cells on the superficial layers of
skin are peeled off.
5.1.8. Fairness products and effective evaluation:
Many times it is observed that fairness products do not give the desired results or
sometimes result in adverse effects. The reason is, most of the fairness actives when not
used appropriately as per the root cause of hyper pigmentation can result in no effect or
adverse effect. An active that works for one individual may not work for another
individual. Again, the reason is the root cause of the hyper pigmentation. Hence, it is
important to screen skin lightening actives for various biological mechanisms of action
with respect to efficacy. The screened skin lightening actives should be positioned
accordingly with respect to their specificity in the mode of action for rectifying the
specific cause of pigmentation disorder. The recent development in cosmetic research is
mechanism oriented as shown in the classical example in Fig. 5.1.11, which illustrates
various actives for various modes of action (Ortonne J P and Bissett D L, 2008).
CHAPTER 1 PART II 5.1. INTRODUCTION
215
Figure 5.1.11: Mechanism of action and actives for pigmentation control
From the elaborate research areas in the field of cosmetics, the present work aims
at conclusive research on fairness actives. Emphasis in this study has been laid on various
synthetic and natural fairness actives, their screening through various mechanisms of
melanogenesis by various in vitro technologies and positioning them in accordance to
their specific mode of action for rectifying pigmentation disorders. Based on how an
active works on various pigmentation mechanisms, all the actives can be categorized as
to what sort of skin darkening they can rectify. This can give clarity as to how it can be
recommended and clear claims can be made with respect to its specific mode of action.
The most effective and highly recommended skin lightening active may be the one which
inhibits most of the mechanisms for pigmentation disorders.
It is therefore a primordial need to categorize the actives as per their effect on a
particular pigmentation disorder. The present research work aims towards the appropriate
positioning and promotion of actives for efficacy towards specific pigmentation disorders.
CHAPTER 1 PART II 5.1. INTRODUCTION
216
5.1.9. Objectives of the research work:
• Screening of various actives through in vitro mechanisms for reducing
hyperpigmentation.
• Screening of antioxidants, anti inflammatory and skin conditioning actives
unexplored earlier for skin lightening efficacy through in vitro mechanisms for
reducing hyperpigmentation.
• Positioning the screened actives in accordance to their specific mode of action for
rectifying pigmentation disorders as
Inhibitors of solar lentiges, melasma and over all skin tanning by inhibitors of
tyrosinase enzyme and melanogenesis.
Inhibitors of UV and free radical induced pigmentation by UV protectants and
antioxidants.
Inhibitors of post inflammatory hyperpigmentation like acne marks etc. by anti
inflammatory actives.
Inhibitors of one or more of the above mechanisms of hyperpigmentation
conditions.
Cell proliferation and collagen enhancers.
• Demonstration of synergistic skin lightening effect by physical combination and
chemical conjugation of actives with different mechanisms of action.
• Study of Nutricosmetic potential of antioxidant plant actives, synergistic
antioxidant compositions and nutricosmetic formulations.
CHAPTER 1 PART II 5.2. MATERIALS AND METHODS
217
5.2.1. Materials:
5.2.1.1. Cell lines:
Swiss 3T3 mouse fibroblast cells and B16F1 mouse melanoma cells were procured from
ATCC, Manassas, VA, USA. Normal human dermal fibroblasts (NHDF) were obtained
from PromoCell GmbH, Heidelberg, Germany. Human Osteosarcoma cell lines (HOS)
were obtained from National Center for Cell Science (NCCS), Pune.
5.2.1.2. Culture media, reagents and cell culture microplates:
Dulbecco’s minimum essential medium (DMEM), RPMI 1640 medium, α-Melanocyte
stimulating hormone (α-MSH), 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), 2,7,
dichlorofluorescin diacetate, Ferrous sulphate, 2,2′ -Azobis(2-methylpropionamidine)
dihydrochloride (AAPH), 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid
(trolox), Fluorescein sodium salt (3’,6’-dihydroxy-spiro[isobenzofuran-1[3H], 9’[9H]-
xanthen]-3-one), Hydrogen peroxide solution, Cobalt (II) fluoride tetrahydrate, Picolinic
acid, Gallic acid, Hyaluronic acid potassium salt from from human umbilical cord,
Hyaluronidase from bovine testes, Cetyl pyridinium chloride, Lipopolysaccharide (LPS),
Picric acid, Sirius Red stain and Dimethylsulphoxide (DMSO) were procured from Sigma,
St. Louis MO., USA. NHDF growth medium was obtained from PromoCell GmbH,
Heidelberg, Germany. Foetal bovine serum (FBS) was procured from Gibco, New York,
USA. EnzChek collagenase inhibiton kit and EnzChek elastase inhibiton kit was obtained
from Molecular Probes, Life Technologies Corporation, California, USA. Tumor necrosis
factor (TNF) α Elisa kit was obtained from R&D systems Inc, Minneapolis, USA.
Forskolin was obtained from the Phytochemistry department of Sami Labs. Neutral red
stain was procured from Himedia Laboratories, Mumbai, India. 96 well and 24 well clear
microplates and 96 well black plates were procured from BD Biosciences, New Jersey,
USA.
5.2. MATERIAL AND METHODS
CHAPTER 1 PART II 5.2. MATERIALS AND METHODS
218
5.2.1.3. Ultraviolet irradiation (UV) source:
Three G15T8E UV B lamps having 14.7W lamp wattage, 0.3A lamp current, 55V lamp
voltage, UV output of 3.1W and with an intensity of 33.3 µW cm-2 were obtained from
Sankyo Denki Co., Ltd, Japan and used as the source of UV irradiation.
5.2.1.4. Test materials:
Ascorbic acid, Octylmethoxycinnamate (OMC), Tetrahydrocurcumin, Glabridin,
Artocarpin, Artocarpus lakoocha heart wood extracts containing varying concentrations
of Oxyresveratrol, Dihydro-oxyresveratrol, Resveratrol, Pterostilbene, 3-Hydroxy
Pterostilbene, Gnetol, Amla extract, Hydroxychavicol, Citrullus colocynthis extract, Oat
ceramides, Apple ceramides, liquid endosperm of Coconut, Galanga extract and
Pomergranate fruit and rind extracts were obtained as mentioned in Chapter 4 Part II,
4.2.1.4.
Arbutin: Arbutin (4-Hydroxyphenyl-β-D-glucopyranoside or Hydroquinone β-D-
glucopyranoside) is off white colored water soluble powder obtained from Sigma
chemicals and used for validation studies in the present research.
Kojic acid: Kojic acid (2-Hydroxymethyl-5-hydroxy-γ-pyrone, 5-Hydroxy-2-
hydroxymethyl-4H-4-pyranone) is white colored water soluble powder obtained from
Sigma chemicals and used for validation studies in the present research.
Coenzyme Q10 (Co Q10): Co Q10 also known as Ubiquinone 10 is yellow colored
powder and an endogenous antioxidant obtained from Sigma chemicals and used for
studies in combination with other actives.
Piperlongumine: Piperlongumine was isolated by the ethanolic extraction of Piper
longum roots.
Thymohydroquinone: Thymohydroquinone was isolated from Nigella sativa (Black
cumin) seed extract by alcoholic extraction.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
219
Eugenia jambolana (Jamun) extract: Jamun extract was made by the hydroalcoholic
extraction of jamun fruit pulp.
Avenanthramides: Different types of Avenanthramides (Av-A, Av-B, Av-C) were
isolated from Avena sativa (Oat) seed kernels by hydroalcoholic extraction.
Asiaticosides: Centella asiatica extract contining Asiaticosides was isolated by the
ethanolic extraction of Centella asiatica plant.
Oleanolic acid: Oleanolic acid was isolated by the ethanolic extraction of Salvia
officinalis (Salvia) leaves.
Soya isoflavones: Soya bean extract containing 40% Soya isoflavones, genistein and
daidzein.
Tetrahydropiperine (THP): THP was obtained from the chemistry dept. of Sami Labs
Limited.
Coriandrum sativum (Coriander) seed oil: Coriander seed oil from Coriandrum sativum
seeds was extracted by carbondioxide by super critical fluid extraction.
Nelumbo nucifera (Lotus) seed extract: Lotus seed extract was prepared by water
extraction of lotus seeds.
Coffea arabica (Coffee) bean extract: Coffee bean extract containing chlorogenic acid
was prepared by water extraction of coffee beans.
Theobroma cacao (Cocoa) bean extract: Cocoa bean extract containing polyphenols
was prepared by water extraction of Cocoa beans.
Camellia simensis (Green tea) extract: Green tea extract containing polyphenols was
prepared by water extraction of Green tea leaves.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
220
Vitis vinifera (Grape) seed extract: Grape seed extract containing polyphenols was
prepared by water extraction of Grape seeds.
Rosmarinic acid: Rosmarinic acid was obtained by hydroalcoholic extraction of
Rosmarinus officinalis leaves.
Saffron: Saffron is prepared by the alcoholic extraction of Crocus sativus flowers.
Ocimum sanctum (Tulsi) extract: Tulsi extract was prepared by hydroalcoholic
extraction of Tulsi leaves.
Morinda citrifolia (Indian mulberry) extract: Mulberry extract was prepared by
ethanolic extraction of Mulberry fruits.
Garcinol: Garcinol is isolated by alcoholic extraction of Garcinia cambogia fruits.
Mangostin: Mangostin is isolated by alcoholic extraction of Garcinia mangostana fruits.
Acetyl-11-keto-beta-boswellic acid (AKBBA): AKBBA was obtained by solvent
extraction of Boswellia serrata gum resin.
Bacillus coagulans culture supernatant: During the expontential phase of the growth of
Bacillus coagulans, the culture medium was taken in aseptic conditions and centrifuged
to remove the cell debris. The culture supernatant thus obtained was used in the present
study.
Oleanoyl peptide: Oleanoyl peptide is the pentapeptide conjugate of oleanolic acid and
was chemically synthesized by conjugating Oleanolic acid to Lys-Thr-Thr-Lys-Ser
pentapeptide. Similaryl a peptide of Thiodipropionic acid and Lys-Thr-Thr-Lys-Ser
pentapeptide was made by chemical conjugation. These pentapeptide conjugates were
used for study of efficacy of actives in chemical conjugation with each other. A
conjugate of Kojic acid with Acetyl-11-keto-beta-boswellic acid (AKBBA) and a
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
221
conjugate of Kojic acid with Oleanolic acid were also made by chemical synthesis and
used for study of efficacy of actives in chemical conjugation with each other.
5.2.2. Methods:
5.2.2.1. Cell culture:
Swiss 3T3 fibroblast cells and B16F1 mouse melanoma cells were cultured in DMEM
supplemented with 10% FBS. Normal Human dermal fibroblasts (NHDF) were cultured
in NHDF growth medium supplemented with 2% FBS. The confluent cultures are
harvested by trypsinization and expanded during two more passages before they were
used for the experiments. Medium and other culture components were renewed after 48–
72 h. All cell cultures were maintained in a humidified atmosphere at 37°C in 95% air
and 5% CO2. Experiments were conducted on 24 hour monolayers of cell cultures which
were obtained by incubating the cells seeded in 96 well plates in a humidified atmosphere
at 37°C in 95% air and 5% CO2 in ThermoForma CO2 incubator for 24 hours.
5.2.2.2. Sample preparation for animal cell based assays:
Samples were prepared in appropriate vehicle. Water soluble samples were prepared in
double distilled autoclaved sterile water. Samples not soluble in water were prepared in
DMSO. DMSO was used at 0.5 to 1% in the appropriate growth medium, where there
was no effect of DMSO on the growing cells. All the prepared samples in the appropriate
vehicle were sterilized by passing through 0.22µm filter, before sample treatment to the
cells. Samples were used at non cytotoxic concentrations. The data obtained within the
maximal non cytotoxic concentrations and the maximal efficacy obtained is represented
in the results.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
222
5.2.2.3. Inhibition of melanin formation:
The following methods are used for screening actives that can directly inhibit or prevent
melanin formation,
5.2.2.3.1. Tyrosinase inhibition:
Pigmentation is a multistep process critically dependent on the functional integrity of
tyrosinase, the rate-limiting enzyme in melanin synthesis. Biosynthesis of melanin is
initiated by the catalytic oxidation of tyrosine to 3,4 dihydroxy phenylalanine (dopa) by
tyrosinase. Subsequent reactions happen spontaneously eventually resulting in the
synthesis of melanin. Under in vitro conditions, tyrosinase enzyme acts on L- Tyrosine
forming a pink colored complex. This pink color intensity formed during the reaction is
quenched in the presence of the inhibitor.
Figure 5.2.1: Principle of Tyrosinase assay
The assay is performed in a 96 well clear microtitre plate. Varying concentrations of the
samples in suitable vehicle (PBS or 0.2% DMSO that does not afftect the enzyme
activity) are pre incubated with 40 units of Mushroom Tyrosinase enzyme at 37oC for 10
minutes. The reaction is initiated by adding 0.7mM L- Tyrosine disodium and the
absorbance is read after 10 minutes of incubation at 37oC in FluostarOptima microplate
reader at 492nm (Choi J et al., 2010). The dose dependent inhibitory activity of samples
is calculated and the results are expressed as IC50 values using Graphpad prism software.
The percentage of inhibition of tyrosianse is calculated as follows,
% Inhibition = [(C-T) / C] X 100
Where C = absorbance due to tyrosinase activity in the absence of inhibitor
T = absorbance due to tyrosinase activity in the presence of inhibitor
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
223
IC50 value is the concentration required for 50% inhibition of the tyrosinase activity and
hence, lower IC50 value indicates better tyrosianse inhibitory potential.
5.2.2.3.2. Inhibition of α-MSH induced melanogenesis in B16F1 mouse melanoma
cell line:
Melanin synthesis can be directly studied in live animal cells. B16F1 mouse melanoma
cells were seeded in a 6 well microtiter plate at a seeding density of 5000 cells per well in
2ml DMEM medium per well. After 24 hours of incubation in a CO2 incubator, melanin
production is induced by 0.6nM ∝-MSH by replacing the medium with medium
containing ∝-MSH. The cells were then treated with varying concentrations of sample
over a period of 9 days with renewal of ∝-MSH containing medium and sample at
regular intervals of 3 days. Control wells were maintained without sample treatment and
only with the vehicle used for sample preparation. After the incubation period, the
medium was removed and the cells were scraped and washed in PBS. Thereafter, melanin
was extracted by 1N NaOH in boiling water bath for 5 minutes. The absorbance of the
melanin extract was read at 405nm in a microplate reader (Chamberlin et al., 2004). The
inhibitory effect of the sample is calculated based on the decrease of melanin formation.
A B C A) B16F1 mouse melanoma cells; B) α-MSH induced melanogenesis in B16F1 mouse melanoma cells; C) Reduction in α-MSH induced melanogenesis in B16F1 mouse melanoma cells on sample treatment Figure 5.2.2: α-MSH induced melanogenesis in B16F1 mouse melanoma cells
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
224
The dose dependent inhibitory activity of samples is calculated and the results are
expressed as IC50 values using Graphpad prism software. The percentage of inhibition of
melanin is calculated as follows,
% Inhibition = [(C-T) / C] X 100
Where C = absorbance due to melanin in the absence of inhibitor
T = absorbance due to melanin in the presence of inhibitor
IC50 value is the concentration required for 50% inhibition of the melanin formation and
hence, lower IC50 value indicates better melanin inhibitory potential.
5.2.2.3.3. Inhibition of cAMP induced melanogenesis in B16F1 mouse melanoma cell
line:
Cyclic adenosine monophosphate (cAMP) is another inducer of melanin synthesis.
Forskolin which is known to induce melanin through cAMP pathway was used to induce
melanin in B16F1 mouse melanoma cells. B16F1 cells were seeded in a 6 well microtiter
plate at a seeding density of 5000 cells per well in 2ml DMEM medium per well. After
24 hours of incubation in a CO2 incubator, melanin production is induced by 7.3µM
forskolin though cAMP pathway by replacing the medium with medium containing
forskolin. The cells were then treated with varying concentrations of sample over a period
of 9 days with renewal of forskolin containing medium and sample at regular intervals of
3 days. Control wells were maintained without sample treatment and only with the
vehicle used for sample preparation. After the incubation period, the medium was
removed and the cells were scraped and washed in PBS. Thereafter, melanin was
extracted by 1N NaOH in boiling water bath for 5 minutes. The absorbance of the
melanin extract was read at 405nm in a microplate reader. The inhibitory effect of the
sample is calculated based on the decrease of melanin formation.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
225
A B C A) B16F1 mouse melanoma cells; B) cAMP induced melanogenesis in B16F1 mouse melanoma cells; C) Reduction in cAMP induced melanogenesis in B16F1 mouse melanoma cells on sample treatment Figure 5.2.3: cAMP induced melanogenesis in B16F1 mouse melanoma cells
The dose dependent inhibitory activity of samples is calculated and the results are
expressed as IC50 values using Graphpad prism software. The percentage of inhibition of
melanin is calculated as follows,
% Inhibition = [(C-T) / C] X 100
Where C = absorbance due to melanin in the absence of inhibitor
T = absorbance due to melanin in the presence of inhibitor
IC50 value is the concentration required for 50% inhibition of the melanin formation and
hence, lower IC50 value indicates better melanin inhibitory potential.
5.2.2.3.4. UV B protection potential:
Protection from UV exposure prevents melanin synthesis by preventing UV stress in the
cells. Swiss 3T3 mouse fibroblast cells were used for UV protection studies. The cells
were seeded with a seeding density of 3000 cells per well of a 96 well plate. Confluent
monolayers of Swiss 3T3 fibroblast cells were initially treated with varying
concentrations of test sample and vehicle (control) in the culture medium and exposed to
UV B irradiation of 0.036 J cm-2 to determine the highest non cytotoxic concentration at
which the sample provides maximum UV protection. 0.036 J cm-2 was standardized as
the UV dosage required for causing approximately 50% cell death to the cell cultures in
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
226
the absence of protection. A control plate was also maintained under similar conditions
without UV exposure which can only give observations on the cytotoxic potential of the
sample. For each concentration, 6 replicates were maintained and the analysis was
performed twice such that the ‘n’ value is 12. After UV exposure, the medium was
replaced with fresh medium without sample and the cells were incubated in a CO2
incubator for 48 hrs. The cells were then developed by NRU staining technique to
analyze the cell viability. The cells were incubated with 0.003% solution of neutral red
prepared in pre warmed DMEM medium for 3 hrs at 370C in CO2 incubator. The excess
dye was then washed off with phosphate buffer saline (PBS). The lysosomal dye was
extracted in 100µl of developer solution consisting of 25ml of water, 24.5ml of ethanol
and 0.5ml of glacial acetic acid at RT for 20 min. The optical density (OD) was read at
492 nm using a microplate reader.
The percentage reduction in UV induced cytotoxicity i.e., the percentage of UV
protection was calculated with respect to the cytotoxicity in exposed cells as compared to
that of the unexposed cells in the presence and absence of sample.
A B A) Swiss 3T3 mouse fibroblasts exposed to UV showing cell death; B) Sample treated Swiss 3T3 mouse fibroblasts exposed to UV showing no cell death Figure 5.2.4: UV protection in Swiss 3T3 mouse fibroblast cells
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
227
% UV induced cytotoxicity in cells without sample treatment (U1) = [(C1-T1) /
C1] X 100
C1 = Absorbance due to cell viability in unexposed cells.
T1 = Absorbance due to cell viability in UV exposed cells.
% UV induced cytotoxicity in sample treated cells (U2) = [(C2-T2) / C2] X 100
C2 = Absorbance due to cell viability in unexposed sample treated cells.
T2 = Absorbance due to cell viability in UV exposed sample treated cells.
% UV protection = [(U1-U2) / U1] X 100
U1 = % UV induced cytotoxicity in cells without sample treatment.
U2 = % UV induced cytotoxicity in samples treated cells.
The dose dependent UV protection conferred by the samples is calculated and the results
are expressed as EC50 values using Graphpad prism software. EC50 value is the effective
concentration required for 50% protection from UV induced cytotoxicity and hence,
lower EC50 value indicates better melanin inhibitory potential.
The following methods are used for screening actives that can inhibit melanin
formation by inhibiting stress conditions due to free radicals and inflammatory markers.
Free radicals generated in the body due to stress conditions like UV exposure, pollution,
unhealthy food habits and ageing, primarily damage the skin. Free radicals trigger
inflammatory markers that eventually cause skin damage. As a result, excess melanin is
produced in a defense mechanism, resulting in pigmentation. Hence, antioxidant and anti
inflammatory properties are indirect yet desirable mechanisms for effective skin
lightening.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
228
5.2.2.4. Antioxidant potential:
The following methods are used for screening actives that can inhibit melanin formation
indirectly by inhibiting the free radical stress,
5.2.2.4.1. DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) scavenging assay:
The DPPH assay is often used to evaluate the ability of antioxidants to scavenge free
radicals which are known to be a major factor in biological damages caused by oxidative
stress. This assay is known to give reliable information concerning the antioxidant ability
of the tested compounds (Huang D et al., 2005). The assay is based on the color change
of the stable free radical DPPH from purple to yellow as the radical is quenched by the
antioxidant (Karagozler A A et al., 2008).
Figure 5.2.5: Priniciple of DPPH scavenging assay
The assay mixture tubes containing 1.5 ml of 0.1mM DPPH methanolic solution and
varying concentrations of the sample in a total volume of 3 ml were incubated at 37° C
for 30 minutes in a shaking water bath. The reduction in absorbance which is directly
proportional to the radical scavenging is measured spectrophotometrically at 517 nm. The
dose dependent free radical scavenging activity of samples is calculated and the results
are expressed as SC50 values using Graphpad prism software.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
229
The percentage of scavenging is calculated as follows,
% scavenging = [(C-T) / C] X 100
Where C = absorbance in the absence of inhibitor
T = absorbance in the presence of inhibitor
SC50 value is the concentration required for 50% scavenging of free radicals and hence,
lower SC50 value indicates better antioxidant potential.
5.2.2.4.2. Oxygen Radical Absorbance Capacity (ORAC):
Oxygen Radical Absorbance Capacity (ORAC) antioxidant Assay can be used to
determine the total antioxidant capacity of biological fluids, cells, and tissue. It can also
be used to assay the antioxidant activity of naturally occurring or synthetic compounds
for various applications. The assay measures the loss of fluorescein (3’,6’-dihydroxy-
spiro[isobenzofuran-1[3H], 9’[9H]-xanthen]-3-one) fluorescence over time due to
peroxyl-radical formation by the breakdown of 2,2’-azobis-2-methyl-propanimidamide,
dihydrochloride (AAPH). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid
(Trolox), a water soluble vitamin E analog, serves as a positive control inhibiting
fluorescein decay in a dose dependent manner. The ORAC assay is a kinetic assay
measuring fluroescein decay and antioxidant protection over time. The antioxidant
activity of samples can be normalized to equivalent Trolox units to quantify the
composite antioxidant activity present.
A peroxyl radical (ROO-) is formed from the breakdown of AAPH at 37 °C.
The peroxyl radical can oxidize fluorescein to generate a product without fluorescence.
Antioxidants supress this reaction by a hydrogen atom transfer mechanism, inhibiting the
oxidative degradation of the fluorescein signal. The fluorescence signal is measured over
30 minutes by excitation at 485 nm, emission at 538 nm. The concentration of antioxidant
in the test sample is proportional to the fluorescence intensity through the course of the
assay and is assessed by comparing the net area under the curve to that of a known
antioxidant, trolox.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
230
AAPH → ROO-
Fluorescein ---------------------→ Non fluorescent product
[Antioxidants inhibit the oxidation of fluorescein by hydrogen atom transfer]
Antioxidant capacity relating to trolox = Sum sample - Sum blank / Sum standard - Sum blank
Figure 5.2.6: Priniciple of ORAC assay
Varying concentrations of sample in suitable vehicle (PBS or 0.03% DMSO that does not
afftect the fluorescence intensity) were pipetted into each well of a black microplate
containing 10X10-2M 2,2′ -Azobis(2-methylpropionamidine) dihydrochloride (AAPH)
made in 75mM potassium phosphate buffer (pH 7.4) and 4.8X10-7M disodium
fluorescein dye. 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox)
standard from 12.5 – 200µM was also kept under similar conditions. Fluorescence
readings were taken in a Fluostar Optima Microplate Reader at 485/520nm after every 1
minute for 35 minutes (f1……..f35). The final ORAC values were calculated by using a
quadratic regression equation (Y = a + bX + cX2) between the trolox concentration (Y)
ROS
Fluorescent probe +
buffer
Fluorescent probe +
trolox
Fluorescent probe +
sample
Loss of fluorescence Loss of fluorescence Loss of fluorescence
Sum blank Sum standard Sum sample
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
231
(µM) and the net area under the Fluorescence decay curve (X) and were expressed as
micromoles of trolox equivalents per gram (TE/g) or liter of sample.
The Area under curve AUC = (1 + f1/f0 + f2/f0 + …. + f35/f0.) –------- eq 1
Where f0 is the initial fluorescence reading at 0 min and f1 is the fluorescence reading
after 1min.
The data were analyzed by applying eq 1. The net AUC was obtained by subtracting the
AUC of the blank from that of the sample. The value calculated using the net AUC of the
sample and the quadratic regression equation was divided by the weight of the sample in
grams or liter (Ou B et al., 2001). Higher ORAC value indicates better antioxidant
potential.
5.2.2.4.3. Hydroxyl Radical Averting Capacity (HORAC):
Hydroxyl Radical Averting Capacity (HORAC) is also an antioxidant assay similar to
ORAC with the only difference that HORAC is specific for Hydroxyl radicals and the
standard used is Gallic acid. Hydroxyl radical averting capacity is assessed using
fluorescein as the fluorescent probe. The hydroxyl radical is generated by a Cobalt Co
(II)-mediated reaction. The fluorescent decay curve of fluorescein dye is monitored in the
presence and absence of the inhibitor and the area under curve (AUC) is integrated. Net
AUC is calculated which is an index of hydroxyl radical is averting capacity which is
expressed in Gallic acid equivalents per gram (GAE/g) of test compound.
Varying concentrations of sample in suitable vehicle (PBS or 0.009% DMSO that
does not afftect the fluorescence intensity) were pipetted into each well containing
0.09µM disodium fluorescein dye. Immediately 20µl of 30% H2O2 is added into all wells,
and initial fluorescence reading (F0) is taken at 485/520 ex/em wavelength. Then 5µl of
219.79 µM cobalt solution and 522 µM Picolinic acid was pipetted into all the wells and
the fluorescence readings are taken immediately until 35mins (F1, F2, F3------------F35). Gallic
acid standard was also kept under similar conditions. Fluorescence readings were taken
in a Fluostar Optima Microplate Reader at 485/520nm. The final HORAC values were
calculated by using a quadratic regression equation (Y = a + bX + cX2) between the Gallic
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
232
acid concentration (Y) (µM) and the net area under the Fluorescence decay curve (X) and
were expressed as micromoles of Gallic acid equivalents per gram or liter of sample.
The Area under curve AUC = (1 + f1/f0 + f2/f0 + …. + f35/f0.) ------ eq 1
Where f0 is the initial fluorescence reading at 0 min and f1 is the fluorescence reading
after 1min.
The data were analyzed by applying eq 1. The net AUC was obtained by subtracting the
AUC of the blank from that of the sample. The value calculated using the net AUC of the
sample and the quadratic regression equation was divided by the weight of the sample in
gram or liter. The final value obtained is the HORAC value of the sample expressed as
µmol GAE/g (Ou B et al., 2002). Higher HORAC value indicates better antioxidant
potential.
5.2.2.4.4. Reactive Oxygen Species (ROS) scavenging potential:
The generation processes of reactive oxygen species can be monitored using the
luminescence analysis or also fluorescence methods . The intracellular ROS generation of
cells can be investigated using the 2’,7’-dichlorfluorescein-diacetate (DCFH-DA) as a
well-established compound to detect and quantify intracellular produced H2O2 (Cathcart
R et al., 1983). The conversion of the nonfluorescent 2’,7’ - dichlorfluorescein – diacetate
(DCFH-DA) to the highly fluoresecent compound 2’,7’-dichlorfluorescein (DCF)
happens in several steps. First, DCFH-DA is transported across the cell membrane and
deacetylated by esterases to form the non-fluorescent 2’,7’-dichlorfluorescein (DCFH).
This compound is trapped inside of the cells. Next, DCFH is converted to DCF through
the action of peroxid rated by the presence of peroxidase (LeBel C P et al., 1992). Swiss
3T3 mouse fibroblast cells were used to determine the ROS scavenging potential of
samples. The confluent cells were trypsinized and seeded in a 96 well black microplates
at a seeding density of 105 cells per well in PBS. The cells were treated with varying
concentrations of sample in suitable vehicle (PBS or 0.2% DMSO that does not afftect
the fluorescence intensity). Control cells were treated with vehicle used for sample
preparation. 100µl of 0.002% solution of 2,7-dichlorofluorescein diacetate dye was added
and ROS generation was enhanced by subjecting the cells to a chemical stress using
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
233
2.5µM FeSO4. After incubating the cells for 1 hour at 37°C, the ROS generated was
determined by taking fluorescence readings measured at wavelength Ex/Em 485/520 nm
in a microplate reader.
Figure 5.2.7: Priniciple of ROS scavenging assay
The fluorescence readings are directly proportional to the ROS generated and the ROS
scavenging effect of samples was calculated as the percentage scavenging with respect to
the control cells. The dose dependent ROS scavenging activity of samples is calculated
and the results are expressed as SC50 values using Graphpad prism software. The
percentage of scavenging is calculated as follows,
% scavenging = [(C-T) / C] X 100
Where C = Fluorescence due to ROS generated in the absence of inhibitor
T = Fluorescence due to ROS generated in the presence of inhibitor
SC50 value is the concentration required for 50% scavenging of ROS and hence, lower
SC50 value indicates better antioxidant potential.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
234
5.2.2.5. Anti inflammatory potential:
The following methods are used for screening actives that can inhibit melanin formation
indirectly by inhibiting the inflammatory stress,
5.2.2.5.1. Collagenase inhibitory potential:
Collagenase inhibitory potential of samples was determined by using Molecular Probes
EnzChek® Collagenase Assay Kit that provides high sensitivity required for screening
inhibitors in a high-throughput format. The EnzChek kit contains DQgelatin, fluorescein
conjugated gelatin. This substrate is efficiently digested by most of the gelatinases and
collagenases to yield highly fluorescent peptides. The increase in fluorescence is
proportional to proteolytic activity and can be monitored with a fluorescence microplate
reader. The reduction in fluourescence is directly proportional to the collagenase
inhibitory activity of the sample. Collagenase used for the assay is purified from
Clostridium histolyticum. Using 100 µg/mL DQ gelatin and a 30 minute incubation
period, the assay can detect the activity of this enzyme down to a final concentration of 2
× 10-3 U/mL (7 ng protein/mL), where one unit is defined as the amount of enzyme
required to liberate 1 µmole of L-leucine equivalents from collagen in 5 hours at 37°C,
pH 7.5. Varying concentrations of sample in suitable vehicle (PBS or 2% DMSO that
does not afftect the fluorescence intensity) were pre-incubated for 10 minutes with 12.5
µg/ml substrate, DQ gelatin (from pig skin), fluorescein conjugate and then 0.2U/ml of
Collagenase Type IV from Clostridium histolyticum enzyme was added. The
fluorescence intensity was measured after 30 minutes (Em: 485nm and Ex: 520nm.) in
microplate reader. The dose dependent inhibitory activity of samples is calculated and the
results are expressed as IC50 values using Graphpad prism software. The percentage of
inhibition of collagenase is calculated as follows,
% Inhibition = [(C-T) / C] X 100
Where C = absorbance due to collagenase activity in the absence of inhibitor
T = absorbance due to collagenase activity in the presence of inhibitor
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
235
IC50 value is the concentration required for 50% inhibition of the collagenase activity and
hence, lower IC50 value indicates better collagenase inhibitory potential.
5.2.2.5.2. Elastase inhibitory potential:
Elastase inhibitory potential of samples was determined by using Molecular Probes
EnzChek® Elastase Assay Kit that provides high sensitivity required for screening
inhibitors in a high-throughput format. The EnzChek kit contains DQelastin, fluorescein
conjugated soluble bovine neck ligament elastin. This substrate is efficiently digested by
elastase to yield highly fluorescent peptides. The increase in fluorescence is proportional
to proteolytic activity and can be monitored with a fluorescence microplate reader. The
reduction in fluourescence is directly proportional to the elastase inhibitory activity of the
sample. Elastase used for the assay is purified from procine pancreas.
Varying concentrations of sample in suitable vehicle (PBS or 2% DMSO that
does not afftect the fluorescence intensity) were pre-incubated for 10 minutes with the
substrate, 25µg/ml of DQ Elastin (from bovine neck ligament) fluorescein conjugate and
0.1U/ml porcine pancreatic elastase enzyme was added. The fluorescence intensity was
measured after 30 minutes (Em: 485nm and Ex: 520nm) in microplate reader. The dose
dependent inhibitory activity of samples is calculated and the results are expressed as
IC50 values using Graphpad prism software. The percentage of inhibition of elastase is
calculated as follows,
% Inhibition = [(C-T) / C] X 100
Where C = absorbance due to elastase activity in the absence of inhibitor
T = absorbance due to elastase activity in the presence of inhibitor
IC50 value is the concentration required for 50% inhibition of the elastase activity and
hence, lower IC50 value indicates better elastase inhibitory potential.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
236
5.2.2.5.3. Hyaluronidase inhibitory potential:
Hyaluronic acid when incubated with hyaluronidase enzyme solution in the presence and
absence of inhibitor and the unreacted hyaluronic acid gets precipitated with cetyl
pyridinium chloride. The precipitate blocks the transmittance and therefore a decrease in
the absorbance correlates with the amount of digested hyaluronic acid. Hyaluronidase
specifically cleaves the β1,4-glycosidic bond of hyaluronic acid. Hyaluronic acid
substrate (0.3%) was made in 300mM sodium phosphate buffer pH 5.35. Hyaluronidase
(10U/ml) was made in 20mM Sodium phosphate Buffer pH 7.00. Hyaluronidase enzyme
and various concentrations of the sample in suitable vehicle (PBS or 0.1% DMSO that
does not afftect the fluorescence intensity) are pre-incubated at 370C for 10 min. Then the
hyaluronic acid substrate is added and the reaction mixture is incubated for 45 min at
370C. The reaction mix is added to cetyl pyridinium chloride (1%). The absorbance of
undigested hyaluronic acid is read spectrophotometrically at 600nm (Tung J S et al.,
1994). The absorbance of undigested hyaluronic acid after treatment with the sample is
directly proportional to the inhibition of hyaluronidase. The dose dependent inhibitory
activity of samples is calculated and the results are expressed as IC50 values using
Graphpad prism software. The percentage of inhibition of hyaluronidse is calculated as
follows,
% Inhibition = (EC-EA)-(EC- (T-TC)) X 100 (EC-EA)
EC – Absorbance due to undigested hyaluronic acid in the absence of enzyme and
inhibitor.
EA – Absorbance after digestion of hyaluronic acid in the presence of enzyme.
T – Absorbance due to undigested hyaluronic acid in the presence of enzyme and
inhibitor.
TC – Absorbance of the inhibitor alone.
IC50 value is the concentration required for 50% inhibition of the hyaluronidase activity
and hence, lower IC50 value indicates better hyaluronidase inhibitory potential.
CHAPTER 1 PART II 5.2. MATERIALS AND METHODS
237
Figure 5.2.8: Hyaluronidase activity on Hyaluronic acid
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
238
5.2.2.5.4. TNF α inhibitory potential:
For TNF α inhibitory study, human whole blood is used. In whole blood assay,
monocytes appear to be the main source of TNF-α on Lipopolysaccharide (LPS)
stimulation. Monocyte and macrophages are a major source of TNF-α in addition to other
cell types like eosinophils, mast cells, peripheral lymphocytes and granulocytes. The
activation of inflammatory cells is influenced by the intracellular levels of c-AMP which
are regulated by the phosphodiesterase isoenzyme. LPS is the most potent stimulus of
TNF-α production in human blood. After stimulation the assay employs the quantitative
sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-α
has been pre-coated onto a microplate. TNF-α present in the sample is bound by the
immobilized antibody. After washing away any unbound substances, an enzyme-linked
polyclonal antibody specific for TNF-α is added. Following a wash to remove any
unbound antibody-enzyme reagent, a substrate solution is added to the wells and colour
develops in proportion to the amount of TNF-α bound in the initial step. The colour
development is stopped and the intensity of the colour is directly proportional to the
TNF-α content.
Heparinized blood from healthy donors was diluted 1:3 in RPMI 1640 culture
medium containing 10% FBS. Diluted blood samples were pre-incubated with varying
concentrations of sample in suitable vehicle (PBS or 0.1% DMSO) for 1 hr at 370C in an
incubator with 5 % CO2. 0.1% DMSO was used as vehicle for water insoluble samples.
After pre-incubation, the whole blood cells were stimulated by 1ng/mL LPS for the
release of TNF α from the macrophages by incubating for 5 hr at 370C in an incubator
with 5 % CO2. The samples were then centrifuged at 3000 g for 3 minutes at 40C and the
supernatant was assayed for TNF α content by using the TNF α Elisa kit. 200µL of
supernatant from all the tubes was transferred into microtiter plate in respective wells
(Pre-coated mouse monoclonal antibody microplate) followed by the addition of 50µLof
assay diluents in all the wells. After incubation for 2 hr at room temperature, the wells
were washed thoroughly with wash buffer provided and then 200µL of conjugate was
added to each well. The plate was incubated for 2 hr at room temperature. After washing
again, 200 µL of substrate solution was added to each well and incubated for 20 minutes
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
239
at RT. 50µL of Stop Solution to each well which will cause the colour change in the
wells from blue to yellow. The optical density was read at 450nm which is directly
proportional to TNF α content and the percentage of inhibition of TNF α content on
treatment with sample was calculated with respect to that of the untreated cells. The dose
dependent inhibitory activity of samples is calculated and the results are expressed as
IC50 values using Graphpad prism software. The percentage of inhibition of elastase is
calculated as follows,
% Inhibition = [(C-T) / C] X 100
Where C = absorbance due to TNF α in the absence of inhibitor
T = absorbance due to TNF α in the presence of inhibitor
IC50 value is the concentration required for 50% inhibition of TNF α and hence, lower
IC50 value indicates better TNF α inhibitory potential.
5.2.2.6. Cell rejuvenation:
Cell rejuvenation does not directly influence skin lightening but in combination with
pigment inhibitory mechanism a continuous repleneshing of new and lightened skin cells
will help in giving a bright skin tone. The following methods were used to study the cell
rejuvenation potential of the samples,
5.2.2.6.1. Cell proliferation enhancement:
Swiss 3T3 mouse fibroblast cells were used for cell proliferation studies. The cells were
seeded with a seeding density of 3000 cells per well of a 96 well plate. Confluent
monolayers of Swiss 3T3 fibroblast cells were initially treated with varying non cytotoxic
concentrations of test sample and vehicle (control) in the culture medium. For each
concentration, 6 replicates were maintained and the analysis was performed twice such
that the ‘n’ value is 12. After sample treatment, the cells were incubated in a CO2
incubator for 72 hrs. The cells were then developed by NRU staining technique to
analyze the cell viability. The cells were incubated with 0.003% solution of neutral red
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
240
prepared in pre warmed DMEM medium for 3 hrs at 370C in CO2 incubator. The excess
dye was then washed off with phosphate buffer saline (PBS). The lysosomal dye was
extracted in 100µl of developer solution consisting of 25ml of water, 24.5ml of ethanol
and 0.5ml of glacial acetic acid at RT for 20 min. The optical density (OD) was read at
492 nm using a microplate reader (Repetto G et al., 2008). The percentage enhancement
in cell growth with respect to the untreated cells considering the OD of untreated cells as
optimal under normal conditions is calculated as follows,
% enhancement in cell growth = [(100/C) X T] – 100
Where C = absorbance due to cell growth in untreated cells
T = absorbance due to cell growth in sample treated cells
5.2.2.6.2. Scratch wound closure assay:
Swiss 3T3 mouse fibroblast cells were used for Scratch wound closure assay. The cells
were seeded with a seeding density of 20000 cells per well of a 6 well plate. Confluent
monolayers of Swiss 3T3 fibroblast cells were wounded by scratching along the diameter
of the well of the plate using a 1ml micropipette tip having about 0.2µm diameter
resulting in 0.2µm scratch width. The wounded monolayers were then treated with
varying non cytotoxic concentrations of test sample and vehicle (control) in the culture
medium. For each concentration, 2 replicates were maintained and the analysis was
performed twice such that the ‘n’ value is 4. After sample treatment, the cells were
incubated in a CO2 incubator for 72 hrs. The reduction in scratch width is then measured
using a microscopic scale (Walter M N et al., 2010).
The percentage wound closure with respect to the untreated cells is calculated as
follows,
% wound closure = [(C-T) / C] X 100
Where C = C1 – C2
T = T1 – T2
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
241
Where,
C1 = scratch width at 0 hrs in untreated cells
C2 = scratch width after 72 hrs in untreated cells
T1 = scratch width at 0 hrs in sample treated cells
T2 = scratch width after 72 hrs in sample treated cells
A B A) Wounded monolayer of cells; B) Cells proliferating into the wounded area
Figure 5.2.9: Scratch wound closure assay
5.2.2.7. Collagen enhancement:
Collagen enhancement also does not directly influence skin lightening but it facilitates
the repair of skin damage due to various stress condiitons and as the skin damage gets
repaired the stress induced melanogenesis also will subside.
5.2.2.7.1. Collagen enhancement determination by Sirius Red staining:
Collagen enhancement was determined by using Sirius Red stain that binds with a greater
specificity to Collagen type I and Collagen type III of the extracellular matrix. The stain
bound to the collagen is dissolved and the optical density (OD) is measured
spectrophotometrically using a Fluostar optima microtiter plate reader at 544 nm. The
OD of the stain bound to collagen is directly proportional to the collagen content in the
cells (Tullberg-Reinert H and Jundt G, 1999). Human osteosarcoma cells from human
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
242
bone were used for collagen enhancement studies. The cells were seeded with a seeding
density of 10000 cells per well of a 24 well plate. Confluent monolayers of cells were
initially treated with varying non cytotoxic concentrations of test sample and vehicle
(control) in the culture medium. For each concentration, 4 replicates were maintained and
the analysis was performed twice such that the ‘n’ value is 8. After sample treatment, the
cells were incubated in a CO2 incubator for 48 hrs. The cells were then developed by
Sirius red staining technique to analyze the collagen enhancement. The cells were washed
extensively with PBS. The cells were fixed using Bouin’s fluid containing 1.3% picric
acid, 35% formaldehyde and glacial acetic acid in 15:5:1 ration by incubating with 1ml of
Bouin’s fluid per well for 1 hr at RT. The fixative is then removed by suction with
micropipette and the cells were washed under running tap water for 15 minutes. After air
drying the culture plate, the cells were stained using 0.1% Sirius red stain in 1.3% picric
acid. 1ml per well Sirius red stain was added and the cells were incubated for 1 hr under
mild shaking of 70 RPM at RT in Orbitek Shaker. The stain was then removed by suction
and the cells were extensively washed with 0.01N HCl to remove unbound dye. The dye
bound to collagen was then dissolved in 0.2ml of 0.1N NaOH per well for 30 minutes
under mild shaking of 70 RPM in Orbitek Shaker at RT. The dye was then transferred to
96 well microplate and the OD was read at 544nm in Fluostar Optima microplate reader.
The percentage enhancement in collagen with respect to the untreated cells considering
the OD of untreated cells as optimal under normal conditions is calculated as follows,
% enhancement in cell growth = [(100/C) X T] – 100
Where C = absorbance due to collagen in untreated cells
T = absorbance due to collagen in sample treated cells
5.2.2.7.2. Collagen enhancement determination by Flow cytometry:
For more sensitivity, collagen enhancement was also determined by Flow cytometry
also in Swiss 3T3 mouse fibroblast cells (Chanvorachote P et al., 2009). The cells
grown confluently in 25 cm2 flasks were washed with cold PBS and fixed for 3 minutes
with a fixing reagent containing 4% formaldehyde in PBS.
CHAPTER 5 PART II 5.2. MATERIALS AND METHODS
243
Cells were then washed with Tris buffer saline (TBS) and incubated in permeation
solution (1% Triton X-100 in PBS) at RT for 5 minutes. After washing with TBS, the
cells were blocked with a blocking reagent (2.5% FBS in TBS) for 30 minutes at RT
and further incubated with pro-collagen type I rabbit polyclonal antibody for 1 hour at
RT. After washing with TBST (TBS containing Tween) for 10 minutes, the cells were
incubated with secondary antibody, FITC-coupled anti-rabbit, for 1 hour with gentle
rocking at RT. They were then washed, trypsinized and resuspended in PBS and
immediately analyzed by flow cytometry using an excitation wavelength at 488nm and
emission wavelength at 520nm using FACSort, Becton Dickinson, Rutherford, NJ).
CHAPTER 1 PART II 5.3. RESULTS AND DISCUSSION
244
Approaches for skin whitening have broadened widely in the recent years. The utilization
of single agents inhibiting tyrosinase is in many cases extended to the use of complex
mixtures that target different mechanism like tyrosinase expression, melanogenesis,
antioxidant and anti-inflammatory effects (Ortonne J P and Bissett D L, 2008). Although
skin hyperpigmentation is a common concern, the causes for hyperpigmentation are not
the same and therefore, the approaches for reducing hyperpigmentation can also not be
the same. In the present study, various actives were screened through various in vitro
systems to position them specifically for various hyperpigmentation disorders.
Postitioning is broadly described as classifying skin lightening actives with respect to one
or more of the hyperpigmentation mechanisms that can be specifically rectified by them.
All the actives were screened broadly through mechanisms that directly inhibit melanin
formation and through mechanisms that indirectly influence melanin formation.
5.3.1. Screening of “actives” through various skin lightening mechanisms:
5.3.1.1. Screening of Reference standards through various skin lightening
mechanisms:
Most skin lightening products currently used contain ingredients like Arbutin,
hydroquinone, ascorbic acid, kojic acid etc. that act as direct inhibitors of tyrosinase, the
enzyme present in melanocytes, the skin pigment cells that make melanin. They are used
as reference standards for tyrosinase inhibition (Issa R A et al., 2008). However, there is
evidence to suggest that certain skin lightening actives like hydroquinone can be harmful
(Olumide Y M et al., 2008). Hydroquinone has now been banned in Europe and in many
other countries. In the present study, Arbutin, Kojic acid and Ascorbic acid are
considered as reference standards for comparative analysis with other actives.
Antioxidants play a major role in skin lightening. Tripeptide Glutathione is one such skin
lightener which exerts its efficacy through its significant antioxidant potential. It is an
oral cosmetic (nutricosmetic). It inhibits melanin induced by MSH with an IC50 of 25 ±
3.25µg/ml and melanin induced by cAMP with an IC50 of 200 ± 28µg/ml.
5.3. RESULTS AND DISCUSSION
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
245
5.3.1.1.1. Arbutin: Table 5.3.1: Skin lightening potential of Arbutin
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
IC50 194 ± 25 IC50 100 ± 15 IC50 100 ± 18 Nil
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 500 ± 25 Nil Nil Nil
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α
inhibition
Nil Nil Nil Nil
Table 5.3.2: Effect of Arbutin on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Potential inhibitor
of tyrosinase &
melanogenesis
Pigmentation due to excessive sun
exposure
No effect No significant
protection from UV
exposure
Pigmentation due to free radical
damage
No effect No significant free
radical scavenging
activity
Pigmentation due to inflammatory
responses like pimple marks, scars
due to wounds etc.
No effect
No significant anti
inflammatory
activity
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
246
5.3.1.1.2. Kojic Acid: Table 5.3.3: Skin lightening potential of Kojic acid
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
Induced Melanin
UV protection
IC50 7 ± 1.5 IC50 100 ± 19 Nil Nil
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 500 ± 32 Nil Nil Nil
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α
inhibition
Nil Nil Nil Nil
Table 5.3.4: Effect of Kojic acid on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Potential inhibitor of
tyrosinase &
melanogenesis
Pigmentation due to excessive sun
exposure
No effect No significant
protection from UV
exposure
Pigmentation due to free radical
damage
No effect No significant free
radical scavenging
activity
Pigmentation due to inflammatory
responses like pimple marks, scars
due to wounds etc.
No effect
No significant anti
inflammatory
activity
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
247
5.3.1.1.3. Ascorbic acid: Table 5.3.5: Skin lightening potential of Ascorbic acid
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
IC50 9.33 ± 1.6 IC50 25 ± 6 IC50 100 ± 16 Not significant
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 1.93 ± 0.3 IC50 10 ± 2.3 3400 ± 220 Nil
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α
inhibition
Nil Nil Nil Nil
Table 5.3.6: Effect of Ascorbic acid on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Potential inhibitor of
tyrosinase &
melanogenesis
Pigmentation due to sun exposure Not
significant
No significant
protection from UV
induced cell damage
Pigmentation due free radical damage Significant Potential scavenger
of free radicals
Pigmentation due inflammatory
responses like pimple marks, scars due
to wounds etc.
No effect No significant anti
inflammatory activity
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
248
5.3.1.2. Screening of “actives” known for skin lightening through various skin
lightening mechanisms:
5.3.1.2.1. Tetrahydrocurcumin (THC) from Curcuma longa root extract: Table 5.3.7: Skin lightening potential of THC
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
IC50 1.77 ± 0.3 IC50 3.2 ± 1.1 IC50 4 ± 1.3 Not significant
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 0.93 ± 0.3 IC50 1.2 ± 0.2 10,815 ± 468 2715 ± 126
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
Nil Nil Nil IC50 81 ± 6
Table 5.3.8: Effect of THC on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Potential inhibitor of
tyrosinase &
melanogenesis
Pigmentation due to sun exposure Not
significant
No significant
protection from UV
Pigmentation due free radical damage Significant Antioxidant
Pigmentation due inflammatory
responses like pimple marks, scars
due to wounds etc.
Significant Significant inhibitor
of inflammation
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
249
5.3.1.2.2. Glabridin from Glycyrrhiza glabra (Licorice) root extract: Table 5.3.9: Skin lightening potential of Glabridin
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
IC50 0.25 ± 0.05 IC50 3.5 ± 1 IC50 3.8 ± 1.2 Not significant
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 100 ± 15 IC50 0.25 ± 0.02 7550 ± 520 1129 ± 209
Anti inflammatory potential
Collagenase
inhibition (µg/ml)
Elastase inhibition
(µg/ml)
Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
IC50 50 ± 5 IC50 55 ± 6 Nil IC50 100 ± 9
Table 5.3.10: Effect of Glabridin on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Potential inhibitor of
tyrosinase &
melanogenesis
Pigmentation due to sun exposure Not
significant
No significant
protection from UV
induced cell damage
Pigmentation due free radical damage Significant Potential scavenger
of free radicals
Pigmentation due inflammatory
responses like pimple marks, scars
due to wounds etc.
Significant Significant inhibitor
of inflammatoty
markers and
inflammary enzymes
CHAPTER 1 PART II 5.3. RESULTS AND DISCUSSION
250
5.3.1.2.3. Artocarpin from Artocarpus lakoocha heartwood extract:
Table 5.3.11: Skin lightening potential of Artocarpin
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
(µg/ml)
IC50 1.3 ± 0.2 IC50 2.5 ± 0.2 IC50 1 ± 0.2 EC50 20 ± 4
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 44 ± 0.2 Not significant 3859 ± 0.2 1121 ± 214
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition
(µg/ml)
Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
Not significant IC50 8.8 ± 5 Nil IC50 90 ± 3
Table 5.3.12: Effect of Artocarpin on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Potential inhibitor of
tyrosinase &
melanogenesis
Pigmentation due to sun exposure Significant Significant
protection from UV
induced cell damage
Pigmentation due free radical damage Significant Antioxidant
Pigmentation due inflammatory
responses like pimple marks, scars
due to wounds etc.
Significant Significant inhibitor
of inflammation
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
251
5.3.1.2.4. Stilbenes from plant extracts:
Various stilbene compounds from different plant sources have been screened through
skin lightening models.
5.3.1.2.4.1 Artocarpus lakoocha heartwood extracts containing varying
concentrations of Oxyresveratrol:
The extract with higher content of Oxyresveratrol was not as effective as the extract
containing lower concentration of Oxyresveratrol with respect to melanin inhibition and
anti inflammatory activity (Table 5.3.13). Therefore, it clearly indicates that only the
combination of various actives of the extract is bringing about the significant melanin
inhibition and anti inflammatory activity. The melanogenesis inhibitory potential and anti
inflammatory potential of the Artocarpus lakoocha extract is conferred by the synergistic
combination of Oxyresveratrol & other actives of the extract like Artocarpin etc.
However, the antioxidant potential and UV protection potential of the composition from
Artocarpus lakoocha is conferred by the oxyresveratrol content in the extract as the
antioxidant and UV protection potential increased with the increasing percentage of
Oxyresveratrol (Table 5.3.13). It was also observed that higher concentrations of
Oxyresveratrol caused reduced ROS scavenging potential due to prooxidant effect of
Oxyresveratrol.
Hence, it is clearly understood by the present in vitro studies that the melanin
inhibitory and anti inflammatory potential of the Artocarpus lakoocha extract is
conferred by the synergistic combination of Oxyresveratrol & other actives of the extract
like Artocarpin etc, while the antioxidant potential and UV protection potential is
conferred exclusively by the Oxyresveratrol content in the extract. It was also observed
that hydrogenation of Oxyresveratrol to Dihydro-oxyresveratrol resulted in increase in
melanin inhibitory potential but a decrease in UV protection potential (Table 5.3.15).
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
252
Table 5.3.13: Skin lightening potential of Oxyresveratrol from Artocarpus lakoocha extract
Inhibition of melanin formation
Active Tyrosinase
inhibition
(µg/ml)
Inhibition of
MSH induced
Melanin (µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
(µg/ml)
Oxy 20% IC50 0.48 ± 0.055 IC50 3.45 ± 1.1 IC50 4.35 ± 1.3 EC50 100 ± 9.2
Oxy 30% IC50 0.41 ± 0.05 IC50 3.56 ± 0.9 IC50 5 ± 1.8 EC50 100 ± 9.4
Oxy 50% IC50 0.11 ± 0.023 IC50 12 ± 3.2 IC50 12 ± 2.8 EC50 75 ± 8.3
Oxy 80% IC50 0.087 ± 0.02 IC50 10 ± 2.4 IC50 11 ± 2.9 EC50 50 ± 6.1
Oxy 90% IC50 0.05 ± 0.019 IC50 12 ± 2.6 IC50 12 ± 2.7 EC50 50 ± 6.3
Antioxidant potential
Active DPPH
scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
Oxy 20% IC50 3.1 ± 0.8 IC50 10 ± 2.5 8999 ± 443 4756 ± 298
Oxy 30% IC50 3.7 ± 0.92 IC50 10 ± 1.9 11,370 ± 1220 4776 ± 326
Oxy 50% IC50 2.5 ± 0.76 IC50 10 ± 2.8 15,582 ± 1236 4896 ± 428
Oxy 80% IC50 2.6 ± 0.82 Not significant 18,673 ± 1432 4923 ± 392
Oxy 90% IC50 2.7 ± 0.96 Not significant 21,549 ± 1896 5728 ± 432
Anti inflammatory potential
Active Collagenase
inhibition
(µg/ml)
Elastase
inhibition (µg/ml)
Hyaluronidase
inhibition (µg/ml)
TNF α
inhibition
(µg/ml)
Oxy 20% IC50 15 ± 2 IC50 22 ± 4 IC50 260 ± 13 IC50 70 ± 8
Oxy 30% IC50 17 ± 4 IC50 27 ± 3 IC50 300 ± 15 IC50 55 ± 6
Oxy 50% IC50 31 ± 5 IC50 50 ± 2 IC50 250 ± 10 IC50 62 ± 5
Oxy 80% IC50 58 ± 6 IC50 65 ± 8 IC50 500 ± 22 IC50 69 ± 7
Oxy 90% IC50 92 ± 8 IC50 120 ± 11 IC50 500 ± 25 IC50 65 ± 9
Oxy: Oxyresveratrol
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
253
Table 5.3.14: Effect of Oxyresveratrol on Skin pigmentation disorders Pigmentation
disorder
Effect Rationale
Pigmentation due to
biological imbalances
of body such as over
expression of
hormones and
enzymes
Significant.
Artocarpus
lakoocha extract
containing 20% &
30% oxyresveratrol
has better potential.
Potential inhibitor
of tyrosinase and
melanogenesis
Pigmentation due to
sun exposure
Significant.
Artocarpus
lakoocha extract
containing 80% &
90% oxyresveratrol
has better potential.
Significant
protection against
UV induced cell
death.
Pigmentation due to
free radical damage
Significant.
Artocarpus
lakoocha extract
containing 80% &
90% oxyresveratrol
has better potential.
Significant
scavenger of free
radicals
Pigmentation due
inflammatory
responses like pimple
marks, scars due to
wounds etc.
Significant.
Artocarpus
lakoocha extract
containing 20% &
30% oxyresveratrol
has better potential.
Potential inhibitor
of inflammatory
markers and
inflammatory
enzymes
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
254
5.3.1.2.4.2. Dihydro-oxyresveratrol from Artocarpus lakoocha heartwood extract:
Table 5.3.15: Skin lightening potential of Dihydro-oxyresveratrol
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
Induced Melanin
(µg/ml)
UV protection
IC50 0.03 ± 0.01 IC50 1.63 ± 0.5 IC50 2 ± 0.6 Not significant
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 5.37 ± 1.6 IC50 3.125 ± 0.9 21,549 ± 2022 3097 ± 423
Anti inflammatory potential
Collagenase
inhibition (µg/ml)
Elastase inhibition
(µg/ml)
Hyaluronidase
inhibition (µg/ml)
TNF α inhibition
(µg/ml)
IC50 150 ± 22 IC50 166 ± 17 IC50 147 ± 19 IC50 40 ± 3
Table 5.3.16: Effect of Dihydro-oxyresveratrol on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and
enzymes
Significant Significant inhibitor
of tyrosinase and
melanogenesis
Pigmentation due to sun exposure Not
significant
No significant
protection from UV
Pigmentation due to free radical
damage
Significant Antioxidant
Pigmentation due inflammatory
responses like pimple marks,
scars due to wounds etc.
Significant Significant inhibitor
of inflammatory
markers
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
255
5.3.1.2.4.3. Resveratrol from Polygonum cuspidatum root extract:
Table 5.3.17: Skin lightening potential of Resveratrol
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
Inhibition of cAMP
Induced Melanin
UV protection
(µg/ml)
IC50 5.5 ± 1.2 IC50 2.5 ± 0.8 IC50 3.5 ± 0.91 EC50 30 ± 7
Antioxidant potential
DPPH scavenging ROS scavenging ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 3 ± 1.1 Not significant 25,223 ± 2312 10,000 ± 598
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
Nil Not significant Nil IC50 75 ± 5
Table 5.3.18: Effect of Resveratrol on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as
over expression of hormones
and enzymes
Significant Potential inhibitor of
tyrosinase &
melanogenesis
Pigmentation due to sun
exposure
Significant Significant inhibitor
of cell damage due to
UV exposure
Pigmentation due free radical
damage
Significant Antioxidant
Pigmentation due inflammatory
responses like pimple marks,
scars due to wounds etc.
Significant Significant inhibitor
of inflammatory
markers
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
256
5.3.1.2.4.4. Pterostilbene from Pterocarpus marsupium heartwood extract:
Table 5.3.19: Skin lightening potential of Pterostilbene
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
(µg/ml)
IC50 7.8 ± 1.5 IC50 0.5 ± 0.08 IC50 0.7 ± 0.06 EC50 30 ± 7.2
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 4.9 ± 1.3 IC50 3 ± 0.92 12,508 ± 998 6283 ± 664
Anti inflammatory potential
Collagenase
inhibition (µg/ml)
Elastase inhibition
(µg/ml)
Hyaluronidase
inhibition (µg/ml)
TNF α inhibition
(µg/ml)
IC50 125 ± 14 IC50 50 ± 4 Nil IC50 150 ± 12
Table 5.3.20: Effect of Pterostilbene on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and
enzymes
Significant Significant inhibitor
of tyrosinase and
melanogenesis
Pigmentation due to sun exposure Significant Significant protection
from UV
Pigmentation due to free radical
damage
Significant Antioxidant
Prevention of pigmentation due
inflammatory responses like pimple
marks, scars due to wounds etc.
Significant Significant inhibitor
of inflammation
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
257
Hydroxy Pterostilbene (3HPT) from Pterocarpus marsupium-׀3 .5.3.1.2.4.5
heartwood extract:
Table 5.3.21: Skin lightening potential of 3HPT
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
IC50 2 ± 0.3 IC50 0.7 ± 0.08 IC50 0.5 ± 0.1 Nil
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 1.34 ± 0.3 IC50 5 ± 1.1 13,334 ± 1142 4700 ± 598
Anti inflammatory potential
Collagenase
inhibition (µg/ml)
Elastase inhibition
(µg/ml)
Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
IC50 90 ± 3 IC50 82 ± 9 Nil IC50 158 ± 11
Table 5.3.22: Effect of 3HPT on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Significant inhibitor
of tyrosinase and
melanogenesis
Pigmentation due to sun exposure No effect No significant UV
protection
Pigmentation due free radical damage Significant Antioxidant
Prevention of pigmentation due
inflammatory responses like pimple
marks, scars due to wounds etc.
Significant Significant inhibitor
of inflammation
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
258
5.3.1.2.4.6. Gnetol from Gnetum gnemon:
Table 5.3.23: Skin lightening potential of Gnetol
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
Inhibition of cAMP
induced Melanin
UV protection
(µg/ml)
IC50 149 ± 22 Not significant Not significant EC50 25 ± 5
Antioxidant potential
DPPH scavenging ROS scavenging ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 1.55 ± 0.9 IC50 3.5 ± 1.2 14,322 ± 1233 5000 ± 968
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
Not significant Not significant Not significant Not significant
Table 5.3.24: Effect of Gnetol on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and
enzymes
Mild Inhibits only
tyrosinase
Pigmentation due to sun exposure Significant Significant
protection from
UV
Pigmentation due free radical
damage
Significant Antixidant
Pigmentation due inflammatory
responses like pimple marks,
scars due to wounds etc.
No effect Not an inhibitor
of inflammation
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
259
5.3.1.2.5. Emblica officinalis (Amla) fruit extract containing β-Glucogallin:
For several decades, the fruits of Amla had been claimed to be a rich source of Ascorbic
acid and further its high antioxidant potential was attributed to the presence of ascorbic
acid (Scartezzini P et al., 2006). It was then reported that low molecular hydrolysable
tannins (emblicanins A and B) contribute to the antioxidant potential of Amla
(Pozharitskava O N et al., 2007).
Table 5.3.25: Skin lightening potential of Amla extract
Inhibition of melanin formation
Active Tyrosinase
inhibition (µg/ml)
Inhibition of
MSH induced
Melanin (µg/ml)
Inhibition of
cAMP
induced Melanin
(µg/ml)
UV
protection
(µg/ml)
β-Glucogallin 10%
IC50 200 ± 35 IC50 12 ± 3.6 IC50 14 ± 4.3 IC50 20 ± 3.3
β-Glucogallin 99%
IC50 500 ± 33 IC50 14 ± 2.4 IC50 15 ± 3.9 IC50 25 ± 3.1
Antioxidant potential
Active DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol
GAE/g)
β-Glucogallin 10%
IC50 1.7 ± 0.2 IC50 2.5 ± 1.2 2862 ± 339 1474 ± 212
β-Glucogallin 99%
IC50 0.92 ± 0.1 IC50 2.5 ± 0.9 4436 ± 446 4660 ± 189
Anti inflammatory potential
Active
Collagenase
inhibition (µg/ml)
Elastase
inhibition
Hyaluronidase
inhibition
TNF α
inhibition
β-Glucogallin 10%
IC50 500 ± 22 Nil Not significant Not significant
β-Glucogallin 99%
Nil Nil Not significant Not significant
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
260
However, recent studies have confirmed that only trace amounts of Ascorbic acid are
found in Amla extract and the earlier reported antioxidant hydrolysable tannins,
emblicanins A and B, correspond to 1-O-galloyl- β-D-glucose (β-glucogallin) and mucic
acid 1,4-lactone 5-O-gallate respectively (Majeed M et al., 2009). The trace amount of
free Ascorbic acid in Amla extract suggests that the antioxidant effects exhibited by
Amla fruits are due to gallic acid esters (Majeed M et al., 2009). Due to the presence of
only trace amounts of Ascorbic acid in the fruits of Emblica officinalis, β-glucogallins
could be the active that significantly contributes to the efficacy of Amla extract. Amla
extract containing higher concentration of β-glucogallin had higher antioxidant potential
(Table 5.3.25)
Table 5.3.26: Effect of Amla extract on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to
biological imbalances
of body such as over
expression of hormones
and enzymes
Significant Significant
inhibitor of
melanogenesis
Pigmentation due to sun
exposure
Significant. Significant
protection against
UV induced cell
death.
Pigmentation due to
free radical damage
Significant. Amla
extract containing
99% β-Glucogallin
has better
potential.
Significant
scavenger of free
radicals
Pigmentation due
inflammatory responses
like pimple marks, scars
due to wounds etc.
Mild Not a significant
inhibitor of
inflammatory
markers
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
261
5.3.1.2.6. Piperlongumine from Piper longum root extract:
Table 5.3.27: Skin lightening potential of Piperlongumine
Inhibition of melanin formation
Tyrosinase inhibition Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
Nil IC50 0.625 ± 0.23 IC50 0.5 ± 0.21 Nil
Antioxidant potential
DPPH scavenging ROS scavenging ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 22 ± 4.3 mg/ml Nil Nil Nil
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
Nil Nil Nil IC50 230 ± 25
Table 5.3.28: Effect of Piperlongumine on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as
over expression of hormones
and enzymes
Significant Significant
inhibitor of
melanogenesis
Pigmentation due to sun
exposure
No effect No protection from
UV damage
Pigmentation due free radical
damage
Mild Mild scavenger of
free radicals
Pigmentation due inflammatory
responses like pimple marks,
scars due to wounds etc.
Mild Inhibitor of
inflammatory
markers
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
262
5.3.1.3. Screening of “actives” known for antioxidant and anti inflammatory activity
but unexplored for skin lightening efficacy:
5.3.1.3.1. Hydroxychavicol from Piper betle leaf extract: Table 5.3.29: Skin lightening potential of Hydroxychavicol
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
IC50 8 ± 1.5 IC50 1.3 ± 0.2 IC50 2.5 ± 0.4 Nil
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 0.5 ± 0.1 IC50 10 ± 1.2 29,728 ± 2860 2376 ± 293
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
Not significant Not significant Not significant IC50 89 ± 8
Table 5.3.30: Effect of Hydroxychavicol on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Potential inhibitor of
tyrosinase &
melanogenesis
Pigmentation due to sun exposure No effect No UV protection
Pigmentation due free radical damage Significant Antioxidant
Pigmentation due inflammatory
responses like pimple marks, scars
due to wounds etc.
Significant Significant inhibitor
of inflammation
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
263
5.3.1.3.2. Thymohydroquinone from Nigella sativa (Black cumin) seed extract:
Table 5.3.31: Skin lightening potential of Thymohydroquinone
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
Induced Melanin
(µg/ml)
UV protection
IC50 0.905 ± 0.2 IC50 0.2 ± 0.05 IC50 0.3 ± 0.07 Nil
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 84 ± 11 IC50 20 ± 5.6 5899 ± 369 2000 ± 136
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
Not significant Not significant Not significant IC50 3 ± 0.8
Table 5.3.32: Effect of Thymohydroquinone on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Significant inhibitor
of tyrosinase and
melanogenesis
Prevention of pigmentation due to sun
exposure
Nil No UV protection
Prevention of pigmentation due to free
radical damage
Significant Antioxidant
Prevention of pigmentation due
inflammatory responses like pimple
marks, scars due to wounds etc.
Significant Significant inhibitor
of inflammatory
markers
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
264
5.3.1.3.3. Eugenia jambolana (Jamun) fruit extract:
Table 5.3.33: Skin lightening potential of Jamun extract
Inhibition of melanin formation
Tyrosinase inhibition Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
Not significant IC50 5 ± 1.2 IC50 7 ± 1.4 Nil
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 6.1 ± 2.1 IC50 10 ± 3.2 9000 ± 890 1000 ± 262
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
Not significant Not significant Not significant Not significant
Table 5.3.34: Effect of Jamun extract on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and
enzymes
Significant Significant inhibitor
of melanogenesis
Pigmentation due to sun exposure No effect No UV protection
Pigmentation due to free radical
damage
Significant Antioxidant
Pigmentation due inflammatory
responses like pimple marks,
scars due to wounds etc.
No effect Not an inhibitor of
inflammation
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
265
5.3.1.3.4. Avenanthramides from Avena sativa (Oat) seed kernel extract:
Other than oat ceramides and betaglucans, oats are also the source of Avenanthramides
a type of oat phytoalexins that exist predominantly in the groats of oat seeds. Among a
group of at least 25 avenanthramides that differ in the substituents on the cinnamic acid
and anthranilic acid rings, three are predominant in oat grain: Avenanthramide C or Av-C,
Avenanthramide B or Av-B and Avenanthramide A or Av-A (Fig. 2.36). In vitro
experiments indicate that they have significant antioxidant activities, with Bc > Bf > Bp
(Peterson D M et al., 2002).
In the present research, on screening though various models for skin lightening
mechanisms, it was found that amongst the three avenanthramides, Avenanthramide C or
Av-C showed superior efficacy.
Figure 5.3.1: Avenanthramides A, B and C
It was observed that the presence of ‘OH’ group in Avenanthramide C at R3 position
instead of ‘H’ or ‘OCH3’ groups as in Avenanthramide A and Avenanthramide B
respectively, contributed significantly to the superior skin lightening efficacy of
Avenanthramide C in comparison to other Avenanthramide compounds.
CHAPTER 1 PART II 5.3. RESULTS AND DISCUSSION
266
Table 5.3.35: Skin lightening potential of Avenanthramide C (Av-C)
Inhibition of melanin formation
Tyrosinase inhibition Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
Not significant IC50 20 ± 5.6 IC50 25 ± 6.2 Nil
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 1.2 ± 0.2 IC50 10 ± 3.8 22,352 ± 1926 5000 ± 432
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
Not significant Not significant Not significant IC50 30 ± 2
Table 5.3.36: Effect of Avenanthramide C (Av-c) on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and
enzymes
Significant Inhibitor of
melanogenesis
Pigmentation due to sun
exposure
No effect No UV protection
Pigmentation due to free radical
damage
Significant Antioxidant
Pigmentation due inflammatory
responses like pimple marks,
scars due to wounds etc.
Significant Inhibitor of
inflammatory
markers
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
267
5.3.1.3.5. Colocynthin from Citrullus colocynthis fruit extract:
Table 5.3.37: Skin lightening potential of Colocynthin
Inhibition of melanin formation
Tyrosinase inhibition
(µg/ml)
Inhibition of MSH
induced Melanin
(µg/ml)
Inhibition of cAMP
induced Melanin
(µg/ml)
UV protection
(µg/ml)
IC50 >100 IC50 22 ± 2.3 IC50 20 ± 3.2 EC50 100 ± 9
Antioxidant potential
DPPH scavenging
(µg/ml)
ROS scavenging
(µg/ml)
ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
IC50 50 ± 6.2 IC50 10 ± 3.1 Not significant Not significant
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
(µg/ml)
Not significant Not significant Not significant IC50 50 ± 4
Table 5.3.38: Effect of Colocynthin on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological
imbalances of body such as over
expression of hormones and enzymes
Significant Inhibitor of
tyrosinase and
melanogenesis
Pigmentation due to sun exposure Significant Protects against UV
induced cell death
Pigmentation due to free radical
damage
Significant Antioxidant
Pigmentation due inflammatory
responses like pimple marks, scars
due to wounds etc.
Significant Significant inhibitor
of inflammatory
markers
CHAPTER 1 PART II 5.3. RESULTS AND DISCUSSION
268
5.3.1.4. Screening of “actives” known for skin conditioning and UV protection but
unexplored for skin lightening efficacy:
5.3.1.4.1. Ceramides from Avena sativa (Oat) extract: Table 5.3.39: Skin lightening potential of Oat ceramides
Inhibition of melanin formation
Tyrosinase inhibition Inhibition of MSH
induced Melanin
Inhibition of cAMP
induced Melanin
UV protection
(µg/ml)
Nil Upto 20 µg/ml, 25%
inhibition
Upto 20 µg/ml, 35%
inhibition
EC50 50 ± 5
Antioxidant potential
DPPH scavenging ROS scavenging ORAC
(µmol TE/g)
HORAC
(µmol GAE/g)
Nil Nil Nil Nil
Anti inflammatory potential
Collagenase
inhibition
Elastase inhibition Hyaluronidase
inhibition
TNF α inhibition
Not significant Not significant Not significant Not significant
Table 5.3.40: Effect of Oat ceramides on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to biological imbalances
of body such as over expression of
hormones and enzymes
Mild Inhibitor of
melanogenesis
Pigmentation due to sun exposure Significant Protects from UV
damage
Pigmentation due free radical damage No effect Not an antioxidant
Pigmentation due inflammatory responses
like pimple marks, scars due to wounds
No effect Not an inhibition of
inflammation
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
269
5.3.1.4.2. Ceramides from Malus domestica (Apple) extract:
Table 5.3.41: Skin lightening potential of Apple ceramides
Inhibition of melanin formation
Active Tyrosinase
inhibition
Inhibition of
MSH induced
Melanin
Inhibition of
cAMP
induced Melanin
UV
protection
(µg/ml)
Apple peel
ceramides
Upto 30 µg/ml,
30% inhibition
Upto 20 µg/ml,
15% inhibition
Upto 20 µg/ml,
25% inhibition
Not
significant
Apple fruit
ceramides
Upto 70 µg/ml,
30% inhibition
IC50 12 ± 3.2
µg/ml
IC50 10 ± 2.9
µg/ml
EC50 100 ± 8
Antioxidant potential
Active DPPH
scavenging
ROS scavenging ORAC
(µmol TE/g)
HORAC
(µmol
GAE/g)
Apple peel
ceramides
Not significant Nil Not significant Not
significant
Apple fruit
ceramides
Not significant Upto 10 µg/ml,
10% scavenging
Not significant Not
significant
Anti inflammatory potential
Active Collagenase
inhibition
(µg/ml)
Elastase
inhibition
(µg/ml)
Hyaluronidase
inhibition
TNF α
inhibition
Apple peel
ceramides
IC50 25 ± 3 IC50 200 ± 12 Not significant Not
significant
Apple fruit
ceramides
Nil Nil Not significant Not
significant
It was observed that the apple fruit ceramides had better melanogenesis inhibitory and
UV protection efficacy whereas the apple peel ceramides had better anti inflammatory
efficacy.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
270
Table 5.3.42: Effect of Apple ceramides on Skin pigmentation disorders Pigmentation disorder Effect Rationale
Pigmentation due to
biological imbalances of
body such as over
expression of hormones
and enzymes
Apple fruit ceramides
- significant
Apple peel ceramides
- mild
Inhibitor of
tyrosinase and
melanogenesis
Pigmentation due to sun
exposure
Apple fruit ceramides
– significant
Apple peel ceramides
– Not significant
Significant protection
against UV induced
cell death
Pigmentation due to free
radical damage
Not significant Not significant
scavenger of free
radicals
Pigmentation due
inflammatory responses
like pimple marks, scars
due to wounds etc.
Apple peel ceramides
- significant
Apple fruit ceramides
– Not significant
Inhibitor of
inflammatory
enzymes
CHAPTER 1 PART II 5.3. RESULTS AND DISCUSSION
271
5.3.2. Skin lightening actives ranked as per their efficacy with respect to each
mechanism of action:
Due to the strict safety concerns of the cosmetic industry, the search for new, natural skin
lighteners and their specific mode of action is of utmost importance in field of cosmetic
research. The mode of action of various natural skin lightening actives has been described
in detail below.
5.3.2.1. Inhibitors of Solar lentiges and skin tanning:
One of the most obvious cellular targets for depigmenting agents is the enzyme
tyrosinase. The scientific literature on tyrosinase inhibition shows that a large majority of
the work has been conducted since 2000 and has mostly been devoted to the search for
new depigmenting agents (Smit N et al., 2009). The main manifestations of excess
tyrosinase activity are solar lentiges and skin tanning. Solar lentiges are 1 mm to several
cm in size and are brown to black colored macules occurring in the skin epidermis. Solar
lentiges are found on the UV exposed areas of the body such as the face, dorsum of the
hand, extensor fore arm and upper back. Significant tyrosinase inhibitors can inhibit
tyrosinase thereby decreasing the number of TYR-positive cells and melanin production
per length of the dermal/epidermal interface. Solar lentiges and skin tanning can be
reduced by tyrosinase inhibitors. The best synthetic tyrosinase inhibitors are ranked in
Table 5.3.43, based on their IC50 values in inhibiting tyrosinase enzyme. Lower IC50
value indicates better tyrosinase inhibitory potential.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
272
Table 5.3.43: Significant inhibitors of tyrosinase - Inhibitors of Solar lentiges and skin tanning
Group Rank Plant extract Active IC50 (µg/ml)
I
1 Artocarpus lakoocha Dihydro-oxyresveratrol (DHO) 0.03 ** 2 Oxyresveratrol (OXY) 50-90% 0.1 ** 3 Glycyrrhiza glabra Glabridin (GLAB) 0.25 ** 4 Artocarpus lakoocha Oxyresveratrol (OXY) 20-30% 0.5 **
II
5 Nigella sativa Thymohydroquinone (THQ) 1.0 ** 6 Artocarpus lakoocha Artocarpin (ART) 1.3 ** 7 Curcuma longa Tetrahydrocurcuminoids (THC) 2.0 ** 8 Pterocarpus marsupium 3-Hydroxypterostilbene (3HPT) 2.0 ** 9 Artocarpus lakoocha Resveratrol (RES) 5.5 * 10 Synthetic (from Sigma) Kojic acid (KA) 7.0 11 Pterocarpus marsupium Pterostilbene (PTR) 7.8 12 Piper betle Hydroxychavicol (HC) 8.0 13 Synthetic (from Sigma) Ascorbic acid (ASC) 9.33
III 14 Gnetum sp. Gnetol (GN) 149 15 Synthetic (from Sigma) Arbutin (ARB) 194 16 Emblica officinalis β glucogallin (BG) 200
Group I - Actives with high efficacy; Group II - Actives with moderate efficacy; Group III - actives with mild efficacy. * P value < 0.1 and ** P value < 0.05 as compared to Kojic acid best known for tyrosinase inhibition
Actives with IC50 values ≤ 0.5 are grouped under Tyrosinase inhibitors I which represent
actives with high efficacy. Actives with IC50 values ≤ 10 are grouped under Tyrosinase
inhibitors II which represent actives with moderate efficacy and actives with IC50 values
≥ 100 are grouped under Tyrosinase inhibitors III which represent actives with mild
efficacy (Fig. 5.3.1).
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
273
Tyrosinase inhibitors I
0
0.1
0.2
0.3
0.4
0.5
0.6
DHO OXY 50-90% GLAB OXY 20-30%
Actives
IC 5
0 in
mic
rogr
am/m
l
Tyrosinase inhibitors II
0
2
4
6
8
10
12
THQ ART THC 3HPT RES KA PTR HC ASC
Actives
IC 5
0 in
mic
rogr
am/m
l
Tyrosinase inhibitors III
0
50
100
150
200
250
GN ARB BG
Actives
IC 5
0 in
mic
rogr
am/m
l
Tyrosinase inhibitors I - Actives with high efficacy; Tyrosinase inhibitors II - Actives with moderate efficacy; Tyrosinase inhibitors III - actives with mild efficacy Figure 5.3.2: Inhibitors of Tyrosinase - Inhibitors of Solar lentiges and skin tanning
5.3.2.2. Inhibitors of Melasma and skin tanning:
The main manifestations of melanogenesis induced by over expression of MSH & cAMP
are melasma and skin tanning. Melasma also occurs during pregnancy, usage of oral
contraceptives, certain anti epileptics etc. Melasma is observed as symmetrical facial
hyperpigmentation involving either epidermis, dermis or both. Significant inhibitors of
melanogenesis can inhibit melasma and skin tanning. The best synthetic melanogenesis
inhibitors are ranked in Table 5.3.44, based on their IC50 values in inhibiting
melanogenesis in B16F1 mouse melanoma cells. Lower IC50 value indicates better
melanogenesis inhibitory potenital.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
274
Table 5.3.44: Significant inhibitors of MSH and cAMP induced melanogenesis - Inhibitors of Melasma and skin tanning
Group Rank Plant extract Active IC50 (µg/ml)
I
1 Nigella sativa Thymohydroquinone (THQ) 0.3 ** 2 Pterocarpus marsupium Pterostilbene (PTR) 0.5 ** 3 Piper longum Piperlongumine (PL) 0.6 ** 4 Pterocarpus marsupium 3-Hydroxypterostilbene (3HPT) 0.7 ** 5 Piper betle Hydroxychavicol (HC) 1.3 ** Artocarpus lakoocha Dihydro Oxyresveratrol (DHO) 1.63 ** 6 Polygonum cuspidatun Resveratrol (RES) 2.5 **
7
Curcuma longa Tetrahydrocurcuminoids(THC) 3 **
Glycyrrhiza glabra Glabridin (GLAB) 3 ** Artocarpus lakoocha Oxyresveratrol (OXY) 20-30% 3 ** Artocarpus lakoocha Artocarpin (ART) 3 **
8 Pterocarpus marsupium Dihydropterostilbene (DPTR) 5 ** Eugenia jambolana Polyphenols (PLY) 5 **
II
9 Artocarpus lakoocha Oxyresveratrol (OXY) 50-90% 10 * 10 Emblica officinalis β glucogallin (BG) 12 *
11 Avena sativa Avenanthramide C (AVN) 20 * Synthetic (from Sigma) Ascorbic acid (ASC) 25
12 Synthetic (from Sigma) Glutathione (GLU) 25
13 Synthetic (from Sigma) Kojic acid (KA) 100 Synthetic (from Sigma) Arbutin (ARB) 100
Group I - Actives with high efficacy; Group II - actives with moderate efficacy. * P value < 0.1 and ** P value < 0.05 as compared to Ascorbic acid best known for melanin inhibition
Actives with IC50 values ≤ 5 are grouped under Melanogenesis inhibitors I which
represent actives with high efficacy and actives with IC50 values ≤ 100 are grouped under
Melanogenesis inhibitors II which represent actives with moderate efficacy (Fig. 5.3.2).
Amongst the above listed actives, Kojic acid has no effect on melanogenesis induced by
cAMP (Table 5.3.3) while Ascorbic acid and Glutathione are less affective for
melanogenesis induced by cAMP (Table 5.3.5).
Interstingly, melanogenesis inhibition potential of Artocarpus lakoocha bark
extract was higher in extracts containing lower concentration of Oxyresveratrol as
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
275
observed in the table. Therefore, the activity is due to the synergy between the various
actives present in the extract and not just by Oxyresveratrol.
Melanogenesis inhibitors I
DHPTPLY
THCGLAB
OXY 20-30%ART
0
1
2
3
4
5
6
THQ PTR PL 3HPT HC DHO RES THC DHPT
Actives
IC 5
0 in
mic
rogr
am/m
l
Melanogenesis inhibitors II
0
20
40
60
80
100
120
OXY50-90%
BG AVN ASC GLU KA ARB
Actives
IC 5
0 in
mic
rogr
am/m
l
Melanogenesis inhibitors I - Actives with high efficacy; Melanogenesis inhibitors II - Actives with moderate efficacy Figure 5.3.3: Inhibitors Melanogenesis - Inhibitors of Melasma and skin tanning As evident in Fig. 5.3.3, all the actives listed in Table 5.3.43 except Piperlongumine are
inhibitors of Tyrosinase which is the enzyme responsible to catalyze the final step in
melanogenesis. Similarly, actives listed in Table 5.3.44, inhibit melanogenesis right at the
initial step of the pathway by inhibiting MSH. All the actives that inhibit MSH induced
melanogenesis are the inhibitors of cAMP induced melanogenesis also except for Kojic
acid. In other words, when melanogenesis is induced due to the mere enhancement in
cAMP levels, Kojic acid is ineffective.
From Table 5.3.43 and Table 5.3.44, it is evident that most of the actives
inhibiting the process of melanogenesis at the initial step which is MSH activity also
inhibit the process at the final step which is tyrosinase activity. However, Piperlongumine
which is a significant inhibitor of melanogenesis inhibits only MSH induced
melanogenesis, clearly indicating that it works only by inhibiting the activity of MSH
initially and not by inhibiting tyrosinase. Melanogenesis is inhibited by Piperlongumine
right at the first step.
However, the degree of activity varied with respect to tyrosinase inhibitory and
melanogenesis inhibitory potential. For example, Dihydrooxyresveratrol has the highest
tyrosinase inhibitory potential whereas Thymohydroquinone has the highest
melanogenesis inhibitory potential.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
276
X - Inhibition Figure 5.3.4: Effect of Tyrosinase and Melanogenesis inhibitors on skin hyperpigmentation
5.3.2.3. Protection from UV exposure:
UV exposure is the main reason for over expression of MSH or cAMP or Tyrosinase
which occur as a response to stress due to UV induced free radical damage on skin. The
process of melanogenesis is enhanced as a defence mechanism as melanin tends to
protect the skin from UV damage. Hence, actives that provide protection from UV can
prevent the whole process of excessive melanogenesis and subsequent skin darkening.
These actives have potential as sunscreens. Actives that act as sunscreens as well as
antioxidants can be used not just for prevention from UV exposure but also scavenge the
αMSH ACTH
MC1-R
PKA
MITF
Tyrosinase
cAMP
1. Melanogenesis 2. Differentiation,
dendrite formation
and proliferation of melanocytes
Actives in Table 5.3.43 except
Piperlongumine
X
Actives in Table 5.3.44 X
Actives in Table 5.3.44
except Kojic acid
X
Skin hyperpigmentation
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
277
free radicals generated due to UV exposure and help in “after sun care” of the skin by
soothing the UV damaged skin and reducing the excessive pigmentation. The actives that
significantly protect from UV are ranked in Table 5.3.45, based on their EC50 values in
preventing cell death due to UV exposure in Swiss 3T3 mouse fibroblast cells. Lower
EC50 value indicates better protection form UV induced cell death. The significant actives
for UV protection are ranked in comparison to the standard product for UV protection
Octyl methoxycinnamate (OMC) for which the EC50 was found to be 100µg/ml (Fig.
5.3.4).
Table 5.3.45: Actives that prevent UV induced cell damage
Rank Plant extract Active EC50 (µg/ml)
1 Emblica officinalis β glucogallin (BG) 20 ** Artocarpus lakoocha Artocarpin (ART) 20 **
2 Gnetum sp. Gnetol (GN) 25 **
3 Pterocarpus marsupium Pterostilbene (PTR) 30 ** Polygonum cuspidatun Resveratrol (RES) 30 **
4 Artocarpus lakoocha Oxyresveratrol (OXY) 80-90% 50 *
Avena sativa (Oat) Oat ceramides (OCR) 50 *
5 Artocarpus lakoocha
Oxyresveratrol (OXY) 50% 75 *
6 Oxyresveratrol (OXY) 20-30% 100
7
Malus domestica (Apple) Apple ceramides (APL) 100
Citrullus colocynthis Colocynthin (CYN) 100
Synthetic Octylmethoxycinnamate (OMC) 100 * P value < 0.1 and ** P value < 0.05 as compared to OMC best known for sunscreen potential
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
278
Actives that prevent UV induced cell damage
0
20
40
60
80
100
120
BG ART GN PTR RES OXY80-90%
OCR OXY50%
OXY20-30%
APL CYN OMC
Actives
EC
50
in m
icro
gram
/ml
Figure 5.3.5: Actives that prevent UV damage
5.3.2.4. Inhibitors of Free radical damage:
Reduction of ROS levels in melanocytes may prevent activation of melanogenesis. In
various studies, extracts from plants or fruit or other species were tested for their
antioxidant capacity by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-
scavenging assay or the oxygen radical absorbance capacity (ORAC) (Rangkadilok et al.,
2007). The process of melanogenesis is enhanced as a defence mechanism as melanin
tends to protect the skin from free radical damage induced by UV exposure. Hence,
antioxidant actives prevent free radical damage that further induces excessive
melanogenesis. Actives that act as sunscreens as well as antioxidants can be used not just
for prevention from UV exposure but also scavenge the free radicals generated due to UV
exposure and help in “after sun care” of the skin by reducing the excessive pigmentation.
Significant antioxidant actives are ranked in Table 5.3.47, based on their IC50 values in
scavenging DPPH, scavenging ROS generation in Swiss 3T3 mouse fibroblast cells and
their high ORAC and HORAC values. Lower IC50 values and higher ORAC and HORAC
values indicate better antioxidant potential. Cumulative antioxidant potential for each
active has been calculated as the ratio of additive ORAC and HORAC values and IC50 for
ROS scavenging potential (Table 5.3.46). Cumulative antioxidant potential = A + B /
C, Where, A = ORAC value in μmol trolox equivalents/g, B = HORAC value in
μmol trolox equivalents/g and C = IC50 for ROS scavenging potential.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
279
Table 5.3.46: Cumulative antioxidant potential of actives
Active ORAC HORAC
ORAC +
HORAC ROS Ratio
Hydroxychavicol 29728 2376 32104 0.5 64208
THC 10000 3000 13000 1 13000
Resveratrol 25000 10000 35000 3 11666
Oxyresveratrol 90% 21000 5723 26723 3 8907
Oxyresveratrol 80% 18673 4923 23596 3 7865
Oxyresveratrol 50% 15582 4896 20478 3 6826
Oxyresveratrol 30% 11370 4776 16146 3 5382
Oxyresveratrol 20% 8999 4756 8999 3 4585
Dihydrooxyresveratrol 21549 3097 26549 3 8215
Glabridin 7550 1129 8679 1 8679
Avenanthramide C 22352 5000 27352 10 2735
Gnetol 14322 5000 19322 3 6440
3HPT 13334 4700 17334 3 6011
Pterostilbene 12508 6233 12508 3 6247
β-glucogallin 99% 4436 4660 9096 2.5 3638
β-glucogallin 10% 2862 1474 4336 2.5 1734
Artocarpin 3859 1121 4980 3 1660
Jamun polyphenols 9000 1000 10000 10 1000
Thymohydroquinone 5899 2000 7899 20 395
Ascorbic acid 3400 Nil 3400 10 340
This calculation is applicable when antioxidant actives have significant potential in all the
mechanisms of antioxidant activity as specified in the above formula. ROS scavenging
potential has been given preference over DPPH scavenging potential for calculating the
cumulative antioxidant potential because ROS scavenging assay is performed under the
typical stress induced conditions in mammalian cells where as DPPH scavenging is a
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
280
chemical based assay which can only be an indicator of antioxidant potential at a
preliminary experimentation level.
Table 5.3.47: Significant inhibitors of free radicals - inhibitors of free radical induced melanogenesis Rank Plant extract Active Cumulative antioxidant units
1 Piper betle Hydroxychavicol 64,208 **
2 Curcuma longa THC 13,000 **
3 Polygonum cuspidatum Resveratrol 11,666 **
4
Artocarpus lakoocha Oxyresveratrol 90% 8907 **
Dihydro Oxyresveratrol 8215 **
Glycyrrhiza glabra Glabridin 8679 ** 5
Artocarpus lakoocha Oxyresveratrol 80% 7865 **
6 Oxyresveratrol 50% 6826 ** 7 Gnetum sp. Gnetol 6440 **
8 Pterocarpus marsupium Pterostilbene 6247 **
9 Pterocarpus marsupium 3HPT 6011 **
10 Artocarpus lakoocha
Oxyresveratrol 30% 5382 **
11 Oxyresveratrol 20% 4585 **
12 Emblica officinalis Β- glucogallin 90% 3638 **
13 Avena sativa Avenanthramide C 2735 **
14 Emblica officinalis Β- glucogallin 10% 1734 *
15 Artocarpus lakoocha Artocarpin 1660 *
16 Eugenia jambolana Polyphenols 1000 *
17 Nigella sativa Thymohydroquinone 395
18 Synthetic Ascorbic acid 340
* P value < 0.1 and ** P value < 0.05 as compared to Ascorbic acid best known for antioxidant potential
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
281
X - Inhibition Figure 5.3.6: Effect of Sunscreens and Antioxidants on Melanogenesis
Unlike in the case of melanogenesis inhibition, UV protection and antioxidant potential
of Artocarpus lakoocha bark extract is conferred by the content of Oxyresveratrol as the
antioxidant and UV protection potential increased with the increasing concentrations of
this active in the extract (Table 5.3.47). Similarly, the antioxidant potential of Amla
extract is conferred by the content of β glucogallin in the extract as the antioxidant
potential increased with the increasing concentrations of this active in the extract (Table
5.3.47). All the actives listed in Table 5.3.45, provide protection from UV damage which
is the main reason for a sequence of reactions and ultimately enhanced melanogenesis as
a response to stress conditions. They act as “sunscreens” by either absorbing or blocking
the UV or inhibiting UV induced adversaries (Fig. 5.3.5). Actives listed in Table 5.3.47,
inhibit UV induced free radical generation which induces melanogenesis (Fig. 5.3.5).
UV
ROS ROS
MSH
cAMP
NO
cGMP
PKG
Melanogenesis
DAG
PKC
Actives in Table 5.3.47
(Antioxidants)
Actives in Table 5.3.47
(Antioxidants)
Actives in Table 5.3.45 (Sunscreens)
X
X
X
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
282
All the antioxidant actives also provided UV protection except for
Hydroxychavicol, 3-Hydroxypterostilbene, Avenanthramides, Jamun polyphenols,
Thymohydroquinone and Ascorbic acid which are significant antioxidants but still could
not prevent UV induced cell death (Table 5.3.45 and Table 5.3.47). Similarly, all actives
with significant UV protection are significant antioxidants except the ceramides from
Malus domestica and Avena sativa (Table 5.3.45 and Table 5.3.47). Therefore all
antioxidants need not necessarily provide UV protection and vice-versa. Actives which
are not antioxidants but still render UV protection act as a shield against UV by
absorbing the UV and blocking UV from reaching the biological system. Antioxidants
combat UV induced free radical damage. Some antioxidants prevent UV damage and
some heal UV damage. Actives that heal UV damage are called “after sun care” actives
which may not prevent UV damage but heal UV damage. “After sun care” actives are
mainly antioxidants that combat UV induced free radical damage. For example,
Glutathione a significant antioxidant could not prevent from UV induced cell death in
vitro. Glutathione is the major antioxidant produced by the cells in the body, for
protection from free radicals (oxygen radicals, oxyradicals). These highly reactive
substances, if left unchecked, will damage or destroy key cell components (e.g.
Membranes, DNA) in microseconds. Oxyradicals are generated in many thousand
mitochondria located inside each cell, where nutrients like glucose are burnt using
oxygen to make energy. Oxyradicals also come from pollutants and UV
radiation. Glutathione not only protects the body form these free radicals but also
recycles other well know antioxidants such as vitamin C and vitamin E, keeping them in
their active state. Glutathione plays a crucial role in maintaining the normal balance
between oxidation and anti-oxidation. This, in turn, regulates many of the cell’s vital
functions, such as the synthesis and repair of DNA after UV damage, the synthesis of
proteins and the activation of regulation of enzymes. Hence, Glutathione can be
affectively used as an “after sun care” product, meaning it can reduce the after effects of
UV damage. Table 5.3.48 represents actives which act as “sunscreens”, “after sun care”
actives and both.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
283
Table 5.3.48: Actives and their mode of action for UV protection
Group Active Prevention from UV
damage – Sunscreens
Healing of UV damage –
“After sun care”
I
β glucogallin 10-99% Significant Significant
Oxyresveratrol 20-
90%
Significant Significant
Artocarpin Significant Significant
Gnetol Significant Significant
Pterostilbene Significant Significant
Resveratrol Significant Significant
Colocynthin Significant Mild
II
Ceramides from
Avena sativa
Significant Nil
Ceramides from
Malus domestica
Significant Nil
III
Tetrahydrocurcumin Not significant Significant
Dihydro-
Oxyresveratrol
Not significant Significant
Glabridin Not significant Significant
Ascorbic acid Not significant Significant
Hydroxychavicol Nil Significant
3HPT Nil Significant
Glutathione Nil Significant
Avenanthramides Nil Significant
Jamun polyphenols Nil Significant
Thymohydroquinone Nil Significant
Group I – Sunscreen and “after sun care” actives; Group II - Sunscreens; Group III - “After sun care” actives
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
284
As evident from Table 5.3.48, actives in group I are significant “sunscreens” as well as
“after sun care” actives. Actives in group II are significant “sunscreens” where as actives
in group III are significant “after sun care” actives. It is also observed that Oxyresveratrol
which is both a significant antioxidant as well as UV protectant, on hydrogenation of
Dihydrooxyresveratrol remained just an antioxidant but could not retain the UV
protection potential. Therefore, unlike Oxyresveratrol which is both a significant
“sunscreen” as well as “after sun care” active, Dihydro-oxyresveratrol is just an “after
sun care” active.
5.3.2.5. Inhibitors of post inflammatory hyperpigmentation:
As known from many cases of post-inflammatory hyperpigmentation, melanogenesis can
be stimulated by some inflammatory mediators. Inhibition of the production of
inflammatory mediators (Il1α and TNF-α) was reported for sea grape (Coccoloba uvifera)
extracts (Silveira J E et al., 2007). Via this indirect way, stimulation of melanogenesis in
the pigment cells could be prevented (Briganti S et al., 2003). Post inflammatory
hyperpigmentation is mainly manifested after resolution of skin problems like acne,
contact dermatitis etc. Post inflammatory hyperpigmentation is observed as discrete
hyper pigmented macules with hazy magins on the skin where melanin production is
increased. Upregulation of inflammatory markers like TNF α and prostaglandins occurs
as a result of skin defence mechanism to pathogens and allergens. However, these
markers tend to damage the skin also and hence even after resolution of skin problems,
the skin remains darkened at the affected areas as dark marks or scars due to the
increased melanin production and melanocyte dendricity in response to the inflammatory
markers. The damage can be at the level of epidermis, dermis or both.
Certain inflammatory enzymes like Elastase, Collagenase and Hyaluronidase are
over expressed due to exposure to certain stress conditions like UV, pollutants, toxins etc.
These are serine protease enzymes that dissociate tissues which contain extensive
intercellular fiber networks and clean up any dead tissue leaving the wound bed ready for
healing. Serine proteases also help in PAR-2 (Protease activated receptor) mediated
transfer of melanosomes from melanocytes to keratinocytes resulting in skin darkening.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
285
Hyaluronidase is the natural protein responsible for hydrolysis of the extracellular
mucopolysaccharide, hyaluronic acid. It acts by modifying the permeability of
intercellular ground substance in connective tissue. The breakdown of the viscous
hyaluronic acid decreases tissue resistance and enhances diffusion of substances between
tissue planes. However, over expression of these enzymes results in inflammation of the
skin leading to skin darkening and break down of skin structural proteins and glycans like
elastin, collagen and hyaluronic acid respectively.
Inflammation is a normal biological mechanism triggered in the body in response
to infection, foreign bodies etc., however overstay of inflammation results in the damage
of healthy tissues such as the skin dermis, eventually resulting in hyper-pigmentation.
Therefore, significant inhibitors of inflammatory markers and enzymes can inhibit
increased melanin production in the dermis at the affected area (Fig. 5.3.6). Anti
inflammatory actives are ranked in the Table 5.3.49, based on their IC50 values in
inhibiting elastase, collagenase, hyaluronidase enzymes and TNFα. Lower IC50 value
indicates better anti inflammatory potenital. TNF α inhibition is given higher preference
over elastase, collagenase and hyaluronidase inhibition as TNF α directly influences
melanogenesis while the inflammatory enzymes mainly affect the skin texture and tone.
However, the cumulative effect of inflammatory markers and enzymes is more significant.
Since anti inflammatory actives are widely used as drugs for therapeutic treatment of
various inflammatory diseases like arthritis etc., a reference standard drug is not used for
comparing the actives being screened for skin lightening efficacy. Actives are considered
significant for anti inflammatory efficacy if IC50 values could be obtained within the
optimal dosages evaluated. Actives with significant inhibition of TNF α and
inflammatory enzymes are preferred over those that are significant inhibitors of TNF α
alone. Actives with significant inhibition of TNF α are preferred over those that are
significant inhibitors of inflammatory enzymes alone.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
286
Table 5.3.49: Significant inhibitors of inflammation – inhibitors of post inflammatory hyperpigmentation Rank Plant extract Active Rationale
1 Artocarpus lakoocha Oxyresveratrol 20-90% Significant inhibitors of Elastase, Collagenase enzymes and TNF α
2 Glycyrrhiza glabra Glabridin 3 Artocarpus lakoocha 3HPT 4 Pterocarpus marsupium Pterostilbene 5 Artocarpus lakoocha Artocarpin 6 Nigella sativa Thymohydroquinone
Significant inhibitors of TNF α
7 Avena sativa Avenanthramide C 8 Artocarpus lakoocha Dihydro Oxyresveratrol 9 Citrullus colocynthis Colocynthin 10 Polygonum cuspidatun Resveratrol 11 Curcuma longa THC 12 Piper betle Hydroxychavicol 13 Piper longum Piperlongumine 14 Malus domestica (peel) Ceramides Significant inhibitor of elastase
and collagenase enzymes
The detailed mechanisms of action of various synthetic skin lightening actives gives an
outlook on which active can be recommended based on its mechanism of action for a
specific kind of skin darkening. The ranking can be useful for recommendation based on
the intensity of the pigmentation. For example, for severe post inflammatory
hyperpigmentation scars, actives of significant anit inflammatory potential like
Oxyresveratrol, Dihydrooxyresveratrol, Hydroxychavicol or 3HPT can be recommended.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
287
X - Inhibition Figure 5.3.7: Effect of inhibitors of inflammation on Scar formation
5.3.3. Skin lightening actives ranked as per their cumulative efficacy for all the major
skin lightening mechanisms:
Next to in vitro, the brownish guinea pig model is used in several skin lightening studies
where the pigmentation is induced by either UV or α-MSH. In case of in vivo studies,
prevention of the induction of pigment by the whitening agents could be demonstrated
using a Minolta chromameter or by histochemical investigations showing a decrease in
DOPA positive cells (Yamakoshi J et al., 2003 and Yoshimura, M et al., 2005). Another
animal model used for whitening studies is the zebrafish that also proved useful for
demonstrating the in vivo toxicity of the whitening agents (Choi T Y et al., 2007 and Kim
J H et al., 2008). So far, only limited numbers of clinical trials with skin whitening agents
Skin exposure to allergins/toxins
Skin sebum infection
Inflammation
Fight against foreign bodies
Healing
Dermal matrix damage and
melanogenesis induction in affected
melanocytes
Overstay of Inflammation
Hyperpigmentation
Scars/marks
Actives in Table 5.3.49
X
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
288
or formulations have been performed (Tengamnuay P et al., 2006 and Chang T S, 2009).
For ethical and economic reasons, the cosmetic industry relies heavily on in vitro studies
and a thorough positioning of skin lightening actives is therefore a significant pre
requisite for the development of affective skin lightening agents.
As evident in Fig. 5.3.7 and Table 5.3.50, Oxyresveratrol, Pteostilbene,
Resveratrol and Artocarpin have potential in all the major skin lightening mechanisms
and can be promising actives for various kinds of hyperpigmentation disorders.
X – Inhibition Figure 5.3.8: Actives with significant efficacy in various skin lightening mechanisms
Table 5.3.50 summarizes the actives and their method of action for skin lightening and
also clearly shows the all rounder actives for all kinds of skin darkening.
Excessive skin pigmentation
Tyrosinase
Free radicals
MSH & cAMP
UV exposure
Inflammation
Oxyresveratrol Artocarpin
Pterostilbene Resveratrol
X
X
X
X
X
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
289
Table 5.3.50: Actives and their cumulative efficacy for all the major skin lightening mechanisms
Active SL Melasma UV Free radical
induced pigmentation
Post inflammatory hyperpigmentation
Oxyresveratrol
Pterostilbene
Resveratrol
Artocarpin Amla extract
Dihydro-oxyresveratrol
Tetrahydrocurcumin
Thymohydroquinone
Hydroxychavicol
Glabridin
3-hydroxypterostilbene
Ascorbic acid
Gnetol
Eugenia jambolana extract
Piperlongumine
Glutathione
Kojic acid
Arbutin
Avenanthramide C
Oat ceramides *
Apple fruit ceramides *
Apple peel ceramides * * *
- Actives with significant effect on all skin lightening mechanisms * - Actvies with Mild activity - Actives with no activity
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
290
5.3.4. Novel actives and observations:
5.3.4.1. Novel skin lightening actives:
Some actives were screened on account of their significant properties such as antioxidant,
anti-inflammatory, skin conditioning and UV protection activities. These actives can have
efficacy for skin lightening also as free radical stress, UV stress and inflammation are
some of the pathways for excessive skin pigmentation. Therefore, in the process of
screening of various actives through various skin lightening mechanisms, some novel
plant actives were observed to have skin lightening potential.
5.3.4.1.1. Thymoquinone from Nigella sativa extract: Thymoquinone has significant
antioxidant and anti inflammatory potential and hence could help in skin lightening as
well. Hence, when studied for melanin inhibition potential, it was found to be a
significant inhibitor of melanin.
5.3.4.1.2. Hydroxychavicol from Piper betle extract: Hydroxychavicol has significant
antioxidant and anti inflammatory potential and hence could help in skin lightening as
well. Hence, when studied for melanin inhibition potential, it was found to be a
significant inhibitor of melanin.
5.3.4.1.3. Eugenia jambolana extract: Eugenia jambolana extract rich in polyphenols
has significant antioxidant potential and hence could help in skin lightening as well.
Hence, when studied for melanin inhibition potential, it was found to be a significant
inhibitor of melanin.
5.3.4.1.4. Avenanthramides from Avena sativa extract: Avenanthramides have
significant antioxidant and anti inflammatory potential and hence could help in skin
lightening as well. Hence, when studied for melanin inhibition potential, it was found to
be a significant inhibitor of melanin. Of all the Avenenthramides screened,
Avenenthramide C (Av-C) showed superior potential for skin lightening.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
291
5.3.4.1.5. Colocynthin from Citrullus colocynthis fruit extract: Colocynthin has
significant antioxidant and anti inflammatory potential and hence could help in skin
lightening as well. Hence, when studied for melanin inhibition potential, it was found to
be a significant inhibitor of melanin.
5.3.4.1.6. Natural ceramides from Avena sativa (Oat) and Malus domestica (Apple)
extract: Ceramides have UV protection potential and they act as potential skin
conditioners by preventing transepidermal water loss from the skin. Hence, if they can
prevent excessive pigmentation by inhibiting melanin they can be unique products for
skin lightening. Hence, when studied for melanin inhibition potential, it was found that
Oat ceramides and Apple peel ceramides were mild inhibitors of melanin. But Apple fruit
ceramides were significant inhibitors of melanin.
5.3.4.2. Novel observations:
In the process of screening of various actives through various skin lightening mechanisms,
actives that conferred efficacy and the synergistic interplay of actives for efficacy could
be determined.
5.3.4.2.1. Oxyresveratrol content in Artocarpus lakoocha heartwood extract and its
effect on skin lightening potential: As discussed earlier, Oxyresveratrol is a significant
tyrosinase and melaogenesis inhibiting active of Artocarpus lakoocha heartwood extract.
Surprisingly it was observed that the melanogenesis inhibitory potential of Artocarpus
lakoocha heartwood extract decreased with the increasing concentration of
Oxyresveratrol. But Oxyresveratrol is known for its skin lightening potential. Therefore it
is a novel observation that the melanogenesis inhibitory potential of Artocarpus lakoocha
heartwood extract is not just because of Oxyresveratrol but due to the synergistic activity
of various actives in the extract. It was also observed that on hydrogenation of
Oxyresveratrol to Dihydrooxyresveratrol, there was an enhancement in tyrosinase and
melanin inhibitory potential but there was a decrease in UV protection potential.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
292
5.3.4.2.2. Pterostilbene and Dihydro-pterostilbene from Pterocarpus marsupium
heartwood extract: Unlike in the case of oxyresveratrol, hydrogenation of actives
having good melanogenesis inhibitory potential does not always enhance the activity. It
was observed that on hydrogenation of Pterostilbene to Dihydro-pterostilbene, the
melanogenesis inhibitory potential was reduced. Dihydro-pterostilbene showed mild
tyrosinase inhibition of 25% at 2.5µg/ml and its IC50 for melanin inhibiton was 5µg/ml.
5.3.4.2.3. β-glucogallin in Emblica officinalis (Amla) fruit extract and its effect on
skin lightening potential: Amla extract had significantly higher skin lightening potential
than ascorbic acid. Hence, unlike what was earlier thought that the skin lightening
potential was conferred by ascorbic acid; it was observed in the present study that the
skin lightening potential was attributed due to the synergistic combination of β-
glucogallin and various other gallates in the extract.
5.3.5. Actives that help in skin lightening through indirect mechanisms of action:
5.3.5.1. Compounds with cell proliferation enhancement potential:
Some of the actives with no significant antioxidant, anti inflammatory, UV protection or
skin lightening potential but yet with a significant cell proliferation potential will have a
very good effect in skin lightening as well by enhancing the cell rejuvenation. When the
cell proliferation enhancer is taken in combination with significant skin lightening actives,
while the actives lighten the skin cells, the cell proliferation enhancer helps in
rejuvenation of the cells, with an effect that the darker skin is continuously replenished
by fresh lightened skin cells. Therefore the process of skin lightening is quickened. For
example, Retinoids influence pigmentation by speeding up turnover in the skin, gradually
eliminating anything sitting on the top layers. This sloughing process automatically
begins to slow down in our mid twenties. Retinoids reverse that effect by producing a
faster rate of cell turnover as well as eliminating abnormal melanin in the top layer of
skin. Retinoids are therfore useful in treating melasma and acne scars by reducing the
amount of excess melanin and distributing it more evenly.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
293
Of all the actives screened for cell proliferation enhancement activity, the following
products showed significant activity.
5.3.5.1.1. Liquid endosperm of Cocos nucifera (coconut): Coconut liquid endosperm
was found to enhance the cell proliferation of Swiss 3T3 mouse fibroblast cells as
observed by SRB staining technique (Table 5.3.51 and Fig. 5.3.8).
Table 5.3.51: Cell proliferation enhancement by Liquid endosperm of Coconut in vitro
Coconut liquid
endosperm
Optical Density due to
viable cells at 492nm
% enhancement in cell proliferation
as compared to control
No treatment 0.139 -
2.5 µg/ml 0.154 11%
5 µg/ml 0.164 18%
Figure 5.3.9: Cell proliferation enhancement by Coconut liquid endosperm in Swiss 3T3 mouse fibroblasts
Untreated cells 2.5µg/ml Coconut liquid endosperm treated cells
5µg/ml Coconut liquid endosperm treated cells
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
294
Coconut liquid endosperm (Freeze dried powder) also showed a significant wound
closure enhancement potential of 25% at a concentration of 5µg/ml in Swiss 3T3 mouse
fibroblast cells as analyzed by the scratch wound closure assay (Fig. 5.3.9). The effect of
liquid endosperm of Coconut as an enhancer of cell proliferation was further confirmed at
a reputed research laboratory. The cell proliferation enhancement by Coconut liquid
endosperm was evident by an in vivo study conducted at Dabur Research Foundation,
Ghaziabad, Uttar Pradesh – Efficacy of Sami Formulations Report (Protocol No.
PR/CR/REP/003-00). The hair growth efficacy of Coconut liquid endosperm was
compared with that of a standard, Minoxidil. 60 days old female C57/BL6 mice were
treated topically with cream containing 2% Coconut liquid endosperm for ten days. On
completion of the studies, 8 mm punch biopsies were taken from the resected dorsal skin
and was processed for histopathological studies to obtain longitudinal and transverse
sections. Digital photomicrographs were taken from representative areas at a fixed
magnification of 100X. Histological evaluation of the skin biopsies showed hair growth
promoting activity of Coconut liquid endosperm with the effect that the hair follicles
were transformed from Telogen to Anagen phase of hair growth in the animals on topical
application of formulation containing Coconut liquid endosperm (Table 5.3.52). Hence,
cell proliferation enhancers like Coconut liquid endosperm can have good effect on skin
lightening by quickening the process in combination with other skin lightening actives.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
295
A) Untreated wounded cell monolayer after 24 hours; B) Untreated wounded cell monolayer after 48 hours; C) Coconut liquid endosperm treated wounded cell monolayer after 24 hours; D) Coconut liquid endosperm treated wounded cell monolayer after 24 hours Figure 5.3.10: Wound closure by Coconut liquid endosperm in Swiss 3T3 mouse fibroblasts
B
C D
A
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
296
Table 5.3.52: Cell proliferation enhancement by Liquid endosperm of Coconut in vivo
Treatment Mean follicle count
in subcutis layer
Average skin
thickness (µm)
% of animals showing
Anagen induction
Cream
(2% Coconut liquid
endosperm)
43.00 ± 12.20
220.15 ± 25.60
71.4
2% Minoxidil
(Ref.Standard) 42.86 ± 13.49 218.75 ± 34.04 71.4
5% Dextrose (inert) 3.43 ± 3.27 156.27 ± 5.14 14.2
An in vivo study conducted at Dabur Research Foundation, Ghaziabad, Uttar Pradesh – Efficacy of Sami
Formulations Report (Protocol No. PR/CR/REP/003-00)
5.3.5.1.2. Probiotic bacteria Bacillus coagulans: Another sample which showed
significant cell proliferation enhancement was the culture supernatant containing Bacillus
coagulans exudates. Probiotics are dietary supplements of live bacteria or yeasts thought
to be healthy for the host organism. According to the currently adopted definition,
Probiotics are: ‘Live microorganisms which when administered in adequate amounts
confer a health benefit on the host’. Lactic acid bacteria are the most common type of
microbes used and has been used in the food industry for many years, because they are
able to convert sugars and other carbohydrates into lactic acid. By lowering the pH, they
may create fewer opportunities for spoilage organisms to grow, creating possible health
benefits. Strains of the genera Lactobacillus and Bifidobacterium, are the most widely
used Probiotic bacteria. Creating a complete aseptic environment in the body can destroy
physiological microflora, which in turn causes health problems because microflora are
important in many levels. The introduction of Probiotic micro-organisms to “reset” the
level of bacteria eliminated, represents a winning strategy today, and is something which
most of the scientific community agrees on, with positive results to show for it. Toxins
are the main cause for the deterioration of skin health. Toxins damage the protective skin
barrier by inducing free radicals and also create fertile conditions in skin for harmful
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
297
bacteria to multiply. The same toxins may also interfere with normal functioning of
organs. This may cause organs to overproduce certain hormones that result in skin
damage. For example, toxins stimulate sebum production and more acne-causing bacteria
proliferate resulting in pimples. The most effective way for good skin is to maintain a
correct balance of intestinal and skin bacteria. Probiotic bacteria limit the growth of
harmful bacteria. Probiotics also neutralize toxins and create an environment lethal to
pathological bacteria. By keeping pathological bacteria at bay and preventing
overproduction of toxins, Probiotics actually eliminate the root cause of skin damage.
Probiotic bacteria prevent the growth of pathological bacteria and also help in skin
rejuvenation. Therefore, probiotic bacteria help in curing skin disorders and maintain
healthy skin. A combination of probiotic bacteria with significant skin lightening actives
can help quicken the process of skin lightening. While the actives lighten the skin cells,
the cell proliferation enhancer helps in rejuvenation of the cells, with an effect that the
darker skin is continuously replenished by fresh lightened skin cells. Therefore, such
probiotic bacteria can be used both as topical cosmetics or as nutricosmetics in
combination with other skin lightening actives for cosmetic benefits.
The culture supernatant of the exponential growth phase of Bacillus coagulans
was studied for its effect on the growth of Swiss 3T3 mouse fibroblast cells. The culture
supernatant was diluted in varying concentrations in mouse fibroblast growth medium
and used for treating the mouse fibroblast cells. It was observed that this culture
supernatant rich in exudates like lactic acid etc. could enhance the cell proliferation of
mouse fibroblast cells by 22% upto a non cytotoxic concentration of 3.125% (Table
5.3.53 and Fig. 5.3.10).
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
298
Table 5.3.53: Effect of Bacillus coagulans on cell proliferation enhancement
Conc. of Bacillus coagulans culture supernatant (%)
% enhancement in cell proliferation
% cytotoxicity
0 0 0 0.018 0 0 0.037 0 0 0.075 0 0 0.15 4.4 0 0.31 4.6 0 0.75 14.4 0 1.5 15.5 0 3.125 22.1 0 6.25 6.1 0 12.5 0 8.3 25 0 26.5 50 0 42.5 100 0 42.5
Cell proliferation enhancement by culture supernatant of Bacillus coagulans in Swiss 3T3 mouse fibroblast cells
-10
-5
0
5
10
15
20
25
Conc. (%)
% e
nhan
cem
ent i
n ce
ll pr
olife
ratio
n
Cell proliferation
Figure 5.3.11: Cell proliferation enhancement by Bacillus coagulans in Swiss 3T3 mouse fibroblasts
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
299
5.3.5.2. Compounds with collagen enhancement potential:
5.3.5.2.1. Oleanoyl peptide: Oleanoyl peptide is a pentapeptide conjugate of Oleanolic
acid, a naturally occurring triterpenoid. Oleanoyl peptide was chemically synthesized by
solution phase method by conjugating Oleanolic acid to Lysine-Threonine-Threonine-
Lysine-Serine pentapeptide (Lys-Thr-Thr-Lys-Ser) pentapeptide. Oleanolic acid is known
for its anti inflammatory, antioxidant, anti microbial and wound healing properties. In the
present study, by Sirius red staining method, it was observed that this peptide helped in
the enhancement of collagen levels in Human osteosarcoma cells by 17% at a
concentration of 1.25µg/ml.
Figure 5.3.12: Structure of Pentapeptide conjugate of Oleanolic acid
5.3.5.2.2. Asiaticosides from Centella asiatica extract: Asiaticosides also enhanced the
precursor of collagen, pro-collagen in Swiss 3T3 fibroblasts as analysed by flow
cytometry. The population of fibroblasts in the assay was gated as the total population
and analyzed for the percentage of population of fibroblasts that showed Fluorescein
isothiocyanate (FITC) labeled pro-collagen. It was observed that while the untreated cells
showed 1.7% FITC labeled pro-collagen, Centella asiatica extract treated cells showed
13.5% FITC labeled pro-collagen showing a clear enhancement in pro-collagen synthesis
on treatment with Centella asiatica extract (Fig. 5.3.11).
CH3 CH3
CH3
CH3
OH
CH3
OCH3
CH3
ONH
NH2
O
NH
OHCH3
O
NH OH
CH3
O NH
NH2
O
NH
OH
OH
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
300
Therefore, the products like Coconut liquid endosperm and probiotic bacteria Bacillus
coagulans help in cell rejuvenation while products like Oleanoyl peptide and
Asciaticosides help in collagen enhancement and indirectly help and quicken the process
of skin lightening.
A
B C
A) Total population of Swiss 3T3 mouse fibroblasts analyzed B) Untreated cells showing 1.7% of the total population containing FITC labeled pro-collagen C) Centella asiatica extract treated cells showing 13.5% of the total population containing FITC labeled pro-collagen. FSC-A: Forward scatter; SSC-A: Side scatter Figure 5.3.13: Pro-collagen enhancement by Centella asiatica extract in Swiss 3T3 mouse fibroblasts
1.7% FITC labelled pro-collagen
13.5% FITC labelled pro-collagen
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
301
5.3.6. Synergistic skin lightening efficacy due to integration of different mechanisms of
action:
In a broad perspective, the term “synergy” refers to the combined or cooperative effects
produced by the relationships among various forces, particles, elements, parts or
individuals in a given context, effects that are not otherwise possible. The term is derived
from the Greek word “synergos”, meaning (i) to work together or (ii) to co-operate.
“Synergistic effects” are unknown, unexpected, unsought for useful phenomena that are
accidentally discovered. Thus the concepts of “synergistic effects/synergy” are included
under “inventions”. The present study supports the integration of various skin lightening
mechanisms for a synergistic skin lightening effect. Skin lightening compositions
comprising combinations of carefully evaluated plant actives with varied mechanisms of
action, showed exponentially enhanced activity due to synergy mediated by the actives.
5.3.6.1. Chemical conjugation of actives with different modes of action showing
enhanced skin lightening potential:
5.3.6.1.1. Indirect and Synergistic skin lightening effect by Oleanoyl peptide:
Short-sequence peptides hold significant potential as skin lightening ingredients and for
treatment of pigmentation disorders. Peptides can promote a faster and more effective
approach to skin brightening and lightening by enhancing skin's natural elasticity and
firmness, cell rejuvenation and synthesis of skin structural proteins. In this process the
dull and darker superficial skin is constantly replaced by new skin cells by exfoliation of
old keratin. A combination of skin lightening actives with peptides can therefore be more
effective and faster in the skin lightening process as peptides help in clearance of stagnant
melanin to brighten skin. Certain natural peptides in the body like glutathione directly act
on pigmentation and bring about skin lightening. Peptides, short polymers of amino acids
linked by peptide bonds, have been shown to counteract the degradation of dermal
collagen, resulting in a significant change in the appearance of moderate to deep lines and
wrinkles. As collagen fibrils are broken down through natural biological process, peptide
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
302
portions or by-products of this catabolic process act as signals or messenger molecules in
the formation of new collagen fibrils by the fibroblasts. Cosmetically interesting
activities such as stimulation of collagen synthesis, chemotaxis, anti-stinging effects and
others, can be observed and substantiated with chemically modified peptide sequences.
Long chain fatty acid conjugates of peptides improve skin penetration, specific activity
and economic feasibility of these molecules (Lintner K and Peschard O, 2000).
Olenoylpeptide is one such peptide, a conjugate of Oleanolic acid and Lysine-
Threonine-Threonine-Lysine-Serine pentapeptide (Lys-Thr-Thr-Lys-Ser). Due to
the integrated skin lightening mechanisms of individual actives that are conjugated
together, it has the following properties,
• Inhibition of free radicals that induce skin darkening
• Inhibition of inflammatory markers that cause skin darkening
• Inhibition of collagenase and elastase that degrade the skin
• Enhancement of collagen synthesis that helps heal and rejuvenate the skin
• Although it does not directly inhibit melanogenesis, under in vivo conditions it
helps in skin lightening
Activity of Oleanolic acid: An anti inflammatory active with no melanogenesis
inhibitory potential.
Activity of Lys-Thr-Thr-Lys-Ser pentapeptide: Identification and incorporation of
very specific peptide sequences has demonstrated biological activity in relationship to
collagen formation in both in vitro and ex vivo human tests. Extracellular matrix
production can be improved by chemically synthesized subfragments of type I collagen
carboxy propeptide (Katayama K et al., 1991). The molecule shown to have this
signaling power is a pro-collagen I terminal sequence consisting of the amino acids;
“Lysine - Threonine - Threonine - Lysine – Serine” pentapeptide a fragment of
procollagen I increases the production of Collagen IV and glycosaminoglycan and helps
in wrinkle reduction and improving the skin tone. It has no effect on inflammation or
melanogenesis.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
303
Activity of the conjugate, oleanoylpeptide: A significant anti inflammatory and
antioxidant active as compared to its parent actives, Oeanolic acid and Lys-Thr-Thr-Lys-
Ser pentapeptide (Table 5.3.54). However, there was no significant melanogenesis
inhibitory potential.
Table 5.3.54: Anti inflammatory and antioxidant activity of the conjugate, Oleanoyl peptide in comparison to the individual actives conjugated
Compound
Elastase
inhibition
(IC50 µg/ml)
Collagenase
inhibition
(IC50 µg/ml)
TNF α
inhibition
(IC50 µg/ml)
Antioxidant
activity - DPPH
Scavenging
Oleanoyl peptide 143 119 21 29% inhibition
at 300 µg/ml
Oleanolic acid Nil 129 17% inhibition
at 100 µg/ml Nil
Pentapeptide (Lys-
Thr-Thr-Lys-Ser) Nil
24% inhibition at
500 µg/ml Nil Nil
Rationale for skin lightening activity of Oleanoylpeptide: In Oleanoylpeptide, the
cosmetic approach is two pronged, collagen production being boosted by the
pentapeptide and inflammation being reduced by Oleanolic acid. Moreover,
Oleanoylpeptide showed anti inflammatory potential through pathways like TNF α
inhibition, elastase and collagenase inhibition. Interestingly, although neither Oleanolic
acid, pentapeptide nor the Oleanoylpeptide had direct inhibitory effect on MSH induced
melanogenesis, due to the two pronged cosmetic approach, Oleanoylpeptide could reduce
stress induced pigmentation such as freckles and under eye dark circles which occurs due
to inflammation. This was observed in the in house study for 4 weeks with 17 volunteers
on usage of a formulation of 0.1% Oleanoyl peptide in a cream base (Fig 5.3.12).
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
304
A B
C D
E F
G H
A) Under eye dark circles before treatment B) Reduction in under eye dark circles on treatment with Oleanoyl peptide C) Dark spots on forehead and freckles on nose before treatment D) Reduction in dark spots and freckles on treatment with Oleanoyl peptide E) Under eye darkness and wrinkles before treatment F) Reduction in under eye darkness and wrinkles on treatment with Oleanoyl peptide G) Under eye darkness, wrinkles and skin thinning before treatment H) Reduction in under eye darkness and wrinkles and improvement of under eye skin tone on treatment with Oleanoyl peptide Figure 5.3.14: Effect of Oleanoyl peptide on various hyperpigmentation conditions in human volunteers
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
305
This peptide of Oleanolic acid is novel and significant in activity as it is not obvious that
any active conjugated to Lys-Thr-Thr-Lys-Ser pentapeptide will have activity. The below
example of a peptide of Thiodipropionic acid and Lys-Thr-Thr-Lys-Ser pentapeptide did
not show a significant enhancement in skin lightening potential (Table 5.3.55).
5.3.6.1.2. A peptide of Thiodipropionic acid and Lys-Thr-Thr-Lys-Ser pentapeptide:
In the peptide, only Thiodipropionic acid is an inhibitor of tyrosinase, whereas Lys-Thr-
Thr-Lys-Ser pentapeptide has no effect on tyrosinase. Lys-Thr-Thr-Lys-Ser pentapeptide
in conjugation with various actives like palmitic acid (saturated lipophillic fatty acid) and
other triterpenoids like Oleanolic acid etc, showed significant cosmetic benefits as
described earlier. However Lys-Thr-Thr-Lys-Ser pentapeptide in conjugation with
Thiodipropionic acid, just retained the anti tyrosinase potential of Thiodipropionic acid
and did not show significant enhancement in the anti tyrosinase activity (Table 5.3.55).
Table 5.3.55: Skin lightening potential by a peptide of Thiodipropionic acid and Lys-Thr-Thr-Lys-Ser pentapeptide
Active Characteristic activity Tyrosinase inhibition
Thiodipropionic acid Tyrosinase inhibition 22% inhibition at 100µg/ml
Lys-Thr-Thr-Lys-Ser
pentapeptide
Collagen enhancement Nil
Conjugate of Thiodipropionic
acid and Lys-Thr-Thr-Lys-
Ser pentapeptide
Tyrosinase inhibition
Collagen enhancement
31% inhibition at 100µg/ml
5.3.6.1.3. A conjugate of Kojic acid and Acetyl-11-keto-beta-boswellic acid
(AKBBA):
In the conjugate, only Kojic acid is an inhibitor of melanogenesis through MSH and
tyrosianse inhibitory pathways with mild antioxidant potential, whereas AKBBA is an
anti inflammatory active with no effect on melanogenesis. Kojic acid individually could
inhibit melanogenesis by 32% at 80µg/ml; however, in a chemical conjugation with
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
306
AKBBA, it could inhibit melanogenesis by 32% at 2.5µg/ml (Table 5.3.56). Although
AKBBA is not an inhibitor of melanogenesis, its anti inflammatory activity that added on
to the melanogenesis inhibitory pathways of Kojic acid, enhanced the melanogenesis
inhibitory potential. On the whole, the conjugate has properties that kojic acid did not,
like better inhibition of MSH induced melanogenesis due to the added anti inflammatory
properties of AKBBA.
Table 5.3.56: Synergistic skin lightening potential by a conjugate of Kojic acid and AKBBA
Active Characteristic activity Melanin inhibition
Kojic acid Melanin inhibition
Mild antioxidant:
DPPH scavenging: IC50 is 500µg/ml.
32% at 80µg/ml
AKBBA Anti inflammatory potential:
TNF α inhibition - IC50 is 100µg/ml
Hyaluronidase inhibition - IC50 is 63µg/ml
Collagenase inhibition - IC50 is 250µg/ml
Nil
Conjugate of Kojic acid
and AKBBA
Melanin inhibition
Anti inflammatory potential
32% at 2.5µg/ml
5.3.6.1.4. A conjugate of Oleanolic acid and Kojic acid:
In the conjugate, only Kojic acid is an inhibitor of melanogenesis through MSH and
tyrosianse inhibitory pathways with mild antioxidant potential, whereas Oleanolic acid is
an anti inflammatory active with no effect on melanogenesis. Kojic acid individually
could inhibit melanogenesis with an IC50 of 100µg/ml; however, in a chemical
conjugation with Oleanolic acid, it could inhibit melanogenesis with an IC50 of 5µg/ml
(Table 5.3.57). Although Oleanolic acid is not an inhibitor of melanogenesis, its anti
inflammatory activity added on to the melanogenesis inhibitory pathways of Kojic acid
and enhanced the melanogenesis inhibitory potential. On the whole, the conjugate has
properties that kojic acid did not, like better inhibition of MSH induced melanogenesis
due to the added anti inflammatory properties of Oleanolic acid.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
307
Table 5.3.57: Synergistic skin lightening potential by a conjugate of Kojic acid and Oleanolic acid
Active Characteristic activity Melanin inhibition
Kojic acid Melanin inhibition
Mild antioxidant:
DPPH scavenging: IC50 is 500µg/ml.
50% at 100µg/ml
Oleanolic acid Anti inflammatory potential:
Collagenase inhibition - IC50 is 129µg/ml
Nil
Conjugate of Kojic acid
and Oleanolic acid
Melanin inhibition
Anti inflammatory potential
50% at 5µg/ml
5.3.6.2. Physical combination of actives with different modes of action showing
enhanced skin lightening potential:
5.3.6.2.1. Composition containing 50% THC and 50% Glabridin:
Both THC and Glabridin are good inhibitors of tyrosinase and melanogenesis in
mammalian cells. But only THC is a significant antioxidant. So in a cell system the
antioxidant potential added up to melanogenesis inhibitory pathways and enhanced the
pigmentation reduction in mammalian melanocytes (Table 5.3.58 and Fig. 5.3.13).
0
0.5
1
1.5
2
2.5
3
IC50 in microgram/ml
Tyrosinaseinhibition
Melanin inhibition
Synergistic skin lightening potential by THC and Glabridin
THCGlabridinTHC + Glabridin (1:1)
Figure 5.3.15: Synergistic efficacy of THC and Glabridin
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
308
Table 5.3.58: Synergistic skin lightening potential by a combination of THC and Glabridin
Active Characteristic activity Melanin inhibition
THC Melanin inhibition
Significant antioxidant potential:
DPPH scavenging activity - IC50 is 1.2
µg/ml
ORAC – 10,000 µmol trolox equivalents/g
Anti tyrosinase activity –
IC50 is 2 µg/ml
Melanin inhibition - IC50 is
3 µg/ml
Glabridin Melanin inhibition
Mild antioxidant potential:
DPPH scavenging activity - IC50 is 49
µg/ml
ORAC – 3256 µmol trolox equivalents/g
Anti tyrosinase activity –
IC50 is 0.25 µg/ml
Melanin inhibition - IC50 is
3 µg/ml
THC +
Glabridin
1:1
Melanin inhibition
Significant antioxidant potential:
DPPH scavenging activity - IC50 is 0.97
µg/ml
ORAC – 10,000 µmol trolox equivalents/g
Anti tyrosinase activity –
IC50 is 0.068 µg/ml
Melanin inhibition - IC50 is
1 µg/ml
5.3.6.2.2. Composition containing 0.25% AKBBA, 0.5% THC and
0.1%Tetrahydropiperine (THP):
In the composition, only THC is an inhibitor of melanogenesis, whereas AKBBA is an
anti inflammatory active and THP is a cell permeation enhancer with no effect on
melanogenesis. Although THC is a significant inhibitor of melanogenesis and free
radicals, it alone may not have a significant effect in the cell pigmentation at a
concentration of 0.5% in combination with inert actives with respect to melanogenesis
inhibition. Theoretically, since 100% THC had an IC50 of 3µg/ml, 0.5% THC should
have an IC50 of >100µg/ml. However, the IC50 obtained for the composition is 10µg/ml
(Table 5.3.59). So in a cell system the anti inflammatory potential and enhanced
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
309
bioavailability of the actives to the target sites of a cell added up to melanogenesis
inhibitory pathways and enhanced the pigmentation reduction in mammalian melanocytes.
Table 5.3.59: Synergistic skin lightening potential by a combination of AKBBA, THC and THP
Active Characteristic activity Melanin inhibition
0.25% AKBBA Anti inflammatory potential Nil
0.5% THC Melanin inhibition
Antioxidant activity
IC50 >100µg/ml
0.1% THP A significant cell penetration enhancer.
THP enhanced the permeation of a diterpene
forskolin by 12 times in 60 minutes across rat skin.
Nil
0.25% AKBBA +
0.5% THC +
0.1% THP
Melanin inhibition
Antioxidant activity
Anti inflammatory potential
Enhanced bio availability
IC50 10µg/ml
5.3.6.2.3. Composition containing 0.2% THC, 0.2% Glabridin, 1% AKBBA and
0.1% THP:
In the composition, only THC and Glabridin are the inhibitors of melanogenesis, whereas
AKBBA is an anti inflammatory active and THP is a cell permeation enhancer with no
effect on melanogenesis. Although THC and Glabridin are significant inhibitors of
melanogenesis of equal potential, they alone may not have a significant effect in the cell
pigmentation at a concentration of 0.2% each in combination with inert actives with
respect to melanogenesis inhibition. Theoretically, since 100% THC & Glabridin had an
IC50 of 3µg/ml, 0.2% THC & Glabridin should have an IC50 of >100µg/ml. However, the
IC50 obtained for the composition is 20µg/ml (Table 5.3.60). So in a cell system the anti
inflammatory potential and enhanced bioavailability of the actives to the target sites of a
cell added up to melanogenesis inhibitory pathways and enhanced the pigmentation
reduction in mammalian melanocytes.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
310
Table 5.3.60: Synergistic skin lightening potential by a combination of THC, Glabridin, AKBBA and THP
Active Characteristic activity Melanin inhibition
0.2% THC Melanin inhibition
Antioxidant activity
IC50 >100µg/ml
0.2% Glabridin Melanin inhibition IC50 >100µg/ml
1% AKBBA Anti inflammatory potential Nil
0.1% THP A significant cell penetration
enhancer.
Nil
0.2% THC + 0.2% Glabridin +
1% AKBBA + 0.1% THP
Melanin inhibition
Antioxidant activity
Anti inflammatory potential
Enhanced bio availability
IC50 20µg/ml
5.3.6.2.4. Composition containing 0.5% THC, 0.5% Glabridin, 0.1% Galanga
extract and 0.1% THP:
In the composition, only THC and Glabridin are the inhibitors of melanogenesis, whereas
Galanga extract is a UV protectant with no significant effect on melanogenesis. Although
THC and Glabridin are significant inhibitors of melanogenesis of equal potential, they
alone may not have a significant effect in the cell pigmentation at a concentration of 0.5%
each in combination with inert actives with respect to melanogenesis inhibition.
Theoretically, since 100% THC and Glabridin had an IC50 of 3µg/ml, 0.5% THC and
Glabridin should have an IC50 of >100µg/ml. However, the IC50 obtained for the
composition is 12.5µg/ml (Table 5.3.61). So in a cell system the protection from UV
induced melanogenesis and enhanced bioavailability of the actives to the target sites of a
cell added up to melanogenesis inhibitory pathways and enhanced the pigmentation
reduction in mammalian melanocytes.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
311
Table 5.3.61: Synergistic skin lightening potential by a combination of THC, Glabridin, Galanga extract and THP
Active Characteristic activity Melanin inhibition
0.5% THC Melanin inhibition
Antioxidant activity
IC50 >100µg/ml
0.5% Glabridin Melanin inhibition IC50 >100µg/ml
0.1% Galanga extract Inhibitor of UV induced
cell damage
Nil
0.1% THP A significant cell
penetration enhancer.
Nil
0.5% THC + 0.5% Glabridin +
0.1% Galanga extract + 0.1%
THP
Melanin inhibition
Antioxidant activity
Anti inflammatory potential
Enhanced bio availability
IC50 12.5µg/ml
5.3.6.2.5. Composition containing 0.2% THC, 0.1% Coenzyme Q10, 1% Coconut
liquid endosperm, 0.5% Soya isoflavones, 0.1% Tetrahydropiperine (THP):
In the composition, only THC is the inhibitor of melanogenesis, whereas CoQ10 acts as
an antioxidant, Coconut liquid endosperm and Soya isoflavones help in cell proliferation
and cell conditioning and THP helps in better bioavailability of actives in the cells with
no significant effect on melanogenesis. Although THC is a significant inhibitor of
melanogenesis, it alone may not have a significant effect in the cell pigmentation at a
concentration of 0.2% in combination with inert actives with respect to melanogenesis
inhibition. Theoretically, since 100% THC inhibited 30% melanogenesis at 2µg/ml, 0.2%
THC should have inhibited 30% melanogenesis at >200µg/ml. However, the composition
inhibited 30% of melanogenesis at 20µg/ml (Table 5.3.62). So in a cell system the
antioxidant potential, cell conditioning and enhanced bioavailability of the actives to the
target sites of a cell added up to melanogenesis inhibitory pathways and enhanced the
pigmentation reduction in mammalian melanocytes.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
312
Table 5.3.62: Synergistic skin lightening potential by a combination of THC, Coenzyme Q10 (CoQ10), Coconut liquid endosperm, Soya isoflavones and THP
Active Characteristic activity Melanin inhibition
0.2% THC Melanin inhibition
Antioxidant activity
Inhibits 30%
melanogenesis at conc.
>200µg/ml
0.1% CoQ10 Antioxidant activity:
Naturally found in the mitochondria involved in
neutralizing free radicals.
Nil
1% Coconut
liquid endosperm
Cell proliferation enhancer:
18% enhancement at 5µg/ml
Nil
0.5% Soya
isoflavones
(genistein &
diadzein)
Phytoestrogenic properties:
They stimulate fibroblasts to make collagen and
hyaluronic acid which are essential for good skin
tone. Soya isoflavones also have the ability to
prevent UV damage.
Nil
0.1% THP A significant cell penetration enhancer. Nil
0.2% THC +
0.1% CoQ10 +
1% Coconut
liquid endosperm
+ 0.5% Soya
isoflavones +
0.1% THP
Melanin inhibition
Antioxidant activity
Cell growth and conditioning
Enhanced bio availability
Inhibits 30%
melanogenesis at conc.
20µg/ml
5.3.6.2.6. Composition containing 0.5% THC, 0.2% Centella asiatica extract, 0.5%
Soya isoflavones, 0.1% THP:
In the composition, only THC is the inhibitor of melanogenesis, whereas Centella
asiatica extract & Soya isoflavones help in cell proliferation, cell conditioning and
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
313
collagen enhancement and THP helps in better bioavailability of actives in the cells with
no significant effect on melanogenesis. Although THC is a significant inhibitor of
melanogenesis, it alone may not have a significant effect in the cell pigmentation at a
concentration of 0.5% in combination with inert actives with respect to melanogenesis
inhibition. Theoretically, since 100% THC inhibited 30% melanogenesis at 2µg/ml, 0.5%
THC should have inhibited 30% melanogenesis at >200µg/ml. However, the composition
inhibited 30% of melanogenesis at 20µg/ml (Table 5.3.63). So in a cell system the
antioxidant potential, cell conditioning and enhanced bioavailability of the actives to the
target sites of a cell added up to melanogenesis inhibitory pathways and enhanced the
pigmentation reduction in mammalian melanocytes.
5.3.6.2.7. Composition containing 0.5% Arbutin, 0.2% Glabridin, 1% AKBBA and
0.3% Coriander seed oil extract:
In the composition, only Arbutin and Glabridin are the inhibitors of melanogenesis,
whereas AKBBA is an anti inflammatory active and Coriander seed oil extract acts as a
cell conditioner with no significant effect on melanogenesis. Although Glabridin and
Arbutin are inhibitors of melanogenesis, Glabridin having better potential, they alone
may not have a significant effect on the cell pigmentation in combination with inert
actives with respect to melanogenesis inhibition. 100% Arbutin can provide 40%
melanogenesis inhibition at a concentration of about 80µg/ml. 100% Glabridin can
provide 40% melanogenesis inhibition at a concentration of about 2µg/ml. Theoretically,
considering the activity of the better potential active, Glabridin, 40% inhibition of
melanogenesis is expected to be attained only at a concentration of >200µg/ml with 0.2%
glabridin in the composition. However, 40% inhibition of melanogenesis obtained with
the composition is 5µg/ml (Table 5.3.64). So in a cell system the anti inflammatory
potential and overall cell conditioning properties added up to melanogenesis inhibitory
pathways and enhanced the pigmentation reduction in mammalian melanocytes.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
314
Table 5.3.63: Synergistic skin lightening potential by a combination of THC, Centella asiatica extract, Soya isoflavones and THP
Active Characteristic activity Melanin inhibition
0.5% THC Melanin inhibition
Antioxidant activity
Inhibits 30% melanogenesis at
>200µg/ml
0.2% Centella
asiatica extract
Anti inflammatory property:
Inhibition of Nitric oxide synthesis
facilitating cell proliferation and wound
healing.
Collagen enhancement:
60% enhancement at 5mg/ml
Nil
0.5% Soya
isoflavones
(genistein &
diadzein)
Phytoestrogenic properties:
They stimulate fibroblasts to make
collagen and hyaluronic acid which are
essential for good skin tone. Soya
isoflavones also have the ability to
prevent UV damage.
Nil
0.1% THP A significant cell penetration enhancer. Nil
0.2% THC +
0.2% Centella
asiatica extract +
+ 0.5% Soya
isoflavones +
0.1% THP
Melanin inhibition
Antioxidant activity
Anti inflammatory activity
Cell growth, conditioning and collagen
enhancement
Enhanced bio availability
Inhibits 30% melanogenesis at
20µg/ml
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
315
Table 5.3.64: Synergistic skin lightening potential by a combination of Arbutin, Glabridin, AKBBA and Coriander seed oil extract
Active Characteristic activity Melanin inhibition
0.5% Arbutin Melanin inhibition
Inhibits 40% melanogenesis at
>200µg/ml 0.2% Glabridin
1% AKBBA Anti inflammatory property Nil
0.3% Coriander
seed oil extract
Skin conditioning:
Coriander seed oil extract contains
petroselinic acid triglycerides with
significant skin conditioning potential.
Nil
0.5% Arbutin +
0.2% Glabridin +
+ 1% AKBBA +
0.3% Coriander
seed oil extract
Melanin inhibition
Anti inflammatory activity
Cell condiitoning
Inhibits 40% melanogenesis at
5µg/ml
5.3.6.2.8. Composition containing 0.5% Arbutin, 0.1% Glabridin and 0.1% THP:
In the composition, both Arbutin and Glabridin are the inhibitors of melanogenesis and
only Glabridin has mild antioxidant and anti inflammatory potential. THP only enhances
the bio availability of actives with no significant effect on melanogenesis. Although
Glabridin and Arbutin are inhibitors of melanogenesis, Glabridin having better potential,
they alone may not have a significant effect on the cell pigmentation in combination with
inert actives with respect to melanogenesis inhibition. 100% Arbutin can provide 40%
melanogenesis inhibition at a concentration of about 80µg/ml. 100% Glabridin can
provide 40% melanogenesis inhibition at a concentration of about 2µg/ml. Theoretically,
considering the activity of the better potential active, glabridin, 40% inhibition of
melanogenesis can be attained at a concentration of >200µg/ml with 0.1% glabridin in
the composition. However, 40% inhibition of melanogenesis obtained with the
composition is 5µg/ml (Table 5.3.65). So in a cell system the anti inflammatory potential
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
316
and enhanced bio availability of the actives to the target sites of a cell added up to
melanogenesis inhibitory pathways and enhanced the pigmentation reduction in
mammalian melanocytes. Moreover, Glabridin has a significant anti inflammatory
potential also. 100% glabridin can give 30% inhibition of elastase at 50µg/ml. So
theoretically 0.1% Glabridin should give 30% inhibition at 50mg/ml, where as the same
activity was observed at 140µg/ml. Therefore, the presence of THP enhanced the
bioavailability of glabridin to show better potential.
Table 5.3.65: Synergistic skin lightening potential by a combination of Arbutin, Glabridin and THP
Active Characteristic activity Melanin inhibition
0.5% Arbutin Melanin inhibition
Inhibits 40% melanogenesis at
>200µg/ml 0.1% Glabridin Melanin inhibition
Anti inflammatory potential
Mild antioxidant
0.1% THP A significant cell penetration enhancer. Nil
0.5% Arbutin +
0.1% Glabridin +
+ 0.1% THP
Melanin inhibition
Enhanced bioavailability
Inhibits 40% melanogenesis at
5µg/ml
5.3.6.3. Multifunctional skin lightening compositions:
Even when synergy with respect to one mechanism of action is not observed or when all
the targets of skinlightening mechanisms are not met by a single active, which is mostly a
common phenomenon, two or more actives which address different mechanisms are
combined into a unique composition to obtain a multifunctional skin lightening
composition. Some of the examples are as follows,
5.3.6.3.1. A composition of THC and Galanga extract (1:1): The composition exhibits
the antioxidant and melanin inhibitory benefits of THC along with the UV protection
benefits of Galanga extract.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
317
Combined activity of the composition:
Antioxidant activity: ORAC Value of 1205µmol trolox equivalents/g, DPPH
scavenging activity with IC50 of 17µg/ml.
Melanin inhibition: IC50 of 20µg/ml.
UV protection: Prevents 50% UV induced cell death at 0.1% concentration.
5.3.6.3.2. A composition of Garcinol and Coconut liquid endosperm (1:1): The
compostion exhibits the antioxidant and anti inflammatory properties of Garcinol along
with the cell proliferation property of Coconut water extract thus fastening the process of
skin lightening.
Combined activity of the composition:
Antioxidant activity: ORAC value of 1983µmol trolox equivalents/g, DPPH scavening
potential with an IC50 of 1.7µg/ml, Lipid peroxidation inhibition with an IC50 of 97µg/ml.
Anti inflammatory activity: Anti Hyaluronidase activity with an IC50 of 4.3µg/ml, Anti
Elastase activity with an IC50 of 300µg/ml, Anti Collagenase activity with an IC50 of
125µg/ml.
Cell proliferation enhancement: 48% enhancement at 0.002% concentration.
5.3.6.3.3. A composition of Lotus seed extract, Coffee bean extract and Coconut
liquid endosperm (1:1:1): The compositon exhibits the antioxidant properties of Coffee
bean extract, anti inflammatory properties of Lotus seed extract along with the cell
proliferation property of Coconut water extract thus fastening the process of skin
lightening.
Combined activity of the composition:
Antioxidant activity: ORAC value of 10,930µmol trolox equivalents/g, HORAC value
of 4770µmol gallic acid equivalents/g, DPPH scavening potential with an IC50 of
0.83µg/ml, Lipid peroxidation inhibition with an IC50 of 347µg/ml.
Anti inflammatory activity: Anti Hyaluronidase activity with an IC50 of 6.7µg/ml, Anti
Elastase activity with an IC50 of 125µg/ml, Anti Collagenase activity with an IC50 of
25µg/ml.
Cell proliferation enhancement: 50% enhancement at 0.002% concentration.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
318
The same composition when Coffee bean extract was replaced with Curry leaf extract
showed similar anti inflammatory, cell proliferation enhancement and antioxidant
potenital. But interestingly Lipid peroxidation inhibition was significantly higher than
that of what was attained when Coffee bean extract was used with an IC50 of 8.3µg/ml.
This activity was therefore exclusively contributed by Curry leaf extract.
5.3.6.3.4. A composition of Mangostin and Coconut water extract (1:1): This
composition exhibits the antioxidant and anti inflammatory properties of Mangostin
along with the cell proliferation property of Coconut water extract thus fastening the
process of skin lightening.
Combined activity of the composition:
Antioxidant activity: DPPH scavening potential with an IC50 of 1.6µg/ml, ROS
scavening potential with an IC50 of 2µg/ml Lipid peroxidation inhibition with an IC50 of
22µg/ml.
Anti inflammatory activity: Anti Hyaluronidase activity with an IC50 of 7.6µg/ml, Anti
Elastase activity with an IC50 of 30µg/ml, Anti Collagenase activity with an IC50 of
50µg/ml.
Cell proliferation enhancement: 40% enhancement at 0.002% concentration.
5.3.7. Nutricosmetics for beauty from within:
Nutricosmetics are the products orally taken to improve health and beauty. Natural foods,
oils and other natural ingredients have become an exciting new trend in beauty and
skincare products. The qualities that make fruits and plants, “superfruits and plants” are
their richness in antioxidant and anti inflammatory actives which is important for body
health, skin health and beauty. Therfore, the search for exotic plants and fruits has always
been in priority for cosmetic research to help heal and beautify the skin. Compounds
without direct effect on melanogenesis but with significant antioxidant potential can help
in skin lightening when taken orally as nutricosmetics. For example, many micronutrients
like vitamins, omega 3 fatty acids, carotenoids, flavonoids etc act as nutricosmetics by
preventing UV induced skin damage, skin pigmentation and also slowdown the process
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
319
of skin ageing. Antioxidants play a major role as nutricosmetics. Examples are tripeptide
Glutathione which is a common nutricosmetic, polyphenols etc. As discussed in the
earlier part of the results, compounds without direct effect on melanogenesis but with
significant anti inflammatory potential can still help in skin lightening when applied
topically. Similarly, it has been observed that compounds without direct effect on
melanogenesis but with significant antioxidant potential can help in skin lightening when
taken internally as nutricosmetics. The tripeptide Glutathione is one of the commonly
used antioxidants for nutricosmetic applications (Puizina-Ivic N et al., 2010). Other
major group of antioxidants, that plays an important role as nutricosmetics are
Polyphenols. Polyphenols are the phenol moiety containing chemical actives from plants.
The largest and best studied polyphenols are the flavonoids, which include several
thousand compounds, like flavonols, flavones, catechins, flavanones, anthocyanidins, and
isoflavonoids. Polyphenol rich products like green tea, grape seed extract, coffee bean
extract etc play a signigficant role as nutricosmetics although they do not exhibit
significant melanogenesis inhibitory potential in vitro.
5.3.7.1. Importance of ORAC for nutricosmetic benefits:
High ORAC foods increase the antioxidant power of human blood by 10 – 25% and
protect the blood vessels and capillaries from oxidative damage, thereby rendering
nutricosmetic benefits on oral intake. Table 5.3.66 shows the ORAC values of top food
supplements. All the mentioned food products in Table 5.3.66 are rich in Polyphenols so
further processing of these natural products enriching them to a higher concentration of
Polyphenols will increase the ORAC values, thereby providing the nutricosmetic benefits
at much lower doses.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
320
Table 5.3.66: ORAC values of top Nutricosmetic food supplements as per the data from the U.S. Dept. of Agriculture & Journal of American Chemical Society
Product ORAC value (µmol trolox equivalents/g)
Unprocessed Cocoa powder 260
Acai berry 185
Dark chocolate 131.2
Prunes 57.7
Raisins 28.3
Blue berries 24
Black berries 20.36
Straw berries 15.4
Spinach 12.6
Broccoli florets 8.9
Red grapes 7.39
Cherries 6.7
5.3.7.1.1. Cocoa bean extract as a nutricosmetic:
From Table 5.3.67, it is evident that the ORAC value of Cocoa bean extract significantly
increased with the increasing concentration of polyphenols. However, there is no
significant increase in the DPPH scavenging potential with the increasing polyphenol
content. Even the HORAC value did not change significantly with the increasing
polyphenol content with a value in the range of 1744 to 1823 µmol gallic acid
equivalents/g for 20 to 50% polyphenol content. Similarly, the ROS scavenging potential
was same with an IC50 of 6.25µg/ml for 20 to 50% polyphenol content. Therefore, ORAC
value is crucial for nutricosmetic benefits. The role of polyphenols for antioxidant
potential is evident with the increasing antioxidant potential in proportion to the
increasing polyphenol content of Cocoa bean extract.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
321
Table 5.3.67: Antioxidant potential of Cocoa bean extract
% Polyphenols DPPH scavenging
(IC50 in µg/ml)
ORAC value (µmol
trolox equivalents/g)
26 2 3635
27 2.7 4069
35 2 4922
37 2 5583
39 1.8 6667
50 1.2 8117
>50 1.2 – 1.7 11000 – 13000
5.3.7.1.2. Coffee bean extract as a nutricosmetic:
The principle constituents of green coffee bean were found to be chlorogenic acid and
caffeine out of which chlorogenic acid neutralizes free radicals and hydroxyl radicals,
both of which can lead to cellular degeneration if left unchecked. In addition synergistic
effects are also present due to the concentrated caffeine content ranging from 3-5%.
Compared to green tea and grape seed extract, green coffee bean extract is twice as
effective in absorbing oxygen free radicals. One of the advantages of using the green
coffee bean extract is that the negative effects of coffee are avoided.
Table 5.3.68: Antioxidant potential of Coffee bean extract
%
Chlorogenic
acid
ORAC value
(µmol trolox
equivalents/g)
HORAC value
(µmol gallic acid
equivalents/g)
DPPH scavenging
(IC50 in µg/ml)
40 6291 2935 1
60 9978 5206 2
65 10930 5891 1.25
80 12636 5621 0.96
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
322
From Table 5.3.68, it is evident that the ORAC value of Coffee bean extract significantly
increased with the increasing concentration of chlorogenic acid. However, there is no
significant increase in the DPPH scavenging potential or HORAC value with increasing
chlorogenic acid content.
5.3.7.1.3. Standardized extracts with “high ORAC value” for nutricosmetic benefits:
Standardized plant extracts with high ORAC value have significant potential for
nutricosmetic applications. Some combinations of actives have shown synergy as
nutricosmetics with enhanced antioxidant potential as observed in Table 5.3.69.
5.3.7.1.3.1. Green tea extract containing 70% Polyphenols:
ORAC value: 6907 µmol trolox equivalents/g. Similarly, Green tea extract with 80%
polyphenols has a higher ORAC value of 8027 µmol trolox equivalents/g, showing that
polyphenols play a significant role in the antioxidant potential of the extract.
Other significant properties of Green tea extract containing 70% Polyphenols:
HORAC value: 7121 µmol gallic acid equivalents/g, DPPH scavenging: IC50 - 3 µg/ml,
ROS scavenging: IC50 - 1 µg/ml, Elastase inhibition: IC50 – 275 µg/ml, Collagenase
inhibition: IC50 – 50 µg/ml, Hyaluronidase inhibition: IC50 – 5µg/ml, Tyrosinase
inhibition: 40% inhibition at 50 µg/ml, Melanin inhibition – 14% inhibition at 10µg/ml.
5.3.7.1.3.2. Grape seed extract containing 50% Polyphenols:
ORAC value: 4528 µmol trolox equivalents/g. Similarly, Grape seed extract with 70%
polyphenols has an ORAC value of 9699 µmol trolox equivalents/g, showing that
polyphenols play a significant role in the antioxidant potential of the extract.
Other significant properties of Grape seed extract containing 50% Polyphenols:
HORAC value: 3254 µmol gallic acid equivalents/g, DPPH scavenging: IC50 - 3 µg/ml,
ROS scavenging: IC50 - 5 µg/ml, Elastase inhibition: IC50 – 9 µg/ml, Collagenase
inhibition: IC50 – 12 µg/ml, Hyaluronidase inhibition: IC50 – 5µg/ml, Tyrosinase
inhibition: IC50 – 28 µg/ml
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
323
5.3.7.1.3.3. Pomegranate extract containing 44% Polyphenols:
ORAC value: 4400 µmol trolox equivalents/g. Similary, Pomegranate extract with 65%
polyphenols has an ORAC value of 5687 µmol trolox equivalents/g, showing that
polyphenols play a significant role in the antioxidant potential of the extract.
Other significant properties of Pomegranate extract containing 44% Polyphenols:
DPPH scavenging: 17% scavenging at 300 µg/ml, Elastase inhibition: IC50 – 500 µg/ml,
Collagenase inhibition: IC50 – 62.5 µg/ml, Hyaluronidase inhibition: IC50 – 0.5µg/ml,
Tyrosinase inhibition: IC50 – 7 µg/ml
5.3.7.1.3.4. Pomegranate rind extract:
ORAC value: 14,087 µmol trolox equivalents/g.
Other parts of the pomegranate fruit also were observed to have significant antioxidant
properties. Pomegranate seed extract has an ORAC value of 3350 and Pomegranate juice
extract has an ORAC value of 400 µmol trolox equivalents/g.
Other significant properties of Pomegranate rind extract:
HORAC value: 12,743 µmol gallic acid equivalents/g, DPPH scavenging: IC50 –
0.4µg/ml, Elastase inhibition: IC50 – 500µg/ml, Collagenase inhibition: IC50 – 62.5µg/ml,
Hyaluronidase inhibition: IC50 – 0.5µg/ml, Tyrosinase inhibition: 12% inhibition at
10µg/ml.
5.3.7.1.3.5. Pomegranate extract standardized to 90% Ellagic acid:
ORAC value: 8299 µmol trolox equivalents/g.
Other significant properties: Melanin inhibition: 16% inhibition of melanin at 1.25
µg/ml, ROS scavenging: IC50 - 20 µg/ml.
It was observed that pomegranate rind extract has the highest antioxidant potential than
that of seeds and juice. Hence, pomegranate rind which is unutilized during consumption
has significant nutricosmetic benefits.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
324
5.3.7.1.3.6. Turmeric extract:
ORAC value: 10,410 µmol trolox equivalents/g
Other significant properties:
HORAC value: 9740 µmol gallic acid equivalents/g, DPPH scavenging: IC50 – 1.8µg/ml,
Tyrosinase inhibition: 22% inhibition at 3µg/ml.
5.3.7.1.3.7. Rosmarinic acid:
Rosmarinus officinalis leaf extract containing 90% Rosmarinic acid:
ORAC value: 14000 µmol trolox equivalents/g
Other significant properties:
HORAC value: 5925 µmol gallic acid equivalents/g, DPPH scavenging: IC50 – 0.5µg/ml,
Collagenase inhibition: IC50 – 250µg/ml, Hyaluronidase inhibition: IC50 – 25µg/ml
Rosmarinus officinalis leaf extract containing 50% Rosmarinic acid:
ORAC value: 10000 µmol trolox equivalents/g
Coleus forskohlii leaf extract containing 90% Rosmarinic acid:
ORAC value: 15000 µmol trolox equivalents/g
Coleus forskohlii leaf extract containing 50% Rosmarinic acid:
ORAC value: 14600 µmol trolox equivalents/g
Rosmarinic acid has a significant role in the antioxidant potential of Coleus forskohlii
leaf extract and Rosmarinus officinalis leaf extract. However, the ORAC value of Coleus
forskohlii leaf extract containing 50% Rosmarinic acid is significantly higher than that of
Rosmarinus officinalis leaf extract containing 50% Rosmarinic acid. Therefore the matrix
components of Coleus forskohlii leaf extract act synergistically in combination with
Rosmarinic acid for an enhanced antioxidant potential.
5.3.7.1.3.8. Saffron:
ORAC value: 344 µmol trolox equivalents/g
Other significant properties:
HORAC value: 1305 µmol gallic acid equivalents/g, Elastase inhibition: IC50 - 125 µg/ml.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
325
5.3.7.1.3.9. Tulsi extract:
ORAC value: 3732 µmol trolox equivalents/g
Other significant properties:
DPPH scavenging: IC50 – 3.8µg/ml
5.3.7.1.3.10. Mulberry extract:
ORAC value: 645 µmol trolox equivalents/g
5.3.7.2. Synergistic antioxidant compositions for nutricosmetic benefits:
Nutricosmetics can be used as a single ingredient or a mixture of ingredients in the form
of capsules, tablets, ready made drinks, powder that can be mixed in drinks etc. In any
formulation the ORAC value of the active ingredient or ingredients gets diluted as per
their concentrations in the carrier or placebo system unless it is a pure mixture of actives.
The above mentioned nutricosmetic actives were combined and analyzed for their
antioxidant potential. It is obvious that most of the combinations will have a cumulative
or additional antioxidant activity of all the actives used in the combination. Such an
additional effect is expected in combinations. However, some of the compositions
showed synergistically enhanced antioxidant potential (Table 5.3.69).
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
326
Table 5.3.69: Synergistic antioxidant compositions
Composition
Activity of actives Activity of composition
Activity obtained on
analysis
Expected
activity
(Additional
effect)
Synergistic activity
(Higher than the
expected additional
activity)
ORAC value (µmol trolox equivalents/g)
Rosmarinic acid &
Chlorogenic acid
(1:3)
Rosmarinic acid – 14,000
Chlorogenic acid – 9978 10,984 16,595
Rosmarinic acid &
THC (1:1)
Rosmarinic acid – 14,000
THC – 9244 10,433 16,859
Rosmarinic acid, THC
& Chlorogenic acid
(1:1:1)
Rosmarinic acid – 14,000
THC - 9244
Chlorogenic acid – 9978
11,074 13,931
Turmeric extract &
Green tea extract (1:1)
Turmeric extract –
10,410
Green tea extract – 8027
9000 14,418
5.3.7.3. Nutricosmetic formulations:
For oral applications, the nutricosmetic compositons should be diluted with excepients
and formulated as tablets, capsules or powders for consumption. Since the concentration
of the actives in formulations will be less, the ORAC value will also be less. However
3,000 to 5,000 ORAC units per day are required to have a significant impact on plasma
and tissue antioxidant capacity. Therefore, the ORAC value can be potentiated by
increasing the servings. The whole concept of nutricosmetics is about attaining beauty
from within naturally and without any feel of undergoing a scheduled medication. Tablets
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
327
or capsules can be used directly whereas the powdered formulations can be mixed in
water or other beverages for consumption. For example, though carriers like certain
beverages, ice creams, shakes, soups, pre mix powders etc. containing mostly sugars,
flavors, creams, gums, citric acid etc., do not have nutricosmetic benefits, on mixing a
certain minimal dosage of nuticosmetic actives into these carriers increases the likeability
factor of the preparations as well as its nutricosmetic benefits. Some of the examples are,
5.3.7.3.1. Health drink syrup that can be mixed in milk or water:
In a composition containing nutricosmetic actives like Mulberry extract, Amla extract,
Aloe vera, Grape seed extract and Green tea extract, the ORAC value as observed in
Table 5.3.70 was 62µmol trolox equivalents/g. The ORAC value can be increased to
3100 µmol trolox equivalents/g by having 10 servings of 5gm each per day to attain the
minimal ORAC requirement per day.
Table 5.3.70: Nutricosmetic healthdrink syrup Nutricosmetic
active
Concentration
(%)
ORAC (µmol trolox
equivalents/g)
Recommended Servings of
5g /day
Mulberry extract 2
62 10
Amla extract 2
Aloe vera extract 0.5
Grape seed extract 0.5
Green tea extract 1
5.3.7.3.2. Enriched Green coffee that can be mixed in milk or water:
In a composition containing nutricosmetic actives like Green coffee bean extract, Grape
seed extract and Amla extract in instant coffee, the ORAC value as observed in Table
5.3.71 was 1751µmol trolox equivalents/g. The ORAC value can be increased to 3502
µmol trolox equivalents/g by having a serving of 2gm per day in milk or water to attain
the minimal ORAC requirement per day. Coffee which is the most popular beverage can
be made a health drink with cosmetic benefits by adding nutricosmetic actives into it.
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
328
Table 5.3.71: Enriched coffee as a nutricosmetic
Nutricosmetic
active
Concentrati
on (%)
ORAC (µmol trolox
equivalents/g)
Recommended
Servings of 2g /day
Coffee bean extract 0.92
1751 1 Grape seed extract 2.4
Amla extract 1.5
Instant coffee base 95.24
5.3.7.3.3. Enriched Chocolate that can be mixed in milk:
In a composition containing nutricosmetic actives like Cocoa polyphenols in Cocoa
powder, the ORAC value as observed in Table 5.3.72 was 200µmol trolox equivalents/g.
The ORAC value can be increased to 3000 µmol trolox equivalents/g by having 3
servings of 5gm per day in milk to attain the minimal ORAC requirement per day.
Chocolate drink which is the most popular beverage can be made into a health drink with
cosmetic benefits by adding nutricosmetic actives into it.
Table 5.3.72: Enriched chocolate as nutricosmetic
Nutricosmetic
active
Concentrati
on (%)
ORAC (µmol trolox
equivalents/g)
Recommended
Servings of 5g /day
Cocoa polyphenols 0.66 200 3 Cocoa powder 6.5
5.3.7.3.4. Enriched Tea that can be mixed in water:
In a composition containing nutricosmetic actives like Amla extract, Green tea extract
and Tulsi extract the ORAC value as observed in Table 5.3.73 was 161µmol trolox
equivalents/g. The ORAC value can be increased to 3220 µmol trolox equivalents/g by
having 4 servings of 5gm per day in water to attain the minimal ORAC requirement per
CHAPTER 5 PART II 5.3. RESULTS AND DISCUSSION
329
day. Tea which is a very common beverage can be made a health drink with cosmetic
benefits by adding nutricosmetic actives into it.
Table 5.3.73: Enriched green tea as nutricosmetic Nutricosmetic
active
Concentration
(%)
ORAC (µmol trolox
equivalents/g)
Recommended Servings
of 5g /day
Amla extract 2
161 4 Tulsi extract 1
Amla extract 2
CHAPTER 1 PART II 5.4. CONCLUSION OF PART II
330
Various actives were screened through different in vitro mechanisms for pigmentation
and were positioned in accordance to their specific mode of action for rectifying
pigmentation disorders. Hence, the actives can be recommended for skin lightening based
on the root cause of pigmentation.
In the process of screening, some novel skin lightening actives and some extracts
with synergistic combination of actives have been observed. Thymohydroquinone from
Nigella sativa seed extract, Hydroxychavicol from Piper betle leaf extract,
Avenanthramides from oat kernel extract, ceramides from apple fruits and oats and
Eugenia jambolana extract were found to have significant skin lightening potential. The
skin lightening property of Amla extract and Artocarpus lakoocha extract was not
conferred exclusively by Ascorbic acid and Oxyresveratrol respectively but due to the
synergistic combination of various components of the two extracts.
Synergistic effect of various biological mechanisms for enhanced skin lightening
potential has been demonstrated by chemical conjugations of actives like Oleanoyl
peptide. Similarly the synergistic effect of various biological mechanisms for enhanced
skin lightening potential has been demonstrated by physical combination of actives. The
study emphasizes the integration of various mechanisms of skin lightening for a
synergistic effect.
Combination of antioxidant actives were shown to have synergistic potential and
can be useful as nutricosmetics. Actives like Rosmarinic acid that naturally existed in
combination with leaf matrix components of Coleus forskohlii, showed synergistic
antioxidant activity significantly higher than that of Rosmarinic acid alone. It has also
been shown that although some actives do not directly inhibit melanogenesis, they help in
skin lightening by other mechanisms of action like antioxidant potential, collagen
enhancement and anti inflammatory potential and can also be used as nutricosmetics.
5.4. CONCLUSION OF PART II