part ii screening of actives through …shodhganga.inflibnet.ac.in/bitstream/10603/70415/8/chapter...

CHAPTER 1 PART II 5.1. INTRODUCTION

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There are many fairness products that inhibit melanogenesis and are in great demand. But

there are some facts that are often ignored; fairness is not a measure of skin health.

Complexion that is clear, bright, and glows with health is the hallmark of beautiful skin.

What skin needs is just “adequate protection, cleansing and nourishment” by topical and

internal care (Fig. 5.1.1). That is all the pampering that skin needs for attaining “Bright &

Glowing” sheen.

Figure 5.1.1: Skin care topically and internally

Healthy Skin

Topical Care Internal Care

Protection by Sunscreen

Cleansing by Astringent

Nourishment by Antioxidant rich

Moisturizers, conditioners &

cell rejuvenators

Protection by Antioxidants

Cleansing by Antioxidants that

detoxify blood

Nourishment by nutrition rich diet

& supplements (Nutricosmetics)

PART II

SCREENING OF ACTIVES THROUGH VARIOUS

MECHANISMS OF MELANOGENESIS AND POSITIONING

THEM IN ACCORDANCE TO THEIR SPECIFIC MODE OF

ACTION IN RECTIFYING PIGMENTATION DISORDERS

5.1. INTRODUCTION


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5.1.1. Skin color and melanin:

All organisms, from simple invertebrates to complex human beings, exist in different

colors and patterns, which arise from the unique distribution of pigments throughout the

body. Human skin exists in a wide range of colors and gradations, ranging from white to

brown to black. Pigmentation is highly heritable, being regulated by genetic,

environmental, and endocrine factors that modulate the amount, type, and distribution of

melanins in the skin, hair, and eyes.

Melanin is a chemically inert and stable pigment, which is produced deep inside

the skin but is displayed as a mosaic at the surface of the body. Melanin is therefore

responsible for the most striking polymorphic traits of humans and for the most obvious

and thoroughly discussed aspect of human geographical variability: skin color. In

addition to its roles in heat regulation and color variation, melanin protects against

Ultraviolet radiation (UV), environmental factors etc., and thus is an important defense

system in human skin. Melanin plays a major photoprotective role in human skin by

absorbing, scattering, photo-oxidizing, and scavenging free radicals and acting as a

pseudo-dismutase to minimize the toxic effects of ROS and to prevent damage to DNA,

proteins, and cell membrane lipids (Pathak M A and Fitzpatrick T B, 1993). It is known

that UV-A produces harmful oxygen species such as O.2-, .OH, and 1O2 and that melanin

interacts with them, thus protecting the skin against the damage that could occur (Pathak

M A and Fitzpatrick T B, 1993; Pathak, M A and Stratton K, 1968).

5.1.2. Skin and stress:

Being the largest organ of the body that is always under the influence of internal and

external factors, the skin often reacts to those agents by modifying the constitutive

pigmentation pattern. Minor changes in the physiological status of the human body or

exposure to harmful external factors can affect pigmentation patterns either in transitory

or permanent manners. Understanding the mechanisms by which different factors and

compounds affect melanogenesis is of great interest pharmaceutically (as therapy for

pigmentary diseases like vitiligo) and cosmeceutically (e.g., to design depigmentation

products with potential to reduce skin darkening). Other than genetic factors, many


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factors like endocrine factors that induce temporary (e.g., during pregnancy) or

permanent (e.g., during ageing) changes in skin color, environmental factors (e.g., UV,

pollution), certain drugs, and chemical compounds, etc. play an important role in skin

pigmentation.

Undesirable excess pigmentation can be prevented before it manifests in

permanent manner. Skin pigmentation is the result of the intricate cellular and molecular

interactions between melanocytes and keratinocytes, which together compose the

epidermal melanin unit. All of the other types of cells distributed within different layers

of the skin and the intracellular signaling pathways often overlapping and involving cross-

talking also play a role in skin pigmentation. The skin reacts to stress through all its

cellular and molecular components, which form a complicated, sophisticated, and highly

sensitive signaling network.

5.1.3. Skin structure and functions:

An understanding of skin structure is prerequisite to understand pigmentation

mechanisms. The skin plays an extremely important role, providing a vast physical

barrier against mechanical, chemical, and microbial factors that may affect the

physiological status of the body (Haake A and Holbrook K, 1999). In addition to those

functions, the skin also acts as an immune network and, through its pigments, provides a

unique defense system against UV radiation (UV) (Pathak M A, 1995). Thus,

melanocytes transfer melanosomes through their dendrites to keratinocytes, where they

form the melanin caps that reduce UV-induced DNA damage in human epidermis. The

skin’s layers are represented by the epidermis, the dermis, and the hypodermis, the latter

consisting of fatty tissue that connects the dermis to underlying skeletal components. The

structure of skin in illustrated and discussed in detail (Fig. 5.1.2).

5.1.3.1. Epidermis: The epidermis is an external, stratified epithelium devoid of blood or

nerve supplies of 5–100 µm thickness (which can reach 600 µm on palms and soles)

(Tobin D J, 2006). It is composed of several distinct cell populations; keratinocytes and


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melanocytes are the main constituents, of which the first comprise 95% of the epidermis

and are arranged in four layers, as shown in Fig. 5.1.2.

A) Different layers and components of skin B) Layers of the epidermis Figure 5.1.2: Structure of skin

Stratum basale (also known as the stratum germinativum) is a single layer of cells

attached to a noncellular basement membrane that separates the epidermis from the

dermis. The stratum basale consists mostly of basal keratinocytes, which have stem cell-

like properties, and at least two different types of neural crest-derived cells: Merkel cells

(neuroendocrine cells responsible for the transmission of touch sensation through the

cutaneous nerves) and melanocytes.

Stratum spinosum contains irregular polyhedral keratinocytes with some limited

capacity for cell division. Also found here are the bone marrow-derived sentinel cells of

the immune system called Langerhans’ cells, which represent the antigen-presenting cells


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of the skin and play a vital role in immunological reactions such as allergic contact

dermatitis.

Stratum granulosum contains flattened, polyhedral nondividing keratinocytes

producing granules of a protein called keratinohyalin. These granules increase in size and

number as the cell nuclei gradually degenerate and the cells die. These cells flatten as

dividing cells underneath them progressively push them toward the skin surface.

Stratum corneum contains nonviable, but biochemically active cells called

corneocytes. The keratinocytes continue to differentiate as they move from the basal layer

to the stratum corneum, the result being cornified cells that contain abundant keratin and

lack cytoplasmic organelles. It is these cornified cells that provide a barrier against the

physical and chemical agents in the environment that may adversely affect the body.

More specifically, this epidermal barrier functions to reduce transepidermal water loss

from within and to prevent invasion by infectious agents and noxious substances from

without (Elias P M, 2005).

5.1.3.2. Dermis: The dermis is a 2 to 4 mm-thick layer of connective tissue and

fibroblasts that houses the neural, vascular, lymphatic, and secretory apparatus of the skin.

The main cell type, fibroblasts, is required for synthesis and degradation of the

extracellular matrix (ECM) (Haake A and Holbrook K, 1999). This matrix is a complex

structure composed of highly organized collagen, elastic, and reticular fibers. The dermis

also hosts multifunctional cells of the immune system such as macrophages and mast

cells, the latter being able to trigger allergic reactions by secreting bioactive mediators

such as histamine. Structures within the dermis include: 1) Excretory and secretory glands

(sebaceous, eccrine, and apocrine). Sebaceous glands secrete triglyceride and cholesterol-

rich sebum that lubricate the skin and keep it supple and waterproof. They are often

associated with hair shafts. 2) Hair follicles and nails: in addition to generating the hair

shaft, the hair follicle provides a protective niche to several stem cell populations in the

skin, including keratinocyte stem cells, melanocyte stem cells, a population of epidermal

neural crest stem cells, and the dermal stem cell compartment, known as the dermal

papilla (Cotsarelis G et al., 1990 and Ito M et al., 2005). These stem cells are required

most visibly during wound healing. 3) Sensory nerve receptors of Merkel and Meissner’s


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corpuscles (for touch), Pacinian corpuscles (for pressure), and Ruffini corpuscles

(mechano-receptors). As illustrated in Fig. 5.1.3, dermis contains various skin structural

proteins that confer integrity to the skin. In the dermis, collagen provides the skin with

tensile strength and tissue integrity whereas elastin provides elasticity and resiliency.

Besides collagen and elastic fibers, the dermis contains the extrafibrillar matrix, which is

extracellular and composed of a complex mixture of proteoglycans, glycoproteins,

glycosaminoglycans, water, and hyaluronic acid. The most significant

glycosaminoglycans, which bind to proteins to form the proteoglycans of the skin, are

chondroitin sulfate, dermatan sulfate, keratin sulfate, heparan sulfate, and heparin. The

most important proteoglycans of the skin are versicans, which are involved in assuring

the tightness of the skin, and perlecan, found in basement membranes. Glycoproteins,

such as laminins, matrilins, fibronectin, tenascins, etc., are involved in cell adhesion, cell

migration, and cell-cell communication, which are extremely important processes taking

place in the skin.

A B A) Collagen and elastic fibers in dermis B) Stratified epidermis and vascular dermis Figure 5.1.3: Skin structural proteins

Epidermis

Skin structural proteins

in dermis


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5.1.4. Synthesis and distribution of melanin in skin under normal conditions:

Melanin biosynthesis is a complex pathway that appears in highly specialized cells, called

melanocytes, within membrane-bound organelles referred to as melanosomes (Hearing V

J, 1997). Melanosomes are transferred via dendrites to surrounding keratinocytes, where

they play a critical role in photoprotection. The anatomical relationship between

keratinocytes and melanocytes is known as "the epidermal melanin unit" and it has been

estimated that each melanocyte is in contact with 40 keratinocytes in the basal and

suprabasal layers (Fitzpatrick T B and Breathnach A S, 1963). Several important steps

must occur for the proper synthesis and distribution of melanin, as described briefly

below (Boissy R E and Nordlund J J, 1997).

5.1.4.1. The development of melanocyte precursor cells (melanoblasts) and their

migration from the neural crest to peripheral sites:

Prospective melanocytes, known as melanoblasts, derive from the neural crest beginning

in the second month of human embryonic life and migrate throughout the mesenchyme of

the developing embryo. They reach specific target sites, mainly the dermis, epidermis,

and hair follicles, the uveal tract of the eye, the stria vasculare, the vestibular organ and

the endolymphatic sac of the ear, and leptomeninges of the brain. In humans, this

migration process takes place between the 10th and the 12th wk of development for the

dermis and 2 wk later for the epidermis (Haake A and Holbrook K, 1999).

5.1.4.2. Differentiation of melanoblasts into melanocytes:

Once melanoblasts have reached their final destinations, they differentiate into

melanocytes, which at about the sixth month of fetal life are already established at

epidermal-dermal junction sites (Haake A and Holbrook K, 1999).


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5.1.4.3. Survival and proliferation of melanocytes:

Melanocytes have been identified within fetal epidermis as early as 50 days of gestation.

Dermal melanocytes decrease in number during gestation and virtually disappear by birth,

whereas epidermal melanocytes established at the epidermal-dermal junction continue to

proliferate and start to produce melanin. 5.1.4.4. Formation of melanosomes and production of melanins:

Once established in situ, melanocytes start producing melanosomes, highly organized

elliptic membrane-bound organelles in which melanin synthesis takes place.

Melanosomes are typically divided into four maturation stages (I–IV) determined by their

structure and the quantity, quality, and arrangement of the melanin produced (Seiji M et

al., 1963 and Kushimoto T et al., 2001). Nascent melanosomes are assembled in the

perinuclear region near the Golgi stacks, receiving all enzymatic and structural proteins

required for melanogenesis. Stage I melanosomes are spherical vacuoles lacking

tyrosinase (TYR) activity (the main enzyme involved in melanogenesis) and have no

internal structural components. However, TYR can be detected in the Golgi vesicles, and

it has been shown that it is subsequently trafficked to stage II melanosomes. At this point,

the presence and correct processing of Pmel17, an important melanosomal structural

protein, determine the transformation of stage I melanosomes to elongated, fibrillar

organelles known as stage II melanosomes (Kushimoto T et al., 2001 and Berson J F et

al., 2001); they contain tyrosinase and exhibit minimal deposition of melanin. After this,

melanin synthesis starts and the pigment is uniformly deposited on the internal fibrils, at

which time the melanosomes are termed as stage III. Their last developmental stage (IV)

is detected in highly pigmented melanocytes; these melanosomes are either elliptical or

ellipsoidal, electron-opaque due to complete melanization, and have minimal TYR

activity. The developmental stages detailed above refer mainly to eu-melanosomes

(containing black-brown pigments); however, they are quite similar to pheo-melanosomes

(containing yellow-reddish melanin), the only difference being that the latter remain

round and are not fibrillar during maturation.


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Within melanosomes, at least three enzymes are absolutely required to synthesize

different types of melanin. While tyrosinase is responsible for the critical steps of

melanogenesis, tyrosinase-related protein 1 (TYRP1) and DOPAchrome tautomerase

(DCT) are further involved in modifying the melanin into different types.

TYR (monophenol, 3,4-ß-dihydroxyphenylalanine oxygen oxidoreductase, EC

1.14.18.1) is a single chain type I membrane glycoprotein catalyzing the hydroxylation of

tyrosine to β-3,4-dihydroxyphenylalanine (DOPA) (which is the initial rate-limiting step

in melanogenesis) and the subsequent oxidation of DOPA to DOPAquinone. TYR,

TYRP1, and DCT share numerous structural similarities and follow quite similar

biosynthetic, processing, and trafficking pathways (Hearing V J and Tsukamoto K, 1991).

Their maturation is assisted by chaperones, calnexin being the most important one due to

its involvement in the correct folding of tyrosinase (Halaban R et al., 1997; Branza-

Nichita N et al., 1999 and Branza-Nichita N et al., 2000). The subsequent metabolism of

DOPA and its derivatives by various melanocyte-specific enzymes, including TYRP1 and

DCT, results in the synthesis of eumelanin, a black-brown pigment. The synthesis of

pheomelanin involves the production of cysteinyldopa conjugates from DOPAquinone

after the production of DOPA from tyrosine. TYRP1 is important for the correct

trafficking of tyrosinase to melanosomes (Toyofuku K et al., 2001), and DCT also seems

to be involved in the detoxification processes (Urabe K et al., 1994) taking place within

melanosomes.

Melanins are polymorphous and multifunctional biopolymers that include

eumelanin, pheomelanin, mixed melanins (a combination of the two), and neuromelanin.

Mammalian melanocytes produce two chemically distinct types of melanin pigments:

black-brown eumelanin and yellow-reddish pheomelanin (Prota G, 1992). Although they

contain a common arrangement of repeating units linked by carbon-carbon bonds,

melanin pigments differ from each other with respect to their chemical, structural, and

physical properties. Eumelanin is a highly heterogeneous polymer consisting of 5,6-

dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units in

reduced or oxidized states, as detailed above; pheomelanin consists mainly of sulfur-

containing benzothiazine derivatives (Ito S et al., 2000). Due to their chemical structure,

both eumelanin and pheomelanin are involved in binding to cations, anions, drugs, and


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chemicals, etc., and therefore play an important protective role within melanocytes

(Nordlund J. J, 1985). Neuromelanin, which is produced in dopaminergic neurons of the

human substantia nigra, can also chelate redox active metals (Cu, Mn, Cr) and toxic

metals (Cd, Hg, Pb), and thus protects against their ability to promote neurodegeneration

(Zecca L et al., 1996).

Given their complexity, melanosomes can be used as a model to study organelle

biogenesis, protein trafficking and processing, organelle movement, and cell-cell

interactions (like those occurring during melanin transfer between melanocytes and

keratinocytes) (Hearing V J, 2000). Therefore, even minor changes in the cellular

environment affect melanosomes and pigmentation. Numerous intrinsic and extrinsic

factors, including body distribution, ethnicity/gender differences, variable hormone-

responsiveness, genetic defects, hair cycle-dependent changes, age, UV-R, climate/season,

toxin, pollutants, chemical exposure and infestations, are responsible for a whole range of

responses in melanosome structure and distribution under different types of stress.

Cutaneous pigmentation is the outcome of two important events: the synthesis of

melanin by melanocytes and the transfer of melanosomes to surrounding keratinocytes

(Fitzpatrick T B and Szabo G, 1959). Although the number of melanocytes in human skin

of all types is essentially constant, the number, size, and manner in which melanosomes

are distributed within keratinocytes vary. The melanin content of human melanocytes is

heterogeneous not only between different skin types but also between different sites of

the skin from the same individual. This heterogeneity is highly regulated by gene

expression, which controls the overall activity and expression of melanosomal proteins

within individual melanocytes (Sturm R A et al., 1998). It has been shown that

melanocytes with low melanin content synthesize TYR more slowly and degrade it more

quickly than melanocytes with a higher melanin content and TYR activity (Halaban R et

al., 1983). In general, highly pigmented skin contains numerous single large melanosomal

particles (0.5–0.8 mm in diameter), which are ellipsoidal and intensely melanotic (stage

IV). Lighter pigmentation is associated with smaller (0.3–0.5 mm in diameter) and less

dense melanosomes (stages II and III), which are clustered in membrane-bound groups

(Toda K et al., 1972). These distinct patterns of melanosome type and distribution are


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present at birth and are not determined by external factors (such as sun exposure). They

are responsible for the wide variety of skin complexions.

5.1.4.5. Epidermal melanin unit and the involvement of keratinocytes in melanin

production:

Epidermal melanin unit is a functional and structural complex within the epidermis

consisting of two cell types: melanocytes and keratinocytes. The variation in skin color

among various races is determined mainly by the number, melanin content, and

distribution of melanosomes produced and transferred by each melanocyte to a cluster of

keratinocytes surrounding it (Jimbow K et al., 1976). Once in keratinocytes, the melanin

granules accumulate above the nuclei and absorb harmful UV before it can reach the

nucleus and damage the DNA. When melanin is produced and distributed properly in the

skin, dividing cells are protected at least in part from mutations that might otherwise be

caused by harmful UV (Kobayashi N et al., 1998). The melanocyte-keratinocyte complex

responds quickly to a wide range of environmental stimuli, often in paracrine and/or

autocrine manners and further triggers various molecular responses as illustrated in Fig. 5.

Thus, melanocytes respond to UV, melanocyte-stimulating hormone (MSH), endothelins,

growth factors, cytokines, etc. After UV-R exposure, melanocytes increase their

expression of proopiomelanocortin (POMC, the precursor of MSH) and its receptor

melanocortin 1 receptor (MC1-R), TYR and TYRP1, protein kinase C (PKC), and other

signaling factors (Chakraborty A K et al., 1996 and Funasaka Y et al., 1998). On the

other hand, it is known that UV stimulates the production of endothelin-1 (ET-1) by

keratinocytes and that those factors can then act in a paracrine manner to stimulate

melanocyte function (Tada A et al., 1998 and Abdel-Malek Z et al., 2000). ET-1 is a 2l

amino acid peptide with vasoactive properties first isolated from endothelial cells and

later found to be synthesized and secreted by keratinocytes as well (Imokawa G et al.,

1992; Yohn J J et al., 1993 and Hara M et al., 1995), particularly after exposure to UV-R

(Imokawa G et al., 1992; Yohn J J et al., 1993 and Hara M et al., 1995). The overall

effect of ET-1 is the increase of melanocyte dendricity and the enhancement of

melanocyte migration and melanization (Hara M et al., 1995). Binding of ET-1 to its G


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protein-coupled receptor (ETBR) on melanocytes activates a cascade of signaling

pathways, resulting in mobilization of intracellular calcium, activation of PKC, elevation

of cAMP levels, and activation of mitogen-activated protein kinase (MAPK) (Swope V B

et al., 1995 and Imokawa G et al., 1996). UV stimulates keratinocytes to produce ET-1

and also induces interleukin-1 (IL-1) production in these cells. IL-l is known to induce

ET-1 in keratinocytes in an autocrine manner. Therefore, it has been suggested that these

intracellular events in keratinocytes lead to increased TYR mRNA, protein, and enzymatic

activity in neighboring melanocytes as well as to an increase in melanocyte number

(Imokawa G et al., 1995).

In addition to keratinocytes, fibroblasts, and possibly other cells in the skin

produce cytokines, growth factors, and inflammatory mediators that can increase melanin

production and/or stimulate melanin transfer to keratinocytes by melanocytes. Melanocyte

growth factors affect not only the growth and pigmentation of melanocytes but also their

shape, dendricity, adhesion to matrix proteins, and mobility.

α-MSH, Adenocorticotropic hormone (ACTH), basic fibroblast growth factor

(bFGF), nerve growth factor (NGF), endothelins, granulocyte-macrophage colony-

stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and hepatocyte growth

factor (HGF) are keratinocyte-derived factors that are thought to be involved in the

regulation of the proliferation and/or differentiation of melanocytes (Hirobe T, 2005),

some acting through receptor-mediated signaling pathways (Fig. 5). It has been shown

that in human epidermis, -MSH (Chakraborty A K et al., 1996 and Slominski A et al.,

2000) and ACTH (Chakraborty A K et al., 1996; Slominski A et al., 2000 and

Wakamatsu K et al., 1997) are produced in and released by keratinocytes and are

involved in regulating melanogenesis and/or melanocyte dendrite formation. -MSH and

ACTH bind to a melanocyte-specific receptor, MC1-R (Cone R D et al., 1996), which

activates adenylate cyclase through G-protein, which then elevates cAMP from adenosine

triphosphate (Im S et al., 1998). Cyclic AMP exerts its effect in part through protein

kinase A (PKA) (Insel P A et al., 1975), which phosphorylates and activates the cAMP

response element binding protein (CREB) that binds to the cAMP response element

(CRE) present in the M promoter of the microphthalmia-associated transcription factor

(MITF) gene (Busca R and Ballotti R, 2000 and Tachibana M, 2000). The increase in


194

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


195

signaling pathways including the cAMP/PKA, PKC, and tyrosine kinase pathways

(Costin G E and Hearing VJ, 2007). Therefore, the mechanisms by which various factors

increase skin pigmentation are closely inter-related and briefly illustrated in Fig. 5.1.5.

Figure 5.1.5: Summarized mechanism of Skin pigmentation

5.1.5. Melanogenesis and its importance for cosmetic purposes:

• Melanogenesis is a normal biological mechanism of skin defense from UV,

pollution and other forms of stress that skin undergoes.

• Hence, a safe & effective fairness product is one that does not alter any normal

biological mechanism.

• The cause and mechanism of melanognesis & skin darkening has to be understood

before developing any fairness product.

Activation of MITF cAMP / α-MSH

Activation of Tyrosinase genes

Activation of Tyrosinase

Melanogenesis

Melanin transfer from MELANOCYTES to

peripheral KERATINOCYTES

Pigmentation

Serine protease & PAR 2 activation

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Costin%20GE%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Hearing%20VJ%22%5BAuthor%5D�


196

• Pigmentation disorders like excess pigmentation and uneven pigmentation should

only be rectified by fairness products. Fairness products are expected to bring

back the skin color to normal as per the genetic predisposition of the person.

• Fairness products should only work on pigmentation disorders and not on normal

pigmentation mechanism. They should normalize pigmentation disorders like:

Excess or uneven pigmentation due to skin damage by UV & pollution, Acne

marks and under eye dark circles.

• Similarly tanning products are also in demand for cosmetic purposes. Skin

tanning is also a way of protecting fair-skinned people from skin cancer caused by

exposure to sunlight.

5.1.6. Hyperpigmentation:

There are numerous internal and external stresses that affect human skin pigmentation.

The list is fairly long, so the present study focuses on the common stress conditions

whose mechanisms of action are known to some extent or are currently under

investigation and whose use may affect the discovery of new approaches to reduce

hyperpigmentation. The common external factors are UV radiation that causes tanning

and photoageing; drugs, chemicals, etc. and internal factors are hormonal influences and

inflammation that cause postinflammatory hyperpigmentation.

5.1.6.1. Hyperpigmentation induced by external factors:

5.1.6.1.1. UV influence on human pigmentation:

The skin responds to UV exposure by developing two defensive barriers: thickening of

the stratum corneum and the elaboration of a melanin filter in cells of the epidermis. The

palms and soles are the regions with the thickest stratum corneum, and they are

exceptionally resistant to UV damage. UV triggers various mechanisms in the skin

keratinocytes and melanocytes (Fig. 5.1.6). The keratins and proteins within the stratum

corneum act mainly by scattering and absorbing the UV. UV sets in action an integrated

mechanism for increase in the number of melanocytes as well as stimulation of melanin


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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 19–50% of the solar UV-A can reach the depth of

melanocytes, whereas only 9–14% of solar UV-B reaches these cells. Therefore, UV-A


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stimulates melanin pigmentation, but the resultant tan appears to be transient and less

protective against UV-induced injury than tans generated after UV-B exposure. UV-B is

responsible for causing the sunburn reaction within the skin and is absorbed mainly by

the epidermis and upper dermis. Like UV-A, UV-B stimulates the production of melanin,

which constitutes the basis for tanning. UV-B has great potential to induce erythema, and

therefore its influence on the skin has been thoroughly investigated in vitro and in vivo

(Robins A H, 1991). One role of melanin in the skin is to neutralize the ROS generated by

a variety of factors, including UV-B (Nordlund J J, 1985), therefore functioning like a

natural sunscreen. The influence of UV on human pigmentation from the perspective of

tanning as well as photoageing is a perfect example of factors sharing intracellular

pathways with slightly different end results on the skin.

5.1.6.1.1.1. Tanning response to UV: The tanning response has been shown to have two

distinct phases, termed immediate pigment darkening and delayed tanning. Both have

strong genetic determinants and are generally more pronounced in individuals with dark

baseline (constitutive) pigmentation (Gilchrest B A et al., 1996).

Immediate tanning is a quick but transient brownish tan that follows the exposure

of skin to UV-A or visible light. It begins immediately after exposure, reaches a

maximum within 1–2 h, then fades between 3 and 24 h after exposure (Gilchrest B A et

al., 1996). Immediate tanning reaction is based on the photoxidation of preexisting

melanin, melanin precursors, or even of other epidermal constituents and/or their

redistribution in the epidermis.

Delayed tanning gives rise to a durable tan induced by repeated exposure mainly

to UV-B. It is a gradual process in which the skin starts darkening 48–72 h after

irradiation, reaches a maximum 3 wk after exposure, and the skin does not return to its

original melanin content until 8–10 months later (Gilchrest B A et al., 1996). Delayed

tanning is dependent on both qualitative and quantitative changes within melanocytes,

which enlarge in size, increase their dendricity, and develop a diffuse distribution of thick

filaments in their cell bodies. Therefore, delayed tanning is due to an increase in

melanocyte numbers and melanogenesis.


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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 7-

dehydrocholesterol with subsequent processing to various photoproducts and the

biologically active 1 , 25-dihydroxy-vitamin D3, and releases diacylglycerol (DAG),

which in turn activates PKC, among other possible roles in signal transduction (Nishizuka

Y, 1986; Fig. 5). It was observed that addition of DAG to cultured human melanocytes

increases their melanin content several fold within 24 h (Gordon P R and Gilchrest B A,

1989), and subsequent work demonstrated that UV-R acts synergistically with DAG to

enhance melanogenesis (Friedmann P S et al., 1990). Direct melanogenic effects of UV

on melanocytes might also involve the production of Nitric oxide (NO), which is

considered a major intra- and intercellular messenger molecule. NO elicits its effects

through the activation of a soluble guanylate cyclase, leading to an increase in

intracellular cyclic guanosine monophosphate (cGMP) content and the activation of

cGMP-dependent protein kinase. Furthermore, it has been shown that UV-R increases

both NO and cGMP production, suggesting they are both required for UV-B-induced

melanogenesis (Fig. 5.1.6).

5.1.6.1.2. The action of drugs, chemicals, etc., on human skin pigmentation:

Numerous common drugs can stimulate human skin hyperpigmentation such as certain

antibiotics (sulfonamides and tetracyclines), diuretics, nonsteroidal antiinflammatory

drugs, pain relievers, and some psychoactive medications. The use of oral contraceptives

has been associated with the development of discoloration of the cheeks, forehead, and

nose (Goh C L and Dlova C N, 1999) similar to chloasma with increased melanogenesis

and enlarged melanocytes. Certain antiepileptic agents (mainly hydantoins) may also

cause skin hyperpigmentation (Levantine A and Almeyda J, 1973). Their long-term use

induces a brownish coloration of the face and neck, similar to chloasma of pregnancy. It


200

is already known that chloroquine has an affinity for melanin and causes skin

hyperpigmentation. Different studies have detected melanin in the dermis of patients

undergoing chloroquine treatment (Levy H, 1982).

Levodopa, often used to treat Parkinson’s disease, also induces hyperpigmentation

of the skin (Robins A H, 1991). DOPA is normally transformed into melanin within

melanosomes; therefore, DOPA therapy (applied as levodopa treatment) may possibly

enhance melanin biosynthesis. Heavy metals can also elicit hyperpigmentation, which can

arise after the extensive use of drugs containing arsenic, bismuth, gold, or silver

(Molokhia M M and Portnoy B, 1973). The metals are believed to act by binding, and

thereby inactivating, sulfhydryl compounds in the skin that normally inhibit TYR activity.

Removal of this inhibition stimulates melanogenesis. Mercury products inactivate TYR

probably by replacing the essential copper in the enzymatic site of that protein. Some

chemotherapy agents also can cause hyperpigmentation, the most common ones being

cyclophosphamide, 5-fluorouracil, doxorubicin, daunorubicin, and bleomycin. Their

mechanisms of action are currently unknown but may involve direct toxicity, stimulation

of melanocytes, and/or inflammation.

5.1.6.2. Hyperpigmentation induced by internal factors:

5.1.6.2.1. Hormonal influence on human skin pigmentation:

Hyperpigmentation is sometimes seen during pregnancy and this condition is called

melasma, chloasma, or mask of pregnancy; it occurs mainly on the cheeks, upper lip, chin,

and forehead. It is characterized by a symmetrical hypermelanosis with an irregular

coloration, ranging from light brown to gray and dark brown. Although melasma is

usually associated with pregnancy, multiple other factors can contribute to its

development including UV exposure, hormone therapy, estrogen-containing oral

contraceptives, genetic influences, certain cosmetics, endocrine or hepatic dysfunction,

and selected antiepileptic drugs (Table 5.1.1). Of the environmental sources, UV is the

most influential (Ortonne J P et al., 2003 and Barankin B et al., 2002).

The areas of hyperpigmentation seen in melasma exhibit increased deposition of

melanin in the epidermis and dermis (Kang W H et al., 2002 and Grimes P E et al., 2005).


201

No increase in the number of melanocytes in those areas is observed, but the melanocytes

are larger, more dendritic, and show increased melanogenesis, producing especially

eumelanin (Grimes P E et al., 2005). Studies confirmed an increased number of

melanosomes in keratinocytes, melanocytes, and dendrites in lesional skin compared with

nonlesional skin. During pregnancy (especially in the third trimester), elevated levels of

estrogen, progesterone, and MSH have often been found in association with melasma

(Smith A G et al., 1977 and Parker F, 1981). TYR activity increases and cellular

proliferation is reduced after treatment of melanocytes in culture with ß-estradiol (Ranson

M et al., 1988). Sex steroids increase transcription of genes encoding melanogenic

enzymes in normal human melanocytes, especially those for DCT and TYR

(Kippenberger S et al., 1998). These results are consistent with the significant increases in

melanin synthesis and TYR activity reported for normal human melanocytes under

similar conditions in culture (McLeod S D et al., 1994). It is known that estrogens

improve skin moisture and also increase its thickness and collagen content. Therefore,

estrogen plays a key role in skin ageing homeostasis given the fact that skin appearance

declines quickly in the postmenopausal years. Despite the knowledge that estrogens have

such important effects on skin, their cellular and molecular mechanisms of action are still

poorly understood and their influence on pigmentation is still far from clear.

Examination of the effects of estrogen treatment on TYR activity has revealed a

stimulation of this melanogenic enzyme (Ranson M et al., 1988 and Kippenberger S et al.,

1998). It was recently demonstrated that androgens modulate TYR activity via regulation

of cAMP, a key regulator of skin pigmentation (Tadokoro T et al., 2003). The sum of

these studies emphasizes the importance of both sex hormones in regulating skin

pigmentation.

5.1.6.2.2. Postinflammatory hyperpigmentation of the skin:

Postinflammatory hyperpigmentation is manifested by discrete, hyperpigmented macules

with hazy, feathered margins, which may involve the epidermis and/or dermis. This

usually develops after resolution of inflammatory skin eruptions like acne, contact

dermatitis, or atopic dermatitis. Postinflammatory hyperpigmentation is more common in


202

patients with darker skin and, at the cellular level, is characterized by a normal number of

melanocytes that have increased melanin production (Table 5.1.1).

Arachidonate-derived chemical mediators, especially leukotrienes and

thromboxanes may be responsible for the induction of post inflammatory

hyperpigmentation of the skin because they can stimulate normal human melanocytes in

vitro. These cells become swollen and more dendritic with increased amounts of

immunoreactive TYR. Such morphological changes are thought to be required for the

transfer of melanosomes to surrounding keratinocytes. Those effects were stronger than

that elicited by PGE2, which, together with PGE1 and PGD2, are known to be important

endogenous regulators of inflammatory diseases in the skin and to stimulate mammalian

pigment cells in vitro (Tomita Y et al., 1987) and in vivo (Nordlund J J et al., 1986).

Despite the common frequency of skin hyperpigmentation following inflammation, the

mechanisms responsible for melanin synthesis have not yet been completely clarified, but

some data have became available recently, as follows.

In the skin, PGs (especially PGE2, PGF2 , and small quantities of prostacyclin)

are produced (Pentland A P and Mahoney M G, 1990) and rapidly released by

keratinocytes after UV-R (Hanson D and DeLeo V, 1990 and Pentland A P et al., 1990).

They are chronically present in inflammatory skin lesions and are involved in wound

healing (Pentland A P et al., 1987). UV-R stimulates production of PGF2 by

melanocytes, which in turn stimulates the activity and expression of TYR, suggesting that

PGF2 could act as an autocrine factor for melanocyte differentiation (Scott G et al.,

2005).

On the other hand, PAR-2 is an important factor regulating skin pigmentation

because its activation in keratinocytes stimulates their uptake of melanosomes through

phagocytosis. It has been reported that activation of PAR-2 in keratinocytes stimulates the

release of PGE2 and PGF2 , which act as paracrine factors that stimulate melanocyte

dendricity (Scott G et al., 2004). Melanocyte dendrite formation has been linked to the

cAMP-dependent activation of Rac and the inhibition of Rho (Busca R et al., 1998; Scott

G, 2002 and Scott G and Leopardi S, 2003). However, recent studies demonstrated that

neither PGE2 nor PGF2 stimulates cAMP in melanocytes, thus demonstrating that these

PGs stimulate dendrite formation in a cAMP-independent manner (Scott G et al., 2004).


203

These data suggest that PAR-2 mediates cutaneous pigmentation through regulation of

melanosome uptake and production of PGs, which act as paracrine factors to stimulate

melanocyte dendricity.

All the inflammatory factors and pathways described above interact within the

skin; the final result is an increase of TYR activity and melanocyte dendricity, which

promotes the production of melanin and its distribution to keratinocytes. Therefore,

different factors are responsible for increasing human skin pigmentation via various

intracellular pathways. Table 5.1.1 summarizes the various conditions of

hyperpigmentation, their characteristics and causative factors. Table 5.1.2 summarizes

some of the internal or external stresses and the secondary messengers and effectors that

are involved.


204

Tabl

e 5.

1.1:

Sum

mar

y of

hyp

erpi

gmen

tatio

n co

nditi

ons,

caus

ativ

e fa

ctor

s, cl

inic

al fe

atur

es a

nd c

ellu

lar c

hara

cter

istic

s

Mol

ecul

ar m

arke

rs a

ffec

ted

1. In

crea

sed

TYR

-pos

itive

cel

ls p

er le

ngth

of t

he

derm

al/e

pide

rmal

inte

rfac

e co

mpa

red

with

un

affe

cted

skin

. 2.

Ker

atin

ocyt

es’ p

oten

tial t

o pr

oduc

e ET

-1 is

si

gnifi

cant

ly h

ighe

r com

pare

d w

ith u

naff

ecte

d sk

in.

3. T

NF-

α is

up-

regu

late

d in

the

SL le

sion

al

epid

erm

is.

1. H

igh

leve

ls o

f pro

gest

eron

e, e

stro

gen,

and

M

SH.

2. In

crea

sed

trans

crip

tion

of g

enes

enc

odin

g D

CT,

TY

R.

3. M

elan

ocyt

es a

re la

rger

and

mor

e de

ndrit

ic.

1. P

GE2

and

PG

F2 sy

nthe

sis i

s up-

regu

late

d;

they

act

as p

arac

rine

fact

ors w

hich

stim

ulat

e m

elan

ocyt

e de

ndric

ity.

2. L

euko

trien

es, T

NF-

α an

d th

rom

boxa

nes m

ay

be re

spon

sibl

e fo

r the

indu

ctio

n of

pos

t-in

flam

mat

ory

hype

rpig

men

tatio

n.

Incr

ease

d R

OS

gene

ratio

n an

d su

bseq

uent

cel

l da

mag

e re

sulti

ng in

ove

r exp

ress

ion

of

Tyro

sina

se, M

SH &

cA

MP

as a

stre

ss re

spon

se.

Cel

lula

r ch

arac

teri

stic

s

1. In

crea

sed

mel

anin

pr

oduc

tion.

2.

Slig

ht

incr

ease

in

num

ber o

f m

elan

ocyt

es.

1. In

crea

sed

mel

anin

pr

oduc

tion.

2.

Nor

mal

nu

mbe

r of

mel

anoc

ytes

1.

Incr

ease

d m

elan

in

prod

uctio

n.

2. N

orm

al

num

ber o

f m

elan

ocyt

es.

Tem

pora

ry

incr

ease

in

mel

anin

pr

oduc

tion

Clin

ical

feat

ures

1. C

ircum

scrib

ed, b

row

n to

bl

ack

mac

ules

. 2.

Ran

ge fr

om <

1 m

m to

se

vera

l cm

. 3.

Occ

ur in

epi

derm

is.

4. F

ound

on

UV

-exp

osed

are

as

of th

e bo

dy su

ch a

s the

face

, do

rsum

of t

he h

and,

ext

enso

r fo

rear

m a

nd u

pper

bac

k.

1. S

ymm

etric

faci

al

hype

rpig

men

tatio

n.

2. M

ay in

volv

e ep

ider

mis

, de

rmis

or b

oth.

1.

Dis

cret

e hy

perp

igm

ente

d m

acul

es w

ith h

azy

mar

gins

. 2.

May

invo

lve

epid

erm

is,

derm

is o

r bot

h.

Loss

in sk

in g

low

& ta

nnin

g on

U

V-e

xpos

ed a

reas

of t

he b

ody

such

as t

he fa

ce, d

orsu

m o

f the

ha

nd, e

xten

sor f

orea

rm a

nd

uppe

r bac

k.

Cau

sativ

e fa

ctor

(s)

Indu

ced

by U

V

Sun

expo

sure

, pr

egna

ncy,

or

al

cont

race

ptiv

es,

anti-

epile

ptic

s et

c.

Dev

elop

s aft

er

reso

lutio

n of

ac

ne, c

onta

ct

derm

atiti

s, et

c.

Exp

osur

e to

U

V &

pol

lutio

n

Hyp

erpi

gmen

tatio

n di

sord

er

Sola

r le

ntig

ines

(S

L)

Mel

asm

a

Post

-in

flam

mat

ory

hype

r pi

gmen

tatio

n

Skin

tann

ing


205

Table 5.1.2: Summary of external and internal stress increasing human skin pigmentation and their intracellular secondary messengers and effectors

Stress Secondary messenger Secondary effector UV induces the production of: NO cGMP Protein kinase G (PKG) ET-1 DAG Protein kinase C (PKC) -MSH, ACTH, PGE2 cAMP Protein kinase A (PKA)

Hormones (non classical pathway)

cAMP Mitogen activated protein kinase (MAPK)

Inflammation Inositol 1,4,5 triphosphate MAPK/PKC

5.1.7. Skin lightening:

Skin lightening effect is brought about effectively by a synchronized combination of

various biological mechanisms in skin cells. Main targets like Tyrosinase inhibition and

Melanogenesis inhibition when supported by Antioxidant and Anti inflammatory

mechanisms exert a positive effect on skin cells, and that is a crucial step in creating or

maintaining light pigmented healthy and conditioned skin.

5.1.7.1. Major targets for skin lightening:

Given the complexity of skin and the pathways involved in regulating melanogenesis, one

can assume that stimulating or inhibiting more than one pathway affected by stress would

lead to synergistic effects in increasing or decreasing pigmentation. Shedding light on the

molecular mechanisms underlying hyperpigmentation induced by internal or external

factors, research can be applied to various ends like finding new technologies or

compounds that could decrease pigmentation. Fairness products are expected to rectify

stress related abnormalities in pigmentation mechanism as describe in Table 5.1.1.

Therefore, understanding the mechanisms by which different compounds affect

melanogenesis is of great interest pharmaceutically and cosmeceutically. Table 5.1.3

summarizes the major targets for skin lightening. Table 5.1.4 summarizes the melanin

inhibitory pathways that result in adverse effects.


206

5.1.7.1.1. Peptide hormone (Melanocyte stimulating hormone - MSH) inhibition for

skin lightening:

The Melanocyte-stimulating hormones are a class of peptide hormones that in nature are

produced by cells in the intermediate lobe of the pituitary gland. They stimulate the

production and release of melanin (melanogenesis) by melanocytes in skin and hair.

An increase in MSH will cause a darkening in humans. Melanocyte-stimulating hormone

increases in humans during pregnancy. This, along with increased estrogens, causes

increased pigmentation in pregnant women. Melanocyte-stimulating hormone belongs to

a group called the melanocortins. This group includes ACTH, α-MSH, β-MSH and γ-

MSH; these peptides are all cleavage products of a large precursor peptide called pro-

opiomelanocortin (POMC). α-MSH is the most important melanocortin for pigmentation.

Hence, inhibition of α-MSH is one of the major targets for skin lightening.


207

Table 5.1.3: Major Targets for skin lightening

Target Mechanism Remarks

Peptide hormone (MSH) inhibition

MSH induces melanogenesis in melanocytes.

Inhibitors of MSH induced melanogenesis are potential skin lighteners.

Tyrosinase inhibition

Tyrosinase expression results in melanin formation.

Inhibitors of Tyrosinase are potential skin lighteners.

Protease inhibition Serine proteases induce pigmentation through inflammation.

Inhibitors of serine proteases (elastase, collagenase and hyaluronidase) have potential for skin lightening.

Anti inflammatory potential

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

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

Antioxidant potential

Free radical damage induces pigmentation.

Antioxidants have skin lightening potential.

UV protection UV induces pigmentation through NO and free radical induction.

UV protectants inhibit NO and ROS induced melanognesis.

cAMP induced melanogenesis

cAMP upregulates melanin production by PKC pathway.

Inhibitors of cAMP induced melanogenesis have potential for skin lightening

Melanin transfer in melanocyte-keratinocyte coculture

Endothelin induces transfer of melanin from melanocytes to keratinocytes

Endothelin antagonists inhibit melanin migration to upper keratinocyte layers. Hence inhibitors of melanin migration in cocultures are good skin lighteners.

MITF activation. Upregulation of MITF expression mediates melanogenesis stimulated by cAMP.

Inhibitors of MITF have good skin lightening potential.


208

Table 5.1.4: Melanin inhibitory targets that result in adverse effects

Target Mechanism Remarks

Phenylalanine hydroxylase inhibition

Phenylalanine hydroxylase catalyses the formation of tyrosine which is the precursor of melanin.

Inhibitors of Phenylalanine hydroxylase inhibit the melanin formation pathway. Tyrosine is required for other metabolic pathways. Inactive phenylalanine hydroxylase results in disorders like phenylketonuria etc.

Inhibition of tyrosinase glycosylation

Tyrosinase gets glycosylated in the endoplasmic reticulum and gets into its active form.

Inhibition of glycosylation of tyrosinase results in the formation of inactive tyrosinase that cannot catalyze the formation of melanin. Inhibition of tyrosinase glycosylation results in albinism.

5.1.7.1.2. Tyrosinase inhibition for skin lightening:

Tyrosinase (monophenol, l-dopa:oxygen oxidoreductase, EC 1.14.18.1) is a copper-

containing enzyme present in plant and animal tissues that catalyzes the production of

melanin and other pigments by oxidation of phenols such as tyrosine. Tyrosinases from

different species are diverse in terms of their structural properties, tissue distribution and

cellular location. Human tyrosinase is a transmembrane protein. In humans, tyrosinase is

sorted into melanosomes and the catalytically active domain of the protein resides within

melanosomes. Only a small enzymatically non-essential part of the protein extends into

the cytoplasm. As described earlier and represented in fig, the gene for Tyrosinase is

regulated by the Microphthalmia-associated transcription factor (MITF). Preventing

the maturation or intracellular trafficking of tyrosinase is an alternative way to reduce the

effect of the enzyme on pigmentation (Halaban R et al., 1983; Petrescu S M et al., 1997

and Francis E et al., 2003). Various natural extracts can also influence tyrosinase mRNA

at the transcription level; also mRNA of the other tyrosinase-related proteins or MITF

can be affected (Lee M H et al., 2006; Kim J H et al., 2008 and Zi S X et al., 2009).

Hence, Inhibition of Tyrosinase and/or MITF is one of the major targets for skin

lightening.


209

Figure 5.1.7: Metabolic pathway of Tyrosine conversion to Melanin

Pigmentation is a multistep process critically dependent on the functional integrity of

tyrosinase, the rate-limiting enzyme in melanin synthesis. As illustrated in Fig. 5.1.7,

biosynthesis of melanin is initiated by the catalytic oxidation of tyrosine to 3,4 dihydroxy

phenylalanine (dopa) by tyrosinase. Subsequent reactions happen spontaneously where

tyrosine catalyzes the dehydrogenation of dopa to dopaquinone and 5,6-dihydroxyindole

to indole-5,6-quinone, key reactions in melanin biosynthesis (Fitzpatrick T B et al., 1949;

Hearing V J and Ekel T M, 1976; Korner A and Pawelek J, 1982 and Tripathi R K et al.,


210

1992), eventually resulting in the synthesis of melanin. The amino acid sequences

deduced from human and mouse tyrosinase (TYR and Tyr, respectively) cDNAs predict a

type I membrane glycoprotein with an N-terminal signal sequence and catalytic copper

binding regions with conserved positions of histidine and cysteine residues (Kwon B S et

al., 1987; Kwon B S et al., 1989; Muller G et al., 1988; Yamamoto H et al., 1989 and

Bouchard B et al., 1989). The 60-kDa tyrosinase core polypeptide is modified in the

endoplasmic reticulum (ER) by cotranslational addition of multiple N-linked glycans,

producing the 70-kDa species (Halaban R et al., 1983 and Halaban R et al., 1984).

Complex sugar modifications in the Golgi apparatus further increases tyrosinase's

molecular mass to 80 kDa, the size of the mature wild-type (WT) isoform (Halaban R et

al., 1983; Halaban R et al., 1984 and Halaban R et al., 1997). In normal melanocytes the

70-kDa protein eventually is released from this complex and proceeds to the Golgi

apparatus en route to the melanosomes, the site of melanin synthesis. Tyrosinase is a

melanocyte-specific enzyme critical for the synthesis of melanin, a process normally

restricted to a post-Golgi compartment termed the melanosome. Therefore, inhibition of

tyrosinase activity but not the inhibition of tyrosinase formation at the molecular level is

a major target for skin lightening.

Loss-of-function mutations in tyrosinase are the cause of albinism, demonstrating

the importance of the enzyme in pigmentation. Mutations in tyrosinase are the cause of

classic type I oculocutaneous albinism, an autosomal recessive genetic disorder

characterized by the absence of melanin in melanocytes (Oetting W S and King R A,

1999). Trafficking of albino tyrosinase from the endoplasmic reticulum (ER) to the Golgi

apparatus and beyond is disrupted. Albinism, at least in part, is an ER retention disease.

Mutant proteins, representatives of the albino phenotype, are retained in the ER bound to

calnexin and calreticulin and are not released to the targeted organelle, the melanosome.

Albinism is a disease associated with retention of malfolded protein in the ER that

includes cystic fibrosis and emphysema (Callea F et al., 1992; Sifers R N, 1995;

Kuznetsov G and Nigam S K, 1998 and Kopito R R, 1999). TYR(R402Q)/Tyr(H402A)

gene mutations behaved like the much-studied CFTR(ΔF508) mutation that is responsible

for the large majority of cases of cystic fibrosis (Kopito R R, 1999), and the model

trafficking thermosensitive protein vesicular stomatitis virus G protein (tsO45 strain)


211

(Presley J F et al., 1997). Curcumin and its derivative (tetrahydrocurcumin) are reported

to have a very significant tyrosinase inhibitory activity. Curcumin and its derivatives are

also reported to rescue for CFTR (ΔF508) mutation for the treatment of cystic fibrosis

(Lipecka J et al., 2006 and Patent No: 7521580). This indicates that although cucumin

and its derivatives do inhibit tyrosinase, they do not have any effect on the gene and

protein mechanisms responsible for normal tyrosinase formation and in fact they could

probable have a positive effect on the molecular events resulting in albinism.

5.1.7.1.3. Serine protease inhibition for skin lightening:

Serine proteases or serine endopeptidases are proteases (enzymes that cut peptide bonds

in proteins) in which one of the amino acids at the active site is serine. Serine protease

activated receptor, PAR-2 regulates pigmentation by affecting keratinocyte phagocytosis.

PAR-2 activation increases the ability of keratinocytes to ingest melanosomes, resulting

in skin darkening. Inhibition of PAR-2 activation by serine protease inhibitors reduces

pigment transfer and leads to depigmentation. Inhibition of PAR-2 activation also

prevents UVB induced pigmentation and reduces tanning. Protease activated receptor 2

(PAR-2) is important for melanosomal transfer from melanocytes to keratinocytes and

this transfer can be used as a target for skin lightening (Sharlow E R et al., 2000; Seiberg

M et al., 2000 and Seiberg M, 2001). Hence, Serine protease inhibition is also one of the

targets for skin lightening.

5.1.7.1.4. Inhibition of free radicals and inflammation for skin lightening:

Free radical damage can also induce pigmentation. Free radicals generated in the body

due to stress conditions like UV exposure, pollution, unhealthy food habits and ageing,

primarily damage the skin. As described in detail earlier, free radicals trigger

inflammatory markers that eventually cause skin damage. As a result, excess melanin is

produced in a defense mechanism, resulting in pigmentation. Hence, antioxidant and anti

inflammatory properties are desirable for effective skin lightening. Topically-applied

antioxidants do have merit for all skin types to keep skin healthy and help prevent sun

damage and improve cell function. Antioxidants have been conclusively shown to exert a

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Seiberg%20M%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Seiberg%20M%22%5BAuthor%5D�


212

positive effect on reducing skin irritation and inflammation, and that is a crucial step in

creating or maintaining healthy, vibrant skin and, therefore potentially reducing wrinkles. Hence, Anti oxidant and anti inflammatory actives play a significant role in healthy skin

(Rasik A M and Shukla A, 2000 and Kalka K et al., 2000). Although all antioxidant and

anti inflammatory actives do not necessarily inhibit melanin synthesis directly, they do

have a positive synergistic effect for skin lightening. For example, Glutathione is a

significant antioxidant and not a direct inhibitor of melanin synthesis. However, when

taken internally as a nutricosmetic, it helps in skin lightening.

5.1.7.1.5. UV protection to reduce skin darkening:

As described in detail earlier and in the illustrations in Fig. 5.1.8, UV exposure leads to

free radical damage and excessive pigmentation due to the migration of mature

melanosomes from melanocytes to keratinocytes, as a defense mechanism. Hence, UV

protection is important to prevent skin darkening.

A B A) Transfer of mature melanosomes to keratinocytes B) Melanocyte surrounded by keratinocytes, melanin synthesis and release of melanin granules in keratinocytes Figure 5.1.8: Melanin synthesis in melanocytes and transfer to keratinocytes

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Rasik%20AM%22%5BAuthor%5D�

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http://www.ncbi.nlm.nih.gov/pubmed?term=%22Kalka%20K%22%5BAuthor%5D�


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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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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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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.


216

5.1.9. Objectives of the research work:

• Screening of various actives through in vitro mechanisms for reducing

hyperpigmentation.

• Screening of antioxidants, anti inflammatory and skin conditioning actives

unexplored earlier for skin lightening efficacy through in vitro mechanisms for

reducing hyperpigmentation.

• Positioning the screened actives in accordance to their specific mode of action for

rectifying pigmentation disorders as

Inhibitors of solar lentiges, melasma and over all skin tanning by inhibitors of

tyrosinase enzyme and melanogenesis.

Inhibitors of UV and free radical induced pigmentation by UV protectants and

antioxidants.

Inhibitors of post inflammatory hyperpigmentation like acne marks etc. by anti

inflammatory actives.

Inhibitors of one or more of the above mechanisms of hyperpigmentation

conditions.

Cell proliferation and collagen enhancers.

• Demonstration of synergistic skin lightening effect by physical combination and

chemical conjugation of actives with different mechanisms of action.

• Study of Nutricosmetic potential of antioxidant plant actives, synergistic

antioxidant compositions and nutricosmetic formulations.

CHAPTER 1 PART II 5.2. MATERIALS AND METHODS

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5.2.1. Materials:

5.2.1.1. Cell lines:

Swiss 3T3 mouse fibroblast cells and B16F1 mouse melanoma cells were procured from

ATCC, Manassas, VA, USA. Normal human dermal fibroblasts (NHDF) were obtained

from PromoCell GmbH, Heidelberg, Germany. Human Osteosarcoma cell lines (HOS)

were obtained from National Center for Cell Science (NCCS), Pune.

5.2.1.2. Culture media, reagents and cell culture microplates:

Dulbecco’s minimum essential medium (DMEM), RPMI 1640 medium, α-Melanocyte

stimulating hormone (α-MSH), 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), 2,7,

dichlorofluorescin diacetate, Ferrous sulphate, 2,2′ -Azobis(2-methylpropionamidine)

dihydrochloride (AAPH), 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid

(trolox), Fluorescein sodium salt (3’,6’-dihydroxy-spiro[isobenzofuran-1[3H], 9’[9H]-

xanthen]-3-one), Hydrogen peroxide solution, Cobalt (II) fluoride tetrahydrate, Picolinic

acid, Gallic acid, Hyaluronic acid potassium salt from from human umbilical cord,

Hyaluronidase from bovine testes, Cetyl pyridinium chloride, Lipopolysaccharide (LPS),

Picric acid, Sirius Red stain and Dimethylsulphoxide (DMSO) were procured from Sigma,

St. Louis MO., USA. NHDF growth medium was obtained from PromoCell GmbH,

Heidelberg, Germany. Foetal bovine serum (FBS) was procured from Gibco, New York,

USA. EnzChek collagenase inhibiton kit and EnzChek elastase inhibiton kit was obtained

from Molecular Probes, Life Technologies Corporation, California, USA. Tumor necrosis

factor (TNF) α Elisa kit was obtained from R&D systems Inc, Minneapolis, USA.

Forskolin was obtained from the Phytochemistry department of Sami Labs. Neutral red

stain was procured from Himedia Laboratories, Mumbai, India. 96 well and 24 well clear

microplates and 96 well black plates were procured from BD Biosciences, New Jersey,

USA.

5.2. MATERIAL AND METHODS


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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 µW cm-2 were obtained from

Sankyo Denki Co., Ltd, Japan and used as the source of UV irradiation.

5.2.1.4. Test materials:

Ascorbic acid, Octylmethoxycinnamate (OMC), Tetrahydrocurcumin, Glabridin,

Artocarpin, Artocarpus lakoocha heart wood extracts containing varying concentrations

of Oxyresveratrol, Dihydro-oxyresveratrol, Resveratrol, Pterostilbene, 3-Hydroxy

Pterostilbene, Gnetol, Amla extract, Hydroxychavicol, Citrullus colocynthis extract, Oat

ceramides, Apple ceramides, liquid endosperm of Coconut, Galanga extract and

Pomergranate fruit and rind extracts were obtained as mentioned in Chapter 4 Part II,

4.2.1.4.

Arbutin: Arbutin (4-Hydroxyphenyl-β-D-glucopyranoside or Hydroquinone β-D-

glucopyranoside) is off white colored water soluble powder obtained from Sigma

chemicals and used for validation studies in the present research.

Kojic acid: Kojic acid (2-Hydroxymethyl-5-hydroxy-γ-pyrone, 5-Hydroxy-2-

hydroxymethyl-4H-4-pyranone) is white colored water soluble powder obtained from

Sigma chemicals and used for validation studies in the present research.

Coenzyme Q10 (Co Q10): Co Q10 also known as Ubiquinone 10 is yellow colored

powder and an endogenous antioxidant obtained from Sigma chemicals and used for

studies in combination with other actives.

Piperlongumine: Piperlongumine was isolated by the ethanolic extraction of Piper

longum roots.

Thymohydroquinone: Thymohydroquinone was isolated from Nigella sativa (Black

cumin) seed extract by alcoholic extraction.


219

Eugenia jambolana (Jamun) extract: Jamun extract was made by the hydroalcoholic

extraction of jamun fruit pulp.

Avenanthramides: Different types of Avenanthramides (Av-A, Av-B, Av-C) were

isolated from Avena sativa (Oat) seed kernels by hydroalcoholic extraction.

Asiaticosides: Centella asiatica extract contining Asiaticosides was isolated by the

ethanolic extraction of Centella asiatica plant.

Oleanolic acid: Oleanolic acid was isolated by the ethanolic extraction of Salvia

officinalis (Salvia) leaves.

Soya isoflavones: Soya bean extract containing 40% Soya isoflavones, genistein and

daidzein.

Tetrahydropiperine (THP): THP was obtained from the chemistry dept. of Sami Labs

Limited.

Coriandrum sativum (Coriander) seed oil: Coriander seed oil from Coriandrum sativum

seeds was extracted by carbondioxide by super critical fluid extraction.

Nelumbo nucifera (Lotus) seed extract: Lotus seed extract was prepared by water

extraction of lotus seeds.

Coffea arabica (Coffee) bean extract: Coffee bean extract containing chlorogenic acid

was prepared by water extraction of coffee beans.

Theobroma cacao (Cocoa) bean extract: Cocoa bean extract containing polyphenols

was prepared by water extraction of Cocoa beans.

Camellia simensis (Green tea) extract: Green tea extract containing polyphenols was

prepared by water extraction of Green tea leaves.


220

Vitis vinifera (Grape) seed extract: Grape seed extract containing polyphenols was

prepared by water extraction of Grape seeds.

Rosmarinic acid: Rosmarinic acid was obtained by hydroalcoholic extraction of

Rosmarinus officinalis leaves.

Saffron: Saffron is prepared by the alcoholic extraction of Crocus sativus flowers.

Ocimum sanctum (Tulsi) extract: Tulsi extract was prepared by hydroalcoholic

extraction of Tulsi leaves.

Morinda citrifolia (Indian mulberry) extract: Mulberry extract was prepared by

ethanolic extraction of Mulberry fruits.

Garcinol: Garcinol is isolated by alcoholic extraction of Garcinia cambogia fruits.

Mangostin: Mangostin is isolated by alcoholic extraction of Garcinia mangostana fruits.

Acetyl-11-keto-beta-boswellic acid (AKBBA): AKBBA was obtained by solvent

extraction of Boswellia serrata gum resin.

Bacillus coagulans culture supernatant: During the expontential phase of the growth of

Bacillus coagulans, the culture medium was taken in aseptic conditions and centrifuged

to remove the cell debris. The culture supernatant thus obtained was used in the present

study.

Oleanoyl peptide: Oleanoyl peptide is the pentapeptide conjugate of oleanolic acid and

was chemically synthesized by conjugating Oleanolic acid to Lys-Thr-Thr-Lys-Ser

pentapeptide. Similaryl a peptide of Thiodipropionic acid and Lys-Thr-Thr-Lys-Ser

pentapeptide was made by chemical conjugation. These pentapeptide conjugates were

used for study of efficacy of actives in chemical conjugation with each other. A

conjugate of Kojic acid with Acetyl-11-keto-beta-boswellic acid (AKBBA) and a


221

conjugate of Kojic acid with Oleanolic acid were also made by chemical synthesis and

used for study of efficacy of actives in chemical conjugation with each other.

5.2.2. Methods:

5.2.2.1. Cell culture:

Swiss 3T3 fibroblast cells and B16F1 mouse melanoma cells were cultured in DMEM

supplemented with 10% FBS. Normal Human dermal fibroblasts (NHDF) were cultured

in NHDF growth medium supplemented with 2% FBS. The confluent cultures are

harvested by trypsinization and expanded during two more passages before they were

used for the experiments. Medium and other culture components were renewed after 48–

72 h. All cell cultures were maintained in a humidified atmosphere at 37°C in 95% air

and 5% CO2. Experiments were conducted on 24 hour monolayers of cell cultures which

were obtained by incubating the cells seeded in 96 well plates in a humidified atmosphere

at 37°C in 95% air and 5% CO2 in ThermoForma CO2 incubator for 24 hours.

5.2.2.2. Sample preparation for animal cell based assays:

Samples were prepared in appropriate vehicle. Water soluble samples were prepared in

double distilled autoclaved sterile water. Samples not soluble in water were prepared in

DMSO. DMSO was used at 0.5 to 1% in the appropriate growth medium, where there

was no effect of DMSO on the growing cells. All the prepared samples in the appropriate

vehicle were sterilized by passing through 0.22µm filter, before sample treatment to the

cells. Samples were used at non cytotoxic concentrations. The data obtained within the

maximal non cytotoxic concentrations and the maximal efficacy obtained is represented

in the results.


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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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223

IC50 value is the concentration required for 50% inhibition of the tyrosinase activity and

hence, lower IC50 value indicates better tyrosianse inhibitory potential.

5.2.2.3.2. Inhibition of α-MSH induced melanogenesis in B16F1 mouse melanoma

cell line:

Melanin synthesis can be directly studied in live animal cells. B16F1 mouse melanoma

cells were seeded in a 6 well microtiter plate at a seeding density of 5000 cells per well in

2ml DMEM medium per well. After 24 hours of incubation in a CO2 incubator, melanin

production is induced by 0.6nM ∝-MSH by replacing the medium with medium

containing ∝-MSH. The cells were then treated with varying concentrations of sample

over a period of 9 days with renewal of ∝-MSH containing medium and sample at

regular intervals of 3 days. Control wells were maintained without sample treatment and

only with the vehicle used for sample preparation. After the incubation period, the

medium was removed and the cells were scraped and washed in PBS. Thereafter, melanin

was extracted by 1N NaOH in boiling water bath for 5 minutes. The absorbance of the

melanin extract was read at 405nm in a microplate reader (Chamberlin et al., 2004). The

inhibitory effect of the sample is calculated based on the decrease of melanin formation.

A B C A) B16F1 mouse melanoma cells; B) α-MSH induced melanogenesis in B16F1 mouse melanoma cells; C) Reduction in α-MSH induced melanogenesis in B16F1 mouse melanoma cells on sample treatment Figure 5.2.2: α-MSH induced melanogenesis in B16F1 mouse melanoma cells


224

The dose dependent inhibitory activity of samples is calculated and the results are

expressed as IC50 values using Graphpad prism software. The percentage of inhibition of

melanin is calculated as follows,


Where C = absorbance due to melanin in the absence of inhibitor

T = absorbance due to melanin in the presence of inhibitor

IC50 value is the concentration required for 50% inhibition of the melanin formation and

hence, lower IC50 value indicates better melanin inhibitory potential.

5.2.2.3.3. Inhibition of cAMP induced melanogenesis in B16F1 mouse melanoma cell

line:

Cyclic adenosine monophosphate (cAMP) is another inducer of melanin synthesis.

Forskolin which is known to induce melanin through cAMP pathway was used to induce

melanin in B16F1 mouse melanoma cells. B16F1 cells were seeded in a 6 well microtiter

plate at a seeding density of 5000 cells per well in 2ml DMEM medium per well. After

24 hours of incubation in a CO2 incubator, melanin production is induced by 7.3µM

forskolin though cAMP pathway by replacing the medium with medium containing

forskolin. The cells were then treated with varying concentrations of sample over a period

of 9 days with renewal of forskolin containing medium and sample at regular intervals of

3 days. Control wells were maintained without sample treatment and only with the

vehicle used for sample preparation. After the incubation period, the medium was

removed and the cells were scraped and washed in PBS. Thereafter, melanin was

extracted by 1N NaOH in boiling water bath for 5 minutes. The absorbance of the

melanin extract was read at 405nm in a microplate reader. The inhibitory effect of the

sample is calculated based on the decrease of melanin formation.


225

A B C A) B16F1 mouse melanoma cells; B) cAMP induced melanogenesis in B16F1 mouse melanoma cells; C) Reduction in cAMP induced melanogenesis in B16F1 mouse melanoma cells on sample treatment Figure 5.2.3: cAMP induced melanogenesis in B16F1 mouse melanoma cells

The dose dependent inhibitory activity of samples is calculated and the results are

expressed as IC50 values using Graphpad prism software. The percentage of inhibition of

melanin is calculated as follows,


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


226

the absence of protection. A control plate was also maintained under similar conditions

without UV exposure which can only give observations on the cytotoxic potential of the

sample. For each concentration, 6 replicates were maintained and the analysis was

performed twice such that the ‘n’ value is 12. After UV exposure, the medium was

replaced with fresh medium without sample and the cells were incubated in a CO2

incubator for 48 hrs. The cells were then developed by NRU staining technique to

analyze the cell viability. The cells were incubated with 0.003% solution of neutral red

prepared in pre warmed DMEM medium for 3 hrs at 370C in CO2 incubator. The excess

dye was then washed off with phosphate buffer saline (PBS). The lysosomal dye was

extracted in 100µl of developer solution consisting of 25ml of water, 24.5ml of ethanol

and 0.5ml of glacial acetic acid at RT for 20 min. The optical density (OD) was read at

492 nm using a microplate reader.

The percentage reduction in UV induced cytotoxicity i.e., the percentage of UV

protection was calculated with respect to the cytotoxicity in exposed cells as compared to

that of the unexposed cells in the presence and absence of sample.

A B A) Swiss 3T3 mouse fibroblasts exposed to UV showing cell death; B) Sample treated Swiss 3T3 mouse fibroblasts exposed to UV showing no cell death Figure 5.2.4: UV protection in Swiss 3T3 mouse fibroblast cells


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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.


229

The percentage of scavenging is calculated as follows,

% scavenging = [(C-T) / C] X 100

Where C = absorbance in the absence of inhibitor

T = absorbance in the presence of inhibitor

SC50 value is the concentration required for 50% scavenging of free radicals and hence,

lower SC50 value indicates better antioxidant potential.

5.2.2.4.2. Oxygen Radical Absorbance Capacity (ORAC):

Oxygen Radical Absorbance Capacity (ORAC) antioxidant Assay can be used to

determine the total antioxidant capacity of biological fluids, cells, and tissue. It can also

be used to assay the antioxidant activity of naturally occurring or synthetic compounds

for various applications. The assay measures the loss of fluorescein (3’,6’-dihydroxy-

spiro[isobenzofuran-1[3H], 9’[9H]-xanthen]-3-one) fluorescence over time due to

peroxyl-radical formation by the breakdown of 2,2’-azobis-2-methyl-propanimidamide,

dihydrochloride (AAPH). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid

(Trolox), a water soluble vitamin E analog, serves as a positive control inhibiting

fluorescein decay in a dose dependent manner. The ORAC assay is a kinetic assay

measuring fluroescein decay and antioxidant protection over time. The antioxidant

activity of samples can be normalized to equivalent Trolox units to quantify the

composite antioxidant activity present.

A peroxyl radical (ROO-) is formed from the breakdown of AAPH at 37 °C.

The peroxyl radical can oxidize fluorescein to generate a product without fluorescence.

Antioxidants supress this reaction by a hydrogen atom transfer mechanism, inhibiting the

oxidative degradation of the fluorescein signal. The fluorescence signal is measured over

30 minutes by excitation at 485 nm, emission at 538 nm. The concentration of antioxidant

in the test sample is proportional to the fluorescence intensity through the course of the

assay and is assessed by comparing the net area under the curve to that of a known

antioxidant, trolox.


230

AAPH → ROO-

Fluorescein ---------------------→ Non fluorescent product

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

Antioxidant capacity relating to trolox = Sum sample - Sum blank / Sum standard - Sum blank

Figure 5.2.6: Priniciple of ORAC assay

Varying concentrations of sample in suitable vehicle (PBS or 0.03% DMSO that does not

afftect the fluorescence intensity) were pipetted into each well of a black microplate

containing 10X10-2M 2,2′ -Azobis(2-methylpropionamidine) dihydrochloride (AAPH)

made in 75mM potassium phosphate buffer (pH 7.4) and 4.8X10-7M disodium

fluorescein dye. 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox)

standard from 12.5 – 200µM was also kept under similar conditions. Fluorescence

readings were taken in a Fluostar Optima Microplate Reader at 485/520nm after every 1

minute for 35 minutes (f1……..f35). The final ORAC values were calculated by using a

quadratic regression equation (Y = a + bX + cX2) between the trolox concentration (Y)

ROS

Fluorescent probe +

buffer

Fluorescent probe +

trolox

Fluorescent probe +

sample

Loss of fluorescence Loss of fluorescence Loss of fluorescence

Sum blank Sum standard Sum sample


231

(µM) and the net area under the Fluorescence decay curve (X) and were expressed as

micromoles of trolox equivalents per gram (TE/g) or liter of sample.

The Area under curve AUC = (1 + f1/f0 + f2/f0 + …. + f35/f0.) –------- eq 1

Where f0 is the initial fluorescence reading at 0 min and f1 is the fluorescence reading

after 1min.

The data were analyzed by applying eq 1. The net AUC was obtained by subtracting the

AUC of the blank from that of the sample. The value calculated using the net AUC of the

sample and the quadratic regression equation was divided by the weight of the sample in

grams or liter (Ou B et al., 2001). Higher ORAC value indicates better antioxidant

potential.

5.2.2.4.3. Hydroxyl Radical Averting Capacity (HORAC):

Hydroxyl Radical Averting Capacity (HORAC) is also an antioxidant assay similar to

ORAC with the only difference that HORAC is specific for Hydroxyl radicals and the

standard used is Gallic acid. Hydroxyl radical averting capacity is assessed using

fluorescein as the fluorescent probe. The hydroxyl radical is generated by a Cobalt Co

(II)-mediated reaction. The fluorescent decay curve of fluorescein dye is monitored in the

presence and absence of the inhibitor and the area under curve (AUC) is integrated. Net

AUC is calculated which is an index of hydroxyl radical is averting capacity which is

expressed in Gallic acid equivalents per gram (GAE/g) of test compound.

Varying concentrations of sample in suitable vehicle (PBS or 0.009% DMSO that

does not afftect the fluorescence intensity) were pipetted into each well containing

0.09µM disodium fluorescein dye. Immediately 20µl of 30% H2O2 is added into all wells,

and initial fluorescence reading (F0) is taken at 485/520 ex/em wavelength. Then 5µl of

219.79 µM cobalt solution and 522 µM Picolinic acid was pipetted into all the wells and

the fluorescence readings are taken immediately until 35mins (F1, F2, F3------------F35). Gallic

acid standard was also kept under similar conditions. Fluorescence readings were taken

in a Fluostar Optima Microplate Reader at 485/520nm. The final HORAC values were

calculated by using a quadratic regression equation (Y = a + bX + cX2) between the Gallic

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232

acid concentration (Y) (µM) and the net area under the Fluorescence decay curve (X) and

were expressed as micromoles of Gallic acid equivalents per gram or liter of sample.

The Area under curve AUC = (1 + f1/f0 + f2/f0 + …. + f35/f0.) ------ eq 1

Where f0 is the initial fluorescence reading at 0 min and f1 is the fluorescence reading

after 1min.

The data were analyzed by applying eq 1. The net AUC was obtained by subtracting the

AUC of the blank from that of the sample. The value calculated using the net AUC of the

sample and the quadratic regression equation was divided by the weight of the sample in

gram or liter. The final value obtained is the HORAC value of the sample expressed as

µmol GAE/g (Ou B et al., 2002). Higher HORAC value indicates better antioxidant

potential.

5.2.2.4.4. Reactive Oxygen Species (ROS) scavenging potential:

The generation processes of reactive oxygen species can be monitored using the

luminescence analysis or also fluorescence methods . The intracellular ROS generation of

cells can be investigated using the 2’,7’-dichlorfluorescein-diacetate (DCFH-DA) as a

well-established compound to detect and quantify intracellular produced H2O2 (Cathcart

R et al., 1983). The conversion of the nonfluorescent 2’,7’ - dichlorfluorescein – diacetate

(DCFH-DA) to the highly fluoresecent compound 2’,7’-dichlorfluorescein (DCF)

happens in several steps. First, DCFH-DA is transported across the cell membrane and

deacetylated by esterases to form the non-fluorescent 2’,7’-dichlorfluorescein (DCFH).

This compound is trapped inside of the cells. Next, DCFH is converted to DCF through

the action of peroxid rated by the presence of peroxidase (LeBel C P et al., 1992). Swiss

3T3 mouse fibroblast cells were used to determine the ROS scavenging potential of

samples. The confluent cells were trypsinized and seeded in a 96 well black microplates

at a seeding density of 105 cells per well in PBS. The cells were treated with varying

concentrations of sample in suitable vehicle (PBS or 0.2% DMSO that does not afftect

the fluorescence intensity). Control cells were treated with vehicle used for sample

preparation. 100µl of 0.002% solution of 2,7-dichlorofluorescein diacetate dye was added

and ROS generation was enhanced by subjecting the cells to a chemical stress using

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233

2.5µM FeSO4. After incubating the cells for 1 hour at 37°C, the ROS generated was

determined by taking fluorescence readings measured at wavelength Ex/Em 485/520 nm

in a microplate reader.

Figure 5.2.7: Priniciple of ROS scavenging assay

The fluorescence readings are directly proportional to the ROS generated and the ROS

scavenging effect of samples was calculated as the percentage scavenging with respect to

the control cells. The dose dependent ROS scavenging activity of samples is calculated

and the results are expressed as SC50 values using Graphpad prism software. The

percentage of scavenging is calculated as follows,

% scavenging = [(C-T) / C] X 100

Where C = Fluorescence due to ROS generated in the absence of inhibitor

T = Fluorescence due to ROS generated in the presence of inhibitor

SC50 value is the concentration required for 50% scavenging of ROS and hence, lower

SC50 value indicates better antioxidant potential.


234

5.2.2.5. Anti inflammatory potential:

The following methods are used for screening actives that can inhibit melanin formation

indirectly by inhibiting the inflammatory stress,

5.2.2.5.1. Collagenase inhibitory potential:

Collagenase inhibitory potential of samples was determined by using Molecular Probes

EnzChek® Collagenase Assay Kit that provides high sensitivity required for screening

inhibitors in a high-throughput format. The EnzChek kit contains DQgelatin, fluorescein

conjugated gelatin. This substrate is efficiently digested by most of the gelatinases and

collagenases to yield highly fluorescent peptides. The increase in fluorescence is

proportional to proteolytic activity and can be monitored with a fluorescence microplate

reader. The reduction in fluourescence is directly proportional to the collagenase

inhibitory activity of the sample. Collagenase used for the assay is purified from

Clostridium histolyticum. Using 100 µg/mL DQ gelatin and a 30 minute incubation

period, the assay can detect the activity of this enzyme down to a final concentration of 2

× 10-3 U/mL (7 ng protein/mL), where one unit is defined as the amount of enzyme

required to liberate 1 µmole of L-leucine equivalents from collagen in 5 hours at 37°C,

pH 7.5. Varying concentrations of sample in suitable vehicle (PBS or 2% DMSO that

does not afftect the fluorescence intensity) were pre-incubated for 10 minutes with 12.5

µg/ml substrate, DQ gelatin (from pig skin), fluorescein conjugate and then 0.2U/ml of

Collagenase Type IV from Clostridium histolyticum enzyme was added. The

fluorescence intensity was measured after 30 minutes (Em: 485nm and Ex: 520nm.) in

microplate reader. The dose dependent inhibitory activity of samples is calculated and the

results are expressed as IC50 values using Graphpad prism software. The percentage of

inhibition of collagenase is calculated as follows,


Where C = absorbance due to collagenase activity in the absence of inhibitor

T = absorbance due to collagenase activity in the presence of inhibitor


235

IC50 value is the concentration required for 50% inhibition of the collagenase activity and

hence, lower IC50 value indicates better collagenase inhibitory potential.

5.2.2.5.2. Elastase inhibitory potential:

Elastase inhibitory potential of samples was determined by using Molecular Probes

EnzChek® Elastase Assay Kit that provides high sensitivity required for screening

inhibitors in a high-throughput format. The EnzChek kit contains DQelastin, fluorescein

conjugated soluble bovine neck ligament elastin. This substrate is efficiently digested by

elastase to yield highly fluorescent peptides. The increase in fluorescence is proportional

to proteolytic activity and can be monitored with a fluorescence microplate reader. The

reduction in fluourescence is directly proportional to the elastase inhibitory activity of the

sample. Elastase used for the assay is purified from procine pancreas.

Varying concentrations of sample in suitable vehicle (PBS or 2% DMSO that

does not afftect the fluorescence intensity) were pre-incubated for 10 minutes with the

substrate, 25µg/ml of DQ Elastin (from bovine neck ligament) fluorescein conjugate and

0.1U/ml porcine pancreatic elastase enzyme was added. The fluorescence intensity was

measured after 30 minutes (Em: 485nm and Ex: 520nm) in microplate reader. The dose

dependent inhibitory activity of samples is calculated and the results are expressed as

IC50 values using Graphpad prism software. The percentage of inhibition of elastase is

calculated as follows,


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.


236

5.2.2.5.3. Hyaluronidase inhibitory potential:

Hyaluronic acid when incubated with hyaluronidase enzyme solution in the presence and

absence of inhibitor and the unreacted hyaluronic acid gets precipitated with cetyl

pyridinium chloride. The precipitate blocks the transmittance and therefore a decrease in

the absorbance correlates with the amount of digested hyaluronic acid. Hyaluronidase

specifically cleaves the β1,4-glycosidic bond of hyaluronic acid. Hyaluronic acid

substrate (0.3%) was made in 300mM sodium phosphate buffer pH 5.35. Hyaluronidase

(10U/ml) was made in 20mM Sodium phosphate Buffer pH 7.00. Hyaluronidase enzyme

and various concentrations of the sample in suitable vehicle (PBS or 0.1% DMSO that

does not afftect the fluorescence intensity) are pre-incubated at 370C for 10 min. Then the

hyaluronic acid substrate is added and the reaction mixture is incubated for 45 min at

370C. The reaction mix is added to cetyl pyridinium chloride (1%). The absorbance of

undigested hyaluronic acid is read spectrophotometrically at 600nm (Tung J S et al.,

1994). The absorbance of undigested hyaluronic acid after treatment with the sample is

directly proportional to the inhibition of hyaluronidase. The dose dependent inhibitory

activity of samples is calculated and the results are expressed as IC50 values using

Graphpad prism software. The percentage of inhibition of hyaluronidse is calculated as

follows,

% Inhibition = (EC-EA)-(EC- (T-TC)) X 100 (EC-EA)

EC – Absorbance due to undigested hyaluronic acid in the absence of enzyme and

inhibitor.

EA – Absorbance after digestion of hyaluronic acid in the presence of enzyme.

T – Absorbance due to undigested hyaluronic acid in the presence of enzyme and

inhibitor.

TC – Absorbance of the inhibitor alone.

IC50 value is the concentration required for 50% inhibition of the hyaluronidase activity

and hence, lower IC50 value indicates better hyaluronidase inhibitory potential.


237

Figure 5.2.8: Hyaluronidase activity on Hyaluronic acid


238

5.2.2.5.4. TNF α inhibitory potential:

For TNF α inhibitory study, human whole blood is used. In whole blood assay,

monocytes appear to be the main source of TNF-α on Lipopolysaccharide (LPS)

stimulation. Monocyte and macrophages are a major source of TNF-α in addition to other

cell types like eosinophils, mast cells, peripheral lymphocytes and granulocytes. The

activation of inflammatory cells is influenced by the intracellular levels of c-AMP which

are regulated by the phosphodiesterase isoenzyme. LPS is the most potent stimulus of

TNF-α production in human blood. After stimulation the assay employs the quantitative

sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-α

has been pre-coated onto a microplate. TNF-α present in the sample is bound by the

immobilized antibody. After washing away any unbound substances, an enzyme-linked

polyclonal antibody specific for TNF-α is added. Following a wash to remove any

unbound antibody-enzyme reagent, a substrate solution is added to the wells and colour

develops in proportion to the amount of TNF-α bound in the initial step. The colour

development is stopped and the intensity of the colour is directly proportional to the

TNF-α content.

Heparinized blood from healthy donors was diluted 1:3 in RPMI 1640 culture

medium containing 10% FBS. Diluted blood samples were pre-incubated with varying

concentrations of sample in suitable vehicle (PBS or 0.1% DMSO) for 1 hr at 370C in an

incubator with 5 % CO2. 0.1% DMSO was used as vehicle for water insoluble samples.

After pre-incubation, the whole blood cells were stimulated by 1ng/mL LPS for the

release of TNF α from the macrophages by incubating for 5 hr at 370C in an incubator

with 5 % CO2. The samples were then centrifuged at 3000 g for 3 minutes at 40C and the

supernatant was assayed for TNF α content by using the TNF α Elisa kit. 200µL of

supernatant from all the tubes was transferred into microtiter plate in respective wells

(Pre-coated mouse monoclonal antibody microplate) followed by the addition of 50µLof

assay diluents in all the wells. After incubation for 2 hr at room temperature, the wells

were washed thoroughly with wash buffer provided and then 200µL of conjugate was

added to each well. The plate was incubated for 2 hr at room temperature. After washing

again, 200 µL of substrate solution was added to each well and incubated for 20 minutes


239

at RT. 50µL of Stop Solution to each well which will cause the colour change in the

wells from blue to yellow. The optical density was read at 450nm which is directly

proportional to TNF α content and the percentage of inhibition of TNF α content on

treatment with sample was calculated with respect to that of the untreated cells. The dose

dependent inhibitory activity of samples is calculated and the results are expressed as

IC50 values using Graphpad prism software. The percentage of inhibition of elastase is

calculated as follows,


Where C = absorbance due to TNF α in the absence of inhibitor

T = absorbance due to TNF α in the presence of inhibitor

IC50 value is the concentration required for 50% inhibition of TNF α and hence, lower

IC50 value indicates better TNF α inhibitory potential.

5.2.2.6. Cell rejuvenation:

Cell rejuvenation does not directly influence skin lightening but in combination with

pigment inhibitory mechanism a continuous repleneshing of new and lightened skin cells

will help in giving a bright skin tone. The following methods were used to study the cell

rejuvenation potential of the samples,

5.2.2.6.1. Cell proliferation enhancement:

Swiss 3T3 mouse fibroblast cells were used for cell proliferation studies. The cells were

seeded with a seeding density of 3000 cells per well of a 96 well plate. Confluent

monolayers of Swiss 3T3 fibroblast cells were initially treated with varying non cytotoxic

concentrations of test sample and vehicle (control) in the culture medium. For each

concentration, 6 replicates were maintained and the analysis was performed twice such

that the ‘n’ value is 12. After sample treatment, the cells were incubated in a CO2

incubator for 72 hrs. The cells were then developed by NRU staining technique to

analyze the cell viability. The cells were incubated with 0.003% solution of neutral red


240

prepared in pre warmed DMEM medium for 3 hrs at 370C in CO2 incubator. The excess

dye was then washed off with phosphate buffer saline (PBS). The lysosomal dye was

extracted in 100µl of developer solution consisting of 25ml of water, 24.5ml of ethanol

and 0.5ml of glacial acetic acid at RT for 20 min. The optical density (OD) was read at

492 nm using a microplate reader (Repetto G et al., 2008). The percentage enhancement

in cell growth with respect to the untreated cells considering the OD of untreated cells as

optimal under normal conditions is calculated as follows,

% enhancement in cell growth = [(100/C) X T] – 100

Where C = absorbance due to cell growth in untreated cells

T = absorbance due to cell growth in sample treated cells

5.2.2.6.2. Scratch wound closure assay:

Swiss 3T3 mouse fibroblast cells were used for Scratch wound closure assay. The cells

were seeded with a seeding density of 20000 cells per well of a 6 well plate. Confluent

monolayers of Swiss 3T3 fibroblast cells were wounded by scratching along the diameter

of the well of the plate using a 1ml micropipette tip having about 0.2µm diameter

resulting in 0.2µm scratch width. The wounded monolayers were then treated with

varying non cytotoxic concentrations of test sample and vehicle (control) in the culture

medium. For each concentration, 2 replicates were maintained and the analysis was

performed twice such that the ‘n’ value is 4. After sample treatment, the cells were

incubated in a CO2 incubator for 72 hrs. The reduction in scratch width is then measured

using a microscopic scale (Walter M N et al., 2010).

The percentage wound closure with respect to the untreated cells is calculated as

follows,

% wound closure = [(C-T) / C] X 100

Where C = C1 – C2

T = T1 – T2

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241

Where,

C1 = scratch width at 0 hrs in untreated cells

C2 = scratch width after 72 hrs in untreated cells

T1 = scratch width at 0 hrs in sample treated cells

T2 = scratch width after 72 hrs in sample treated cells

A B A) Wounded monolayer of cells; B) Cells proliferating into the wounded area

Figure 5.2.9: Scratch wound closure assay

5.2.2.7. Collagen enhancement:

Collagen enhancement also does not directly influence skin lightening but it facilitates

the repair of skin damage due to various stress condiitons and as the skin damage gets

repaired the stress induced melanogenesis also will subside.

5.2.2.7.1. Collagen enhancement determination by Sirius Red staining:

Collagen enhancement was determined by using Sirius Red stain that binds with a greater

specificity to Collagen type I and Collagen type III of the extracellular matrix. The stain

bound to the collagen is dissolved and the optical density (OD) is measured

spectrophotometrically using a Fluostar optima microtiter plate reader at 544 nm. The

OD of the stain bound to collagen is directly proportional to the collagen content in the

cells (Tullberg-Reinert H and Jundt G, 1999). Human osteosarcoma cells from human

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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 Bouin’s fluid containing 1.3% picric

acid, 35% formaldehyde and glacial acetic acid in 15:5:1 ration by incubating with 1ml of

Bouin’s fluid per well for 1 hr at RT. The fixative is then removed by suction with

micropipette and the cells were washed under running tap water for 15 minutes. After air

drying the culture plate, the cells were stained using 0.1% Sirius red stain in 1.3% picric

acid. 1ml per well Sirius red stain was added and the cells were incubated for 1 hr under

mild shaking of 70 RPM at RT in Orbitek Shaker. The stain was then removed by suction

and the cells were extensively washed with 0.01N HCl to remove unbound dye. The dye

bound to collagen was then dissolved in 0.2ml of 0.1N NaOH per well for 30 minutes

under mild shaking of 70 RPM in Orbitek Shaker at RT. The dye was then transferred to

96 well microplate and the OD was read at 544nm in Fluostar Optima microplate reader.

The percentage enhancement in collagen with respect to the untreated cells considering

the OD of untreated cells as optimal under normal conditions is calculated as follows,

% enhancement in cell growth = [(100/C) X T] – 100

Where C = absorbance due to collagen in untreated cells

T = absorbance due to collagen in sample treated cells

5.2.2.7.2. Collagen enhancement determination by Flow cytometry:

For more sensitivity, collagen enhancement was also determined by Flow cytometry

also in Swiss 3T3 mouse fibroblast cells (Chanvorachote P et al., 2009). The cells

grown confluently in 25 cm2 flasks were washed with cold PBS and fixed for 3 minutes

with a fixing reagent containing 4% formaldehyde in PBS.


243

Cells were then washed with Tris buffer saline (TBS) and incubated in permeation

solution (1% Triton X-100 in PBS) at RT for 5 minutes. After washing with TBS, the

cells were blocked with a blocking reagent (2.5% FBS in TBS) for 30 minutes at RT

and further incubated with pro-collagen type I rabbit polyclonal antibody for 1 hour at

RT. After washing with TBST (TBS containing Tween) for 10 minutes, the cells were

incubated with secondary antibody, FITC-coupled anti-rabbit, for 1 hour with gentle

rocking at RT. They were then washed, trypsinized and resuspended in PBS and

immediately analyzed by flow cytometry using an excitation wavelength at 488nm and

emission wavelength at 520nm using FACSort, Becton Dickinson, Rutherford, NJ).

CHAPTER 1 PART II 5.3. RESULTS AND DISCUSSION

244

Approaches for skin whitening have broadened widely in the recent years. The utilization

of single agents inhibiting tyrosinase is in many cases extended to the use of complex

mixtures that target different mechanism like tyrosinase expression, melanogenesis,

antioxidant and anti-inflammatory effects (Ortonne J P and Bissett D L, 2008). Although

skin hyperpigmentation is a common concern, the causes for hyperpigmentation are not

the same and therefore, the approaches for reducing hyperpigmentation can also not be

the same. In the present study, various actives were screened through various in vitro

systems to position them specifically for various hyperpigmentation disorders.

Postitioning is broadly described as classifying skin lightening actives with respect to one

or more of the hyperpigmentation mechanisms that can be specifically rectified by them.

All the actives were screened broadly through mechanisms that directly inhibit melanin

formation and through mechanisms that indirectly influence melanin formation.

5.3.1. Screening of “actives” through various skin lightening mechanisms:

5.3.1.1. Screening of Reference standards through various skin lightening

mechanisms:

Most skin lightening products currently used contain ingredients like Arbutin,

hydroquinone, ascorbic acid, kojic acid etc. that act as direct inhibitors of tyrosinase, the

enzyme present in melanocytes, the skin pigment cells that make melanin. They are used

as reference standards for tyrosinase inhibition (Issa R A et al., 2008). However, there is

evidence to suggest that certain skin lightening actives like hydroquinone can be harmful

(Olumide Y M et al., 2008). Hydroquinone has now been banned in Europe and in many

other countries. In the present study, Arbutin, Kojic acid and Ascorbic acid are

considered as reference standards for comparative analysis with other actives.

Antioxidants play a major role in skin lightening. Tripeptide Glutathione is one such skin

lightener which exerts its efficacy through its significant antioxidant potential. It is an

oral cosmetic (nutricosmetic). It inhibits melanin induced by MSH with an IC50 of 25 ±

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

5.3. RESULTS AND DISCUSSION


245

5.3.1.1.1. Arbutin: Table 5.3.1: Skin lightening potential of Arbutin

Inhibition of melanin formation


(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

IC50 194 ± 25 IC50 100 ± 15 IC50 100 ± 18 Nil


DPPH scavenging

(µg/ml)

ROS scavenging ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 500 ± 25 Nil Nil Nil


Collagenase

inhibition

Elastase inhibition Hyaluronidase

inhibition

TNF α

inhibition

Nil Nil Nil Nil

Table 5.3.2: Effect of Arbutin on Skin pigmentation disorders Pigmentation disorder Effect Rationale

Pigmentation due to biological

imbalances of body such as over

expression of hormones and enzymes

Significant Potential inhibitor

of tyrosinase &

melanogenesis

Pigmentation due to excessive sun

exposure

No effect No significant

protection from UV

exposure

Pigmentation due to free radical

damage

No effect No significant free

radical scavenging

activity

Pigmentation due to inflammatory

responses like pimple marks, scars

due to wounds etc.

No effect

No significant anti

inflammatory

activity


246

5.3.1.1.2. Kojic Acid: Table 5.3.3: Skin lightening potential of Kojic acid



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

Induced Melanin

UV protection

IC50 7 ± 1.5 IC50 100 ± 19 Nil Nil


DPPH scavenging

(µg/ml)

ROS scavenging ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 500 ± 32 Nil Nil Nil


Collagenase

inhibition


inhibition

TNF α

inhibition

Nil Nil Nil Nil

Table 5.3.4: Effect of Kojic acid on Skin pigmentation disorders Pigmentation disorder Effect Rationale




Significant Potential inhibitor of

tyrosinase &

melanogenesis

Pigmentation due to excessive sun

exposure

No effect No significant

protection from UV

exposure


damage

No effect No significant free

radical scavenging

activity

Pigmentation due to inflammatory


due to wounds etc.

No effect

No significant anti

inflammatory

activity


247

5.3.1.1.3. Ascorbic acid: Table 5.3.5: Skin lightening potential of Ascorbic acid



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

IC50 9.33 ± 1.6 IC50 25 ± 6 IC50 100 ± 16 Not significant


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 1.93 ± 0.3 IC50 10 ± 2.3 3400 ± 220 Nil


Collagenase

inhibition


inhibition

TNF α

inhibition

Nil Nil Nil Nil

Table 5.3.6: Effect of Ascorbic acid on Skin pigmentation disorders Pigmentation disorder Effect Rationale





tyrosinase &

melanogenesis

Pigmentation due to sun exposure Not

significant

No significant

protection from UV

induced cell damage

Pigmentation due free radical damage Significant Potential scavenger

of free radicals

Pigmentation due inflammatory

responses like pimple marks, scars due

to wounds etc.

No effect No significant anti

inflammatory activity


248

5.3.1.2. Screening of “actives” known for skin lightening through various skin

lightening mechanisms:

5.3.1.2.1. Tetrahydrocurcumin (THC) from Curcuma longa root extract: Table 5.3.7: Skin lightening potential of THC



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

IC50 1.77 ± 0.3 IC50 3.2 ± 1.1 IC50 4 ± 1.3 Not significant


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 0.93 ± 0.3 IC50 1.2 ± 0.2 10,815 ± 468 2715 ± 126


Collagenase

inhibition


inhibition

TNF α inhibition

(µg/ml)

Nil Nil Nil IC50 81 ± 6

Table 5.3.8: Effect of THC on Skin pigmentation disorders Pigmentation disorder Effect Rationale





tyrosinase &

melanogenesis


significant

No significant

protection from UV

Pigmentation due free radical damage Significant Antioxidant



due to wounds etc.

Significant Significant inhibitor

of inflammation


249

5.3.1.2.2. Glabridin from Glycyrrhiza glabra (Licorice) root extract: Table 5.3.9: Skin lightening potential of Glabridin



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

IC50 0.25 ± 0.05 IC50 3.5 ± 1 IC50 3.8 ± 1.2 Not significant


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 100 ± 15 IC50 0.25 ± 0.02 7550 ± 520 1129 ± 209


Collagenase

inhibition (µg/ml)

Elastase inhibition

(µg/ml)

Hyaluronidase

inhibition

TNF α inhibition

(µg/ml)

IC50 50 ± 5 IC50 55 ± 6 Nil IC50 100 ± 9

Table 5.3.10: Effect of Glabridin on Skin pigmentation disorders Pigmentation disorder Effect Rationale





tyrosinase &

melanogenesis


significant

No significant

protection from UV

induced cell damage

Pigmentation due free radical damage Significant Potential scavenger

of free radicals



due to wounds etc.


of inflammatoty

markers and

inflammary enzymes


250

5.3.1.2.3. Artocarpin from Artocarpus lakoocha heartwood extract:

Table 5.3.11: Skin lightening potential of Artocarpin



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

(µg/ml)

IC50 1.3 ± 0.2 IC50 2.5 ± 0.2 IC50 1 ± 0.2 EC50 20 ± 4


DPPH scavenging

(µg/ml)

ROS scavenging ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 44 ± 0.2 Not significant 3859 ± 0.2 1121 ± 214


Collagenase

inhibition

Elastase inhibition

(µg/ml)

Hyaluronidase

inhibition

TNF α inhibition

(µg/ml)

Not significant IC50 8.8 ± 5 Nil IC50 90 ± 3

Table 5.3.12: Effect of Artocarpin on Skin pigmentation disorders Pigmentation disorder Effect Rationale





tyrosinase &

melanogenesis

Pigmentation due to sun exposure Significant Significant

protection from UV

induced cell damage




due to wounds etc.


of inflammation


251

5.3.1.2.4. Stilbenes from plant extracts:

Various stilbene compounds from different plant sources have been screened through

skin lightening models.

5.3.1.2.4.1 Artocarpus lakoocha heartwood extracts containing varying

concentrations of Oxyresveratrol:

The extract with higher content of Oxyresveratrol was not as effective as the extract

containing lower concentration of Oxyresveratrol with respect to melanin inhibition and

anti inflammatory activity (Table 5.3.13). Therefore, it clearly indicates that only the

combination of various actives of the extract is bringing about the significant melanin

inhibition and anti inflammatory activity. The melanogenesis inhibitory potential and anti

inflammatory potential of the Artocarpus lakoocha extract is conferred by the synergistic

combination of Oxyresveratrol & other actives of the extract like Artocarpin etc.

However, the antioxidant potential and UV protection potential of the composition from

Artocarpus lakoocha is conferred by the oxyresveratrol content in the extract as the

antioxidant and UV protection potential increased with the increasing percentage of

Oxyresveratrol (Table 5.3.13). It was also observed that higher concentrations of

Oxyresveratrol caused reduced ROS scavenging potential due to prooxidant effect of

Oxyresveratrol.

Hence, it is clearly understood by the present in vitro studies that the melanin

inhibitory and anti inflammatory potential of the Artocarpus lakoocha extract is

conferred by the synergistic combination of Oxyresveratrol & other actives of the extract

like Artocarpin etc, while the antioxidant potential and UV protection potential is

conferred exclusively by the Oxyresveratrol content in the extract. It was also observed

that hydrogenation of Oxyresveratrol to Dihydro-oxyresveratrol resulted in increase in

melanin inhibitory potential but a decrease in UV protection potential (Table 5.3.15).


252

Table 5.3.13: Skin lightening potential of Oxyresveratrol from Artocarpus lakoocha extract


Active Tyrosinase

inhibition

(µg/ml)

Inhibition of

MSH induced

Melanin (µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

(µg/ml)

Oxy 20% IC50 0.48 ± 0.055 IC50 3.45 ± 1.1 IC50 4.35 ± 1.3 EC50 100 ± 9.2

Oxy 30% IC50 0.41 ± 0.05 IC50 3.56 ± 0.9 IC50 5 ± 1.8 EC50 100 ± 9.4

Oxy 50% IC50 0.11 ± 0.023 IC50 12 ± 3.2 IC50 12 ± 2.8 EC50 75 ± 8.3

Oxy 80% IC50 0.087 ± 0.02 IC50 10 ± 2.4 IC50 11 ± 2.9 EC50 50 ± 6.1

Oxy 90% IC50 0.05 ± 0.019 IC50 12 ± 2.6 IC50 12 ± 2.7 EC50 50 ± 6.3


Active DPPH

scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

Oxy 20% IC50 3.1 ± 0.8 IC50 10 ± 2.5 8999 ± 443 4756 ± 298

Oxy 30% IC50 3.7 ± 0.92 IC50 10 ± 1.9 11,370 ± 1220 4776 ± 326

Oxy 50% IC50 2.5 ± 0.76 IC50 10 ± 2.8 15,582 ± 1236 4896 ± 428

Oxy 80% IC50 2.6 ± 0.82 Not significant 18,673 ± 1432 4923 ± 392

Oxy 90% IC50 2.7 ± 0.96 Not significant 21,549 ± 1896 5728 ± 432


Active Collagenase

inhibition

(µg/ml)

Elastase

inhibition (µg/ml)

Hyaluronidase

inhibition (µg/ml)

TNF α

inhibition

(µg/ml)

Oxy 20% IC50 15 ± 2 IC50 22 ± 4 IC50 260 ± 13 IC50 70 ± 8

Oxy 30% IC50 17 ± 4 IC50 27 ± 3 IC50 300 ± 15 IC50 55 ± 6

Oxy 50% IC50 31 ± 5 IC50 50 ± 2 IC50 250 ± 10 IC50 62 ± 5

Oxy 80% IC50 58 ± 6 IC50 65 ± 8 IC50 500 ± 22 IC50 69 ± 7

Oxy 90% IC50 92 ± 8 IC50 120 ± 11 IC50 500 ± 25 IC50 65 ± 9

Oxy: Oxyresveratrol


253

Table 5.3.14: Effect of Oxyresveratrol on Skin pigmentation disorders Pigmentation

disorder

Effect Rationale

Pigmentation due to

biological imbalances

of body such as over

expression of

hormones and

enzymes

Significant.

Artocarpus

lakoocha extract

containing 20% &

30% oxyresveratrol

has better potential.

Potential inhibitor

of tyrosinase and

melanogenesis

Pigmentation due to

sun exposure

Significant.

Artocarpus

lakoocha extract

containing 80% &

90% oxyresveratrol


Significant

protection against

UV induced cell

death.

Pigmentation due to

free radical damage

Significant.

Artocarpus

lakoocha extract

containing 80% &

90% oxyresveratrol


Significant

scavenger of free

radicals

Pigmentation due

inflammatory

responses like pimple

marks, scars due to

wounds etc.

Significant.

Artocarpus

lakoocha extract

containing 20% &

30% oxyresveratrol


Potential inhibitor

of inflammatory

markers and

inflammatory

enzymes


254

5.3.1.2.4.2. Dihydro-oxyresveratrol from Artocarpus lakoocha heartwood extract:

Table 5.3.15: Skin lightening potential of Dihydro-oxyresveratrol



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

Induced Melanin

(µg/ml)

UV protection

IC50 0.03 ± 0.01 IC50 1.63 ± 0.5 IC50 2 ± 0.6 Not significant


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 5.37 ± 1.6 IC50 3.125 ± 0.9 21,549 ± 2022 3097 ± 423


Collagenase

inhibition (µg/ml)

Elastase inhibition

(µg/ml)

Hyaluronidase

inhibition (µg/ml)

TNF α inhibition

(µg/ml)

IC50 150 ± 22 IC50 166 ± 17 IC50 147 ± 19 IC50 40 ± 3

Table 5.3.16: Effect of Dihydro-oxyresveratrol on Skin pigmentation disorders Pigmentation disorder Effect Rationale



expression of hormones and

enzymes


of tyrosinase and

melanogenesis


significant

No significant

protection from UV


damage

Significant Antioxidant


responses like pimple marks,

scars due to wounds etc.


of inflammatory

markers


255

5.3.1.2.4.3. Resveratrol from Polygonum cuspidatum root extract:

Table 5.3.17: Skin lightening potential of Resveratrol



(µg/ml)

Inhibition of MSH

induced Melanin

Inhibition of cAMP

Induced Melanin

UV protection

(µg/ml)

IC50 5.5 ± 1.2 IC50 2.5 ± 0.8 IC50 3.5 ± 0.91 EC50 30 ± 7


DPPH scavenging ROS scavenging ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 3 ± 1.1 Not significant 25,223 ± 2312 10,000 ± 598


Collagenase

inhibition


inhibition

TNF α inhibition

(µg/ml)

Nil Not significant Nil IC50 75 ± 5

Table 5.3.18: Effect of Resveratrol on Skin pigmentation disorders Pigmentation disorder Effect Rationale


imbalances of body such as

over expression of hormones

and enzymes


tyrosinase &

melanogenesis

Pigmentation due to sun

exposure


of cell damage due to

UV exposure

Pigmentation due free radical

damage






of inflammatory

markers


256

5.3.1.2.4.4. Pterostilbene from Pterocarpus marsupium heartwood extract:

Table 5.3.19: Skin lightening potential of Pterostilbene



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

(µg/ml)

IC50 7.8 ± 1.5 IC50 0.5 ± 0.08 IC50 0.7 ± 0.06 EC50 30 ± 7.2


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 4.9 ± 1.3 IC50 3 ± 0.92 12,508 ± 998 6283 ± 664


Collagenase

inhibition (µg/ml)

Elastase inhibition

(µg/ml)

Hyaluronidase

inhibition (µg/ml)

TNF α inhibition

(µg/ml)

IC50 125 ± 14 IC50 50 ± 4 Nil IC50 150 ± 12

Table 5.3.20: Effect of Pterostilbene on Skin pigmentation disorders Pigmentation disorder Effect Rationale




enzymes


of tyrosinase and

melanogenesis

Pigmentation due to sun exposure Significant Significant protection

from UV


damage


Prevention of pigmentation due

inflammatory responses like pimple

marks, scars due to wounds etc.


of inflammation


257

Hydroxy Pterostilbene (3HPT) from Pterocarpus marsupium-׀3 .5.3.1.2.4.5

heartwood extract:

Table 5.3.21: Skin lightening potential of 3HPT



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

IC50 2 ± 0.3 IC50 0.7 ± 0.08 IC50 0.5 ± 0.1 Nil


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 1.34 ± 0.3 IC50 5 ± 1.1 13,334 ± 1142 4700 ± 598


Collagenase

inhibition (µg/ml)

Elastase inhibition

(µg/ml)

Hyaluronidase

inhibition

TNF α inhibition

(µg/ml)

IC50 90 ± 3 IC50 82 ± 9 Nil IC50 158 ± 11

Table 5.3.22: Effect of 3HPT on Skin pigmentation disorders Pigmentation disorder Effect Rationale





of tyrosinase and

melanogenesis

Pigmentation due to sun exposure No effect No significant UV

protection






of inflammation


258

5.3.1.2.4.6. Gnetol from Gnetum gnemon:

Table 5.3.23: Skin lightening potential of Gnetol



(µg/ml)

Inhibition of MSH

induced Melanin

Inhibition of cAMP

induced Melanin

UV protection

(µg/ml)

IC50 149 ± 22 Not significant Not significant EC50 25 ± 5



(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 1.55 ± 0.9 IC50 3.5 ± 1.2 14,322 ± 1233 5000 ± 968


Collagenase

inhibition


inhibition

TNF α inhibition

Not significant Not significant Not significant Not significant

Table 5.3.24: Effect of Gnetol on Skin pigmentation disorders Pigmentation disorder Effect Rationale




enzymes

Mild Inhibits only

tyrosinase

Pigmentation due to sun exposure Significant Significant

protection from

UV


damage

Significant Antixidant




No effect Not an inhibitor

of inflammation


259

5.3.1.2.5. Emblica officinalis (Amla) fruit extract containing β-Glucogallin:

For several decades, the fruits of Amla had been claimed to be a rich source of Ascorbic

acid and further its high antioxidant potential was attributed to the presence of ascorbic

acid (Scartezzini P et al., 2006). It was then reported that low molecular hydrolysable

tannins (emblicanins A and B) contribute to the antioxidant potential of Amla

(Pozharitskava O N et al., 2007).

Table 5.3.25: Skin lightening potential of Amla extract


Active Tyrosinase

inhibition (µg/ml)

Inhibition of

MSH induced

Melanin (µg/ml)

Inhibition of

cAMP

induced Melanin

(µg/ml)

UV

protection

(µg/ml)

β-Glucogallin 10%

IC50 200 ± 35 IC50 12 ± 3.6 IC50 14 ± 4.3 IC50 20 ± 3.3

β-Glucogallin 99%

IC50 500 ± 33 IC50 14 ± 2.4 IC50 15 ± 3.9 IC50 25 ± 3.1


Active DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol

GAE/g)

β-Glucogallin 10%

IC50 1.7 ± 0.2 IC50 2.5 ± 1.2 2862 ± 339 1474 ± 212

β-Glucogallin 99%

IC50 0.92 ± 0.1 IC50 2.5 ± 0.9 4436 ± 446 4660 ± 189


Active

Collagenase

inhibition (µg/ml)

Elastase

inhibition

Hyaluronidase

inhibition

TNF α

inhibition

β-Glucogallin 10%

IC50 500 ± 22 Nil Not significant Not significant

β-Glucogallin 99%

Nil Nil Not significant Not significant


260

However, recent studies have confirmed that only trace amounts of Ascorbic acid are

found in Amla extract and the earlier reported antioxidant hydrolysable tannins,

emblicanins A and B, correspond to 1-O-galloyl- β-D-glucose (β-glucogallin) and mucic

acid 1,4-lactone 5-O-gallate respectively (Majeed M et al., 2009). The trace amount of

free Ascorbic acid in Amla extract suggests that the antioxidant effects exhibited by

Amla fruits are due to gallic acid esters (Majeed M et al., 2009). Due to the presence of

only trace amounts of Ascorbic acid in the fruits of Emblica officinalis, β-glucogallins

could be the active that significantly contributes to the efficacy of Amla extract. Amla

extract containing higher concentration of β-glucogallin had higher antioxidant potential

(Table 5.3.25)

Table 5.3.26: Effect of Amla extract on Skin pigmentation disorders Pigmentation disorder Effect Rationale

Pigmentation due to

biological imbalances

of body such as over

expression of hormones

and enzymes

Significant Significant

inhibitor of

melanogenesis


exposure

Significant. Significant

protection against

UV induced cell

death.

Pigmentation due to

free radical damage

Significant. Amla

extract containing

99% β-Glucogallin

has better

potential.

Significant

scavenger of free

radicals

Pigmentation due

inflammatory responses

like pimple marks, scars

due to wounds etc.

Mild Not a significant

inhibitor of

inflammatory

markers


261

5.3.1.2.6. Piperlongumine from Piper longum root extract:

Table 5.3.27: Skin lightening potential of Piperlongumine


Tyrosinase inhibition Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

Nil IC50 0.625 ± 0.23 IC50 0.5 ± 0.21 Nil



(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 22 ± 4.3 mg/ml Nil Nil Nil


Collagenase

inhibition


inhibition

TNF α inhibition

(µg/ml)

Nil Nil Nil IC50 230 ± 25

Table 5.3.28: Effect of Piperlongumine on Skin pigmentation disorders Pigmentation disorder Effect Rationale


imbalances of body such as

over expression of hormones

and enzymes


inhibitor of

melanogenesis


exposure

No effect No protection from

UV damage


damage

Mild Mild scavenger of

free radicals




Mild Inhibitor of

inflammatory

markers


262

5.3.1.3. Screening of “actives” known for antioxidant and anti inflammatory activity

but unexplored for skin lightening efficacy:

5.3.1.3.1. Hydroxychavicol from Piper betle leaf extract: Table 5.3.29: Skin lightening potential of Hydroxychavicol



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

IC50 8 ± 1.5 IC50 1.3 ± 0.2 IC50 2.5 ± 0.4 Nil


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 0.5 ± 0.1 IC50 10 ± 1.2 29,728 ± 2860 2376 ± 293


Collagenase

inhibition


inhibition

TNF α inhibition

(µg/ml)

Not significant Not significant Not significant IC50 89 ± 8

Table 5.3.30: Effect of Hydroxychavicol on Skin pigmentation disorders Pigmentation disorder Effect Rationale





tyrosinase &

melanogenesis

Pigmentation due to sun exposure No effect No UV protection




due to wounds etc.


of inflammation


263

5.3.1.3.2. Thymohydroquinone from Nigella sativa (Black cumin) seed extract:

Table 5.3.31: Skin lightening potential of Thymohydroquinone



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

Induced Melanin

(µg/ml)

UV protection

IC50 0.905 ± 0.2 IC50 0.2 ± 0.05 IC50 0.3 ± 0.07 Nil


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 84 ± 11 IC50 20 ± 5.6 5899 ± 369 2000 ± 136


Collagenase

inhibition


inhibition

TNF α inhibition

(µg/ml)

Not significant Not significant Not significant IC50 3 ± 0.8

Table 5.3.32: Effect of Thymohydroquinone on Skin pigmentation disorders Pigmentation disorder Effect Rationale





of tyrosinase and

melanogenesis

Prevention of pigmentation due to sun

exposure

Nil No UV protection

Prevention of pigmentation due to free

radical damage






of inflammatory

markers


264

5.3.1.3.3. Eugenia jambolana (Jamun) fruit extract:

Table 5.3.33: Skin lightening potential of Jamun extract



induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

Not significant IC50 5 ± 1.2 IC50 7 ± 1.4 Nil


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 6.1 ± 2.1 IC50 10 ± 3.2 9000 ± 890 1000 ± 262


Collagenase

inhibition


inhibition

TNF α inhibition


Table 5.3.34: Effect of Jamun extract on Skin pigmentation disorders Pigmentation disorder Effect Rationale




enzymes


of melanogenesis

Pigmentation due to sun exposure No effect No UV protection


damage





No effect Not an inhibitor of

inflammation


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.


266

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



induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

Not significant IC50 20 ± 5.6 IC50 25 ± 6.2 Nil


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 1.2 ± 0.2 IC50 10 ± 3.8 22,352 ± 1926 5000 ± 432


Collagenase

inhibition


inhibition

TNF α inhibition

(µg/ml)


Table 5.3.36: Effect of Avenanthramide C (Av-c) on Skin pigmentation disorders Pigmentation disorder Effect Rationale




enzymes

Significant Inhibitor of

melanogenesis


exposure

No effect No UV protection


damage






inflammatory

markers


267

5.3.1.3.5. Colocynthin from Citrullus colocynthis fruit extract:

Table 5.3.37: Skin lightening potential of Colocynthin



(µg/ml)

Inhibition of MSH

induced Melanin

(µg/ml)

Inhibition of cAMP

induced Melanin

(µg/ml)

UV protection

(µg/ml)

IC50 >100 IC50 22 ± 2.3 IC50 20 ± 3.2 EC50 100 ± 9


DPPH scavenging

(µg/ml)

ROS scavenging

(µg/ml)

ORAC

(µmol TE/g)

HORAC

(µmol GAE/g)

IC50 50 ± 6.2 IC50 10 ± 3.1 Not significant Not significant


Collagenase

inhibition


inhibition

TNF α inhibition

(µg/ml)


Table 5.3.38: Effect of Colocynthin on Skin pigmentation disorders Pigmentation disorder Effect Rationale





tyrosinase and

melanogenesis

Pigmentation due to sun exposure Significant Protects against UV

induced cell death


damage




due to wounds etc.


of inflammatory

markers


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



induced Melanin

Inhibition of cAMP

induced Melanin

UV protection

(µg/ml)

Nil Upto 20 µg/ml, 25%

inhibition

Upto 20 µg/ml, 35%

inhibition

EC50 50 ± 5



(µmol TE/g)

HORAC

(µmol GAE/g)

Nil Nil Nil Nil


Collagenase

inhibition


inhibition

TNF α inhibition


Table 5.3.40: Effect of Oat ceramides on Skin pigmentation disorders Pigmentation disorder Effect Rationale

Pigmentation due to biological imbalances

of body such as over expression of

hormones and enzymes

Mild Inhibitor of

melanogenesis

Pigmentation due to sun exposure Significant Protects from UV

damage

Pigmentation due free radical damage No effect Not an antioxidant

Pigmentation due inflammatory responses

like pimple marks, scars due to wounds

No effect Not an inhibition of

inflammation


269

5.3.1.4.2. Ceramides from Malus domestica (Apple) extract:

Table 5.3.41: Skin lightening potential of Apple ceramides


Active Tyrosinase

inhibition

Inhibition of

MSH induced

Melanin

Inhibition of

cAMP

induced Melanin

UV

protection

(µg/ml)

Apple peel

ceramides

Upto 30 µg/ml,

30% inhibition

Upto 20 µg/ml,

15% inhibition

Upto 20 µg/ml,

25% inhibition

Not

significant

Apple fruit

ceramides

Upto 70 µg/ml,

30% inhibition

IC50 12 ± 3.2

µg/ml

IC50 10 ± 2.9

µg/ml

EC50 100 ± 8


Active DPPH

scavenging

ROS scavenging ORAC

(µmol TE/g)

HORAC

(µmol

GAE/g)

Apple peel

ceramides

Not significant Nil Not significant Not

significant

Apple fruit

ceramides

Not significant Upto 10 µg/ml,

10% scavenging

Not significant Not

significant


Active Collagenase

inhibition

(µg/ml)

Elastase

inhibition

(µg/ml)

Hyaluronidase

inhibition

TNF α

inhibition

Apple peel

ceramides

IC50 25 ± 3 IC50 200 ± 12 Not significant Not

significant

Apple fruit

ceramides

Nil Nil Not significant Not

significant

It was observed that the apple fruit ceramides had better melanogenesis inhibitory and

UV protection efficacy whereas the apple peel ceramides had better anti inflammatory

efficacy.


270

Table 5.3.42: Effect of Apple ceramides on Skin pigmentation disorders Pigmentation disorder Effect Rationale

Pigmentation due to

biological imbalances of

body such as over

expression of hormones

and enzymes

Apple fruit ceramides

- significant

Apple peel ceramides

- mild

Inhibitor of

tyrosinase and

melanogenesis


exposure


– significant


– Not significant

Significant protection

against UV induced

cell death

Pigmentation due to free

radical damage

Not significant Not significant

scavenger of free

radicals

Pigmentation due

inflammatory responses

like pimple marks, scars

due to wounds etc.


- significant


– Not significant

Inhibitor of

inflammatory

enzymes


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.


272

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

Group Rank Plant extract Active IC50 (µg/ml)

I

1 Artocarpus lakoocha Dihydro-oxyresveratrol (DHO) 0.03 ** 2 Oxyresveratrol (OXY) 50-90% 0.1 ** 3 Glycyrrhiza glabra Glabridin (GLAB) 0.25 ** 4 Artocarpus lakoocha Oxyresveratrol (OXY) 20-30% 0.5 **

II

5 Nigella sativa Thymohydroquinone (THQ) 1.0 ** 6 Artocarpus lakoocha Artocarpin (ART) 1.3 ** 7 Curcuma longa Tetrahydrocurcuminoids (THC) 2.0 ** 8 Pterocarpus marsupium 3-Hydroxypterostilbene (3HPT) 2.0 ** 9 Artocarpus lakoocha Resveratrol (RES) 5.5 * 10 Synthetic (from Sigma) Kojic acid (KA) 7.0 11 Pterocarpus marsupium Pterostilbene (PTR) 7.8 12 Piper betle Hydroxychavicol (HC) 8.0 13 Synthetic (from Sigma) Ascorbic acid (ASC) 9.33

III 14 Gnetum sp. Gnetol (GN) 149 15 Synthetic (from Sigma) Arbutin (ARB) 194 16 Emblica officinalis β glucogallin (BG) 200

Group I - Actives with high efficacy; Group II - Actives with moderate efficacy; Group III - actives with mild efficacy. * P value < 0.1 and ** P value < 0.05 as compared to Kojic acid best known for tyrosinase inhibition

Actives with IC50 values ≤ 0.5 are grouped under Tyrosinase inhibitors I which represent

actives with high efficacy. Actives with IC50 values ≤ 10 are grouped under Tyrosinase

inhibitors II which represent actives with moderate efficacy and actives with IC50 values

≥ 100 are grouped under Tyrosinase inhibitors III which represent actives with mild

efficacy (Fig. 5.3.1).


273

Tyrosinase inhibitors I

0

0.1

0.2

0.3

0.4

0.5

0.6

DHO OXY 50-90% GLAB OXY 20-30%

Actives

IC 5

0 in

mic

rogr

am/m

l

Tyrosinase inhibitors II

0

2

4

6

8

10

12

THQ ART THC 3HPT RES KA PTR HC ASC

Actives

IC 5

0 in

mic

rogr

am/m

l

Tyrosinase inhibitors III

0

50

100

150

200

250

GN ARB BG

Actives

IC 5

0 in

mic

rogr

am/m

l

Tyrosinase inhibitors I - Actives with high efficacy; Tyrosinase inhibitors II - Actives with moderate efficacy; Tyrosinase inhibitors III - actives with mild efficacy Figure 5.3.2: Inhibitors of Tyrosinase - Inhibitors of Solar lentiges and skin tanning

5.3.2.2. Inhibitors of Melasma and skin tanning:

The main manifestations of melanogenesis induced by over expression of MSH & cAMP

are melasma and skin tanning. Melasma also occurs during pregnancy, usage of oral

contraceptives, certain anti epileptics etc. Melasma is observed as symmetrical facial

hyperpigmentation involving either epidermis, dermis or both. Significant inhibitors of

melanogenesis can inhibit melasma and skin tanning. The best synthetic melanogenesis

inhibitors are ranked in Table 5.3.44, based on their IC50 values in inhibiting

melanogenesis in B16F1 mouse melanoma cells. Lower IC50 value indicates better

melanogenesis inhibitory potenital.


274

Table 5.3.44: Significant inhibitors of MSH and cAMP induced melanogenesis - Inhibitors of Melasma and skin tanning

Group Rank Plant extract Active IC50 (µg/ml)

I

1 Nigella sativa Thymohydroquinone (THQ) 0.3 ** 2 Pterocarpus marsupium Pterostilbene (PTR) 0.5 ** 3 Piper longum Piperlongumine (PL) 0.6 ** 4 Pterocarpus marsupium 3-Hydroxypterostilbene (3HPT) 0.7 ** 5 Piper betle Hydroxychavicol (HC) 1.3 ** Artocarpus lakoocha Dihydro Oxyresveratrol (DHO) 1.63 ** 6 Polygonum cuspidatun Resveratrol (RES) 2.5 **

7

Curcuma longa Tetrahydrocurcuminoids(THC) 3 **

Glycyrrhiza glabra Glabridin (GLAB) 3 ** Artocarpus lakoocha Oxyresveratrol (OXY) 20-30% 3 ** Artocarpus lakoocha Artocarpin (ART) 3 **

8 Pterocarpus marsupium Dihydropterostilbene (DPTR) 5 ** Eugenia jambolana Polyphenols (PLY) 5 **

II

9 Artocarpus lakoocha Oxyresveratrol (OXY) 50-90% 10 * 10 Emblica officinalis β glucogallin (BG) 12 *

11 Avena sativa Avenanthramide C (AVN) 20 * Synthetic (from Sigma) Ascorbic acid (ASC) 25

12 Synthetic (from Sigma) Glutathione (GLU) 25

13 Synthetic (from Sigma) Kojic acid (KA) 100 Synthetic (from Sigma) Arbutin (ARB) 100

Group I - Actives with high efficacy; Group II - actives with moderate efficacy. * P value < 0.1 and ** P value < 0.05 as compared to Ascorbic acid best known for melanin inhibition

Actives with IC50 values ≤ 5 are grouped under Melanogenesis inhibitors I which

represent actives with high efficacy and actives with IC50 values ≤ 100 are grouped under

Melanogenesis inhibitors II which represent actives with moderate efficacy (Fig. 5.3.2).

Amongst the above listed actives, Kojic acid has no effect on melanogenesis induced by

cAMP (Table 5.3.3) while Ascorbic acid and Glutathione are less affective for

melanogenesis induced by cAMP (Table 5.3.5).

Interstingly, melanogenesis inhibition potential of Artocarpus lakoocha bark

extract was higher in extracts containing lower concentration of Oxyresveratrol as


275

observed in the table. Therefore, the activity is due to the synergy between the various

actives present in the extract and not just by Oxyresveratrol.

Melanogenesis inhibitors I

DHPTPLY

THCGLAB

OXY 20-30%ART

0

1

2

3

4

5

6

THQ PTR PL 3HPT HC DHO RES THC DHPT

Actives

IC 5

0 in

mic

rogr

am/m

l

Melanogenesis inhibitors II

0

20

40

60

80

100

120

OXY50-90%

BG AVN ASC GLU KA ARB

Actives

IC 5

0 in

mic

rogr

am/m

l

Melanogenesis inhibitors I - Actives with high efficacy; Melanogenesis inhibitors II - Actives with moderate efficacy Figure 5.3.3: Inhibitors Melanogenesis - Inhibitors of Melasma and skin tanning As evident in Fig. 5.3.3, all the actives listed in Table 5.3.43 except Piperlongumine are

inhibitors of Tyrosinase which is the enzyme responsible to catalyze the final step in

melanogenesis. Similarly, actives listed in Table 5.3.44, inhibit melanogenesis right at the

initial step of the pathway by inhibiting MSH. All the actives that inhibit MSH induced

melanogenesis are the inhibitors of cAMP induced melanogenesis also except for Kojic

acid. In other words, when melanogenesis is induced due to the mere enhancement in

cAMP levels, Kojic acid is ineffective.

From Table 5.3.43 and Table 5.3.44, it is evident that most of the actives

inhibiting the process of melanogenesis at the initial step which is MSH activity also

inhibit the process at the final step which is tyrosinase activity. However, Piperlongumine

which is a significant inhibitor of melanogenesis inhibits only MSH induced

melanogenesis, clearly indicating that it works only by inhibiting the activity of MSH

initially and not by inhibiting tyrosinase. Melanogenesis is inhibited by Piperlongumine

right at the first step.

However, the degree of activity varied with respect to tyrosinase inhibitory and

melanogenesis inhibitory potential. For example, Dihydrooxyresveratrol has the highest

tyrosinase inhibitory potential whereas Thymohydroquinone has the highest

melanogenesis inhibitory potential.


276

X - Inhibition Figure 5.3.4: Effect of Tyrosinase and Melanogenesis inhibitors on skin hyperpigmentation

5.3.2.3. Protection from UV exposure:

UV exposure is the main reason for over expression of MSH or cAMP or Tyrosinase

which occur as a response to stress due to UV induced free radical damage on skin. The

process of melanogenesis is enhanced as a defence mechanism as melanin tends to

protect the skin from UV damage. Hence, actives that provide protection from UV can

prevent the whole process of excessive melanogenesis and subsequent skin darkening.

These actives have potential as sunscreens. Actives that act as sunscreens as well as

antioxidants can be used not just for prevention from UV exposure but also scavenge the

αMSH ACTH

MC1-R

PKA

MITF

Tyrosinase

cAMP

1. Melanogenesis 2. Differentiation,

dendrite formation

and proliferation of melanocytes

Actives in Table 5.3.43 except

Piperlongumine

X

Actives in Table 5.3.44 X

Actives in Table 5.3.44

except Kojic acid

X

Skin hyperpigmentation


277

free radicals generated due to UV exposure and help in “after sun care” of the skin by

soothing the UV damaged skin and reducing the excessive pigmentation. The actives that

significantly protect from UV are ranked in Table 5.3.45, based on their EC50 values in

preventing cell death due to UV exposure in Swiss 3T3 mouse fibroblast cells. Lower

EC50 value indicates better protection form UV induced cell death. The significant actives

for UV protection are ranked in comparison to the standard product for UV protection

Octyl methoxycinnamate (OMC) for which the EC50 was found to be 100µg/ml (Fig.

5.3.4).

Table 5.3.45: Actives that prevent UV induced cell damage

Rank Plant extract Active EC50 (µg/ml)

1 Emblica officinalis β glucogallin (BG) 20 ** Artocarpus lakoocha Artocarpin (ART) 20 **

2 Gnetum sp. Gnetol (GN) 25 **

3 Pterocarpus marsupium Pterostilbene (PTR) 30 ** Polygonum cuspidatun Resveratrol (RES) 30 **

4 Artocarpus lakoocha Oxyresveratrol (OXY) 80-90% 50 *

Avena sativa (Oat) Oat ceramides (OCR) 50 *

5 Artocarpus lakoocha

Oxyresveratrol (OXY) 50% 75 *

6 Oxyresveratrol (OXY) 20-30% 100

7

Malus domestica (Apple) Apple ceramides (APL) 100

Citrullus colocynthis Colocynthin (CYN) 100

Synthetic Octylmethoxycinnamate (OMC) 100 * P value < 0.1 and ** P value < 0.05 as compared to OMC best known for sunscreen potential


278

Actives that prevent UV induced cell damage

0

20

40

60

80

100

120

BG ART GN PTR RES OXY80-90%

OCR OXY50%

OXY20-30%

APL CYN OMC

Actives

EC

50

in m

icro

gram

/ml

Figure 5.3.5: Actives that prevent UV damage

5.3.2.4. Inhibitors of Free radical damage:

Reduction of ROS levels in melanocytes may prevent activation of melanogenesis. In

various studies, extracts from plants or fruit or other species were tested for their

antioxidant capacity by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-

scavenging assay or the oxygen radical absorbance capacity (ORAC) (Rangkadilok et al.,

2007). The process of melanogenesis is enhanced as a defence mechanism as melanin

tends to protect the skin from free radical damage induced by UV exposure. Hence,

antioxidant actives prevent free radical damage that further induces excessive

melanogenesis. Actives that act as sunscreens as well as antioxidants can be used not just

for prevention from UV exposure but also scavenge the free radicals generated due to UV

exposure and help in “after sun care” of the skin by reducing the excessive pigmentation.

Significant antioxidant actives are ranked in Table 5.3.47, based on their IC50 values in

scavenging DPPH, scavenging ROS generation in Swiss 3T3 mouse fibroblast cells and

their high ORAC and HORAC values. Lower IC50 values and higher ORAC and HORAC

values indicate better antioxidant potential. Cumulative antioxidant potential for each

active has been calculated as the ratio of additive ORAC and HORAC values and IC50 for

ROS scavenging potential (Table 5.3.46). Cumulative antioxidant potential = A + B /

C, Where, A = ORAC value in μmol trolox equivalents/g, B = HORAC value in

μmol trolox equivalents/g and C = IC50 for ROS scavenging potential.


279

Table 5.3.46: Cumulative antioxidant potential of actives

Active ORAC HORAC

ORAC +

HORAC ROS Ratio

Hydroxychavicol 29728 2376 32104 0.5 64208

THC 10000 3000 13000 1 13000

Resveratrol 25000 10000 35000 3 11666

Oxyresveratrol 90% 21000 5723 26723 3 8907

Oxyresveratrol 80% 18673 4923 23596 3 7865

Oxyresveratrol 50% 15582 4896 20478 3 6826

Oxyresveratrol 30% 11370 4776 16146 3 5382

Oxyresveratrol 20% 8999 4756 8999 3 4585

Dihydrooxyresveratrol 21549 3097 26549 3 8215

Glabridin 7550 1129 8679 1 8679

Avenanthramide C 22352 5000 27352 10 2735

Gnetol 14322 5000 19322 3 6440

3HPT 13334 4700 17334 3 6011

Pterostilbene 12508 6233 12508 3 6247

β-glucogallin 99% 4436 4660 9096 2.5 3638

β-glucogallin 10% 2862 1474 4336 2.5 1734

Artocarpin 3859 1121 4980 3 1660

Jamun polyphenols 9000 1000 10000 10 1000

Thymohydroquinone 5899 2000 7899 20 395

Ascorbic acid 3400 Nil 3400 10 340

This calculation is applicable when antioxidant actives have significant potential in all the

mechanisms of antioxidant activity as specified in the above formula. ROS scavenging

potential has been given preference over DPPH scavenging potential for calculating the

cumulative antioxidant potential because ROS scavenging assay is performed under the

typical stress induced conditions in mammalian cells where as DPPH scavenging is a


280

chemical based assay which can only be an indicator of antioxidant potential at a

preliminary experimentation level.

Table 5.3.47: Significant inhibitors of free radicals - inhibitors of free radical induced melanogenesis Rank Plant extract Active Cumulative antioxidant units

1 Piper betle Hydroxychavicol 64,208 **

2 Curcuma longa THC 13,000 **

3 Polygonum cuspidatum Resveratrol 11,666 **

4

Artocarpus lakoocha Oxyresveratrol 90% 8907 **

Dihydro Oxyresveratrol 8215 **

Glycyrrhiza glabra Glabridin 8679 ** 5

Artocarpus lakoocha Oxyresveratrol 80% 7865 **

6 Oxyresveratrol 50% 6826 ** 7 Gnetum sp. Gnetol 6440 **

8 Pterocarpus marsupium Pterostilbene 6247 **

9 Pterocarpus marsupium 3HPT 6011 **

10 Artocarpus lakoocha

Oxyresveratrol 30% 5382 **

11 Oxyresveratrol 20% 4585 **

12 Emblica officinalis Β- glucogallin 90% 3638 **

13 Avena sativa Avenanthramide C 2735 **

14 Emblica officinalis Β- glucogallin 10% 1734 *

15 Artocarpus lakoocha Artocarpin 1660 *

16 Eugenia jambolana Polyphenols 1000 *

17 Nigella sativa Thymohydroquinone 395

18 Synthetic Ascorbic acid 340

* P value < 0.1 and ** P value < 0.05 as compared to Ascorbic acid best known for antioxidant potential


281

X - Inhibition Figure 5.3.6: Effect of Sunscreens and Antioxidants on Melanogenesis

Unlike in the case of melanogenesis inhibition, UV protection and antioxidant potential

of Artocarpus lakoocha bark extract is conferred by the content of Oxyresveratrol as the

antioxidant and UV protection potential increased with the increasing concentrations of

this active in the extract (Table 5.3.47). Similarly, the antioxidant potential of Amla

extract is conferred by the content of β glucogallin in the extract as the antioxidant

potential increased with the increasing concentrations of this active in the extract (Table

5.3.47). All the actives listed in Table 5.3.45, provide protection from UV damage which

is the main reason for a sequence of reactions and ultimately enhanced melanogenesis as

a response to stress conditions. They act as “sunscreens” by either absorbing or blocking

the UV or inhibiting UV induced adversaries (Fig. 5.3.5). Actives listed in Table 5.3.47,

inhibit UV induced free radical generation which induces melanogenesis (Fig. 5.3.5).

UV

ROS ROS

MSH

cAMP

NO

cGMP

PKG

Melanogenesis

DAG

PKC


(Antioxidants)


(Antioxidants)

Actives in Table 5.3.45 (Sunscreens)

X

X

X


282

All the antioxidant actives also provided UV protection except for

Hydroxychavicol, 3-Hydroxypterostilbene, Avenanthramides, Jamun polyphenols,

Thymohydroquinone and Ascorbic acid which are significant antioxidants but still could

not prevent UV induced cell death (Table 5.3.45 and Table 5.3.47). Similarly, all actives

with significant UV protection are significant antioxidants except the ceramides from

Malus domestica and Avena sativa (Table 5.3.45 and Table 5.3.47). Therefore all

antioxidants need not necessarily provide UV protection and vice-versa. Actives which

are not antioxidants but still render UV protection act as a shield against UV by

absorbing the UV and blocking UV from reaching the biological system. Antioxidants

combat UV induced free radical damage. Some antioxidants prevent UV damage and

some heal UV damage. Actives that heal UV damage are called “after sun care” actives

which may not prevent UV damage but heal UV damage. “After sun care” actives are

mainly antioxidants that combat UV induced free radical damage. For example,

Glutathione a significant antioxidant could not prevent from UV induced cell death in

vitro. Glutathione is the major antioxidant produced by the cells in the body, for

protection from free radicals (oxygen radicals, oxyradicals). These highly reactive

substances, if left unchecked, will damage or destroy key cell components (e.g.

Membranes, DNA) in microseconds. Oxyradicals are generated in many thousand

mitochondria located inside each cell, where nutrients like glucose are burnt using

oxygen to make energy. Oxyradicals also come from pollutants and UV

radiation. Glutathione not only protects the body form these free radicals but also

recycles other well know antioxidants such as vitamin C and vitamin E, keeping them in

their active state. Glutathione plays a crucial role in maintaining the normal balance

between oxidation and anti-oxidation. This, in turn, regulates many of the cell’s vital

functions, such as the synthesis and repair of DNA after UV damage, the synthesis of

proteins and the activation of regulation of enzymes. Hence, Glutathione can be

affectively used as an “after sun care” product, meaning it can reduce the after effects of

UV damage. Table 5.3.48 represents actives which act as “sunscreens”, “after sun care”

actives and both.


283

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

Group Active Prevention from UV

damage – Sunscreens

Healing of UV damage –

“After sun care”

I

β glucogallin 10-99% Significant Significant

Oxyresveratrol 20-

90%


Artocarpin Significant Significant

Gnetol Significant Significant

Pterostilbene Significant Significant

Resveratrol Significant Significant

Colocynthin Significant Mild

II

Ceramides from

Avena sativa

Significant Nil

Ceramides from

Malus domestica

Significant Nil

III

Tetrahydrocurcumin Not significant Significant

Dihydro-

Oxyresveratrol

Not significant Significant

Glabridin Not significant Significant

Ascorbic acid Not significant Significant

Hydroxychavicol Nil Significant

3HPT Nil Significant

Glutathione Nil Significant

Avenanthramides Nil Significant

Jamun polyphenols Nil Significant

Thymohydroquinone Nil Significant

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


284

As evident from Table 5.3.48, actives in group I are significant “sunscreens” as well as

“after sun care” actives. Actives in group II are significant “sunscreens” where as actives

in group III are significant “after sun care” actives. It is also observed that Oxyresveratrol

which is both a significant antioxidant as well as UV protectant, on hydrogenation of

Dihydrooxyresveratrol remained just an antioxidant but could not retain the UV

protection potential. Therefore, unlike Oxyresveratrol which is both a significant

“sunscreen” as well as “after sun care” active, Dihydro-oxyresveratrol is just an “after

sun care” active.

5.3.2.5. Inhibitors of post inflammatory hyperpigmentation:

As known from many cases of post-inflammatory hyperpigmentation, melanogenesis can

be stimulated by some inflammatory mediators. Inhibition of the production of

inflammatory mediators (Il1α and TNF-α) was reported for sea grape (Coccoloba uvifera)

extracts (Silveira J E et al., 2007). Via this indirect way, stimulation of melanogenesis in

the pigment cells could be prevented (Briganti S et al., 2003). Post inflammatory

hyperpigmentation is mainly manifested after resolution of skin problems like acne,

contact dermatitis etc. Post inflammatory hyperpigmentation is observed as discrete

hyper pigmented macules with hazy magins on the skin where melanin production is

increased. Upregulation of inflammatory markers like TNF α and prostaglandins occurs

as a result of skin defence mechanism to pathogens and allergens. However, these

markers tend to damage the skin also and hence even after resolution of skin problems,

the skin remains darkened at the affected areas as dark marks or scars due to the

increased melanin production and melanocyte dendricity in response to the inflammatory

markers. The damage can be at the level of epidermis, dermis or both.

Certain inflammatory enzymes like Elastase, Collagenase and Hyaluronidase are

over expressed due to exposure to certain stress conditions like UV, pollutants, toxins etc.

These are serine protease enzymes that dissociate tissues which contain extensive

intercellular fiber networks and clean up any dead tissue leaving the wound bed ready for

healing. Serine proteases also help in PAR-2 (Protease activated receptor) mediated

transfer of melanosomes from melanocytes to keratinocytes resulting in skin darkening.


285

Hyaluronidase is the natural protein responsible for hydrolysis of the extracellular

mucopolysaccharide, hyaluronic acid. It acts by modifying the permeability of

intercellular ground substance in connective tissue. The breakdown of the viscous

hyaluronic acid decreases tissue resistance and enhances diffusion of substances between

tissue planes. However, over expression of these enzymes results in inflammation of the

skin leading to skin darkening and break down of skin structural proteins and glycans like

elastin, collagen and hyaluronic acid respectively.

Inflammation is a normal biological mechanism triggered in the body in response

to infection, foreign bodies etc., however overstay of inflammation results in the damage

of healthy tissues such as the skin dermis, eventually resulting in hyper-pigmentation.

Therefore, significant inhibitors of inflammatory markers and enzymes can inhibit

increased melanin production in the dermis at the affected area (Fig. 5.3.6). Anti

inflammatory actives are ranked in the Table 5.3.49, based on their IC50 values in

inhibiting elastase, collagenase, hyaluronidase enzymes and TNFα. Lower IC50 value

indicates better anti inflammatory potenital. TNF α inhibition is given higher preference

over elastase, collagenase and hyaluronidase inhibition as TNF α directly influences

melanogenesis while the inflammatory enzymes mainly affect the skin texture and tone.

However, the cumulative effect of inflammatory markers and enzymes is more significant.

Since anti inflammatory actives are widely used as drugs for therapeutic treatment of

various inflammatory diseases like arthritis etc., a reference standard drug is not used for

comparing the actives being screened for skin lightening efficacy. Actives are considered

significant for anti inflammatory efficacy if IC50 values could be obtained within the

optimal dosages evaluated. Actives with significant inhibition of TNF α and

inflammatory enzymes are preferred over those that are significant inhibitors of TNF α

alone. Actives with significant inhibition of TNF α are preferred over those that are

significant inhibitors of inflammatory enzymes alone.


286

Table 5.3.49: Significant inhibitors of inflammation – inhibitors of post inflammatory hyperpigmentation Rank Plant extract Active Rationale

1 Artocarpus lakoocha Oxyresveratrol 20-90% Significant inhibitors of Elastase, Collagenase enzymes and TNF α

2 Glycyrrhiza glabra Glabridin 3 Artocarpus lakoocha 3HPT 4 Pterocarpus marsupium Pterostilbene 5 Artocarpus lakoocha Artocarpin 6 Nigella sativa Thymohydroquinone

Significant inhibitors of TNF α

7 Avena sativa Avenanthramide C 8 Artocarpus lakoocha Dihydro Oxyresveratrol 9 Citrullus colocynthis Colocynthin 10 Polygonum cuspidatun Resveratrol 11 Curcuma longa THC 12 Piper betle Hydroxychavicol 13 Piper longum Piperlongumine 14 Malus domestica (peel) Ceramides Significant inhibitor of elastase

and collagenase enzymes

The detailed mechanisms of action of various synthetic skin lightening actives gives an

outlook on which active can be recommended based on its mechanism of action for a

specific kind of skin darkening. The ranking can be useful for recommendation based on

the intensity of the pigmentation. For example, for severe post inflammatory

hyperpigmentation scars, actives of significant anit inflammatory potential like

Oxyresveratrol, Dihydrooxyresveratrol, Hydroxychavicol or 3HPT can be recommended.


287

X - Inhibition Figure 5.3.7: Effect of inhibitors of inflammation on Scar formation

5.3.3. Skin lightening actives ranked as per their cumulative efficacy for all the major

skin lightening mechanisms:

Next to in vitro, the brownish guinea pig model is used in several skin lightening studies

where the pigmentation is induced by either UV or α-MSH. In case of in vivo studies,

prevention of the induction of pigment by the whitening agents could be demonstrated

using a Minolta chromameter or by histochemical investigations showing a decrease in

DOPA positive cells (Yamakoshi J et al., 2003 and Yoshimura, M et al., 2005). Another

animal model used for whitening studies is the zebrafish that also proved useful for

demonstrating the in vivo toxicity of the whitening agents (Choi T Y et al., 2007 and Kim

J H et al., 2008). So far, only limited numbers of clinical trials with skin whitening agents

Skin exposure to allergins/toxins

Skin sebum infection

Inflammation

Fight against foreign bodies

Healing

Dermal matrix damage and

melanogenesis induction in affected

melanocytes

Overstay of Inflammation

Hyperpigmentation

Scars/marks


X


288

or formulations have been performed (Tengamnuay P et al., 2006 and Chang T S, 2009).

For ethical and economic reasons, the cosmetic industry relies heavily on in vitro studies

and a thorough positioning of skin lightening actives is therefore a significant pre

requisite for the development of affective skin lightening agents.

As evident in Fig. 5.3.7 and Table 5.3.50, Oxyresveratrol, Pteostilbene,

Resveratrol and Artocarpin have potential in all the major skin lightening mechanisms

and can be promising actives for various kinds of hyperpigmentation disorders.

X – Inhibition Figure 5.3.8: Actives with significant efficacy in various skin lightening mechanisms

Table 5.3.50 summarizes the actives and their method of action for skin lightening and

also clearly shows the all rounder actives for all kinds of skin darkening.

Excessive skin pigmentation

Tyrosinase

Free radicals

MSH & cAMP

UV exposure

Inflammation

Oxyresveratrol Artocarpin

Pterostilbene Resveratrol

X

X

X

X

X


289

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

Active SL Melasma UV Free radical

induced pigmentation

Post inflammatory hyperpigmentation

Oxyresveratrol

Pterostilbene

Resveratrol

Artocarpin Amla extract

Dihydro-oxyresveratrol

Tetrahydrocurcumin

Thymohydroquinone

Hydroxychavicol

Glabridin

3-hydroxypterostilbene

Ascorbic acid

Gnetol

Eugenia jambolana extract

Piperlongumine

Glutathione

Kojic acid

Arbutin

Avenanthramide C

Oat ceramides *

Apple fruit ceramides *

Apple peel ceramides * * *

- Actives with significant effect on all skin lightening mechanisms * - Actvies with Mild activity - Actives with no activity


290

5.3.4. Novel actives and observations:

5.3.4.1. Novel skin lightening actives:

Some actives were screened on account of their significant properties such as antioxidant,

anti-inflammatory, skin conditioning and UV protection activities. These actives can have

efficacy for skin lightening also as free radical stress, UV stress and inflammation are

some of the pathways for excessive skin pigmentation. Therefore, in the process of

screening of various actives through various skin lightening mechanisms, some novel

plant actives were observed to have skin lightening potential.

5.3.4.1.1. Thymoquinone from Nigella sativa extract: Thymoquinone has significant

antioxidant and anti inflammatory potential and hence could help in skin lightening as

well. Hence, when studied for melanin inhibition potential, it was found to be a

significant inhibitor of melanin.

5.3.4.1.2. Hydroxychavicol from Piper betle extract: Hydroxychavicol has significant

antioxidant and anti inflammatory potential and hence could help in skin lightening as

well. Hence, when studied for melanin inhibition potential, it was found to be a

significant inhibitor of melanin.

5.3.4.1.3. Eugenia jambolana extract: Eugenia jambolana extract rich in polyphenols

has significant antioxidant potential and hence could help in skin lightening as well.

Hence, when studied for melanin inhibition potential, it was found to be a significant

inhibitor of melanin.

5.3.4.1.4. Avenanthramides from Avena sativa extract: Avenanthramides have

significant antioxidant and anti inflammatory potential and hence could help in skin

lightening as well. Hence, when studied for melanin inhibition potential, it was found to

be a significant inhibitor of melanin. Of all the Avenenthramides screened,

Avenenthramide C (Av-C) showed superior potential for skin lightening.


291

5.3.4.1.5. Colocynthin from Citrullus colocynthis fruit extract: Colocynthin has

significant antioxidant and anti inflammatory potential and hence could help in skin

lightening as well. Hence, when studied for melanin inhibition potential, it was found to

be a significant inhibitor of melanin.

5.3.4.1.6. Natural ceramides from Avena sativa (Oat) and Malus domestica (Apple)

extract: Ceramides have UV protection potential and they act as potential skin

conditioners by preventing transepidermal water loss from the skin. Hence, if they can

prevent excessive pigmentation by inhibiting melanin they can be unique products for

skin lightening. Hence, when studied for melanin inhibition potential, it was found that

Oat ceramides and Apple peel ceramides were mild inhibitors of melanin. But Apple fruit

ceramides were significant inhibitors of melanin.

5.3.4.2. Novel observations:

In the process of screening of various actives through various skin lightening mechanisms,

actives that conferred efficacy and the synergistic interplay of actives for efficacy could

be determined.

5.3.4.2.1. Oxyresveratrol content in Artocarpus lakoocha heartwood extract and its

effect on skin lightening potential: As discussed earlier, Oxyresveratrol is a significant

tyrosinase and melaogenesis inhibiting active of Artocarpus lakoocha heartwood extract.

Surprisingly it was observed that the melanogenesis inhibitory potential of Artocarpus

lakoocha heartwood extract decreased with the increasing concentration of

Oxyresveratrol. But Oxyresveratrol is known for its skin lightening potential. Therefore it

is a novel observation that the melanogenesis inhibitory potential of Artocarpus lakoocha

heartwood extract is not just because of Oxyresveratrol but due to the synergistic activity

of various actives in the extract. It was also observed that on hydrogenation of

Oxyresveratrol to Dihydrooxyresveratrol, there was an enhancement in tyrosinase and

melanin inhibitory potential but there was a decrease in UV protection potential.


292

5.3.4.2.2. Pterostilbene and Dihydro-pterostilbene from Pterocarpus marsupium

heartwood extract: Unlike in the case of oxyresveratrol, hydrogenation of actives

having good melanogenesis inhibitory potential does not always enhance the activity. It

was observed that on hydrogenation of Pterostilbene to Dihydro-pterostilbene, the

melanogenesis inhibitory potential was reduced. Dihydro-pterostilbene showed mild

tyrosinase inhibition of 25% at 2.5µg/ml and its IC50 for melanin inhibiton was 5µg/ml.

5.3.4.2.3. β-glucogallin in Emblica officinalis (Amla) fruit extract and its effect on

skin lightening potential: Amla extract had significantly higher skin lightening potential

than ascorbic acid. Hence, unlike what was earlier thought that the skin lightening

potential was conferred by ascorbic acid; it was observed in the present study that the

skin lightening potential was attributed due to the synergistic combination of β-

glucogallin and various other gallates in the extract.

5.3.5. Actives that help in skin lightening through indirect mechanisms of action:

5.3.5.1. Compounds with cell proliferation enhancement potential:

Some of the actives with no significant antioxidant, anti inflammatory, UV protection or

skin lightening potential but yet with a significant cell proliferation potential will have a

very good effect in skin lightening as well by enhancing the cell rejuvenation. When the

cell proliferation enhancer is taken in combination with significant skin lightening actives,

while the actives lighten the skin cells, the cell proliferation enhancer helps in

rejuvenation of the cells, with an effect that the darker skin is continuously replenished

by fresh lightened skin cells. Therefore the process of skin lightening is quickened. For

example, Retinoids influence pigmentation by speeding up turnover in the skin, gradually

eliminating anything sitting on the top layers. This sloughing process automatically

begins to slow down in our mid twenties. Retinoids reverse that effect by producing a

faster rate of cell turnover as well as eliminating abnormal melanin in the top layer of

skin. Retinoids are therfore useful in treating melasma and acne scars by reducing the

amount of excess melanin and distributing it more evenly.


293

Of all the actives screened for cell proliferation enhancement activity, the following

products showed significant activity.

5.3.5.1.1. Liquid endosperm of Cocos nucifera (coconut): Coconut liquid endosperm

was found to enhance the cell proliferation of Swiss 3T3 mouse fibroblast cells as

observed by SRB staining technique (Table 5.3.51 and Fig. 5.3.8).

Table 5.3.51: Cell proliferation enhancement by Liquid endosperm of Coconut in vitro

Coconut liquid

endosperm

Optical Density due to

viable cells at 492nm

% enhancement in cell proliferation

as compared to control

No treatment 0.139 -

2.5 µg/ml 0.154 11%

5 µg/ml 0.164 18%

Figure 5.3.9: Cell proliferation enhancement by Coconut liquid endosperm in Swiss 3T3 mouse fibroblasts

Untreated cells 2.5µg/ml Coconut liquid endosperm treated cells

5µg/ml Coconut liquid endosperm treated cells


294

Coconut liquid endosperm (Freeze dried powder) also showed a significant wound

closure enhancement potential of 25% at a concentration of 5µg/ml in Swiss 3T3 mouse

fibroblast cells as analyzed by the scratch wound closure assay (Fig. 5.3.9). The effect of

liquid endosperm of Coconut as an enhancer of cell proliferation was further confirmed at

a reputed research laboratory. The cell proliferation enhancement by Coconut liquid

endosperm was evident by an in vivo study conducted at Dabur Research Foundation,

Ghaziabad, Uttar Pradesh – Efficacy of Sami Formulations Report (Protocol No.

PR/CR/REP/003-00). The hair growth efficacy of Coconut liquid endosperm was

compared with that of a standard, Minoxidil. 60 days old female C57/BL6 mice were

treated topically with cream containing 2% Coconut liquid endosperm for ten days. On

completion of the studies, 8 mm punch biopsies were taken from the resected dorsal skin

and was processed for histopathological studies to obtain longitudinal and transverse

sections. Digital photomicrographs were taken from representative areas at a fixed

magnification of 100X. Histological evaluation of the skin biopsies showed hair growth

promoting activity of Coconut liquid endosperm with the effect that the hair follicles

were transformed from Telogen to Anagen phase of hair growth in the animals on topical

application of formulation containing Coconut liquid endosperm (Table 5.3.52). Hence,

cell proliferation enhancers like Coconut liquid endosperm can have good effect on skin

lightening by quickening the process in combination with other skin lightening actives.


295

A) Untreated wounded cell monolayer after 24 hours; B) Untreated wounded cell monolayer after 48 hours; C) Coconut liquid endosperm treated wounded cell monolayer after 24 hours; D) Coconut liquid endosperm treated wounded cell monolayer after 24 hours Figure 5.3.10: Wound closure by Coconut liquid endosperm in Swiss 3T3 mouse fibroblasts

B

C D

A


296

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

Treatment Mean follicle count

in subcutis layer

Average skin

thickness (µm)

% of animals showing

Anagen induction

Cream

(2% Coconut liquid

endosperm)

43.00 ± 12.20

220.15 ± 25.60

71.4

2% Minoxidil

(Ref.Standard) 42.86 ± 13.49 218.75 ± 34.04 71.4

5% Dextrose (inert) 3.43 ± 3.27 156.27 ± 5.14 14.2

An in vivo study conducted at Dabur Research Foundation, Ghaziabad, Uttar Pradesh – Efficacy of Sami

Formulations Report (Protocol No. PR/CR/REP/003-00)

5.3.5.1.2. Probiotic bacteria Bacillus coagulans: Another sample which showed

significant cell proliferation enhancement was the culture supernatant containing Bacillus

coagulans exudates. Probiotics are dietary supplements of live bacteria or yeasts thought

to be healthy for the host organism. According to the currently adopted definition,

Probiotics are: ‘Live microorganisms which when administered in adequate amounts

confer a health benefit on the host’. Lactic acid bacteria are the most common type of

microbes used and has been used in the food industry for many years, because they are

able to convert sugars and other carbohydrates into lactic acid. By lowering the pH, they

may create fewer opportunities for spoilage organisms to grow, creating possible health

benefits. Strains of the genera Lactobacillus and Bifidobacterium, are the most widely

used Probiotic bacteria. Creating a complete aseptic environment in the body can destroy

physiological microflora, which in turn causes health problems because microflora are

important in many levels. The introduction of Probiotic micro-organisms to “reset” the

level of bacteria eliminated, represents a winning strategy today, and is something which

most of the scientific community agrees on, with positive results to show for it. Toxins

are the main cause for the deterioration of skin health. Toxins damage the protective skin

barrier by inducing free radicals and also create fertile conditions in skin for harmful


297

bacteria to multiply. The same toxins may also interfere with normal functioning of

organs. This may cause organs to overproduce certain hormones that result in skin

damage. For example, toxins stimulate sebum production and more acne-causing bacteria

proliferate resulting in pimples. The most effective way for good skin is to maintain a

correct balance of intestinal and skin bacteria. Probiotic bacteria limit the growth of

harmful bacteria. Probiotics also neutralize toxins and create an environment lethal to

pathological bacteria. By keeping pathological bacteria at bay and preventing

overproduction of toxins, Probiotics actually eliminate the root cause of skin damage.

Probiotic bacteria prevent the growth of pathological bacteria and also help in skin

rejuvenation. Therefore, probiotic bacteria help in curing skin disorders and maintain

healthy skin. A combination of probiotic bacteria with significant skin lightening actives

can help quicken the process of skin lightening. While the actives lighten the skin cells,

the cell proliferation enhancer helps in rejuvenation of the cells, with an effect that the

darker skin is continuously replenished by fresh lightened skin cells. Therefore, such

probiotic bacteria can be used both as topical cosmetics or as nutricosmetics in

combination with other skin lightening actives for cosmetic benefits.

The culture supernatant of the exponential growth phase of Bacillus coagulans

was studied for its effect on the growth of Swiss 3T3 mouse fibroblast cells. The culture

supernatant was diluted in varying concentrations in mouse fibroblast growth medium

and used for treating the mouse fibroblast cells. It was observed that this culture

supernatant rich in exudates like lactic acid etc. could enhance the cell proliferation of

mouse fibroblast cells by 22% upto a non cytotoxic concentration of 3.125% (Table

5.3.53 and Fig. 5.3.10).


298

Table 5.3.53: Effect of Bacillus coagulans on cell proliferation enhancement

Conc. of Bacillus coagulans culture supernatant (%)

% enhancement in cell proliferation

% cytotoxicity

0 0 0 0.018 0 0 0.037 0 0 0.075 0 0 0.15 4.4 0 0.31 4.6 0 0.75 14.4 0 1.5 15.5 0 3.125 22.1 0 6.25 6.1 0 12.5 0 8.3 25 0 26.5 50 0 42.5 100 0 42.5

Cell proliferation enhancement by culture supernatant of Bacillus coagulans in Swiss 3T3 mouse fibroblast cells

-10

-5

0

5

10

15

20

25

Conc. (%)

% e

nhan

cem

ent i

n ce

ll pr

olife

ratio

n

Cell proliferation

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


299

5.3.5.2. Compounds with collagen enhancement potential:

5.3.5.2.1. Oleanoyl peptide: Oleanoyl peptide is a pentapeptide conjugate of Oleanolic

acid, a naturally occurring triterpenoid. Oleanoyl peptide was chemically synthesized by

solution phase method by conjugating Oleanolic acid to Lysine-Threonine-Threonine-

Lysine-Serine pentapeptide (Lys-Thr-Thr-Lys-Ser) pentapeptide. Oleanolic acid is known

for its anti inflammatory, antioxidant, anti microbial and wound healing properties. In the

present study, by Sirius red staining method, it was observed that this peptide helped in

the enhancement of collagen levels in Human osteosarcoma cells by 17% at a

concentration of 1.25µg/ml.

Figure 5.3.12: Structure of Pentapeptide conjugate of Oleanolic acid

5.3.5.2.2. Asiaticosides from Centella asiatica extract: Asiaticosides also enhanced the

precursor of collagen, pro-collagen in Swiss 3T3 fibroblasts as analysed by flow

cytometry. The population of fibroblasts in the assay was gated as the total population

and analyzed for the percentage of population of fibroblasts that showed Fluorescein

isothiocyanate (FITC) labeled pro-collagen. It was observed that while the untreated cells

showed 1.7% FITC labeled pro-collagen, Centella asiatica extract treated cells showed

13.5% FITC labeled pro-collagen showing a clear enhancement in pro-collagen synthesis

on treatment with Centella asiatica extract (Fig. 5.3.11).

CH3 CH3

CH3

CH3

OH

CH3

OCH3

CH3

ONH

NH2

O

NH

OHCH3

O

NH OH

CH3

O NH

NH2

O

NH

OH

OH


300

Therefore, the products like Coconut liquid endosperm and probiotic bacteria Bacillus

coagulans help in cell rejuvenation while products like Oleanoyl peptide and

Asciaticosides help in collagen enhancement and indirectly help and quicken the process

of skin lightening.

A

B C

A) Total population of Swiss 3T3 mouse fibroblasts analyzed B) Untreated cells showing 1.7% of the total population containing FITC labeled pro-collagen C) Centella asiatica extract treated cells showing 13.5% of the total population containing FITC labeled pro-collagen. FSC-A: Forward scatter; SSC-A: Side scatter Figure 5.3.13: Pro-collagen enhancement by Centella asiatica extract in Swiss 3T3 mouse fibroblasts

1.7% FITC labelled pro-collagen

13.5% FITC labelled pro-collagen


301

5.3.6. Synergistic skin lightening efficacy due to integration of different mechanisms of

action:

In a broad perspective, the term “synergy” refers to the combined or cooperative effects

produced by the relationships among various forces, particles, elements, parts or

individuals in a given context, effects that are not otherwise possible. The term is derived

from the Greek word “synergos”, meaning (i) to work together or (ii) to co-operate.

“Synergistic effects” are unknown, unexpected, unsought for useful phenomena that are

accidentally discovered. Thus the concepts of “synergistic effects/synergy” are included

under “inventions”. The present study supports the integration of various skin lightening

mechanisms for a synergistic skin lightening effect. Skin lightening compositions

comprising combinations of carefully evaluated plant actives with varied mechanisms of

action, showed exponentially enhanced activity due to synergy mediated by the actives.

5.3.6.1. Chemical conjugation of actives with different modes of action showing

enhanced skin lightening potential:

5.3.6.1.1. Indirect and Synergistic skin lightening effect by Oleanoyl peptide:

Short-sequence peptides hold significant potential as skin lightening ingredients and for

treatment of pigmentation disorders. Peptides can promote a faster and more effective

approach to skin brightening and lightening by enhancing skin's natural elasticity and

firmness, cell rejuvenation and synthesis of skin structural proteins. In this process the

dull and darker superficial skin is constantly replaced by new skin cells by exfoliation of

old keratin. A combination of skin lightening actives with peptides can therefore be more

effective and faster in the skin lightening process as peptides help in clearance of stagnant

melanin to brighten skin. Certain natural peptides in the body like glutathione directly act

on pigmentation and bring about skin lightening. Peptides, short polymers of amino acids

linked by peptide bonds, have been shown to counteract the degradation of dermal

collagen, resulting in a significant change in the appearance of moderate to deep lines and

wrinkles. As collagen fibrils are broken down through natural biological process, peptide


302

portions or by-products of this catabolic process act as signals or messenger molecules in

the formation of new collagen fibrils by the fibroblasts. Cosmetically interesting

activities such as stimulation of collagen synthesis, chemotaxis, anti-stinging effects and

others, can be observed and substantiated with chemically modified peptide sequences.

Long chain fatty acid conjugates of peptides improve skin penetration, specific activity

and economic feasibility of these molecules (Lintner K and Peschard O, 2000).

Olenoylpeptide is one such peptide, a conjugate of Oleanolic acid and Lysine-

Threonine-Threonine-Lysine-Serine pentapeptide (Lys-Thr-Thr-Lys-Ser). Due to

the integrated skin lightening mechanisms of individual actives that are conjugated

together, it has the following properties,

• Inhibition of free radicals that induce skin darkening

• Inhibition of inflammatory markers that cause skin darkening

• Inhibition of collagenase and elastase that degrade the skin

• Enhancement of collagen synthesis that helps heal and rejuvenate the skin

• Although it does not directly inhibit melanogenesis, under in vivo conditions it

helps in skin lightening

Activity of Oleanolic acid: An anti inflammatory active with no melanogenesis

inhibitory potential.

Activity of Lys-Thr-Thr-Lys-Ser pentapeptide: Identification and incorporation of

very specific peptide sequences has demonstrated biological activity in relationship to

collagen formation in both in vitro and ex vivo human tests. Extracellular matrix

production can be improved by chemically synthesized subfragments of type I collagen

carboxy propeptide (Katayama K et al., 1991). The molecule shown to have this

signaling power is a pro-collagen I terminal sequence consisting of the amino acids;

“Lysine - Threonine - Threonine - Lysine – Serine” pentapeptide a fragment of

procollagen I increases the production of Collagen IV and glycosaminoglycan and helps

in wrinkle reduction and improving the skin tone. It has no effect on inflammation or

melanogenesis.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Lintner%20K%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Peschard%20O%22%5BAuthor%5D�

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Katayama%20K%22%5BAuthor%5D�


303

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

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

Ser pentapeptide (Table 5.3.54). However, there was no significant melanogenesis

inhibitory potential.

Table 5.3.54: Anti inflammatory and antioxidant activity of the conjugate, Oleanoyl peptide in comparison to the individual actives conjugated

Compound

Elastase

inhibition

(IC50 µg/ml)

Collagenase

inhibition

(IC50 µg/ml)

TNF α

inhibition

(IC50 µg/ml)

Antioxidant

activity - DPPH

Scavenging

Oleanoyl peptide 143 119 21 29% inhibition

at 300 µg/ml

Oleanolic acid Nil 129 17% inhibition

at 100 µg/ml Nil

Pentapeptide (Lys-

Thr-Thr-Lys-Ser) Nil

24% inhibition at

500 µg/ml Nil Nil

Rationale for skin lightening activity of Oleanoylpeptide: In Oleanoylpeptide, the

cosmetic approach is two pronged, collagen production being boosted by the

pentapeptide and inflammation being reduced by Oleanolic acid. Moreover,

Oleanoylpeptide showed anti inflammatory potential through pathways like TNF α

inhibition, elastase and collagenase inhibition. Interestingly, although neither Oleanolic

acid, pentapeptide nor the Oleanoylpeptide had direct inhibitory effect on MSH induced

melanogenesis, due to the two pronged cosmetic approach, Oleanoylpeptide could reduce

stress induced pigmentation such as freckles and under eye dark circles which occurs due

to inflammation. This was observed in the in house study for 4 weeks with 17 volunteers

on usage of a formulation of 0.1% Oleanoyl peptide in a cream base (Fig 5.3.12).


304

A B

C D

E F

G H

A) Under eye dark circles before treatment B) Reduction in under eye dark circles on treatment with Oleanoyl peptide C) Dark spots on forehead and freckles on nose before treatment D) Reduction in dark spots and freckles on treatment with Oleanoyl peptide E) Under eye darkness and wrinkles before treatment F) Reduction in under eye darkness and wrinkles on treatment with Oleanoyl peptide G) Under eye darkness, wrinkles and skin thinning before treatment H) Reduction in under eye darkness and wrinkles and improvement of under eye skin tone on treatment with Oleanoyl peptide Figure 5.3.14: Effect of Oleanoyl peptide on various hyperpigmentation conditions in human volunteers


305

This peptide of Oleanolic acid is novel and significant in activity as it is not obvious that

any active conjugated to Lys-Thr-Thr-Lys-Ser pentapeptide will have activity. The below

example of a peptide of Thiodipropionic acid and Lys-Thr-Thr-Lys-Ser pentapeptide did

not show a significant enhancement in skin lightening potential (Table 5.3.55).

5.3.6.1.2. A peptide of Thiodipropionic acid and Lys-Thr-Thr-Lys-Ser pentapeptide:

In the peptide, only Thiodipropionic acid is an inhibitor of tyrosinase, whereas Lys-Thr-

Thr-Lys-Ser pentapeptide has no effect on tyrosinase. Lys-Thr-Thr-Lys-Ser pentapeptide

in conjugation with various actives like palmitic acid (saturated lipophillic fatty acid) and

other triterpenoids like Oleanolic acid etc, showed significant cosmetic benefits as

described earlier. However Lys-Thr-Thr-Lys-Ser pentapeptide in conjugation with

Thiodipropionic acid, just retained the anti tyrosinase potential of Thiodipropionic acid

and did not show significant enhancement in the anti tyrosinase activity (Table 5.3.55).

Table 5.3.55: Skin lightening potential by a peptide of Thiodipropionic acid and Lys-Thr-Thr-Lys-Ser pentapeptide

Active Characteristic activity Tyrosinase inhibition

Thiodipropionic acid Tyrosinase inhibition 22% inhibition at 100µg/ml

Lys-Thr-Thr-Lys-Ser

pentapeptide

Collagen enhancement Nil

Conjugate of Thiodipropionic

acid and Lys-Thr-Thr-Lys-

Ser pentapeptide


Collagen enhancement

31% inhibition at 100µg/ml

5.3.6.1.3. A conjugate of Kojic acid and Acetyl-11-keto-beta-boswellic acid

(AKBBA):

In the conjugate, only Kojic acid is an inhibitor of melanogenesis through MSH and

tyrosianse inhibitory pathways with mild antioxidant potential, whereas AKBBA is an

anti inflammatory active with no effect on melanogenesis. Kojic acid individually could

inhibit melanogenesis by 32% at 80µg/ml; however, in a chemical conjugation with


306

AKBBA, it could inhibit melanogenesis by 32% at 2.5µg/ml (Table 5.3.56). Although

AKBBA is not an inhibitor of melanogenesis, its anti inflammatory activity that added on

to the melanogenesis inhibitory pathways of Kojic acid, enhanced the melanogenesis

inhibitory potential. On the whole, the conjugate has properties that kojic acid did not,

like better inhibition of MSH induced melanogenesis due to the added anti inflammatory

properties of AKBBA.

Table 5.3.56: Synergistic skin lightening potential by a conjugate of Kojic acid and AKBBA

Active Characteristic activity Melanin inhibition

Kojic acid Melanin inhibition

Mild antioxidant:

DPPH scavenging: IC50 is 500µg/ml.

32% at 80µg/ml

AKBBA Anti inflammatory potential:

TNF α inhibition - IC50 is 100µg/ml

Hyaluronidase inhibition - IC50 is 63µg/ml

Collagenase inhibition - IC50 is 250µg/ml

Nil

Conjugate of Kojic acid

and AKBBA

Melanin inhibition


32% at 2.5µg/ml

5.3.6.1.4. A conjugate of Oleanolic acid and Kojic acid:

In the conjugate, only Kojic acid is an inhibitor of melanogenesis through MSH and

tyrosianse inhibitory pathways with mild antioxidant potential, whereas Oleanolic acid is

an anti inflammatory active with no effect on melanogenesis. Kojic acid individually

could inhibit melanogenesis with an IC50 of 100µg/ml; however, in a chemical

conjugation with Oleanolic acid, it could inhibit melanogenesis with an IC50 of 5µg/ml

(Table 5.3.57). Although Oleanolic acid is not an inhibitor of melanogenesis, its anti

inflammatory activity added on to the melanogenesis inhibitory pathways of Kojic acid

and enhanced the melanogenesis inhibitory potential. On the whole, the conjugate has

properties that kojic acid did not, like better inhibition of MSH induced melanogenesis

due to the added anti inflammatory properties of Oleanolic acid.


307

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


Kojic acid Melanin inhibition

Mild antioxidant:

DPPH scavenging: IC50 is 500µg/ml.

50% at 100µg/ml

Oleanolic acid Anti inflammatory potential:

Collagenase inhibition - IC50 is 129µg/ml

Nil

Conjugate of Kojic acid

and Oleanolic acid

Melanin inhibition


50% at 5µg/ml

5.3.6.2. Physical combination of actives with different modes of action showing

enhanced skin lightening potential:

5.3.6.2.1. Composition containing 50% THC and 50% Glabridin:

Both THC and Glabridin are good inhibitors of tyrosinase and melanogenesis in

mammalian cells. But only THC is a significant antioxidant. So in a cell system the

antioxidant potential added up to melanogenesis inhibitory pathways and enhanced the

pigmentation reduction in mammalian melanocytes (Table 5.3.58 and Fig. 5.3.13).

0

0.5

1

1.5

2

2.5

3

IC50 in microgram/ml

Tyrosinaseinhibition

Melanin inhibition

Synergistic skin lightening potential by THC and Glabridin

THCGlabridinTHC + Glabridin (1:1)

Figure 5.3.15: Synergistic efficacy of THC and Glabridin


308

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


THC Melanin inhibition

Significant antioxidant potential:

DPPH scavenging activity - IC50 is 1.2

µg/ml

ORAC – 10,000 µmol trolox equivalents/g

Anti tyrosinase activity –

IC50 is 2 µg/ml

Melanin inhibition - IC50 is

3 µg/ml

Glabridin Melanin inhibition

Mild antioxidant potential:

DPPH scavenging activity - IC50 is 49

µg/ml

ORAC – 3256 µmol trolox equivalents/g


IC50 is 0.25 µg/ml


3 µg/ml

THC +

Glabridin

1:1

Melanin inhibition

Significant antioxidant potential:

DPPH scavenging activity - IC50 is 0.97

µg/ml

ORAC – 10,000 µmol trolox equivalents/g


IC50 is 0.068 µg/ml


1 µg/ml

5.3.6.2.2. Composition containing 0.25% AKBBA, 0.5% THC and

0.1%Tetrahydropiperine (THP):

In the composition, only THC is an inhibitor of melanogenesis, whereas AKBBA is an

anti inflammatory active and THP is a cell permeation enhancer with no effect on

melanogenesis. Although THC is a significant inhibitor of melanogenesis and free

radicals, it alone may not have a significant effect in the cell pigmentation at a

concentration of 0.5% in combination with inert actives with respect to melanogenesis

inhibition. Theoretically, since 100% THC had an IC50 of 3µg/ml, 0.5% THC should

have an IC50 of >100µg/ml. However, the IC50 obtained for the composition is 10µg/ml

(Table 5.3.59). So in a cell system the anti inflammatory potential and enhanced


309

bioavailability of the actives to the target sites of a cell added up to melanogenesis

inhibitory pathways and enhanced the pigmentation reduction in mammalian melanocytes.

Table 5.3.59: Synergistic skin lightening potential by a combination of AKBBA, THC and THP


0.25% AKBBA Anti inflammatory potential Nil

0.5% THC Melanin inhibition

Antioxidant activity

IC50 >100µg/ml

0.1% THP A significant cell penetration enhancer.

THP enhanced the permeation of a diterpene

forskolin by 12 times in 60 minutes across rat skin.

Nil

0.25% AKBBA +

0.5% THC +

0.1% THP

Melanin inhibition



Enhanced bio availability

IC50 10µg/ml

5.3.6.2.3. Composition containing 0.2% THC, 0.2% Glabridin, 1% AKBBA and

0.1% THP:

In the composition, only THC and Glabridin are the inhibitors of melanogenesis, whereas

AKBBA is an anti inflammatory active and THP is a cell permeation enhancer with no

effect on melanogenesis. Although THC and Glabridin are significant inhibitors of

melanogenesis of equal potential, they alone may not have a significant effect in the cell

pigmentation at a concentration of 0.2% each in combination with inert actives with

respect to melanogenesis inhibition. Theoretically, since 100% THC & Glabridin had an

IC50 of 3µg/ml, 0.2% THC & Glabridin should have an IC50 of >100µg/ml. However, the

IC50 obtained for the composition is 20µg/ml (Table 5.3.60). So in a cell system the anti

inflammatory potential and enhanced bioavailability of the actives to the target sites of a

cell added up to melanogenesis inhibitory pathways and enhanced the pigmentation

reduction in mammalian melanocytes.


310

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




IC50 >100µg/ml

0.2% Glabridin Melanin inhibition IC50 >100µg/ml

1% AKBBA Anti inflammatory potential Nil

0.1% THP A significant cell penetration

enhancer.

Nil

0.2% THC + 0.2% Glabridin +

1% AKBBA + 0.1% THP

Melanin inhibition




IC50 20µg/ml

5.3.6.2.4. Composition containing 0.5% THC, 0.5% Glabridin, 0.1% Galanga

extract and 0.1% THP:

In the composition, only THC and Glabridin are the inhibitors of melanogenesis, whereas

Galanga extract is a UV protectant with no significant effect on melanogenesis. Although

THC and Glabridin are significant inhibitors of melanogenesis of equal potential, they

alone may not have a significant effect in the cell pigmentation at a concentration of 0.5%

each in combination with inert actives with respect to melanogenesis inhibition.

Theoretically, since 100% THC and Glabridin had an IC50 of 3µg/ml, 0.5% THC and

Glabridin should have an IC50 of >100µg/ml. However, the IC50 obtained for the

composition is 12.5µg/ml (Table 5.3.61). So in a cell system the protection from UV

induced melanogenesis and enhanced bioavailability of the actives to the target sites of a

cell added up to melanogenesis inhibitory pathways and enhanced the pigmentation

reduction in mammalian melanocytes.


311

Table 5.3.61: Synergistic skin lightening potential by a combination of THC, Glabridin, Galanga extract and THP




IC50 >100µg/ml

0.5% Glabridin Melanin inhibition IC50 >100µg/ml

0.1% Galanga extract Inhibitor of UV induced

cell damage

Nil

0.1% THP A significant cell

penetration enhancer.

Nil

0.5% THC + 0.5% Glabridin +

0.1% Galanga extract + 0.1%

THP

Melanin inhibition




IC50 12.5µg/ml

5.3.6.2.5. Composition containing 0.2% THC, 0.1% Coenzyme Q10, 1% Coconut

liquid endosperm, 0.5% Soya isoflavones, 0.1% Tetrahydropiperine (THP):

In the composition, only THC is the inhibitor of melanogenesis, whereas CoQ10 acts as

an antioxidant, Coconut liquid endosperm and Soya isoflavones help in cell proliferation

and cell conditioning and THP helps in better bioavailability of actives in the cells with

no significant effect on melanogenesis. Although THC is a significant inhibitor of

melanogenesis, it alone may not have a significant effect in the cell pigmentation at a


inhibition. Theoretically, since 100% THC inhibited 30% melanogenesis at 2µg/ml, 0.2%

THC should have inhibited 30% melanogenesis at >200µg/ml. However, the composition

inhibited 30% of melanogenesis at 20µg/ml (Table 5.3.62). So in a cell system the

antioxidant potential, cell conditioning and enhanced bioavailability of the actives to the

target sites of a cell added up to melanogenesis inhibitory pathways and enhanced the

pigmentation reduction in mammalian melanocytes.


312

Table 5.3.62: Synergistic skin lightening potential by a combination of THC, Coenzyme Q10 (CoQ10), Coconut liquid endosperm, Soya isoflavones and THP




Inhibits 30%

melanogenesis at conc.

>200µg/ml

0.1% CoQ10 Antioxidant activity:

Naturally found in the mitochondria involved in

neutralizing free radicals.

Nil

1% Coconut

liquid endosperm

Cell proliferation enhancer:

18% enhancement at 5µg/ml

Nil

0.5% Soya

isoflavones

(genistein &

diadzein)

Phytoestrogenic properties:

They stimulate fibroblasts to make collagen and

hyaluronic acid which are essential for good skin

tone. Soya isoflavones also have the ability to

prevent UV damage.

Nil

0.1% THP A significant cell penetration enhancer. Nil

0.2% THC +

0.1% CoQ10 +

1% Coconut

liquid endosperm

+ 0.5% Soya

isoflavones +

0.1% THP

Melanin inhibition


Cell growth and conditioning


Inhibits 30%

melanogenesis at conc.

20µg/ml

5.3.6.2.6. Composition containing 0.5% THC, 0.2% Centella asiatica extract, 0.5%

Soya isoflavones, 0.1% THP:

In the composition, only THC is the inhibitor of melanogenesis, whereas Centella

asiatica extract & Soya isoflavones help in cell proliferation, cell conditioning and


313

collagen enhancement and THP helps in better bioavailability of actives in the cells with

no significant effect on melanogenesis. Although THC is a significant inhibitor of

melanogenesis, it alone may not have a significant effect in the cell pigmentation at a


inhibition. Theoretically, since 100% THC inhibited 30% melanogenesis at 2µg/ml, 0.5%

THC should have inhibited 30% melanogenesis at >200µg/ml. However, the composition

inhibited 30% of melanogenesis at 20µg/ml (Table 5.3.63). So in a cell system the

antioxidant potential, cell conditioning and enhanced bioavailability of the actives to the

target sites of a cell added up to melanogenesis inhibitory pathways and enhanced the

pigmentation reduction in mammalian melanocytes.

5.3.6.2.7. Composition containing 0.5% Arbutin, 0.2% Glabridin, 1% AKBBA and

0.3% Coriander seed oil extract:

In the composition, only Arbutin and Glabridin are the inhibitors of melanogenesis,

whereas AKBBA is an anti inflammatory active and Coriander seed oil extract acts as a

cell conditioner with no significant effect on melanogenesis. Although Glabridin and

Arbutin are inhibitors of melanogenesis, Glabridin having better potential, they alone

may not have a significant effect on the cell pigmentation in combination with inert

actives with respect to melanogenesis inhibition. 100% Arbutin can provide 40%

melanogenesis inhibition at a concentration of about 80µg/ml. 100% Glabridin can

provide 40% melanogenesis inhibition at a concentration of about 2µg/ml. Theoretically,

considering the activity of the better potential active, Glabridin, 40% inhibition of

melanogenesis is expected to be attained only at a concentration of >200µg/ml with 0.2%

glabridin in the composition. However, 40% inhibition of melanogenesis obtained with

the composition is 5µg/ml (Table 5.3.64). So in a cell system the anti inflammatory

potential and overall cell conditioning properties added up to melanogenesis inhibitory

pathways and enhanced the pigmentation reduction in mammalian melanocytes.


314

Table 5.3.63: Synergistic skin lightening potential by a combination of THC, Centella asiatica extract, Soya isoflavones and THP




Inhibits 30% melanogenesis at

>200µg/ml

0.2% Centella

asiatica extract

Anti inflammatory property:

Inhibition of Nitric oxide synthesis

facilitating cell proliferation and wound

healing.

Collagen enhancement:

60% enhancement at 5mg/ml

Nil

0.5% Soya

isoflavones

(genistein &

diadzein)

Phytoestrogenic properties:

They stimulate fibroblasts to make

collagen and hyaluronic acid which are

essential for good skin tone. Soya

isoflavones also have the ability to

prevent UV damage.

Nil


0.2% THC +

0.2% Centella

asiatica extract +

+ 0.5% Soya

isoflavones +

0.1% THP

Melanin inhibition


Anti inflammatory activity

Cell growth, conditioning and collagen

enhancement



20µg/ml


315

Table 5.3.64: Synergistic skin lightening potential by a combination of Arbutin, Glabridin, AKBBA and Coriander seed oil extract


0.5% Arbutin Melanin inhibition


>200µg/ml 0.2% Glabridin

1% AKBBA Anti inflammatory property Nil

0.3% Coriander

seed oil extract

Skin conditioning:

Coriander seed oil extract contains

petroselinic acid triglycerides with

significant skin conditioning potential.

Nil

0.5% Arbutin +

0.2% Glabridin +

+ 1% AKBBA +

0.3% Coriander

seed oil extract

Melanin inhibition

Anti inflammatory activity

Cell condiitoning


5µg/ml

5.3.6.2.8. Composition containing 0.5% Arbutin, 0.1% Glabridin and 0.1% THP:

In the composition, both Arbutin and Glabridin are the inhibitors of melanogenesis and

only Glabridin has mild antioxidant and anti inflammatory potential. THP only enhances

the bio availability of actives with no significant effect on melanogenesis. Although

Glabridin and Arbutin are inhibitors of melanogenesis, Glabridin having better potential,

they alone may not have a significant effect on the cell pigmentation in combination with

inert actives with respect to melanogenesis inhibition. 100% Arbutin can provide 40%

melanogenesis inhibition at a concentration of about 80µg/ml. 100% Glabridin can

provide 40% melanogenesis inhibition at a concentration of about 2µg/ml. Theoretically,

considering the activity of the better potential active, glabridin, 40% inhibition of

melanogenesis can be attained at a concentration of >200µg/ml with 0.1% glabridin in

the composition. However, 40% inhibition of melanogenesis obtained with the

composition is 5µg/ml (Table 5.3.65). So in a cell system the anti inflammatory potential


316

and enhanced bio availability of the actives to the target sites of a cell added up to

melanogenesis inhibitory pathways and enhanced the pigmentation reduction in

mammalian melanocytes. Moreover, Glabridin has a significant anti inflammatory

potential also. 100% glabridin can give 30% inhibition of elastase at 50µg/ml. So

theoretically 0.1% Glabridin should give 30% inhibition at 50mg/ml, where as the same

activity was observed at 140µg/ml. Therefore, the presence of THP enhanced the

bioavailability of glabridin to show better potential.

Table 5.3.65: Synergistic skin lightening potential by a combination of Arbutin, Glabridin and THP


0.5% Arbutin Melanin inhibition


>200µg/ml 0.1% Glabridin Melanin inhibition


Mild antioxidant


0.5% Arbutin +

0.1% Glabridin +

+ 0.1% THP

Melanin inhibition

Enhanced bioavailability


5µg/ml

5.3.6.3. Multifunctional skin lightening compositions:

Even when synergy with respect to one mechanism of action is not observed or when all

the targets of skinlightening mechanisms are not met by a single active, which is mostly a

common phenomenon, two or more actives which address different mechanisms are

combined into a unique composition to obtain a multifunctional skin lightening

composition. Some of the examples are as follows,

5.3.6.3.1. A composition of THC and Galanga extract (1:1): The composition exhibits

the antioxidant and melanin inhibitory benefits of THC along with the UV protection

benefits of Galanga extract.


317

Combined activity of the composition:

Antioxidant activity: ORAC Value of 1205µmol trolox equivalents/g, DPPH

scavenging activity with IC50 of 17µg/ml.

Melanin inhibition: IC50 of 20µg/ml.

UV protection: Prevents 50% UV induced cell death at 0.1% concentration.

5.3.6.3.2. A composition of Garcinol and Coconut liquid endosperm (1:1): The

compostion exhibits the antioxidant and anti inflammatory properties of Garcinol along

with the cell proliferation property of Coconut water extract thus fastening the process of

skin lightening.


Antioxidant activity: ORAC value of 1983µmol trolox equivalents/g, DPPH scavening

potential with an IC50 of 1.7µg/ml, Lipid peroxidation inhibition with an IC50 of 97µg/ml.

Anti inflammatory activity: Anti Hyaluronidase activity with an IC50 of 4.3µg/ml, Anti

Elastase activity with an IC50 of 300µg/ml, Anti Collagenase activity with an IC50 of

125µg/ml.

Cell proliferation enhancement: 48% enhancement at 0.002% concentration.

5.3.6.3.3. A composition of Lotus seed extract, Coffee bean extract and Coconut

liquid endosperm (1:1:1): The compositon exhibits the antioxidant properties of Coffee

bean extract, anti inflammatory properties of Lotus seed extract along with the cell

proliferation property of Coconut water extract thus fastening the process of skin

lightening.


Antioxidant activity: ORAC value of 10,930µmol trolox equivalents/g, HORAC value

of 4770µmol gallic acid equivalents/g, DPPH scavening potential with an IC50 of

0.83µg/ml, Lipid peroxidation inhibition with an IC50 of 347µg/ml.



25µg/ml.



318

The same composition when Coffee bean extract was replaced with Curry leaf extract

showed similar anti inflammatory, cell proliferation enhancement and antioxidant

potenital. But interestingly Lipid peroxidation inhibition was significantly higher than

that of what was attained when Coffee bean extract was used with an IC50 of 8.3µg/ml.

This activity was therefore exclusively contributed by Curry leaf extract.

5.3.6.3.4. A composition of Mangostin and Coconut water extract (1:1): This

composition exhibits the antioxidant and anti inflammatory properties of Mangostin

along with the cell proliferation property of Coconut water extract thus fastening the

process of skin lightening.


Antioxidant activity: DPPH scavening potential with an IC50 of 1.6µg/ml, ROS

scavening potential with an IC50 of 2µg/ml Lipid peroxidation inhibition with an IC50 of

22µg/ml.



50µg/ml.


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


319

of skin ageing. Antioxidants play a major role as nutricosmetics. Examples are tripeptide

Glutathione which is a common nutricosmetic, polyphenols etc. As discussed in the

earlier part of the results, compounds without direct effect on melanogenesis but with

significant anti inflammatory potential can still help in skin lightening when applied

topically. Similarly, it has been observed that compounds without direct effect on

melanogenesis but with significant antioxidant potential can help in skin lightening when

taken internally as nutricosmetics. The tripeptide Glutathione is one of the commonly

used antioxidants for nutricosmetic applications (Puizina-Ivic N et al., 2010). Other

major group of antioxidants, that plays an important role as nutricosmetics are

Polyphenols. Polyphenols are the phenol moiety containing chemical actives from plants.

The largest and best studied polyphenols are the flavonoids, which include several

thousand compounds, like flavonols, flavones, catechins, flavanones, anthocyanidins, and

isoflavonoids. Polyphenol rich products like green tea, grape seed extract, coffee bean

extract etc play a signigficant role as nutricosmetics although they do not exhibit

significant melanogenesis inhibitory potential in vitro.

5.3.7.1. Importance of ORAC for nutricosmetic benefits:

High ORAC foods increase the antioxidant power of human blood by 10 – 25% and

protect the blood vessels and capillaries from oxidative damage, thereby rendering

nutricosmetic benefits on oral intake. Table 5.3.66 shows the ORAC values of top food

supplements. All the mentioned food products in Table 5.3.66 are rich in Polyphenols so

further processing of these natural products enriching them to a higher concentration of

Polyphenols will increase the ORAC values, thereby providing the nutricosmetic benefits

at much lower doses.


320

Table 5.3.66: ORAC values of top Nutricosmetic food supplements as per the data from the U.S. Dept. of Agriculture & Journal of American Chemical Society

Product ORAC value (µmol trolox equivalents/g)

Unprocessed Cocoa powder 260

Acai berry 185

Dark chocolate 131.2

Prunes 57.7

Raisins 28.3

Blue berries 24

Black berries 20.36

Straw berries 15.4

Spinach 12.6

Broccoli florets 8.9

Red grapes 7.39

Cherries 6.7

5.3.7.1.1. Cocoa bean extract as a nutricosmetic:

From Table 5.3.67, it is evident that the ORAC value of Cocoa bean extract significantly

increased with the increasing concentration of polyphenols. However, there is no

significant increase in the DPPH scavenging potential with the increasing polyphenol

content. Even the HORAC value did not change significantly with the increasing

polyphenol content with a value in the range of 1744 to 1823 µmol gallic acid

equivalents/g for 20 to 50% polyphenol content. Similarly, the ROS scavenging potential

was same with an IC50 of 6.25µg/ml for 20 to 50% polyphenol content. Therefore, ORAC

value is crucial for nutricosmetic benefits. The role of polyphenols for antioxidant

potential is evident with the increasing antioxidant potential in proportion to the

increasing polyphenol content of Cocoa bean extract.


321

Table 5.3.67: Antioxidant potential of Cocoa bean extract

% Polyphenols DPPH scavenging

(IC50 in µg/ml)

ORAC value (µmol

trolox equivalents/g)

26 2 3635

27 2.7 4069

35 2 4922

37 2 5583

39 1.8 6667

50 1.2 8117

>50 1.2 – 1.7 11000 – 13000

5.3.7.1.2. Coffee bean extract as a nutricosmetic:

The principle constituents of green coffee bean were found to be chlorogenic acid and

caffeine out of which chlorogenic acid neutralizes free radicals and hydroxyl radicals,

both of which can lead to cellular degeneration if left unchecked. In addition synergistic

effects are also present due to the concentrated caffeine content ranging from 3-5%.

Compared to green tea and grape seed extract, green coffee bean extract is twice as

effective in absorbing oxygen free radicals. One of the advantages of using the green

coffee bean extract is that the negative effects of coffee are avoided.

Table 5.3.68: Antioxidant potential of Coffee bean extract

%

Chlorogenic

acid

ORAC value

(µmol trolox

equivalents/g)

HORAC value

(µmol gallic acid

equivalents/g)

DPPH scavenging

(IC50 in µg/ml)

40 6291 2935 1

60 9978 5206 2

65 10930 5891 1.25

80 12636 5621 0.96


322

From Table 5.3.68, it is evident that the ORAC value of Coffee bean extract significantly

increased with the increasing concentration of chlorogenic acid. However, there is no

significant increase in the DPPH scavenging potential or HORAC value with increasing

chlorogenic acid content.

5.3.7.1.3. Standardized extracts with “high ORAC value” for nutricosmetic benefits:

Standardized plant extracts with high ORAC value have significant potential for

nutricosmetic applications. Some combinations of actives have shown synergy as

nutricosmetics with enhanced antioxidant potential as observed in Table 5.3.69.

5.3.7.1.3.1. Green tea extract containing 70% Polyphenols:

ORAC value: 6907 µmol trolox equivalents/g. Similarly, Green tea extract with 80%

polyphenols has a higher ORAC value of 8027 µmol trolox equivalents/g, showing that

polyphenols play a significant role in the antioxidant potential of the extract.

Other significant properties of Green tea extract containing 70% Polyphenols:

HORAC value: 7121 µmol gallic acid equivalents/g, DPPH scavenging: IC50 - 3 µg/ml,

ROS scavenging: IC50 - 1 µg/ml, Elastase inhibition: IC50 – 275 µg/ml, Collagenase

inhibition: IC50 – 50 µg/ml, Hyaluronidase inhibition: IC50 – 5µg/ml, Tyrosinase

inhibition: 40% inhibition at 50 µg/ml, Melanin inhibition – 14% inhibition at 10µg/ml.

5.3.7.1.3.2. Grape seed extract containing 50% Polyphenols:

ORAC value: 4528 µmol trolox equivalents/g. Similarly, Grape seed extract with 70%

polyphenols has an ORAC value of 9699 µmol trolox equivalents/g, showing that


Other significant properties of Grape seed extract containing 50% Polyphenols:

HORAC value: 3254 µmol gallic acid equivalents/g, DPPH scavenging: IC50 - 3 µg/ml,

ROS scavenging: IC50 - 5 µg/ml, Elastase inhibition: IC50 – 9 µg/ml, Collagenase

inhibition: IC50 – 12 µg/ml, Hyaluronidase inhibition: IC50 – 5µg/ml, Tyrosinase

inhibition: IC50 – 28 µg/ml


323

5.3.7.1.3.3. Pomegranate extract containing 44% Polyphenols:

ORAC value: 4400 µmol trolox equivalents/g. Similary, Pomegranate extract with 65%

polyphenols has an ORAC value of 5687 µmol trolox equivalents/g, showing that


Other significant properties of Pomegranate extract containing 44% Polyphenols:

DPPH scavenging: 17% scavenging at 300 µg/ml, Elastase inhibition: IC50 – 500 µg/ml,

Collagenase inhibition: IC50 – 62.5 µg/ml, Hyaluronidase inhibition: IC50 – 0.5µg/ml,

Tyrosinase inhibition: IC50 – 7 µg/ml

5.3.7.1.3.4. Pomegranate rind extract:

ORAC value: 14,087 µmol trolox equivalents/g.

Other parts of the pomegranate fruit also were observed to have significant antioxidant

properties. Pomegranate seed extract has an ORAC value of 3350 and Pomegranate juice

extract has an ORAC value of 400 µmol trolox equivalents/g.

Other significant properties of Pomegranate rind extract:

HORAC value: 12,743 µmol gallic acid equivalents/g, DPPH scavenging: IC50 –

0.4µg/ml, Elastase inhibition: IC50 – 500µg/ml, Collagenase inhibition: IC50 – 62.5µg/ml,

Hyaluronidase inhibition: IC50 – 0.5µg/ml, Tyrosinase inhibition: 12% inhibition at

10µg/ml.

5.3.7.1.3.5. Pomegranate extract standardized to 90% Ellagic acid:

ORAC value: 8299 µmol trolox equivalents/g.

Other significant properties: Melanin inhibition: 16% inhibition of melanin at 1.25

µg/ml, ROS scavenging: IC50 - 20 µg/ml.

It was observed that pomegranate rind extract has the highest antioxidant potential than

that of seeds and juice. Hence, pomegranate rind which is unutilized during consumption

has significant nutricosmetic benefits.


324

5.3.7.1.3.6. Turmeric extract:

ORAC value: 10,410 µmol trolox equivalents/g

Other significant properties:

HORAC value: 9740 µmol gallic acid equivalents/g, DPPH scavenging: IC50 – 1.8µg/ml,

Tyrosinase inhibition: 22% inhibition at 3µg/ml.

5.3.7.1.3.7. Rosmarinic acid:

Rosmarinus officinalis leaf extract containing 90% Rosmarinic acid:

ORAC value: 14000 µmol trolox equivalents/g


HORAC value: 5925 µmol gallic acid equivalents/g, DPPH scavenging: IC50 – 0.5µg/ml,

Collagenase inhibition: IC50 – 250µg/ml, Hyaluronidase inhibition: IC50 – 25µg/ml

Rosmarinus officinalis leaf extract containing 50% Rosmarinic acid:


Coleus forskohlii leaf extract containing 90% Rosmarinic acid:


Coleus forskohlii leaf extract containing 50% Rosmarinic acid:


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:



HORAC value: 1305 µmol gallic acid equivalents/g, Elastase inhibition: IC50 - 125 µg/ml.


325

5.3.7.1.3.9. Tulsi extract:



DPPH scavenging: IC50 – 3.8µg/ml

5.3.7.1.3.10. Mulberry extract:


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).


326

Table 5.3.69: Synergistic antioxidant compositions

Composition

Activity of actives Activity of composition

Activity obtained on

analysis

Expected

activity

(Additional

effect)

Synergistic activity

(Higher than the

expected additional

activity)

ORAC value (µmol trolox equivalents/g)

Rosmarinic acid &

Chlorogenic acid

(1:3)

Rosmarinic acid – 14,000

Chlorogenic acid – 9978 10,984 16,595

Rosmarinic acid &

THC (1:1)


THC – 9244 10,433 16,859

Rosmarinic acid, THC

& Chlorogenic acid

(1:1:1)


THC - 9244

Chlorogenic acid – 9978

11,074 13,931

Turmeric extract &

Green tea extract (1:1)

Turmeric extract –

10,410

Green tea extract – 8027

9000 14,418

5.3.7.3. Nutricosmetic formulations:

For oral applications, the nutricosmetic compositons should be diluted with excepients

and formulated as tablets, capsules or powders for consumption. Since the concentration

of the actives in formulations will be less, the ORAC value will also be less. However

3,000 to 5,000 ORAC units per day are required to have a significant impact on plasma

and tissue antioxidant capacity. Therefore, the ORAC value can be potentiated by

increasing the servings. The whole concept of nutricosmetics is about attaining beauty

from within naturally and without any feel of undergoing a scheduled medication. Tablets


327

or capsules can be used directly whereas the powdered formulations can be mixed in

water or other beverages for consumption. For example, though carriers like certain

beverages, ice creams, shakes, soups, pre mix powders etc. containing mostly sugars,

flavors, creams, gums, citric acid etc., do not have nutricosmetic benefits, on mixing a

certain minimal dosage of nuticosmetic actives into these carriers increases the likeability

factor of the preparations as well as its nutricosmetic benefits. Some of the examples are,

5.3.7.3.1. Health drink syrup that can be mixed in milk or water:

In a composition containing nutricosmetic actives like Mulberry extract, Amla extract,

Aloe vera, Grape seed extract and Green tea extract, the ORAC value as observed in

Table 5.3.70 was 62µmol trolox equivalents/g. The ORAC value can be increased to

3100 µmol trolox equivalents/g by having 10 servings of 5gm each per day to attain the

minimal ORAC requirement per day.

Table 5.3.70: Nutricosmetic healthdrink syrup Nutricosmetic

active

Concentration

(%)

ORAC (µmol trolox

equivalents/g)

Recommended Servings of

5g /day

Mulberry extract 2

62 10

Amla extract 2

Aloe vera extract 0.5

Grape seed extract 0.5

Green tea extract 1

5.3.7.3.2. Enriched Green coffee that can be mixed in milk or water:

In a composition containing nutricosmetic actives like Green coffee bean extract, Grape

seed extract and Amla extract in instant coffee, the ORAC value as observed in Table

5.3.71 was 1751µmol trolox equivalents/g. The ORAC value can be increased to 3502

µmol trolox equivalents/g by having a serving of 2gm per day in milk or water to attain

the minimal ORAC requirement per day. Coffee which is the most popular beverage can

be made a health drink with cosmetic benefits by adding nutricosmetic actives into it.


328

Table 5.3.71: Enriched coffee as a nutricosmetic

Nutricosmetic

active

Concentrati

on (%)

ORAC (µmol trolox

equivalents/g)

Recommended

Servings of 2g /day

Coffee bean extract 0.92

1751 1 Grape seed extract 2.4

Amla extract 1.5

Instant coffee base 95.24

5.3.7.3.3. Enriched Chocolate that can be mixed in milk:

In a composition containing nutricosmetic actives like Cocoa polyphenols in Cocoa

powder, the ORAC value as observed in Table 5.3.72 was 200µmol trolox equivalents/g.

The ORAC value can be increased to 3000 µmol trolox equivalents/g by having 3

servings of 5gm per day in milk to attain the minimal ORAC requirement per day.

Chocolate drink which is the most popular beverage can be made into a health drink with

cosmetic benefits by adding nutricosmetic actives into it.

Table 5.3.72: Enriched chocolate as nutricosmetic

Nutricosmetic

active

Concentrati

on (%)

ORAC (µmol trolox

equivalents/g)

Recommended

Servings of 5g /day

Cocoa polyphenols 0.66 200 3 Cocoa powder 6.5

5.3.7.3.4. Enriched Tea that can be mixed in water:

In a composition containing nutricosmetic actives like Amla extract, Green tea extract

and Tulsi extract the ORAC value as observed in Table 5.3.73 was 161µmol trolox

equivalents/g. The ORAC value can be increased to 3220 µmol trolox equivalents/g by

having 4 servings of 5gm per day in water to attain the minimal ORAC requirement per


329

day. Tea which is a very common beverage can be made a health drink with cosmetic

benefits by adding nutricosmetic actives into it.

Table 5.3.73: Enriched green tea as nutricosmetic Nutricosmetic

active

Concentration

(%)

ORAC (µmol trolox

equivalents/g)

Recommended Servings

of 5g /day

Amla extract 2

161 4 Tulsi extract 1

Amla extract 2

CHAPTER 1 PART II 5.4. CONCLUSION OF PART II

330

Various actives were screened through different in vitro mechanisms for pigmentation

and were positioned in accordance to their specific mode of action for rectifying

pigmentation disorders. Hence, the actives can be recommended for skin lightening based

on the root cause of pigmentation.

In the process of screening, some novel skin lightening actives and some extracts

with synergistic combination of actives have been observed. Thymohydroquinone from

Nigella sativa seed extract, Hydroxychavicol from Piper betle leaf extract,

Avenanthramides from oat kernel extract, ceramides from apple fruits and oats and

Eugenia jambolana extract were found to have significant skin lightening potential. The

skin lightening property of Amla extract and Artocarpus lakoocha extract was not

conferred exclusively by Ascorbic acid and Oxyresveratrol respectively but due to the

synergistic combination of various components of the two extracts.

Synergistic effect of various biological mechanisms for enhanced skin lightening

potential has been demonstrated by chemical conjugations of actives like Oleanoyl

peptide. Similarly the synergistic effect of various biological mechanisms for enhanced

skin lightening potential has been demonstrated by physical combination of actives. The

study emphasizes the integration of various mechanisms of skin lightening for a

synergistic effect.

Combination of antioxidant actives were shown to have synergistic potential and

can be useful as nutricosmetics. Actives like Rosmarinic acid that naturally existed in

combination with leaf matrix components of Coleus forskohlii, showed synergistic

antioxidant activity significantly higher than that of Rosmarinic acid alone. It has also

been shown that although some actives do not directly inhibit melanogenesis, they help in

skin lightening by other mechanisms of action like antioxidant potential, collagen

enhancement and anti inflammatory potential and can also be used as nutricosmetics.

5.4. CONCLUSION OF PART II

part ii screening of actives through …shodhganga.inflibnet.ac.in/bitstream/10603/70415/8/chapter...

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