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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

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.
Topical Care

Protection by
Sunscreen

Internal Care

Healthy Skin

Cleansing by
Astringent

Nourishment by
Antioxidant rich
Moisturizers,
conditioners &
cell rejuvenators
Figure 5.1.1: Skin care topically and internally

Protection by
Antioxidants
Cleansing by
Antioxidants that
detoxify blood

Nourishment by
nutrition rich diet
& supplements
(Nutricosmetics)

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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 crosstalking 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
skins 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 5100 m thickness (which can reach 600 m 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 celllike 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 cholesterolrich 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 Meissners

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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.
Epidermis

Skin structural
proteins
in dermis

A) Collagen and elastic fibers in dermis B) Stratified epidermis and vascular dermis

Figure 5.1.3: Skin structural proteins

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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 (IIV) 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--dihydroxyphenylalanine oxygen oxidoreductase, EC
1.14.18.1) is a single chain type I membrane glycoprotein catalyzing the hydroxylation of
tyrosine to -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; BranzaNichita 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 5,6dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units in
reduced or oxidized states, as detailed above; pheomelanin consists mainly of sulfurcontaining 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 hormoneresponsiveness, 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 (0.50.8 mm in diameter), which are ellipsoidal and intensely melanotic (stage
IV). Lighter pigmentation is associated with smaller (0.30.5 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.
-MSH, Adenocorticotropic hormone (ACTH), basic fibroblast growth factor
(bFGF), nerve growth factor (NGF), endothelins, granulocyte-macrophage colonystimulating 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

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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.

Activation of MITF

cAMP / -MSH

Activation of Tyrosinase genes

Activation of Tyrosinase

Melanogenesis

Serine protease &

PAR 2 activation

Melanin transfer from

MELANOCYTES to
peripheral KERATINOCYTES

Pigmentation

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.

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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

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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 1950% of the solar UV-A can reach the depth of
melanocytes, whereas only 914% of solar UV-B reaches these cells. Therefore, UV-A

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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 12 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 4872 h after
irradiation, reaches a maximum 3 wk after exposure, and the skin does not return to its
original melanin content until 810 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.

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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 7dehydrocholesterol 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

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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 Parkinsons 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).

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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 -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

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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
thromboxanes

chemical

may be responsible

mediators,
for

the

especially

induction

of

leukotrienes
post

and

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).

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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.

Melasma

1. Increased
melanin
production.
2. Normal
number of
melanocytes.

2. Normal
number of
melanocytes

1. Increased
melanin
production.

2. Slight
increase in
number of
melanocytes.

1. Increased
melanin
production.

Cellular
characteristics

Increased ROS generation and subsequent cell

damage resulting in over expression of
Tyrosinase, MSH & cAMP as a stress response.

1. PGE2 and PGF2 synthesis is up-regulated;

they act as paracrine factors which stimulate
melanocyte dendricity.
2. Leukotrienes, TNF- and thromboxanes may
be responsible for the induction of postinflammatory hyperpigmentation.

1. High levels of progesterone, estrogen, and

MSH.
2. Increased transcription of genes encoding
DCT, TYR.
3. Melanocytes are larger and more dendritic.

1. Increased TYR-positive cells per length of the

dermal/epidermal interface compared with
unaffected skin.
2. Keratinocytes potential to produce ET-1 is
significantly higher compared with unaffected
skin.
3. TNF- is up-regulated in the SL lesional
epidermis.

Molecular markers affected

PART II
5.1. INTRODUCTION

Loss in skin glow & tanning on Temporary

Exposure to UV-exposed areas of the body increase in
Skin tanning
UV & pollution such as the face, dorsum of the melanin
hand, extensor forearm and
production
upper back.

1. Discrete hyperpigmented
PostDevelops after macules with hazy margins.
inflammatory resolution of
hyper
acne, contact 2. May involve epidermis,
pigmentation dermatitis, etc. dermis or both.

Sun exposure, 1. Symmetric facial

pregnancy, hyperpigmentation.
oral
contraceptives, 2. May involve epidermis,
anti-epileptics dermis or both.
etc.

Clinical features

Solar
lentigines
(SL)

Causative
factor(s)

1. Circumscribed, brown to
black macules.
2. Range from <1 mm to
several cm.
Induced by UV 3. Occur in epidermis.
4. Found on UV-exposed areas
of the body such as the face,
dorsum of the hand, extensor
forearm and upper back.

Hyperpigmen
tation
disorder

Table 5.1.1: Summary of hyperpigmentation conditions, causative factors, clinical features and cellular characteristics

CHAPTER 1

204

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205

Table 5.1.2: Summary of external and internal stress increasing human skin
pigmentation and their intracellular secondary messengers and effectors
Stress
UV induces the production of:
NO
ET-1
-MSH, ACTH, PGE2
Hormones (non classical
pathway)
Inflammation

Secondary messenger
cGMP
DAG
cAMP
cAMP
Inositol 1,4,5 triphosphate

Secondary effector
Protein kinase G (PKG)
Protein kinase C (PKC)
Protein kinase A (PKA)
Mitogen activated protein
kinase (MAPK)
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.

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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, -MSH, -MSH and MSH; these peptides are all cleavage products of a large precursor peptide called proopiomelanocortin (POMC). -MSH is the most important melanocortin for pigmentation.
Hence, inhibition of -MSH is one of the major targets for skin lightening.

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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.

Serine proteases induce

Protease inhibition pigmentation through
inflammation.

Inhibitors of serine proteases

(elastase, collagenase and
hyaluronidase) have potential for
skin lightening.

Overstay of Inflammatory
response by markers like TNF
Anti inflammatory etc. induces pigmentation by
dermal matrix damage and
potential
induction of pigmentation by
affected melanocytes.

Inhibitors of inflammatory
markers like TNF etc. have
potential for skin lightening.

Antioxidant
potential

Free radical damage induces

pigmentation.

UV protection

UV induces pigmentation through UV protectants inhibit NO and

NO and free radical induction.
ROS induced melanognesis.

cAMP induced
melanogenesis

cAMP upregulates melanin

production by PKC pathway.

Melanin transfer in
Endothelin induces transfer of
melanocytemelanin from melanocytes to
keratinocyte
keratinocytes
coculture

MITF activation.

Antioxidants have skin

lightening potential.

Inhibitors of cAMP induced

melanogenesis have potential for
skin lightening
Endothelin antagonists inhibit
melanin migration to upper
keratinocyte layers. Hence
inhibitors of melanin migration
in cocultures are good skin
lighteners.

Upregulation of MITF expression

Inhibitors of MITF have good
mediates melanogenesis
skin lightening potential.
stimulated by cAMP.

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Table 5.1.4: Melanin inhibitory targets that result in adverse effects

Target

Mechanism

Remarks

Phenylalanine
hydroxylase
inhibition

Inhibitors of Phenylalanine hydroxylase

Phenylalanine
inhibit the melanin formation pathway.
hydroxylase catalyses the
Tyrosine is required for other metabolic
formation of tyrosine
pathways.
Inactive
phenylalanine
which is the precursor of
hydroxylase results in disorders like
melanin.
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 coppercontaining 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.

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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.,

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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(F508) 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)

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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 (F508) 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

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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) 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

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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

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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).

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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.

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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.

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5.2. MATERIAL AND METHODS

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:

Dulbeccos minimum essential medium (DMEM), RPMI 1640 medium, -Melanocyte
stimulating hormone (-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) 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.

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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 W 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--D-glucopyranoside or Hydroquinone -Dglucopyranoside) 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--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.

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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.

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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

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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 37C 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 37C 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 0.22m 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.

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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

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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 -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) B16F1 mouse melanoma cells; B) -MSH induced melanogenesis in B16F1 mouse melanoma cells;
C) Reduction in -MSH induced melanogenesis in B16F1 mouse melanoma cells on sample treatment

Figure 5.2.2: -MSH induced melanogenesis in B16F1 mouse melanoma cells

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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 7.3M
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.

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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

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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 100l 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) 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

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% 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.

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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.

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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-dihydroxyspiro[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
(Trolox),

(AAPH).

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid

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.

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AAPH ROOFluorescein --------------------- Non fluorescent product

[Antioxidants inhibit the oxidation of fluorescein by hydrogen atom transfer]

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

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 200M 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)

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(M) 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
0.09M disodium fluorescein dye. Immediately 20l of 30% H2O2 is added into all wells,
and initial fluorescence reading (F0) is taken at 485/520 ex/em wavelength. Then 5l of
219.79 M cobalt solution and 522 M 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

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acid concentration (Y) (M) 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
mol 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 uorescence methods . The intracellular ROS generation of
cells can be investigated using the 2,7-dichloruorescein-diacetate (DCFH-DA) as a
well-established compound to detect and quantify intracellular produced H2O2 (Cathcart
R et al., 1983). The conversion of the nonuorescent 2,7 - dichloruorescein diacetate
(DCFH-DA) to the highly
uoresecent compound

2,7-dichloruorescein (DCF)

happens in several steps. First, DCFH-DA is transported across the cell membrane and
deacetylated by esterases to form the non-uorescent 2,7-dichloruorescein (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. 100l 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

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2.5M FeSO4. After incubating the cells for 1 hour at 37C, 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.

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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 g/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 mole of L-leucine equivalents from collagen in 5 hours at 37C,
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
g/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

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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, 25g/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.

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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 1,4-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.

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Figure 5.2.8: Hyaluronidase activity on Hyaluronic acid

237

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238

5.2.2.5.4. TNF inhibitory potential:

For TNF inhibitory study, human whole blood is used. In whole blood assay,
monocytes appear to be the main source of TNF- on Lipopolysaccharide (LPS)
stimulation. Monocyte and macrophages are a major source of TNF- 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- production in human blood. After stimulation the assay employs the quantitative
sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-
has been pre-coated onto a microplate. TNF- present in the sample is bound by the
immobilized antibody. After washing away any unbound substances, an enzyme-linked
polyclonal antibody specific for TNF- 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- bound in the initial step. The colour
development is stopped and the intensity of the colour is directly proportional to the
TNF- 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 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 content by using the TNF Elisa kit. 200L 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 50Lof
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 200L of conjugate was
added to each well. The plate was incubated for 2 hr at room temperature. After washing
again, 200 L of substrate solution was added to each well and incubated for 20 minutes

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at RT. 50L 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 content and the percentage of inhibition of TNF 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 in the absence of inhibitor
T = absorbance due to TNF in the presence of inhibitor
IC50 value is the concentration required for 50% inhibition of TNF and hence, lower
IC50 value indicates better TNF 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

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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 100l 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 0.2m diameter
resulting in 0.2m 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

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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) 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

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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 Bouins fluid containing 1.3% picric
acid, 35% formaldehyde and glacial acetic acid in 15:5:1 ration by incubating with 1ml of
Bouins 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.

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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).

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244

5.3. RESULTS AND DISCUSSION

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
3.25g/ml and melanin induced by cAMP with an IC50 of 200 28g/ml.

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5.3.1.1.1. Arbutin:
Table 5.3.1: Skin lightening potential of Arbutin
Inhibition of melanin formation
Tyrosinase inhibition

Inhibition of MSH

Inhibition of cAMP

(g/ml)

induced Melanin

induced Melanin

(g/ml)

(g/ml)

IC50 100 15

IC50 100 18

IC50 194 25

UV protection

Nil

Antioxidant potential
DPPH scavenging

ROS scavenging

(g/ml)
IC50 500 25

Nil

ORAC

HORAC

(mol TE/g)

(mol GAE/g)

Nil

Nil

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Nil

Nil

Hyaluronidase

TNF

inhibition

inhibition

Nil

Nil

Table 5.3.2: Effect of Arbutin on Skin pigmentation disorders

Pigmentation disorder

Effect

Rationale

Pigmentation due to biological

Significant

Potential inhibitor

imbalances of body such as over

of tyrosinase &

expression of hormones and enzymes

melanogenesis

Pigmentation due to excessive sun

No effect

exposure

No significant
protection from UV
exposure

Pigmentation due to free radical

No effect

damage

No significant free
radical scavenging
activity

Pigmentation due to inflammatory

No effect

No significant anti

responses like pimple marks, scars

inflammatory

due to wounds etc.

activity

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5.3.1.1.2. Kojic Acid:

Table 5.3.3: Skin lightening potential of Kojic acid
Inhibition of melanin formation
Tyrosinase inhibition

Inhibition of MSH

Inhibition of cAMP

(g/ml)

induced Melanin

Induced Melanin

UV protection

(g/ml)
IC50 7 1.5

IC50 100 19

Nil

Nil

Antioxidant potential
DPPH scavenging

ROS scavenging

(g/ml)
IC50 500 32

Nil

ORAC

HORAC

(mol TE/g)

(mol GAE/g)

Nil

Nil

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Nil

Nil

Hyaluronidase

TNF

inhibition

inhibition

Nil

Nil

Table 5.3.4: Effect of Kojic acid on Skin pigmentation disorders

Pigmentation disorder

Effect

Rationale

Pigmentation due to biological

Significant

Potential inhibitor of

imbalances of body such as over

tyrosinase &

expression of hormones and enzymes

melanogenesis

Pigmentation due to excessive sun

No effect

exposure

No significant
protection from UV
exposure

Pigmentation due to free radical

No effect

damage

No significant free
radical scavenging
activity

Pigmentation due to inflammatory

No effect

No significant anti

responses like pimple marks, scars

inflammatory

due to wounds etc.

activity

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5.3.1.1.3. Ascorbic acid:

Table 5.3.5: Skin lightening potential of Ascorbic acid
Inhibition of melanin formation
Tyrosinase inhibition

Inhibition of MSH

Inhibition of cAMP

(g/ml)

induced Melanin

induced Melanin

(g/ml)

(g/ml)

IC50 25 6

IC50 100 16

IC50 9.33 1.6

UV protection

Not significant

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 1.93 0.3

IC50 10 2.3

3400 220

Nil

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Nil

Nil

Hyaluronidase

TNF

inhibition

inhibition

Nil

Nil

Table 5.3.6: Effect of Ascorbic acid on Skin pigmentation disorders

Pigmentation disorder

Effect

Rationale

Pigmentation due to biological

Significant

Potential inhibitor of

imbalances of body such as over

tyrosinase &

expression of hormones and enzymes

melanogenesis

Pigmentation due to sun exposure

Not

No significant

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

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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

Inhibition of MSH

Inhibition of cAMP

(g/ml)

induced Melanin

induced Melanin

(g/ml)

(g/ml)

IC50 3.2 1.1

IC50 4 1.3

IC50 1.77 0.3

UV protection

Not significant

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 0.93 0.3

IC50 1.2 0.2

10,815 468

2715 126

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Nil

Nil

Hyaluronidase

TNF inhibition

inhibition

(g/ml)

Nil

IC50 81 6

Table 5.3.8: Effect of THC on Skin pigmentation disorders

Pigmentation disorder

Effect

Rationale

Pigmentation due to biological

Significant

Potential inhibitor of

imbalances of body such as over

tyrosinase &

expression of hormones and enzymes

melanogenesis

Pigmentation due to sun exposure

Not

No significant

significant

protection from UV

Pigmentation due free radical damage

Significant

Antioxidant

Pigmentation due inflammatory

Significant

Significant inhibitor

responses like pimple marks, scars

due to wounds etc.

of inflammation

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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

Inhibition of MSH

Inhibition of cAMP

(g/ml)

induced Melanin

induced Melanin

(g/ml)

(g/ml)

IC50 3.5 1

IC50 3.8 1.2

IC50 0.25 0.05

UV protection

Not significant

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 100 15

IC50 0.25 0.02

7550 520

1129 209

Anti inflammatory potential

Collagenase

Elastase inhibition

Hyaluronidase

TNF inhibition

inhibition (g/ml)

(g/ml)

inhibition

(g/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

Significant

Potential inhibitor of

imbalances of body such as over

tyrosinase &

expression of hormones and enzymes

melanogenesis

Pigmentation due to sun exposure

Not

No significant

significant

protection from UV
induced cell damage

Pigmentation due free radical damage

Significant

Potential scavenger
of free radicals

Pigmentation due inflammatory

Significant

Significant inhibitor

responses like pimple marks, scars

of inflammatoty

due to wounds etc.

markers and
inflammary enzymes

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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

Inhibition of MSH

Inhibition of cAMP

UV protection

(g/ml)

induced Melanin

induced Melanin

(g/ml)

(g/ml)

(g/ml)

IC50 2.5 0.2

IC50 1 0.2

IC50 1.3 0.2

EC50 20 4

Antioxidant potential
DPPH scavenging

ROS scavenging

(g/ml)
IC50 44 0.2

Not significant

ORAC

HORAC

(mol TE/g)

(mol GAE/g)

3859 0.2

1121 214

Anti inflammatory potential

Collagenase

Elastase inhibition

Hyaluronidase

TNF inhibition

inhibition

(g/ml)

inhibition

(g/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

Significant

Potential inhibitor of

imbalances of body such as over

tyrosinase &

expression of hormones and enzymes

melanogenesis

Pigmentation due to sun exposure

Significant

Significant
protection from UV
induced cell damage

Pigmentation due free radical damage

Significant

Antioxidant

Pigmentation due inflammatory

Significant

Significant inhibitor

responses like pimple marks, scars

due to wounds etc.

of inflammation

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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).

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Table 5.3.13: Skin lightening potential of Oxyresveratrol from Artocarpus lakoocha

extract
Inhibition of melanin formation
Active

Tyrosinase

Inhibition of

Inhibition of cAMP

UV protection

inhibition

MSH induced

induced Melanin

(g/ml)

(g/ml)

Melanin (g/ml)

(g/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

ROS scavenging

ORAC

HORAC

scavenging

(g/ml)

(mol TE/g)

(mol GAE/g)

(g/ml)
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

Elastase

Hyaluronidase

inhibition

inhibition (g/ml) inhibition (g/ml)

(g/ml)

TNF
inhibition
(g/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

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253

Table 5.3.14: Effect of Oxyresveratrol on Skin pigmentation disorders

Pigmentation

Effect

Rationale

Pigmentation due to

Significant.

Potential inhibitor

biological imbalances

Artocarpus

of tyrosinase and

of body such as over

lakoocha extract

melanogenesis

expression of

containing 20% &

hormones and

30% oxyresveratrol

enzymes

has better potential.

Pigmentation due to

Significant.

Significant

sun exposure

Artocarpus

protection against

lakoocha extract

UV induced cell

containing 80% &

death.

disorder

90% oxyresveratrol
has better potential.
Pigmentation due to

Significant.

Significant

free radical damage

Artocarpus

scavenger of free

lakoocha extract

radicals

containing 80% &

90% oxyresveratrol
has better potential.
Pigmentation due

Significant.

Potential inhibitor

inflammatory

Artocarpus

of inflammatory

responses like pimple

lakoocha extract

markers and

marks, scars due to

containing 20% &

inflammatory

wounds etc.

30% oxyresveratrol

enzymes

has better potential.

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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

Inhibition of MSH

Inhibition of cAMP

(g/ml)

induced Melanin

Induced Melanin

(g/ml)

(g/ml)

IC50 1.63 0.5

IC50 2 0.6

IC50 0.03 0.01

UV protection

Not significant

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 5.37 1.6

IC50 3.125 0.9

21,549 2022

3097 423

Anti inflammatory potential

Collagenase

Elastase inhibition

Hyaluronidase

TNF inhibition

inhibition (g/ml)

(g/ml)

inhibition (g/ml)

(g/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

Significant

Significant inhibitor

imbalances of body such as over

of tyrosinase and

expression of hormones and

melanogenesis

enzymes
Pigmentation due to sun exposure

Pigmentation due to free radical

Not

No significant

significant

protection from UV

Significant

Antioxidant

Significant

Significant inhibitor

damage
Pigmentation due inflammatory
responses like pimple marks,

of inflammatory

scars due to wounds etc.

markers

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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

Inhibition of MSH

Inhibition of cAMP

UV protection

(g/ml)

induced Melanin

Induced Melanin

(g/ml)

IC50 5.5 1.2

IC50 2.5 0.8

IC50 3.5 0.91

EC50 30 7

Antioxidant potential
DPPH scavenging

IC50 3 1.1

ROS scavenging

Not significant

ORAC

HORAC

(mol TE/g)

(mol GAE/g)

25,223 2312

10,000 598

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Nil

Not significant

Hyaluronidase

TNF inhibition

inhibition

(g/ml)

Nil

IC50 75 5

Table 5.3.18: Effect of Resveratrol on Skin pigmentation disorders

Pigmentation disorder

Effect

Rationale

Pigmentation due to biological

Significant

Potential inhibitor of

imbalances of body such as

tyrosinase &

over expression of hormones

melanogenesis

and enzymes
Pigmentation due to sun

Significant

exposure

Significant inhibitor
of cell damage due to
UV exposure

Pigmentation due free radical

Significant

Antioxidant

Significant

Significant inhibitor

damage
Pigmentation due inflammatory
responses like pimple marks,

of inflammatory

scars due to wounds etc.

markers

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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

Inhibition of MSH

Inhibition of cAMP

UV protection

(g/ml)

induced Melanin

induced Melanin

(g/ml)

(g/ml)

(g/ml)

IC50 0.5 0.08

IC50 0.7 0.06

IC50 7.8 1.5

EC50 30 7.2

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 4.9 1.3

IC50 3 0.92

12,508 998

6283 664

Anti inflammatory potential

Collagenase

Elastase inhibition

Hyaluronidase

TNF inhibition

inhibition (g/ml)

(g/ml)

inhibition (g/ml)

(g/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

Significant

Significant inhibitor

imbalances of body such as over

of tyrosinase and

expression of hormones and

melanogenesis

enzymes
Pigmentation due to sun exposure

Significant

Significant protection
from UV

Pigmentation due to free radical

Significant

Antioxidant

Significant

Significant inhibitor

damage
Prevention of pigmentation due
inflammatory responses like pimple
marks, scars due to wounds etc.

of inflammation

CHAPTER 5

5.3.1.2.4.5.

PART II
5.3. RESULTS AND DISCUSSION

3 -Hydroxy

257

Pterostilbene (3HPT) from Pterocarpus marsupium

heartwood extract:
Table 5.3.21: Skin lightening potential of 3HPT
Inhibition of melanin formation
Tyrosinase inhibition

Inhibition of MSH

Inhibition of cAMP

(g/ml)

induced Melanin

induced Melanin

(g/ml)

(g/ml)

IC50 0.7 0.08

IC50 0.5 0.1

IC50 2 0.3

UV protection

Nil

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 1.34 0.3

IC50 5 1.1

13,334 1142

4700 598

Anti inflammatory potential

Collagenase

Elastase inhibition

Hyaluronidase

TNF inhibition

inhibition (g/ml)

(g/ml)

inhibition

(g/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

Significant

Significant inhibitor

imbalances of body such as over

of tyrosinase and

expression of hormones and enzymes

melanogenesis

Pigmentation due to sun exposure

No effect

No significant UV
protection

Pigmentation due free radical damage

Significant

Antioxidant

Prevention of pigmentation due

Significant

Significant inhibitor

inflammatory responses like pimple

marks, scars due to wounds etc.

of inflammation

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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

Inhibition of MSH

Inhibition of cAMP

UV protection

(g/ml)

induced Melanin

induced Melanin

(g/ml)

IC50 149 22

Not significant

Not significant

EC50 25 5

Antioxidant potential
DPPH scavenging

IC50 1.55 0.9

ROS scavenging

IC50 3.5 1.2

ORAC

HORAC

(mol TE/g)

(mol GAE/g)

14,322 1233

5000 968

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Not significant

TNF inhibition

Hyaluronidase
inhibition

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

Mild

Inhibits only

imbalances of body such as over

tyrosinase

expression of hormones and

enzymes
Pigmentation due to sun exposure

Significant

Significant
protection from
UV

Pigmentation due free radical

Significant

Antixidant

No effect

Not an inhibitor

damage
Pigmentation due inflammatory
responses like pimple marks,
scars due to wounds etc.

of inflammation

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259

5.3.1.2.5. Emblica officinalis (Amla) fruit extract containing -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 of

Inhibition of

UV

inhibition (g/ml)

MSH induced

cAMP

protection

Melanin (g/ml)

induced Melanin

(g/ml)

(g/ml)
-Glucogallin
10%
-Glucogallin
99%

IC50 200 35

IC50 12 3.6

IC50 14 4.3

IC50 20 3.3

IC50 500 33

IC50 14 2.4

IC50 15 3.9

IC50 25 3.1

Antioxidant potential
Active

DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol
GAE/g)

-Glucogallin
10%
-Glucogallin
99%

IC50 1.7 0.2

IC50 2.5 1.2

2862 339

1474 212

IC50 0.92 0.1

IC50 2.5 0.9

4436 446

4660 189

Anti inflammatory potential

Active
-Glucogallin
10%
-Glucogallin
99%

Collagenase

Elastase

Hyaluronidase

TNF

inhibition (g/ml)

inhibition

inhibition

inhibition

IC50 500 22

Nil

Not significant

Nil

Nil

Not significant

Not
significant
Not
significant

CHAPTER 5

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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- -D-glucose (-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, -glucogallins
could be the active that significantly contributes to the efficacy of Amla extract. Amla
extract containing higher concentration of -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

Significant

Significant

biological imbalances

inhibitor of

of body such as over

melanogenesis

expression of hormones
and enzymes
Pigmentation due to sun

Significant.

exposure

Significant
protection against
UV induced cell
death.

Pigmentation due to

Significant. Amla

Significant

free radical damage

extract containing

scavenger of free

99% -Glucogallin

radicals

has better
potential.
Pigmentation due

Mild

Not a significant

inflammatory responses

inhibitor of

like pimple marks, scars

inflammatory

due to wounds etc.

markers

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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

Nil

Inhibition of MSH

Inhibition of cAMP

induced Melanin

induced Melanin

(g/ml)

(g/ml)

IC50 0.625 0.23

IC50 0.5 0.21

UV protection

Nil

Antioxidant potential
DPPH scavenging

IC50 22 4.3 mg/ml

ROS scavenging

Nil

ORAC

HORAC

(mol TE/g)

(mol GAE/g)

Nil

Nil

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Nil

Nil

Hyaluronidase

TNF inhibition

inhibition

(g/ml)

Nil

IC50 230 25

Table 5.3.28: Effect of Piperlongumine on Skin pigmentation disorders

Pigmentation disorder

Effect

Rationale

Pigmentation due to biological

Significant

Significant

imbalances of body such as

inhibitor of

over expression of hormones

melanogenesis

and enzymes
Pigmentation due to sun

No effect

exposure
Pigmentation due free radical

UV damage
Mild

damage
Pigmentation due inflammatory

No protection from

Mild scavenger of
free radicals

Mild

Inhibitor of

responses like pimple marks,

inflammatory

scars due to wounds etc.

markers

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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

Inhibition of MSH

Inhibition of cAMP

(g/ml)

induced Melanin

induced Melanin

(g/ml)

(g/ml)

IC50 1.3 0.2

IC50 2.5 0.4

IC50 8 1.5

UV protection

Nil

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 0.5 0.1

IC50 10 1.2

29,728 2860

2376 293

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Not significant

Not significant

Hyaluronidase

TNF inhibition

inhibition

(g/ml)

Not significant

IC50 89 8

Table 5.3.30: Effect of Hydroxychavicol on Skin pigmentation disorders

Pigmentation disorder

Effect

Rationale

Pigmentation due to biological

Significant

Potential inhibitor of

imbalances of body such as over

tyrosinase &

expression of hormones and enzymes

melanogenesis

Pigmentation due to sun exposure

No effect

No UV protection

Pigmentation due free radical damage

Significant

Antioxidant

Pigmentation due inflammatory

Significant

Significant inhibitor

responses like pimple marks, scars

due to wounds etc.

of inflammation

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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

Inhibition of MSH

Inhibition of cAMP

(g/ml)

induced Melanin

Induced Melanin

(g/ml)

(g/ml)

IC50 0.2 0.05

IC50 0.3 0.07

IC50 0.905 0.2

UV protection

Nil

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 84 11

IC50 20 5.6

5899 369

2000 136

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Not significant

Not significant

Hyaluronidase

TNF inhibition

inhibition

(g/ml)

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

Significant

Significant inhibitor

imbalances of body such as over

of tyrosinase and

expression of hormones and enzymes

melanogenesis

Prevention of pigmentation due to sun

Nil

No UV protection

Significant

Antioxidant

Significant

Significant inhibitor

exposure
Prevention of pigmentation due to free
radical damage
Prevention of pigmentation due
inflammatory responses like pimple

of inflammatory

marks, scars due to wounds etc.

markers

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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

Not significant

Inhibition of MSH

Inhibition of cAMP

induced Melanin

induced Melanin

(g/ml)

(g/ml)

IC50 5 1.2

IC50 7 1.4

UV protection

Nil

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 6.1 2.1

IC50 10 3.2

9000 890

1000 262

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Not significant

TNF inhibition

Hyaluronidase
inhibition

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

Significant

Significant inhibitor

imbalances of body such as over

of melanogenesis

expression of hormones and

enzymes
Pigmentation due to sun exposure

No effect

No UV protection

Pigmentation due to free radical

Significant

Antioxidant

No effect

Not an inhibitor of

damage
Pigmentation due inflammatory
responses like pimple marks,
scars due to wounds etc.

inflammation

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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.

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266

Table 5.3.35: Skin lightening potential of Avenanthramide C (Av-C)

Inhibition of melanin formation
Tyrosinase inhibition

Not significant

Inhibition of MSH

Inhibition of cAMP

induced Melanin

induced Melanin

(g/ml)

(g/ml)

IC50 20 5.6

IC50 25 6.2

UV protection

Nil

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 1.2 0.2

IC50 10 3.8

22,352 1926

5000 432

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Not significant

Not significant

Hyaluronidase

TNF inhibition

inhibition

(g/ml)

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

Significant

Inhibitor of

imbalances of body such as over

melanogenesis

expression of hormones and

enzymes
Pigmentation due to sun

No effect

No UV protection

Significant

Antioxidant

Significant

Inhibitor of

exposure
Pigmentation due to free radical
damage
Pigmentation due inflammatory
responses like pimple marks,

inflammatory

scars due to wounds etc.

markers

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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

Inhibition of MSH

Inhibition of cAMP

UV protection

(g/ml)

induced Melanin

induced Melanin

(g/ml)

(g/ml)

(g/ml)

IC50 22 2.3

IC50 20 3.2

IC50 >100

EC50 100 9

Antioxidant potential
DPPH scavenging

ROS scavenging

ORAC

HORAC

(g/ml)

(g/ml)

(mol TE/g)

(mol GAE/g)

IC50 50 6.2

IC50 10 3.1

Not significant

Not significant

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Not significant

Not significant

Hyaluronidase

TNF inhibition

inhibition

(g/ml)

Not significant

IC50 50 4

Table 5.3.38: Effect of Colocynthin on Skin pigmentation disorders

Pigmentation disorder

Effect

Rationale

Pigmentation due to biological

Significant

Inhibitor of

imbalances of body such as over

tyrosinase and

expression of hormones and enzymes

melanogenesis

Pigmentation due to sun exposure

Significant

Protects against UV
induced cell death

Pigmentation due to free radical

Significant

Antioxidant

Significant

Significant inhibitor

damage
Pigmentation due inflammatory
responses like pimple marks, scars

of inflammatory

due to wounds etc.

markers

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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

Nil

Inhibition of MSH

Inhibition of cAMP

UV protection

induced Melanin

induced Melanin

(g/ml)

Upto 20 g/ml, 25% Upto 20 g/ml, 35% EC50 50 5

inhibition

inhibition

Antioxidant potential
DPPH scavenging

Nil

ROS scavenging

Nil

ORAC

HORAC

(mol TE/g)

(mol GAE/g)

Nil

Nil

Anti inflammatory potential

Collagenase

Elastase inhibition

inhibition
Not significant

Hyaluronidase

TNF inhibition

inhibition
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

Mild

Inhibitor of

of body such as over expression of

melanogenesis

hormones and enzymes

Pigmentation due to sun exposure

Significant

Protects from UV
damage

Pigmentation due free radical damage

No effect

Not an antioxidant

Pigmentation due inflammatory responses

No effect

Not an inhibition of

like pimple marks, scars due to wounds

inflammation

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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 of

Inhibition of

UV

inhibition

MSH induced

cAMP

protection

Melanin

induced Melanin

(g/ml)

Apple peel

Upto 30 g/ml,

Upto 20 g/ml,

Upto 20 g/ml,

Not

ceramides

30% inhibition

15% inhibition

25% inhibition

significant

Apple fruit

Upto 70 g/ml,

IC50 12 3.2

IC50 10 2.9

EC50 100 8

ceramides

30% inhibition

g/ml

g/ml

Antioxidant potential
Active

DPPH

ROS scavenging

scavenging

ORAC

HORAC

(mol TE/g)

(mol
GAE/g)

Apple peel

Not significant

Nil

Not significant

ceramides
Apple fruit

Not
significant

Not significant

ceramides

Upto 10 g/ml, Not significant

Not

10% scavenging

significant

Anti inflammatory potential

Active

Apple peel

Collagenase

Elastase

Hyaluronidase

TNF

inhibition

inhibition

inhibition

inhibition

(g/ml)

(g/ml)

IC50 25 3

IC50 200 12

Not significant

Not

ceramides
Apple fruit
ceramides

significant
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.

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270

Table 5.3.42: Effect of Apple ceramides on Skin pigmentation disorders

Pigmentation disorder

Effect

Rationale

Pigmentation due to

Apple fruit ceramides

Inhibitor of

biological imbalances of

- significant

tyrosinase and

body such as over

melanogenesis

expression of hormones

Apple peel ceramides

and enzymes

- mild

Pigmentation due to sun

Apple fruit ceramides

Significant protection

exposure

significant

against UV induced
cell death

Apple peel ceramides

Not significant

Pigmentation due to free

Not significant

radical damage

Not significant
scavenger of free
radicals

Pigmentation due

Apple peel ceramides

Inhibitor of

inflammatory responses

- significant

inflammatory

like pimple marks, scars

due to wounds etc.

enzymes
Apple fruit ceramides
Not significant

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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.

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272

Table 5.3.43: Significant inhibitors of tyrosinase - Inhibitors of Solar lentiges and skin
tanning
Group Rank

II

III

1
2
3
4
5
6
7

Plant extract
Artocarpus lakoocha
Glycyrrhiza glabra
Artocarpus lakoocha
Nigella sativa
Artocarpus lakoocha
Curcuma longa

Active
Dihydro-oxyresveratrol (DHO)
Oxyresveratrol (OXY) 50-90%
Glabridin (GLAB)
Oxyresveratrol (OXY) 20-30%
Thymohydroquinone (THQ)
Artocarpin (ART)
Tetrahydrocurcuminoids (THC)

IC50
(g/ml)
0.03 **
0.1 **
0.25 **
0.5 **
1.0 **
1.3 **
2.0 **

Pterocarpus marsupium 3-Hydroxypterostilbene (3HPT) 2.0 **

Artocarpus lakoocha

10

Synthetic (from Sigma) Kojic acid (KA)

7.0

11

Pterocarpus marsupium Pterostilbene (PTR)

7.8

12
13

Piper betle
Hydroxychavicol (HC)
Synthetic (from Sigma) Ascorbic acid (ASC)

8.0
9.33

14

Gnetum sp.

149

15

Synthetic (from Sigma) Arbutin (ARB)

16

Emblica officinalis

Resveratrol (RES)

Gnetol (GN)
glucogallin (BG)

5.5 *

194
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
Tyrosinase inhibitors II

Tyrosinase inhibitors I
0.6

12

0.5

10

IC 50 in microgram/ml

IC 50 in microgram/ml

273

0.4
0.3
0.2
0.1
0

8
6
4
2
0

DHO

OXY 50-90%

GLAB

OXY 20-30%

THQ ART THC 3HPT RES

Actives

KA

PTR

HC

ASC

Actives

Tyrosinase inhibitors III

IC 50 in microgram/ml

250
200
150
100
50
0
GN

ARB

BG

Actives

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.

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274

Table 5.3.44: Significant inhibitors of MSH and cAMP induced melanogenesis Inhibitors of Melasma and skin tanning
Group Rank
1
2
3
4
5

8
9

IC50
(g/ml)
Nigella sativa
Thymohydroquinone (THQ)
0.3 **
Pterocarpus marsupium Pterostilbene (PTR)
0.5 **
Piper longum
Piperlongumine (PL)
0.6 **
Pterocarpus marsupium 3-Hydroxypterostilbene (3HPT) 0.7 **
Piper betle
Hydroxychavicol (HC)
1.3 **
Artocarpus lakoocha
Dihydro Oxyresveratrol (DHO) 1.63 **
Polygonum cuspidatun Resveratrol (RES)
2.5 **
Plant extract

Curcuma longa

Tetrahydrocurcuminoids(THC) 3 **

Glycyrrhiza glabra

Glabridin (GLAB)

Artocarpus lakoocha
Artocarpus lakoocha

Oxyresveratrol (OXY) 20-30% 3 **

Artocarpin (ART)
3 **

II

3 **

Pterocarpus marsupium Dihydropterostilbene (DPTR)

5 **

Eugenia jambolana

Polyphenols (PLY)

5 **

Artocarpus lakoocha

Oxyresveratrol (OXY) 50-90% 10 *

10 Emblica officinalis
11

Active

Avena sativa

Synthetic (from Sigma)

12 Synthetic (from Sigma)
Synthetic (from Sigma)
13
Synthetic (from Sigma)

glucogallin (BG)

12 *

Avenanthramide C (AVN)

20 *

Ascorbic acid (ASC)

Glutathione (GLU)
Kojic acid (KA)
Arbutin (ARB)

25
25
100
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

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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

THC
GLAB
OXY 20-30%
ART

5
4

120
IC 50 in microgram/ml

IC 50 in microgram/ml

Melanogenesis inhibitors II
DHPT
PLY

3
2

100
80
60
40
20

1
0

0
THQ

PTR

PL

3HPT

HC

DHO RES

THC DHPT

Actives

OXY
50-90%

BG

AVN

ASC

GLU

KA

ARB

Actives

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

melanogenesis inhibitory potential.

whereas

Thymohydroquinone

has

the

highest

CHAPTER 5

Actives in
Table 5.3.44

PART II
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276

MSH
ACTH
MC1-R

cAMP
Actives in
Table 5.3.44
except
Kojic acid

PKA

MITF

Tyrosinase

Actives in Table
5.3.43
except
Piperlongumine

1. Melanogenesis
2. Differentiation,
dendrite
formation
and proliferation
of melanocytes

Skin
hyperpigmentation
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

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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 100g/ml (Fig.
5.3.4).
Table 5.3.45: Actives that prevent UV induced cell damage
Rank
1
2
3
4
5
6
7

Plant extract
Emblica officinalis
Artocarpus lakoocha
Gnetum sp.

Active
glucogallin (BG)
Artocarpin (ART)
Gnetol (GN)

EC50
(g/ml)
20 **
20 **
25 **

Pterocarpus marsupium Pterostilbene (PTR)

30 **

Polygonum cuspidatun

Resveratrol (RES)

30 **

Artocarpus lakoocha

Oxyresveratrol (OXY) 80-90%

50 *

Avena sativa (Oat)

Oat ceramides (OCR)

50 *

Oxyresveratrol (OXY) 50%

75 *

Oxyresveratrol (OXY) 20-30%

100

Artocarpus lakoocha

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

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278

Actives that prevent UV induced cell damage

EC 50 in microgram/ml

120
100
80
60
40
20
0
BG ART GN

PTR RES OXY OCR OXY OXY APL CYN OMC

8050% 2090%
30%

Actives

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) radicalscavenging 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 mol trolox equivalents/g, B = HORAC value in
mol trolox equivalents/g and C = IC50 for ROS scavenging potential.

CHAPTER 5

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279

Table 5.3.46: Cumulative antioxidant potential of actives

ORAC +
Active

ORAC

HORAC HORAC

ROS

Ratio

Hydroxychavicol

29728

2376

32104

0.5

64208

THC

10000

3000

13000

13000

Resveratrol

25000

10000

35000

11666

Oxyresveratrol 90%

21000

5723

26723

8907

Oxyresveratrol 80%

18673

4923

23596

7865

Oxyresveratrol 50%

15582

4896

20478

6826

Oxyresveratrol 30%

11370

4776

16146

5382

Oxyresveratrol 20%

8999

4756

8999

4585

Dihydrooxyresveratrol 21549

3097

26549

8215

Glabridin

7550

1129

8679

8679

Avenanthramide C

22352

5000

27352

10

2735

Gnetol

14322

5000

19322

6440

3HPT

13334

4700

17334

6011

Pterostilbene

12508

6233

12508

6247

-glucogallin 99%

4436

4660

9096

2.5

3638

-glucogallin 10%

2862

1474

4336

2.5

1734

Artocarpin

3859

1121

4980

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

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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
1 Piper betle

Active
Hydroxychavicol

Cumulative antioxidant units

64,208 **

THC

13,000 **

Curcuma longa

Polygonum cuspidatum Resveratrol

11,666 **
Oxyresveratrol 90%
8907 **
Artocarpus lakoocha
Dihydro Oxyresveratrol 8215 **

Glycyrrhiza glabra
5
6

Artocarpus lakoocha

Glabridin

8679 **

Oxyresveratrol 80%

7865 **

Oxyresveratrol 50%

6826 **

Gnetol

6440 **

Gnetum sp.

Pterocarpus marsupium Pterostilbene

6247 **

Pterocarpus marsupium 3HPT

6011 **

10
11

Artocarpus lakoocha

Oxyresveratrol 30%

5382 **

Oxyresveratrol 20%

4585 **

12

Emblica officinalis

- glucogallin 90%

3638 **

13

Avena sativa

Avenanthramide C

2735 **

14

Emblica officinalis

- 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

UV
X

281

Actives in
Table 5.3.45
(Sunscreens)

X
Actives in
Table 5.3.47
(Antioxidants)

X
ROS

DAG

ROS

Actives in
Table 5.3.47
(Antioxidants)

NO

cGMP

PKC

PKG

MSH

cAMP

Melanogenesis
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 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).

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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 cells 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.

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283

Table 5.3.48: Actives and their mode of action for UV protection

Group

Active

Prevention from UV

Healing of UV damage

damage Sunscreens

After sun care

glucogallin 10-99% Significant

Significant

Oxyresveratrol 20-

Significant

Significant

Artocarpin

Significant

Significant

Gnetol

Significant

Significant

Pterostilbene

Significant

Significant

Resveratrol

Significant

Significant

Colocynthin

Significant

Mild

Ceramides from

Significant

Nil

Significant

Nil

Tetrahydrocurcumin

Not significant

Significant

Dihydro-

Not significant

Significant

Glabridin

Not significant

Significant

Ascorbic acid

Not significant

Significant

Nil

Significant

3HPT

Nil

Significant

Glutathione

Nil

Significant

Avenanthramides

Nil

Significant

Jamun polyphenols

Nil

Significant

Thymohydroquinone Nil

Significant

90%
I

Avena sativa
II

Ceramides from
Malus domestica

Oxyresveratrol

III Hydroxychavicol

Group I Sunscreen and after sun care actives; Group II - Sunscreens; Group III - After sun care
actives

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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 (Il1 and TNF-) 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 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.

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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 TNF. Lower IC50 value
indicates better anti inflammatory potenital. TNF inhibition is given higher preference
over elastase, collagenase and hyaluronidase inhibition as TNF 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 and
inflammatory enzymes are preferred over those that are significant inhibitors of TNF
alone. Actives with significant inhibition of TNF are preferred over those that are
significant inhibitors of inflammatory enzymes alone.

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Table 5.3.49: Significant inhibitors of inflammation inhibitors of post inflammatory

hyperpigmentation
Rank
Plant extract
1 Artocarpus lakoocha
2 Glycyrrhiza glabra
3 Artocarpus lakoocha
4 Pterocarpus marsupium
5 Artocarpus lakoocha
6 Nigella sativa
7 Avena sativa
8 Artocarpus lakoocha
9 Citrullus colocynthis
10 Polygonum cuspidatun
11 Curcuma longa
12 Piper betle
13 Piper longum

Active
Oxyresveratrol 20-90%
Glabridin
3HPT
Pterostilbene
Artocarpin
Thymohydroquinone
Avenanthramide C
Dihydro Oxyresveratrol
Colocynthin
Resveratrol
THC
Hydroxychavicol
Piperlongumine

14 Malus domestica (peel) Ceramides

Rationale
Significant inhibitors of Elastase,
Collagenase enzymes and
TNF

Significant inhibitors of TNF

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.

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Skin sebum
infection

287

Skin exposure to
allergins/toxins

Inflammation
Overstay of
Inflammation
Fight against
foreign bodies

Dermal matrix
damage and
melanogenesis
induction in affected
melanocytes

Actives in Table 5.3.49

Healing
Hyperpigmentation

Scars/marks

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 -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

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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.

MSH & cAMP

X

Free radicals

Tyrosinase

Excessive skin
pigmentation
Oxyresveratrol
Artocarpin
Pterostilbene
Resveratrol

UV exposure

Inflammation
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.

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Table 5.3.50: Actives and their cumulative efficacy for all the major skin lightening
mechanisms

Oxyresveratrol

Free radical
Post inflammatory
induced
hyperpigmentation
pigmentation

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 *

Active

SL

Melasma

UV

- Actives with significant effect on all skin lightening mechanisms

* - Actvies with Mild activity
- Actives with no activity

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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.

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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.

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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 2.5g/ml and its IC50 for melanin inhibiton was 5g/ml.
5.3.4.2.3. -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 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.

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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

Optical Density due to

% enhancement in cell proliferation

endosperm

viable cells at 492nm

as compared to control

No treatment

0.139

2.5 g/ml

0.154

11%

5 g/ml

0.164

18%

Untreated cells

2.5g/ml Coconut liquid

endosperm treated cells

5g/ml Coconut liquid

endosperm treated cells

Figure 5.3.9: Cell proliferation enhancement by Coconut liquid endosperm in Swiss

3T3 mouse fibroblasts

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Coconut liquid endosperm (Freeze dried powder) also showed a significant wound
closure enhancement potential of 25% at a concentration of 5g/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.

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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

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Table 5.3.52: Cell proliferation enhancement by Liquid endosperm of Coconut in vivo

Treatment

Mean follicle count

Average skin

% of animals showing

in subcutis layer

thickness (m)

Anagen induction

43.00 12.20

220.15 25.60

71.4

42.86 13.49

218.75 34.04

71.4

3.43 3.27

156.27 5.14

14.2

Cream
(2% Coconut liquid
endosperm)
2% Minoxidil
(Ref.Standard)
5% Dextrose (inert)

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

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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).

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Table 5.3.53: Effect of Bacillus coagulans on cell proliferation enhancement

Conc. of Bacillus
coagulans culture
supernatant (%)
0
0.018
0.037
0.075
0.15
0.31
0.75
1.5
3.125
6.25
12.5
25
50
100

% enhancement
in cell
proliferation
0
0
0
0
4.4
4.6
14.4
15.5
22.1
6.1
0
0
0
0

% cytotoxicity

0
0
0
0
0
0
0
0
0
0
8.3
26.5
42.5
42.5

Cell proliferation enhancement by culture supernatant of

Bacillus coagulans in Swiss 3T3 mouse fibroblast cells
25

% enhancement in cell
proliferation

20
15
10

Cell proliferation
5
0
-5
-10

Conc. (%)

Figure 5.3.11: Cell proliferation enhancement by Bacillus coagulans in Swiss 3T3

mouse fibroblasts

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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-ThreonineLysine-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 1.25g/ml.
OH
H3C

NH2

CH3
NH
HO
O

CH3

CH3
CH3

NH

NH

O
O

CH3

O
NH

HO
H3C

NH

CH3
H2N

OH

CH3
HO

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).

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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

1.7%
FITC labelled
pro-collagen

13.5%
FITC labelled
pro-collagen

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

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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

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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 LysineThreonine-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.

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Activity of the conjugate, oleanoylpeptide: A significant anti inflammatory and

antioxidant active as compared to its parent actives, Oeanolic acid and Lys-Thr-Thr-LysSer 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
Elastase

Collagenase

TNF

Antioxidant

inhibition

inhibition

inhibition

activity - DPPH

(IC50 g/ml)

(IC50 g/ml)

(IC50 g/ml)

Scavenging

Oleanoyl peptide

143

119

21

Oleanolic acid

Nil

129

Compound

Pentapeptide (LysThr-Thr-Lys-Ser)

Nil

24% inhibition at
500 g/ml

17% inhibition
at 100 g/ml
Nil

29% inhibition
at 300 g/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
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).

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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

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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-ThrThr-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 LysThr-Thr-Lys-Ser pentapeptide
Active

Characteristic activity

Tyrosinase inhibition

Thiodipropionic acid

Tyrosinase inhibition

22% inhibition at 100g/ml

Lys-Thr-Thr-Lys-Ser

Collagen enhancement

Nil

pentapeptide
Conjugate of Thiodipropionic Tyrosinase inhibition
acid and Lys-Thr-Thr-Lys-

31% inhibition at 100g/ml

Collagen enhancement

Ser pentapeptide

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 80g/ml; however, in a chemical conjugation with

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AKBBA, it could inhibit melanogenesis by 32% at 2.5g/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
Kojic acid

Characteristic activity
Melanin inhibition

Melanin inhibition
32% at 80g/ml

Mild antioxidant:
DPPH scavenging: IC50 is 500g/ml.
AKBBA

Anti inflammatory potential:

Nil

TNF inhibition - IC50 is 100g/ml

Hyaluronidase inhibition - IC50 is 63g/ml
Collagenase inhibition - IC50 is 250g/ml
Conjugate of Kojic acid

Melanin inhibition

and AKBBA

Anti inflammatory potential

32% at 2.5g/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 100g/ml; however, in a chemical
conjugation with Oleanolic acid, it could inhibit melanogenesis with an IC50 of 5g/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.

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Table 5.3.57: Synergistic skin lightening potential by a conjugate of Kojic acid and
Oleanolic acid
Active
Kojic acid

Characteristic activity

Melanin inhibition
50% at 100g/ml

Melanin inhibition
Mild antioxidant:
DPPH scavenging: IC50 is 500g/ml.

Oleanolic acid

Nil

Anti inflammatory potential:

Collagenase inhibition - IC50 is 129g/ml

Conjugate of Kojic acid Melanin inhibition

and Oleanolic acid

50% at 5g/ml

Anti inflammatory potential

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).
Synergistic skin lightening potential by
THC and Glabridin
THC
Glabridin
3

THC + Glabridin (1:1)

2.5
2

IC50 in
1.5
microgram/ml
1
0.5
0
Tyrosinase
inhibition

Melanin inhibition

Figure 5.3.15: Synergistic efficacy of THC and Glabridin

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Table 5.3.58: Synergistic skin lightening potential by a combination of THC and

Glabridin
Active
THC

Characteristic activity

Melanin inhibition

Melanin inhibition

Anti tyrosinase activity

Significant antioxidant potential:

IC50 is 2 g/ml

DPPH scavenging activity - IC50 is 1.2

Melanin inhibition - IC50 is

g/ml

3 g/ml

ORAC 10,000 mol trolox equivalents/g

Glabridin

Melanin inhibition

Anti tyrosinase activity

Mild antioxidant potential:

IC50 is 0.25 g/ml

DPPH scavenging activity - IC50 is 49

Melanin inhibition - IC50 is

g/ml

3 g/ml

ORAC 3256 mol trolox equivalents/g

THC +

Melanin inhibition

Anti tyrosinase activity

Glabridin

Significant antioxidant potential:

IC50 is 0.068 g/ml

1:1

DPPH scavenging activity - IC50 is 0.97

Melanin inhibition - IC50 is

g/ml

1 g/ml

ORAC 10,000 mol trolox equivalents/g

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 3g/ml, 0.5% THC should
have an IC50 of >100g/ml. However, the IC50 obtained for the composition is 10g/ml
(Table 5.3.59). So in a cell system the anti inflammatory potential and enhanced

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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

IC50 >100g/ml

Antioxidant activity
0.1% THP

A significant cell penetration enhancer.

Nil

THP enhanced the permeation of a diterpene

forskolin by 12 times in 60 minutes across rat skin.
0.25% AKBBA + Melanin inhibition
0.5% THC +

Antioxidant activity

0.1% THP

Anti inflammatory potential

IC50 10g/ml

Enhanced bio availability

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 3g/ml, 0.2% THC & Glabridin should have an IC50 of >100g/ml. However, the
IC50 obtained for the composition is 20g/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.

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Table 5.3.60: Synergistic skin lightening potential by a combination of THC, Glabridin,

AKBBA and THP
Active
0.2% THC

Characteristic activity
Melanin inhibition

Melanin inhibition
IC50 >100g/ml

Antioxidant activity
0.2% Glabridin

Melanin inhibition

IC50 >100g/ml

1% AKBBA

Anti inflammatory potential

Nil

0.1% THP

significant

cell

penetration Nil

enhancer.
0.2% THC + 0.2% Glabridin + Melanin inhibition
1% AKBBA + 0.1% THP

IC50 20g/ml

Antioxidant activity
Anti inflammatory potential
Enhanced bio availability

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 3g/ml, 0.5% THC and
Glabridin should have an IC50 of >100g/ml. However, the IC50 obtained for the
composition is 12.5g/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.

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Table 5.3.61: Synergistic skin lightening potential by a combination of THC, Glabridin,

Galanga extract and THP
Active
0.5% THC

Characteristic activity
Melanin inhibition

Melanin inhibition
IC50 >100g/ml

Antioxidant activity
0.5% Glabridin

Melanin inhibition

IC50 >100g/ml

0.1% Galanga extract

Inhibitor of UV induced

Nil

cell damage
0.1% THP

A significant cell

Nil

penetration enhancer.
0.5% THC + 0.5% Glabridin +

Melanin inhibition

0.1% Galanga extract + 0.1%

Antioxidant activity

THP

Anti inflammatory potential

IC50 12.5g/ml

Enhanced bio availability

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 2g/ml, 0.2%
THC should have inhibited 30% melanogenesis at >200g/ml. However, the composition
inhibited 30% of melanogenesis at 20g/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.

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Table 5.3.62: Synergistic skin lightening potential by a combination of THC,

Coenzyme Q10 (CoQ10), Coconut liquid endosperm, Soya isoflavones and THP
Active
0.2% THC

Characteristic activity

Melanin inhibition

Melanin inhibition

Inhibits 30%

Antioxidant activity

melanogenesis at conc.
>200g/ml

0.1% CoQ10

Antioxidant activity:

Nil

Naturally found in the mitochondria involved in

neutralizing free radicals.
1% Coconut

Cell proliferation enhancer:

Nil

liquid endosperm 18% enhancement at 5g/ml

0.5% Soya

Phytoestrogenic properties:

isoflavones

They stimulate fibroblasts to make collagen and

(genistein &

hyaluronic acid which are essential for good skin

diadzein)

tone. Soya isoflavones also have the ability to

Nil

prevent UV damage.
0.1% THP

A significant cell penetration enhancer.

Nil

0.2% THC +

Melanin inhibition

Inhibits 30%

0.1% CoQ10 +

Antioxidant activity

melanogenesis at conc.

1% Coconut

Cell growth and conditioning

20g/ml

liquid endosperm Enhanced bio availability

+ 0.5% Soya
isoflavones +
0.1% THP

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

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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 2g/ml, 0.5%
THC should have inhibited 30% melanogenesis at >200g/ml. However, the composition
inhibited 30% of melanogenesis at 20g/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 80g/ml. 100% Glabridin can
provide 40% melanogenesis inhibition at a concentration of about 2g/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 >200g/ml with 0.2%
glabridin in the composition. However, 40% inhibition of melanogenesis obtained with
the composition is 5g/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.

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Table 5.3.63: Synergistic skin lightening potential by a combination of THC, Centella

asiatica extract, Soya isoflavones and THP
Active
0.5% THC

Characteristic activity

Melanin inhibition

Melanin inhibition

Inhibits 30% melanogenesis at

Antioxidant activity

>200g/ml

0.2% Centella

Anti inflammatory property:

Nil

asiatica extract

Inhibition of Nitric oxide synthesis

facilitating cell proliferation and wound
healing.
Collagen enhancement:
60% enhancement at 5mg/ml

0.5% Soya

Phytoestrogenic properties:

isoflavones

They stimulate fibroblasts to make

(genistein &

collagen and hyaluronic acid which are

diadzein)

essential for good skin tone. Soya

Nil

isoflavones also have the ability to

prevent UV damage.
0.1% THP

A significant cell penetration enhancer.

Nil

0.2% THC +

Melanin inhibition

Inhibits 30% melanogenesis at

0.2% Centella

Antioxidant activity

20g/ml

asiatica extract + Anti inflammatory activity

+ 0.5% Soya

Cell growth, conditioning and collagen

isoflavones +

enhancement

0.1% THP

Enhanced bio availability

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Table 5.3.64: Synergistic skin lightening potential by a combination of Arbutin,

Glabridin, AKBBA and Coriander seed oil extract
Active

Characteristic activity

0.5% Arbutin

Melanin inhibition
Inhibits 40% melanogenesis at

Melanin inhibition
0.2% Glabridin

>200g/ml

1% AKBBA

Anti inflammatory property

Nil

0.3% Coriander

Skin conditioning:

Nil

seed oil extract

Coriander seed oil extract contains

petroselinic acid triglycerides with
significant skin conditioning potential.

0.5% Arbutin +

Melanin inhibition

0.2% Glabridin + Anti inflammatory activity

Inhibits 40% melanogenesis at

5g/ml

+ 1% AKBBA + Cell condiitoning

0.3% Coriander
seed oil extract

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 80g/ml. 100% Glabridin can
provide 40% melanogenesis inhibition at a concentration of about 2g/ml. Theoretically,
considering the activity of the better potential active, glabridin, 40% inhibition of
melanogenesis can be attained at a concentration of >200g/ml with 0.1% glabridin in
the composition. However, 40% inhibition of melanogenesis obtained with the
composition is 5g/ml (Table 5.3.65). So in a cell system the anti inflammatory potential

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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 50g/ml. So
theoretically 0.1% Glabridin should give 30% inhibition at 50mg/ml, where as the same
activity was observed at 140g/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

0.5% Arbutin

Melanin inhibition

0.1% Glabridin

Melanin inhibition
Anti inflammatory potential

Melanin inhibition

Inhibits 40% melanogenesis at

>200g/ml

Mild antioxidant
0.1% THP

A significant cell penetration enhancer.

Nil

0.5% Arbutin +

Melanin inhibition

Inhibits 40% melanogenesis at

0.1% Glabridin + Enhanced bioavailability

5g/ml

+ 0.1% THP

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.

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Combined activity of the composition:

Antioxidant activity: ORAC Value of 1205mol trolox equivalents/g, DPPH
scavenging activity with IC50 of 17g/ml.
Melanin inhibition: IC50 of 20g/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 1983mol trolox equivalents/g, DPPH scavening
potential with an IC50 of 1.7g/ml, Lipid peroxidation inhibition with an IC50 of 97g/ml.
Anti inflammatory activity: Anti Hyaluronidase activity with an IC50 of 4.3g/ml, Anti
Elastase activity with an IC50 of 300g/ml, Anti Collagenase activity with an IC50 of
125g/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 10,930mol trolox equivalents/g, HORAC value
of 4770mol gallic acid equivalents/g, DPPH scavening potential with an IC50 of
0.83g/ml, Lipid peroxidation inhibition with an IC50 of 347g/ml.
Anti inflammatory activity: Anti Hyaluronidase activity with an IC50 of 6.7g/ml, Anti
Elastase activity with an IC50 of 125g/ml, Anti Collagenase activity with an IC50 of
25g/ml.
Cell proliferation enhancement: 50% enhancement at 0.002% concentration.

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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 8.3g/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 1.6g/ml, ROS
scavening potential with an IC50 of 2g/ml Lipid peroxidation inhibition with an IC50 of
22g/ml.
Anti inflammatory activity: Anti Hyaluronidase activity with an IC50 of 7.6g/ml, Anti
Elastase activity with an IC50 of 30g/ml, Anti Collagenase activity with an IC50 of
50g/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

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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.

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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 (mol 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 mol gallic acid
equivalents/g for 20 to 50% polyphenol content. Similarly, the ROS scavenging potential
was same with an IC50 of 6.25g/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.

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Table 5.3.67: Antioxidant potential of Cocoa bean extract

% Polyphenols

DPPH scavenging

ORAC

value

(mol

(IC50 in g/ml)

trolox equivalents/g)

26

3635

27

2.7

4069

35

4922

37

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
%

ORAC value

HORAC value

DPPH scavenging

Chlorogenic (mol trolox

(mol gallic acid

(IC50 in g/ml)

acid

equivalents/g)

equivalents/g)

40

6291

2935

60

9978

5206

65

10930

5891

1.25

80

12636

5621

0.96

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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 mol trolox equivalents/g. Similarly, Green tea extract with 80%
polyphenols has a higher ORAC value of 8027 mol 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 mol gallic acid equivalents/g, DPPH scavenging: IC50 - 3 g/ml,
ROS scavenging: IC50 - 1 g/ml, Elastase inhibition: IC50 275 g/ml, Collagenase
inhibition: IC50 50 g/ml, Hyaluronidase inhibition: IC50 5g/ml, Tyrosinase
inhibition: 40% inhibition at 50 g/ml, Melanin inhibition 14% inhibition at 10g/ml.
5.3.7.1.3.2. Grape seed extract containing 50% Polyphenols:
ORAC value: 4528 mol trolox equivalents/g. Similarly, Grape seed extract with 70%
polyphenols has an ORAC value of 9699 mol 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 mol gallic acid equivalents/g, DPPH scavenging: IC50 - 3 g/ml,
ROS scavenging: IC50 - 5 g/ml, Elastase inhibition: IC50 9 g/ml, Collagenase
inhibition: IC50 12 g/ml, Hyaluronidase inhibition: IC50 5g/ml, Tyrosinase
inhibition: IC50 28 g/ml

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5.3.7.1.3.3. Pomegranate extract containing 44% Polyphenols:

ORAC value: 4400 mol trolox equivalents/g. Similary, Pomegranate extract with 65%
polyphenols has an ORAC value of 5687 mol 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 g/ml, Elastase inhibition: IC50 500 g/ml,
Collagenase inhibition: IC50 62.5 g/ml, Hyaluronidase inhibition: IC50 0.5g/ml,
Tyrosinase inhibition: IC50 7 g/ml
5.3.7.1.3.4. Pomegranate rind extract:
ORAC value: 14,087 mol 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 mol trolox equivalents/g.
Other significant properties of Pomegranate rind extract:
HORAC value: 12,743 mol gallic acid equivalents/g, DPPH scavenging: IC50
0.4g/ml, Elastase inhibition: IC50 500g/ml, Collagenase inhibition: IC50 62.5g/ml,
Hyaluronidase inhibition: IC50 0.5g/ml, Tyrosinase inhibition: 12% inhibition at
10g/ml.
5.3.7.1.3.5. Pomegranate extract standardized to 90% Ellagic acid:
ORAC value: 8299 mol trolox equivalents/g.
Other significant properties: Melanin inhibition: 16% inhibition of melanin at 1.25
g/ml, ROS scavenging: IC50 - 20 g/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.

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5.3.7.1.3.6. Turmeric extract:

ORAC value: 10,410 mol trolox equivalents/g
Other significant properties:
HORAC value: 9740 mol gallic acid equivalents/g, DPPH scavenging: IC50 1.8g/ml,
Tyrosinase inhibition: 22% inhibition at 3g/ml.
5.3.7.1.3.7. Rosmarinic acid:
Rosmarinus officinalis leaf extract containing 90% Rosmarinic acid:
ORAC value: 14000 mol trolox equivalents/g
Other significant properties:
HORAC value: 5925 mol gallic acid equivalents/g, DPPH scavenging: IC50 0.5g/ml,
Collagenase inhibition: IC50 250g/ml, Hyaluronidase inhibition: IC50 25g/ml
Rosmarinus officinalis leaf extract containing 50% Rosmarinic acid:
ORAC value: 10000 mol trolox equivalents/g
Coleus forskohlii leaf extract containing 90% Rosmarinic acid:
ORAC value: 15000 mol trolox equivalents/g
Coleus forskohlii leaf extract containing 50% Rosmarinic acid:
ORAC value: 14600 mol 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 mol trolox equivalents/g
Other significant properties:
HORAC value: 1305 mol gallic acid equivalents/g, Elastase inhibition: IC50 - 125 g/ml.

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5.3.7.1.3.9. Tulsi extract:

ORAC value: 3732 mol trolox equivalents/g
Other significant properties:
DPPH scavenging: IC50 3.8g/ml
5.3.7.1.3.10. Mulberry extract:
ORAC value: 645 mol 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).

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Table 5.3.69: Synergistic antioxidant compositions

Activity of actives

Composition

Activity of composition
Expected

Synergistic activity

Activity obtained on

activity

(Higher than the

analysis

(Additional

expected additional

effect)

activity)

ORAC value (mol trolox equivalents/g)

Rosmarinic acid &
Chlorogenic acid
(1:3)

Rosmarinic acid 14,000

Chlorogenic acid 9978

Rosmarinic acid &

Rosmarinic acid 14,000

THC (1:1)

THC 9244

Rosmarinic acid, THC

Rosmarinic acid 14,000

& Chlorogenic acid

THC - 9244

(1:1:1)

Chlorogenic acid 9978

Turmeric extract &

Green tea extract (1:1)

10,984

16,595

10,433

16,859

11,074

13,931

9000

14,418

Turmeric extract
10,410
Green tea extract 8027

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

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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 62mol trolox equivalents/g. The ORAC value can be increased to
3100 mol 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

Concentration ORAC (mol trolox

Recommended Servings of

active

(%)

5g /day

Mulberry extract

Amla extract

Aloe vera extract

0.5

Grape seed extract

0.5

Green tea extract

equivalents/g)

62

10

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 1751mol trolox equivalents/g. The ORAC value can be increased to 3502
mol 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.

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Table 5.3.71: Enriched coffee as a nutricosmetic

Nutricosmetic

Concentrati

ORAC (mol trolox

Recommended

active

on (%)

equivalents/g)

Servings of 2g /day

Coffee bean extract

0.92

Grape seed extract

2.4

Amla extract

1.5

Instant coffee base

95.24

1751

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 200mol trolox equivalents/g.
The ORAC value can be increased to 3000 mol 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

Concentrati

ORAC (mol trolox

Recommended

active

on (%)

equivalents/g)

Servings of 5g /day

Cocoa polyphenols

0.66

Cocoa powder

6.5

200

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 161mol trolox
equivalents/g. The ORAC value can be increased to 3220 mol trolox equivalents/g by
having 4 servings of 5gm per day in water to attain the minimal ORAC requirement per

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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

Concentration ORAC (mol trolox

Recommended Servings

active

(%)

of 5g /day

Amla extract

Tulsi extract

Amla extract

equivalents/g)

161

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5.4. CONCLUSION OF PART II

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.