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BiowhiteTM

Plant active

LA LINEA COSMECEUTICA ANTI-AGE [formulata]IN PRIMA PERSONA DAL DERMO-CHIRURGO ESTETICO

Table of contents

Biowhite

™

Introduction ........................................................................... 2Skin and its environment ..............................................................2Protection against UV ...................................................................2

The melanin ........................................................................... 3The melanogenesis ......................................................................3

Distribution of melanocytes in human Organisation of the melanocytary system Melanins synthesis Biochemical phases in melanin synthesis

Pigmentation disorders .................................................................7

Biowhite™: a demonstrated efficacy ...........................................8Status report on depigmenting reagents: problems and solutions ..............8Results and discussion ................................................................ 10

Anti-tyrosinase activity on isolated enzyme Melanocyte cytotoxicity In vitro study on tyrosinase from cultured melanocytes In vitro study on human melanocyte culture (DOPA-reaction) Ex vivo study on human skin explants In vivo study

Biowhite™: description ............................................................ 17Main components ...................................................................... 17

Saxifrage extracts: Saxifraga sarmentosa Grape extracts: Vitis vinifera Mulberry extracts: Morus bombycis Scutellaria root extracts: Scutellaria baicalensis EDTA

Conclusion ........................................................................... 19

Bibliography ......................................................................... 21


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Biowhite™

Introduction

The skin is the body's first line of defense from external aggressions. It is not simply a passive, sealed barrier but plays an active part in the

synthesis and secretion of molecules with immuno-modulating properties, in particular for protection from solar radiation.

Specific cells called melanocytes protect the body from solar radiation. Melanogenesis is a complex process which produces a dark pigment called melanin. Melanin protects cutaneous structures and delays sunburn (erythema).Before discussing the structure and the exact functions of melanocytes in detail, let us examine how the skin responds to ultra-violet radiation.

In the epidermis, solar radiation defence mechanisms are located at three levels:

• The proteo-hydro-lipidic surface film has a very low photo-protective effect, basically due to urocanic acid - a molecule found in perspiration. This film filters sunlight and absorbs some UVB radiations. The concentration of this substance in the horny layer increases as more perspiration is produced.

• The horny layer has a major photo-protective role: most solar radiation is reflected by the skin. Horny layer thickness varies in different places of the body and increases when exposed to solar radiation to provide additional protection. Hyperkeratosis is the reason why exposure to the

sun for three weeks multiplies the time required for a sunburn to appear by a factor of four.

• Melanic pigmentation is the main and most effective protection from solar radiation. Activated spontaneously in black-skinned subjects, it is stimulated by UV radiation in white-skinned subjects. "Sun-tans" are due to the stimulation of every phase of melanogenesis.

Melanin is a natural solar filter, absorbing and reflecting over 90% of UVs which pass through the horny layer.

However, all melanins do not provide effective solar protection and phæomelanin (the melanin of "red-haired" people) is much less active than eumelanin (found in "black-haired" people). Moreover, phaeomelanin is extremely phototoxic, because like all melanins, it reacts with some forms of free radicals but actually forms more toxic free radicals which can irreversibly damage the genetic material of the keratinocytes(1).

Some disorders caused by melanisation unit malfunction can induce unsightly hyper-pigmentation.

Melanin synthesis inhibitors are particularly interesting in cosmetology, not only for whitening applications - to whiten darkly-pigmented skins or to inhibit hyper-pigmentation in some pathologies, for example - but also for applications designed to lighten complexions and add lustre to skin surfaces.

Structurally, the skin is a complex, vital, organ. It is generally considered to have five main functions:

• A protective function. The skin protects the in-terior medium from aggressions by the external medium,

• An exchange function with the external envi-ronment. Cutaneous tissue is not a totally sealed barrier,

• A sensorial function: the skin feels pressure, cold, heat and pain; the skin is the main compo-nent in the mechanism which maintains body tem-perature constant,

• An immuno-modulating function function throu-gh the synthesis and secretion of soluble messen-gers such as cytokines.

• Metabolic functions - primarily the synthesis of vitamin D.

The most important of these five functions is to pro-tect the body from external aggressions.

The skin is a physical, biochemical and immunolo-gical barrier which resists mechanical aggressions, pollution and solar radiation and prevents the pene-tration of substances from outside the body such as microbes and other antigens.

Skin and its environment

The different epidermal cell populations combine their actions to protect the body from the external environment.

Protection against UV

(1) Wenczl E et al. J Invest Dermatol 1998 ; 111 : 678-682.


Engelhard seeks to present reliable information concerning the composition, properties and use of its products, services and processes. However, all literature, advice and other material concerning any product, service or process or its selection or use is provided WITH NO WARRANTY OF ANY KIND. All sales are subject to Engelhard’s Terms and Conditions of Sale, which are available upon request. ALL IMPLIED WARRANTIES ARE DISCLAIMED. Remedies for any breach and Engelhard’s liability, including that for patent infringement, are limited as provided in Engelhard’s Terms and Conditions of Sale. Engelhard is not liable for consequential, incidental, or special dama-ges. Nothing should be construed as a recommendation or inducement to infringe any patent. No assumption should be made that all safety or environmental protection measures are indicated, or that other measures may not be required.


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March 22, 2006 Biowhite™

Each melanocyte is an isolated cell located in the epidermis supported by the basal membrane. Melanocyte have globular cellular bodies (Figure 1) from which many cell ramifications of different lengths - the dendrites - are deployed.

These penetrate the neighbouring keratinocytes of the basal layer and reach up to the third layer of the mucous Malpighi body.

The usual name given to this structure comprising a melanocyte and 36 associated keratinocytes is the

"epidermal melanisation unit".

The melaninThe melanogenesis

The pigmentary system in man is extremely complex - and explains the wide diversity in human skin coloration.

Melanocytes are the key of this organisation. They derive from the same embryonic tissue as nerve cells.

It is only between the 8th and the 11th week of embryonic life that the melanoblasts, which do not have the definitive shape of melanocytes at that point, migrate towards the territories in the body that they will subsequently occupy (the skin, the retina and hair follicles).

Distribution of melanocytes in humanOnly electron microscopy counts can precisely eva-luate the quantity of melanocytes present in the tis-sue investigated. Dispersed throughout the whole cutaneous surface, they represent 13% of the cell population of the epidermis. They are extremely numerous in zones exposed to sunlight such as the forehead and the cheeks (from 2000 to 2500 per mm2). This count varies from 1000 to 1500 in the rest of the body. It should be noted that subjects with high levels of pigmentation and black-skinned

people have only slightly more melanocytes than people with "red" and "fair" hair. Skin colouring does not depend on the quantity of melanocytes but on the type of melanin the body produces and its loca-tion in the epidermis.

The number of active melanocytes decreases with age (from 10 to 20% every 10 years). This decrease become clearly visible after 40, particularly at the hair roots.

Organisation of the melanocytary system

Inter-cellular organisation

Melanocytes are not attached to neighbouring cells and do not have desmosomes.Recent studies of the "epidermal melanisation unit" show that the melanosomes in the melanocyte den-drites are transferred by phagocytosis into adjacent keratinocytes, including the keratinocytes in the basal membrane.

Basal membrane keratinocytes contain very high quantities of melanosomes. They are organised in a systematic way around the nucleus and more par-

ticularly on the upper pole of the cell, i.e. towards the surface of the tegument. This phenomenon, called "capping", covers and pro-tects the basal membrane keratinocyte cell nuclei (as well as the DNA it contains) from UV radiations.

Any unrepaired alteration to the chromosomes of these keratinocytes has disastrous consequences on cutaneous tissues because they are the only cells which renew the epidermis.

Horry layer

Granular layer

Malpighi

mucous

body

Basale

layer

Lame basale

Figure 1: Melanocyte(2)

(2) Peyrefitte G. Paris : Ed Simep ; 1993.



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After being phagocytosed into the keratinocy-tes, melanosomes are usually digested by the hydrolytic enzymes of these cells. This leads

to release of the melanin grains which can themsel-ves be degraded. Thus, the number of melanosomes decreases in the upper layers of the epidermis and they totally disappear in the horny layer. Observed in red-haired and Caucasian subjects, this pheno-menon is not exactly the same in black-skinned sub-jects.

It would seem that no hydrolytic activity follows melanosome phagocytosis because they are obser-ved entire and intact in the horny layer - making it a continuous filter (Figure 2). In yellow-skinned races, the melanosomal degradation process in the upper layers of the epidermis exists but is slowed.

Intra-cellular organisation

The cytoplasm of the melanocytes contains a system of micro-tubules and micro-filaments which, in some species of animals (e.g. chameleons), are responsible for rapid colour changes but which, in man, are used for melanosome movements in cells and particularly for migration to the ends of the dendrites.

Melanosomes are vesicles which bud from the Golgi apparatus (Figure 3). Melanins are produced in the-se melanosomes by a sequence of complex chemical reactions from an amino-acid - tyrosin - in the pre-sence of an enzyme - the tyrosinase.

Information transmitted by the cell nucleus ins-tructs the ribosomes of the endoplasmic reticulum to synthesise tyrosinase. Tyrosinase is concentrated in the vesicles secreted - sometimes called pre-me-lanosomes. Inactive in the Golgi apparatus, tyrosi-nase is activated when it arrives in the vesicles.

As these vesicles migrate to the skin surface, their appearance changes and their structure, observed with electron microscopy, is increasingly dense. Throughout this migration, the activity of the tyro-sinase decreases but does not completely cease.

In dark-skinned subjects, the size of the egg-sha-ped melanosomes is between 0.5 and 2 µm and in

"red-haired" subjects from 2 to 8 µm. Depending on their degree of maturity, four phases can be obser-ved (Figure 3):

• Phase I melanosomes are round, clear vesicles which contain a disorganised protein-type mate-rial and micro-vesicles of tyrosinase,

• Phase II melanosomes are egg-shaped vesicles. The protein molecules are organised as a spiral of filaments.

• In Phase III the internal structure of the melano-somes becomes denser.

• Phase IV melanosomes are entirely melanised and completely opaque. It is these structures which are transferred to adjacent keratinocytes.• L e s mélanosomes de stade IV, sont entièrement mé-lanisés, totalement opaques. Ce sont ces structu-res qui sont tranférées aux kératinocytes voisins.

Figure 2: Diagram showing variations in the three principle ways in which melanosomes are transferred to keratinocytes(3)

Celtic Caucasian Negroid

Ribosomes

Endoplasmic reticulum

Golgi appaatus

Tyrosinase synthesis Organite synthesis Melanin synthesis

Figure 3: The different phases of melanin synthesis in the melanocyte(2)

I II III IV

(2) Peyrefitte G. Paris : Ed Simep ; 1993.

(3) Fitzpatrick T et al. Tokyo : University of Tokyo Press ; 1974.



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Melanins synthesis

A sequence of complex phases induces the ty-rosin to form polymer structures - melanins - the colour of which depends on their state of

oxidation. Biochemical differences in melanins ex-plain different pigment colours. There are two ma-jor groups of melanins with totally different physical, chemical and biological properties: eumelanins and phaeomelanins.

• Eumelanins are brown or black melanins, with hi-gh molecular weights and a polymerised structure. Insoluble in most known solvents, their structure is complex and has not yet been totally investiga-ted. Formed by the polymerisation of several hun-dreds of oxidised phenol radicals, they become amorphous and generate a totally light-absorbent chemical - which is why they appear to be black. The chemical compounds in this polymer inclu-de metals such as copper and zinc as well as low quantities of sulphur.

• Phaeomelanins are typically yellow to red/brown and are isolated from the hair of red-haired or fair-haired people. Unlike eumelanins, phaeomelanins are soluble in mildly basic media as their degree of polymerisation - and thus their molecular wei-ght - is much lower.

Their sulphur content is also much higher. Although they absorb UV radiations, the protection they afford is approximately 1000 times lower than eumelanins.

Everyone has both types of melanin. Differences in proportions are what create differences in skin and hair coloration.

The colour of normal human skin results from the combination of three colours induced by three types of pigments: red oxidised haemoglobin and blue re-duced haemoglobin in the blood vessels of the dermis and particularly the combination of the two types of melanin described above which are responsible for what is called "constitutive skin pigmentation".

Constitutive skin pigmentation is genetically pro-grammed for each individual. The quality and quan-tity of melanin produced determines the large spec-trum of skin colours.

It should however be noted that only some hundreds of milligrams of melanin differentiate the whitest skins of northern Europeans from the darkest skins encountered in Africa...

Melanisation is the most effective biological solar radiation protection process. Sun-tans are pigmen-tation - called adaptive pigmentation.

When the skin is affected by UV radiation it produ-ces several tens of milligrams of additional melanin as a protection.

There is no change in the quality of the melanin pro-duced but an increase in the quantity.



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Although distinguished by their molecular weights and their general physical proper-ties, eumelanins and phæomelanins are rela-

ted from a biochemical point of view because the first two phases of their biosynthesis are the same (Figure 4):

• L-tyrosin is first oxidised into L-dihydroxyphenyla-lanin (L-Dopa) by the action of the tyrosin-3-hy-droxylase also known as L-tyrosin, tetrahydropte-ridine: oxygen oxydoreductase (E.C 1.14.16.2.).

• The L-Dopa is oxidised into L-Dopaquinone un-der the action of tyrosinase or 1,2-benzene-diol: oxygen oxydoreductase (E.C 1.10.3.1.), an enzy-me which is only activated when Cu2+ ions are present.

After L-Dopaquinone, their metabolic pathway sepa-rate and they either become eumelanins or phaeo-melanins.

• To make eumelanins, L-Dopaquinone is cyclized to form a leucoderivative which itself is oxidised again by the tyrosinase to form a coloured mole-cule called Dopachrome or hallachrome - detecta-ble at 475 nm by spectrophotometry.

Dopachrome is then decarboxylated to form a molecule called indol-5,6-quinone - the basic unit in the polymerised structures of eumelanins.

• Phaeomelanins are formed by a more complex se-ries of reactions which are much less well unders-tood. L-Dopaquinone is modified by a cysteic clus-ter (probably via a glutathion reduction phase) to obtain cysteinyldopa. Cysteinyldopa is then oxi-dised into alanin benzothiazin. Phaeomelanins are characterised by 1-4 benzothiazin units.

• The final organisation of eumelanin and phaeome-lanin polymers is probably due to the action of spe-cific proteins only found in melanic structures.

Figure 4: Melanin biochemical synthesis

Biochemical phases in melanin synthesis



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The main factors in skin coloration are the nature of the melanin, the place where the melanin is concentrated (epidermis or dermis)

and skin vascularisation. Variations observed in melanocytes have little effect on pigmentation which is more directly related to the quantity, type and distribution of melanosomes.

Conversely, pigmentation anomalies (hyper- or hypo-pigmentation) are directly related to the quantity of melanocytes. Among hyper-pigmentations, we list below those for which:

• Melanocytes proliferate correctly: basically these are pigmented spots caused by temporary or per-manent epidermal melanisation unit dysfunction.

Freckles are frequently observed in skin areas exposed to the sun (the face, forearms, hands and legs) in red-haired phototypes. These are eumelanin zones on phaeomelanin backgrounds.

Chloasma - pregnancy mask - is a more or less intense, irregular and symmetrical hyper-pigmentation localised on the forehead, around the eyes and the sides of the face. Chloasma is a hypersecretion of melanin induced by hormonal factors amplified by the effects of the sun.

Diffuse brown hypermelanosis is symptomatic of endocrine system disorders or nutritional anomalies.

Hypermelanosis can follow cutaneous inflammations. This phenomenon is responsible for the pigmentation of scars and pigmentation caused by irritants combined with exposure to sun. Bergamot, frequently used in cosmetics, is a powerful photo-sensitizer and can induce unsightly hyper-pigmentations.

• Melanocytes do not proliferate correctly:

Lentigines are small pigmented spots (0.1 to 0.3 cm) which appear on the mid-part of the face. Lentiginosis can be hereditary and is considered to be a disorder which can appear anywhere on the body and persist throughout the winter.

Solar Lentigo is a wider lesion than a freckle which occurs after serious sunburn on all cutaneous phototypes.

Senile Lentigo is generally observed on the back of the hands of older subjects; solar radiation stimulates its development.

Most frequently observed on the face, Dubreuilh melanosis (or malignant lentigo of the elderly) is a large, pigmented, multi-coloured stain with irregular contours. It is a pre-cancerous condition.

Moles (or naevus) are accumulations of melanocytes in the epidermis and the dermis. Moles have recently received particular attention. Apparently, the number of moles has increased over the last thirty years as have malignant melanomas which are currently considered to be the most rapidly progressing form of cancer.

Malignant melanomas are increasingly well understood as their development can be observed directly. The first signs of malignant tumours of the pigmentary system in healthy skins are the degeneration of an existing naevus or Debreuilh melanosis.

Pigmentation disorders



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Biowhite™: a demonstrated efficacyStatus report on depigmenting reagents: problems and solutions

Some disorders of the melanocyte system, rela-ted to a dysfunction of the melanization me-chanism, can cause particularly unsightly hyper-

pigmentation. It is not surprising, therefor, that de-pigmenting product development has been a major research topic for cosmetics manufacturersgie.

There are four basic approaches to reducing skin pigmentation or preventing excessive pigmentation. These are:

• melanocyte activity reduction by using substan-ces to slow down melanin synthesis by selective cytotoxicity of melanocytes, for example,

• chemical and biological L-Dopa molecule reduc-tion to slow oxidation into a chromophoric com-pound,

• elimination of inflammatory reactions like erythe-mas which occur after UV radiation,

• tyrosinase activity reduction. This can be carried out by inhibiting tyrosinase synthesis - the key en-zyme in melanogenesis - or by using an inhibitor which competes (or does not compete) with tyro-sinase usual substrates.

In the past, the methods used to whiten skin we-re relatively drastic and did not require understan-ding of melanogenesis mechanisms. Chemicals used included hydrogen peroxide, mercurialised amide chlorate, mercaptoamines and many derivatives of phenol - principally hydroquinone and its catechol and alkyl-phenol esters.

These molecules whiten the skin very rapidly by de-grading the organisation of melanin structures. The use of these molecules is based on the observa-tion of extremely rapid skin whitening due to the breakdown of organized melanin structures.

Hydroquinone has a very weak anti-tyrosinase acti-vity and has high cytotoxic activity (CI50 of approxi-mately 100 µg/ml), in normal human melanocyte cultures at concentrations significantly lower than those necessary to inhibit tyrosinase(4).

Other compounds were then developed such as ar-butine, an hydroquinone derivative with low cyto-toxic properties.

However the relatively unspecific action mechanism of these products makes the outcome of their usage somewhat doubtful.

For example, arbutine, which has a weak anti-tyrosi-nase activity - 20% inhibition at a concentration of 100 µm/ml and after 5 days treatment(5) - has even been suspected of increasing pigmentation in normal human melanocytes in culture(6).

For these reasons, more "specific" approaches were developed and the main depigmenting substances developed act on a molecular level inside melano-genesis regulation mechanisms. These substances target tyrosinase, the key enzyme in melanogene-sis, the existence of which was demonstrated in the epidermis by Lerner et al.(7) in 1949. Basically the-se substances are kojic acid, ascorbic acid and their derivatives.

(4) Smith C et al. Pigment Cell Res 1988 ; 1 : 386-389.

(5) Chakraborty AK et al. Pigment Cell Res 1998 ; 11 : 206-212.

(6) Nakajima M et al. Pigment Cell Res 1998 ; 11 : 12-17.

(7) Lerner A et al. J Biol Chem 1949 ; 178 : 185-195.



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Biowhite™: a demonstrated efficacyStatus report on depigmenting reagents: problems and solutions

However, despite their specificity, these pro-ducts have other, major, drawbacks. The high instability of kojic acid which oxidizes rapidly

in complex compounds like cosmetic formulations leads to the appearance of a yellow-brown color which is not exactly the organoleptic property best suited to this field. Additionally, kojic acid, although possessing anti-tyrosinase properties(8) has recently been associated with contact allergy phenomena(9).

Similarly, all work published on the depigmenting activity of ascorbic acid and its derivatives is also inconclusive. In fact, Kameyama et al.(10), reported that ascorbic acid oxidizes and decomposes rapi-dly in an aqueous medium and, for this reason, is not a good depigmenting agent. These authors al-so showed that an ascorbic acid derivative, magne-sium-L-ascorbyl-2-phosphate (VC-PMG), is much mo-re efficacious and powerfully inhibits the formation of melanin in normal human melanocytes in cultu-re(10). However, the authors conclude by an in vi-vo study of the depigmenting activity of VC-PMG in which they report 55% efficacy of this compound in a pathological situation (pregnancy masks and aging spots) and only 12% in a physiological situation.

Additionally, other authors have been able to show that ascorbic acid has pro-oxidizing effects in a me-dium containing copper sulfate and/or metal ions(11) or, further, that through the formation of hydrogen peroxide (H2O2) it is highly cytotoxic in normal hu-man dermal fibroblasts cultures(12).

Apparently then, all depigmenting substances used up until now have major drawbacks. It is these draw-backs which have led to Engelhard's new depigmen-ting substance development program. This program used proven techniques:

1) chemistry for the study of anti-tyrosinase activity,

2) cytochemistry and histochemistry (DOPA- reac-tion) to reveal melanin in cultured melanocytes and in human skin explants respectively.

Development was revolved around four comple-mentary approaches:

1) The development of a stable product with a high anti-tyrosinase activity in an isolated enzyme mo-del and on normal human melanocytes in culture,

2) Confirmation that anti-tyrosinase activity measu-red in the normal human sight melanocyte cultu-res were not caused by a cytotoxic phenomenon,

3) Demonstration of a reduction in melanin in nor-mal human melanocytes in culture in response to anti-tyrosinase activity,

4) Confirmation of melanin synthesis inhibiting acti-vity in an ex vivo model (explants of human skin) which mimic the in vivo situation where several types of cells inter-react.

Our research led to Biowhite™, a new generation of depigmenting substances made from botanical extracts which deliver powerful depigmenting effects at extremely low concentrations.

Very well tolerated in cosmetic applications, its cytotoxicity is 200 times less than hydroquinone.

(8) Kahn V. Pigment Cell Res 1995 ; 8 : 234-240.

(9) Nakagawa M et al. Contact Dermatitis 1995 ; 32 : 9-13.

(10) Kameyama K et al. J Am Acad Dermatol 1996 ; 34 : 29-33.

(11) Tirosh O et al. Free Rad Biol Med 1996 ; 20 : 421-432.

(12) Peterkofsky B et al. J Cell Physiol 1977 ; 90 : 61-70.



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Anti-tyrosinase activity on isolated enzyme

The first experiments to assess the depigmenting power of Biowhite™ were carried out on an isolated enzyme model. Some of the plant extracts used in the study were totally ineffective in inhibiting tyrosinase: this is the case of bergamot and santolin extracts which had no effect at 0.3% or 0.7%

concentration.

Hydroquinone is accepted in the USA as an OTC whitening product, but has no tyrosinase inhibiting action. This molecule works in a completely different way. Its whitening effect is due to its very high cytotoxicity, combined to its ability to avoid black melanine pigment formation.

0.1% ascorbic acid inhibits 66% of tyrosinase activity and this test demonstrates its whitening activity. However, at this concentration, it turns preparations yellow.

Kojic acid used at 0.1 and 1% respectively inhibits 71 and 80% of the tyrosinase activity. The interest of this extremely high activity is however limited by the preparation drawbacks mentioned above.

The inhibiting activity of Biowhite™ on tyrosinase can be detected at a concentration of 0.01%. Biowhite™ inhibits 93% of tyrosinase activity at a concentration of 0.5%.

These studies carried out on purified enzyme fully demonstrate that:

1) the anti-tyrosinase activity of Biowhite™ is extremely powerful (virtually total inhibition at a concentration of 0.5%)

2) Biowhite™ has a much higher depigmenting potential than every other conventional depigmenting substance tested in the same condition.

It was important to confirm that Biowhite™, used at a concentration inducing maximum tyrosinase inhibition (0.5% of Biowhite™ for 93% tyrosinase inhibition) has not cytotoxic for the target cell, the melanocyte.

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Control (0.0%) Bergamote (0.7%) Bergamote (0.3%) Santoline (0.7%) Santoline (0.3%) Hydroquinone (1.6%) Hydroquinone (0.8%) Hydroquinone (0.4%) Ascorbic acid (0.1%) Kojic acid (1.0%) Kojic acid (0.1%) Biowhite™ (100%) Biowhite™ (50%) Biowhite™ (5%) Biowhite™ (1%) Biowhite™ (0.5%) Biowhite™ (0.02%) Biowhite™ (0.015%) Biowhite™ (0.01%) Biowhite™ (0.001%)

Figure 5: % tyrosinase inhibition obtained in vitro for the different tested active ingre-dients

0 10 20 30 40 50 60 70 80 90 100

MATERIALS & METHODSTyrosinase catalyses the for-mation of L-dopaquinone and then Dopachrome from L-Dopa. Dopachrome is a coloured compound and it is possible to observe its appearance by UV-visible spectrophotometry at 475 nm. Comparisons in the rate at which this coloured molecule appears indicate the power of an active to modify enzymatic activity.

Comparison of Dopachrome for-mation rates enables accurate determination of activations and inhibitions obtained with the different molecules tested.The nature and composition of the ingredients used to develop Biowhite™ were selected using this evaluation technique.

Results and discussion

0%0%0%

0%0%0%

0%

0%66%

80%71%

100%

100%

100%97%

93%

46%21,6%

0%0%

Inhibition (%)



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Melanocyte cytotoxicity

In this test, human cultured melanocytes were exposed to increasing concentrations of Biowhite™ for 72 hours. Cell viability was assessed at the end of the period.

Test results show that Biowhite™ has low cytotoxic activity on human melanocytes in culture. In fact, the concentration which leads to the loss of viability of half the cells (CI50) is between 1 and 10%. This result should be compared with the results obtained during tests of anti-tyrosinase activity in which an inhibition of 93% of the enzymatic activity was obtained with a concentration of Biowhite™ of just 0.5%! The depig-menting effect of Biowhite™ was not the result of cytotoxic effect on its target cell.

In 1991, MAEDA & FUKUDA(13) demonstrated that in human cultured melanocytes the CI50 of depigmenting substances conventionally used in cosmetics was much lower than Biowhite™ (hydroquinone: 5.5 10-3 mg/ml; linoleic acid: 2.8 10-3 mg/ml) which means that these substances are much more cytotoxic for their tar-get cell than Biowhite™.

The next step was to find out if Biowhite™ could penetrate the melanocyte and reach the target molecule - tyrosinase.

In vitro study on tyrosinase from cultured melanocytesIn these experiments, normal human melanocytes were cultured in the presence or absence of Biowhite™ during 15 hours, then the tyrosinase activity was measured.

The results obtained show that tyrosinase activity in the melanocyte extracts was strongly inhibited (- 85%) when incubated in a medium containing only 0.1% of Biowhite™.

It should be noted that Biowhite™, used at an extre-mely weak concentration, inhibits tyrosinase activi-ty to a greater degree than the positive control with a 0.02% concentration of ascorbic acid (maximum non-cytotoxic concentration).

In our experimental conditions, 0.1% Biowhite™ penetrates the melanocyte and inter-acts with its target molecule, tyrosinase (85% inhibition).

It is also of interest to note that the concentra-tion of Biowhite™ used here (0.1%) closely corre-lates with the concentration obtained after transcutaneous penetration of approximately 1% of for-mulated substance.

MATERIALS & METHODSMelanocyte cell line G361 (human melanome) was cultu-red in Mac Coy Medium (GIBCO) supplemented with 10% fetal calf serum, 50-UI/ml penicil-lin, 50-µg/ml streptomycin and 2-mM glutamin.

Cell were seeded with 3.105 cells per well, suspended in the medium described pre-viously and Biowhite™ added at different concentrations.After incubating for 72-hours with Biowhite™, cell viabi-lity was evaluated by a protein assay with a BIO-RAD kit.

0

20

40

60

80

100

120

Témoin Biowhite 0.1% VitC 0.02%Control Biowhite™

0.1%Ascorbic acide

0.02%

- 25%Ty

rosi

nasi

c ac

tivi

ty (

%)

- 85%

Figure 6: Anti-tyrosinasic activity of 0.1% Biowhite™ obtained in an in vitro model of enzyme extracted from melanocytes in culture

120

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20

0

(13) Maeda K et al. J Soc Cosmet Chem 1991 ; 42 : 361-368.

MATERIALS & METHODSIntracellular anti-tyrosinasic activity was quantified after extraction of melanocytes. The extracts were incubated with L-DOPA and 3-methyl-2- benzothia-zolinonehydrazone hydrochloride or Besthorn's hydrazone (MBTH), a compound which reacts instanta-neously with dopaquinone to form a coloured compound which is detectable at 505 nm. This extre-mely sensitive method makes it possible to measure the dopaqui-none which is formed following the oxidation of the L-DOPA by the tyrosinase directly. In this study, the melanocytes were isolated from a skin fragment obtained from a 35 years old female sub-ject by abdominal plastic surgery. Biowhite™ was tested in the mela-nocyte culture medium at a final concentration of 0.1% (vol/vol). A negative control by cells untreated with L-DOPA and a positive control of cells treated with ascorbic acid and L-DOPA were performed in parallel. The ascorbic acid was used at a non-cytotoxic concen-tration of 0.02%. The melanocytes were cultured for 15 hours at 37°C with the different tested active compounds, then rinced. Cells were then dissolved in a phosphate buffer containing Triton X-100. The extract obtai-ned was then combined with L-DOPA and MBTH, pre-incuba-ted at 37°C. The absorption rate at 505 nm was monitored for 5 minutes. Comparing the rates at which the absorbing compound occurs at 505 nm with the tes-ted active compounds and with the control makes it possible to determine the inhibition obtained with the tested product extremely accurately. If an active modifies enzymatic activity, the rate at which coloured pigment develops changes.



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RESULTS

The assessed experiments show that the control melanocytes (i.e. untreated with L-DOPA) had little coloration (Figure 7, A).

For the cells incubated with L-DOPA:

• The enzymatic activity of the tyrosinase coloured the positive control melanocytes brown (Figure 7, B).

• Melanocytes treated by a recognised tyrosinase inhibitor such as ascorbic acid totally inhibited the formation of pigments in the melanocytes (Figure 7, C).

• The colour of cells treated with a 0.1% concentration of Biowhite™ is similar to the colour of control cells treated without L-DOPA indicating that Biowhite™ totally inhibited the tyrosinase (Figure 7, D).

OBJECTIVEThe purpose of these experiments was to show that anti-tyrosinase activity of Biowhite™ led to a diminution of the melanic pigments synthesis. For that reason, normal human melanocytes were cultured in the presence or absence of Biowhite™ during 15 hours. At the end of this period, the tyrosinase activity was demonstrated by a cytochemical study (DOPA-reaction).

CONCLUSIONIn conclusion we well show, in cultures of normal human melanocytes, that the anti-tyrosinase activity of Biowhite™ lead to an inhibition of the melanic pigments synthesis.

This inhibition is comparable to the inhibition observed with ascorbic acid, a whitening active not very sta-ble and of which coloured decomposition products are not compatible with the organoleptic properties usually expected for cosmetics.

A B Negative control Positive control

C DPositive control Biowhite™ (0.1%) Ascorbic acide (0.02%)

Figure 7: Anti-tyrosinasic activity of 0.1% Biowhite™ obtained in an in vitro model of human melanocytes in cul-ture (DOPA-reaction)

MATERIALS & METHODSIntracellular tyrosinasic acti-vity was demonstrated by a cytochemical study (DOPA-reaction) in which melanocytes are treated with a tyrosinase substrate, L-DOPA.

For this study, the melano-cytes were isolated from a skin fragment obtained from a 35 year old female subject by abdominal plastic surgery. Biowhite™ was added to the melanocyte culture medium at a final concentration of 0.1% (vol/vol). A negative control by cells untreated with L-DOPA and a positive control of cells treated with ascorbic acid and L-DOPA were performed in parallel. The ascorbic acid was used at a non-cytotoxic concentration of 0.02%.

The melanocytes were cultu-red for 15 hours at 37°C with the test active before being rinsed, fixed and incubated in L-DOPA solution.

The reaction was then stopped by rinsing the cells in PBS and then the melanic pigments, formed by the oxidation of the L-DOPA by the endoge-nous tyrosinase, were observed through a microscope under white light.

The degree of intensity of mela-nocytes coloration indicates the activity of the intracellular tyrosinase directly.

If an active modified the enzy-matic activity of the tyrosinase, this could be seen as a change in the degree of intensity of melanocyte coloration for a given incubation period with L-DOPA.

In vitro study on human melanocyte culture (DOPA-reaction)



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OBJECTIVEThe ultimate aim of this investigation was to confirm that Biowhite™ is able to exert its depigmenting activity in an ex vivo model close to the in vivo situation in which several types of cells inter-react. For this, human skin explants were treated (or not) with Biowhite™ for 15 hours. At the end of this period, intra-melanocyte tyrosinase activity was observed by a histochemical technique.

RESULTS

The results obtained are the following:

The control explant, treated with only L-DOPA shows active melanocytes (Figure 8, A), possessing a strong tyrosinase activity in the basal layer of the epidermis.

Used as a reference, hydroquinone at 0.55% (w/v) completely inhibits the formation of melanin (Figure 8, B). This expected result validate the study.

Biowhite™ at 0.1%, 1% et 10% (v/v) also completely inhibits the histochemical reaction (Figures 8, C, D, E).

CONCLUSIONIn conclusion, in our experimental conditions, Biowhite™ has a very powerful depigmenting effect. In fact, 0.1% of Biowhite™ induces an inhibition level comparable with that obtained with 0.55% of hydro-quinone, a recognized but extremely cytotoxic depigmenting substance.

We have also shown, in a model close to an in vivo situation, that Biowhite™ diffuses into the skin to reach the target cells to exert its depigmenting action.

Ex vivo study on human skin explants

Basal Membrane Melanocytes filled up with melanic pigments (black)

Negative control

A

Positive controlHydroquinone 0.55 % (w/v)

MATERIALS & METHODSThe anti-tyrosinase activity of Biowhite™ was evaluated using an ex vivo model, consisting in human skin explants.

In this assay, the evaluation of intramelanocyte anti-tyrosina-se activity was objectived with an histochimical study based on the explant treatment with a tyrosinase substrate: a L-DOPA solution.

The oxidation of L-DOPA by tyrosinase produces in the melanocyte a black pigment, precursor of melanin, which can easily be detected by optic microscopy. The tyrosinase activity could be measured by the intensity of the coloration obtained.

For this study, the human skin explants were obtained from abdominal plastic surgery of a 27-years old woman.

Biowhite™ was tested at 0.1%, 1% and 10% (v/v) in ultrapur desionized water. A positive control and a negative control were also conducted in the same experiment. Hydroquinone at 0.55% (w/v) was selected as the positive control for the study. Hydroquinone is well known for its strong whitening activity and is classified as an OTC active compound. Even though it has no anti-tyrosi-nase activity, this molecule can inhibit the melanin pigment formation in melanocytes; this behaviour allows the molecule to be recommended for this study.

The different solutions were incubated with L-DOPA (0.05 M) for 15 hours at 37°C.After fixation and inclusion in paraffin, the skin slices were colored with hemalun/eosin/safran and cresyl violet, and observed under white light, with optical microscopy.Melanic pigments, formed by oxidation of exogeneous L-DOPA by endogeneous tyrosi-nase could be measured by the intensity of the colorimetric reaction obtained.

Pictures of skin slices were made to show the results of the experiment.

B

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Figure 8: Anti-tyrosinasic activity of Biowhite™ at 0.1; 1 and 10% obtained in an ex vivo model of human skin explants

Biowhite™ 0.1 % (v/v) Biowhite™ 1 % (v/v)

Biowhite™ 10 % (v/v)



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In vivo study

In order to increase the suitability of Biowhite™ objectivation, a study of its depigmenting activity was performed in vivo, on healthy volunteers. In interesting way, this study was conducted on different healthy volunteers: “caucasian“ and “asiatic“ volunteers. For this study, Engelhard decided to use a completely

innovative method of measurement which will be an extremely useful complement to conventional methods hitherto used for estimating cutaneous pigmentation. This novel method involves analyzing images acquired using a digital camera.

In order to gauge the performance of this new measurement modality and confirm the scientific validity of the results, Engelhard performed parallel measurements using a conventional chromameter.

The image analysis system had two principle advantages:

1) it overcame the problem of differences in skin color encountered when the chromameter probe is placed in contact with the surface of the skin,

2) it could be used to analyze large areas of skin—this is a particularly big advantage when it comes to analyzing hyperpigmented spots.

Both methods gave similar results meaning that the efficacy of Biowhite™, incorporated at 10% into a mask presentation, has been assessed in a very comprehensive fashion.

It should also be noted that, as well as exploring these technical issues, Engelhard has undertaken a rigorous assessment of the lightening and whitening activities of Biowhite™, not only with respect to overall facial complexion but also to hyperpigmented spots because 10 of the 20 “caucasian“ subjects chosen had such blemishes on their faces.

STUDY ON "CAUCASIAN" VOLUNTEERS

20healthy “caucasian” volunteers were included in this study. The whitening power of Biowhite™ on both the whole face and hyperpigmented spots was assessed after a 56-days of treatment (6 applications of mask containing 10% of Biowhite™) using chromametry and image analysis.

Chromametry results

Chromametry measures pigmentation changes by evaluating three different parameters: L for luminosity (from +L for white to -L for black); a for the red-green component (from +a for red to -a for green); and b for the blue-yellow component (from +b for yellow to -b for blue) (see opposite figure). From these three readings, the Individual Typology Angle can also be calculated or ITA.

To summarize:

• An increase in L parameter corresponds to lightening of the skin.

• An increase in a parameter corresponds to an increase in the red component (i.e. increased blood supply).

• An increase in b parameter corresponds to an increase in melanin levels.

• An increase in the ITA value corresponds to lightening of the skin and a reduction in melanin levels.mélaniques.

MATERIALS & METHODS20 “caucasian“ healthy volun-teers were included in this study.

After a ten-minute acclimati-zation period for adaptation to the local temperature, pic-tures of the volunteers’ skin were taken and chromametric measurements were made:

1) On a non-treated, control area of skin,

2) On the area of the face—rigorously defined and delineated—which was to be treated.

When the subject presented hyperpigmented spots, the darkest patch was pictured and specially assessed by chro-mametry.

After 56 days of treatment (a total of six applications of mask containing 10 % of Biowhite™), the same regions were re-photographed and re-measured with the chro-mameter. Differences in pigmentation with respect to t0 were calculated and expressed as a percentage value.

The chromameter used was a Minolta CR-300 (Minolta) and the digital camera was a Sony CCD-RGB type XC-711/711P with a Nikon 60 mm objective lens.

White +L

Green

Blue

Yellow +b

Red +a

Black

Representation of a color space L, a, b



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The results presented in Figure 9 show that, after a 56 days treatment (only six applications of mask), Biowhite™ had induced a significant

reduction in the amount of melanin present (parameter b) both in the face as a whole and also in hyperpigmented spots.

In this study, Biowhite™ also induced significant lightening of hyperpigmented skin blemishes (as indicated by increases in parameter L and the ITA derivative).

On the basis of the results of this series of tests, it can be seen that Biowhite™ has genuine, long term, generalized depigmenting activity as well as being effective against hyperpigmented spots.

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Image analysis

Special software was used to derive parameters L, a and b for each image. The results presented in Figure 10 show that, after 56 days of treatment, Biowhite™ had induced a significant decrease in the amount of melanin present (parameter b) in both normal facial skin and in hyperpigmented spots.

Biowhite™ was also responsible for significant lightening of the hyperpigmented spots.

These observations are entirely consistent with the chromametry data, thereby confirming both the validity of the methods used for evaluation and the results obtained pertaining to the whitening and skin-lightening activity of Biowhite™.

As shown in Figure 11 it can also be seen that image analysis also detects a significant reduction in skin redness (parameter a) which is not detected by chromametry. This effect is again observed in both normal facial skin and hyperpigmented spots.

Therefore, taking the results of all these tests together, it is clear that a series of just six applications of mask containing 10% of Biowhite™ induces:

1) Significant, generalized whitening (parameters b and a)

2) Significant whitening and lightening of hyperpigmented spots of skin (parameters b, a and L, and the ITA value)

Apart from the impressiveness of the actual results on the efficacy of Biowhite™, the innovative methodolo-gical approach adopted by Engelhard resulted in the detection of effects—notably the marked reduction in parameter a—which could not be detected by more conventional techniques like chromametry.

Figure 11: Image analysis to evaluate long term Biowhite™ activity: the red component of skin color

Figure 10: Image analysis to evaluate long term Biowhite™ activity

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STUDY ON "ASIATIC" VOLUNTEERS

10healthy “asiatic” volunteers were included in this study (the low number of volunteers in this study is explained by the difficulty to select “asiatic“ volunteers in France). The whitening power of Biowhite™ was assessed after a 56-days of treatment (6 applications of mask containing 10% of

Biowhite™) using chromametry and image analysis.

Chromametry results

The results presented in Figure 12 show that the chromametry doesn’t show any significant reduction in the amount of melanin present (parameter b) or clearness skin (parameter L and ITA) for the “asiatic” volunteers.

Image analysis

Special software was used to derive parameters L, a and b for each image.

The results presented in Figure 13 show that, after 56 days of treatment (only 6 applications of mask), Biowhite™ had induced a decrease in the amount of melanin present (parameter b) and of the red com-ponent of the skin (parameter a).

Biowhite™ was also responsible for significant lightening of skin (increasing of parameters L and ITA).

Therefore, taking the results of all these tests together, it is clear that a series of only six applications of mask containing 10% of Biowhite™, induces:

1) Significant, generalized whitening (parameters b and a),

2) Significant lightening of “asiatic” volunteers’s skin (parameters L and ITA).

Apart from the impressiveness of the actual results on the efficacy of Biowhite™ on a panel of “caucasian” and “asiatic” volunteers, the image analysis had demonstrated some specific actions of Biowhite™ which could not be detected by more conventional techniques like chromametry.

Figure 12: Chromametric evaluation of long term Biowhite™ activity

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Figure 13: Image analysis to evaluate long term Biowhite™ activity

*: statistically different from t0 (p<0.05)**: statistically different from t0 (p<0.01)

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MATERIALS & METHODS10 “asiatic“ healthy volunteers were included in this study.

After a ten-minute acclimati-zation period for adaptation to the local temperature, pho-tographic images of the volunteers’ skin were taken and chromametric measure-ments were made:

1) On a non-treated, control area of skin,

2) On the area of the face—ri-gorously defined and deli-neated—which was to be treated.

After 56 days of treatment (6 applications of mask contai-ning 10% of Biowhite™), the same regions were re-photo-graphed and re-measured with the chromameter. Differences in pigmentation with respect to t0 were calculated and expressed as a percentage value.

The chromameter used was a Minolta CR-300 (Minolta) and the digital camera was a Sony CCD-RGB type XC-711/711P with a Nikon 60 mm objective lens.

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Biowhite™: descriptionMain components

"Saxifrage" is the name used to describe ap-proximately 370 species of extremely resis-tant perennials found in mountainous and roc-

ky areas. Extracts are obtained from leaves but also from whole plants.

Many traditional pharmacopoeias, including the Chinese pharmacopoeia, describe these plants. Extracts of saxifrage are used to treat contact der-matosis and burns. Potentialised anti-inflammato-ry effects have also been observed with these ex-tracts.

The principle active ingredients include:

• tannins called proanthocyanidins: these poly-mers are made up of epigallocatechin units and have extremely varied molecular masses (and thus a wide range of degrees of polymerisation).

These tannins inhibit lipidic oxidation and are known to trap free radicals extremely effecti-vely.

the saxifrage extracts used in Biowhite™ have clearly demonstrable anti-radical effects on lipi-dic peroxidation obtained in vitro by UV irradia-tion of polyunsaturated fatty acids.

• arbutin - a natural molecule with a structure clo-se to hydroquinone - is perfectly stable and consi-dered to have low cytotoxicity,

• long-chain alcanes (17 to 32 atoms of carbon), terpenes and alcohols (camphene, linalool, bo-reol...).

The saxifrage extracts in Biowhite™ also reduce the mutagenic power of UV radiation.

This has been demonstrated by in vitro studies on Escherichia coli.

Saxifrage extracts: Saxifraga sarmentosa

Grape extracts: Vitis vinifera

These extracts are used for their content in various sugars and for their high concentration in α-hydroxy acids (mainly tartaric acid).

Synergistic action has been observed between this extract and others Biowhite™ components on the inhibi-tion of tyrosinase.



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Mulberry extracts: Morus bombycis

There are 10 different types of mulberry; Morus bombycis is a common specie in the temperate regions of the northern

hemisphere. Described in the traditional Chinese and Japanese pharmacopoeias, extracts of mulberry are obtained from dried bark, roots and, more rarely, leaves. Extracts of mulberry have recognised vasoconstrictor and analgesic properties and are used to treat asthma and bronchitis.

DESHAPANDE et al.(14), KONNO et al.(15), NOMURA et al.(16-18) described the essential components of the plant as:

• Triterpenoides - the most familiar being alpha-amyrin and beta-amyrin.

• Phenylflavone derivatives such as A, B and C kuwanones and morusin. Phenylflavones are anti-inflammatories and the basic molecules which inhibit tyrosinase.

This perennial plant which grows mainly in China, Siberia and Korea, is a member of the Labiatea fa-mily. The dried root of Scutellaria baicalensis is cal-led oughon in chinese medicine and used for its anti-inflammatory properties(19). In cosmetic, oughon is used as a skin whitening active compound.

Representative chemically identified molecules of oughon are flavonoids such as woogonin, baicalin and baicalein. On one hand, these flavonoids ha-ve anti-inflammatory properties and on the other hand, woogonin, baicalin and baicalein have been described for their tyrosinase inhibitory potential.

EDTA

EDTA, Ethylenediaminetetraacetic acid, is widely used in pharmaceutical and cosmetic field as a

chelating agent of metallic ions, and more specifi-cally, as a chelating agent of cupper and iron ions. The Biowhite™ complex contains the disodium form of EDTA.

EDTA inhibit tyrosinase by trapping the metallic co-factor Cu++, needed for the enzymatic oxydation of tyrosine in melanin precursors.

Scutellaria root extracts: Scutellaria baicalensis

(14) Deshpande V et al. Ind J Chem 1976 ; 14 : 647-650.

(15) Konno C et al. Planta Medica 1977 ; 32 : 118-124.

(16) Nomura T et al. Chem Pharm Bull 1 978 ; 26 : 1394-1402.

(17) Nomura T et al. Chem Pharm Bull 1978 ; 26 : 1453-1458.

(18) Nomura T et al. Heterocycles 1979 ; 12 : 1289-1295.

(19) Niwa Y et al. Blood 1984 ; 64 : 994-999.



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ConclusionBiowhite™ represents an optimized depigmen-

ting approach, this active compound:

1) is a strong inhibitor of tyrosinase and there-fore of melanin synthesis,

2) is highly specific because its inhibition of melanin synthesis does not depend on cyto-toxic effects on skin cells,

3) is capable of reaching its cellular and mo-lecular targets, as has been shown experi-mentally in a three-dimensional tissue mo-del.

In vivo, the anti-tyrosinasic activity of 10% Biowhite™ incorporated into a mask, is shown by visible ef-fects:

Biowhite™ shows after just six applications of mask over 56 days, a powerful skin-lightening activity on both normal skin and hyperpigmented spots.

In interesting way, the activities of Biowhite™ can be shown in vivo, as well for "caucasian" volunteers as "asiatic" volunteers.

Biowhite™, the suitability of an innovating objec-tivation for an optimized skin-lightening active compound.



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Bibliography(1) Wenczl E, Van Der Schans G, Roza L, Kolb R, Timmerman A, Smit N, Pavel S, Schothorst A. (Pheo)melanin photosensitives UVA-induced DNA damage in cultured human melanocytes. J Invest Dermatol 1998 ; 111 : 678-682.

(2) Peyrefitte G. Biologie de la peau. Cahier d’esthétique - Cosmétique. Paris : Ed Simep ; 1993.

(3) Fitzpatrick T, Pathak M, Harber L, Seiji M, Kukita A. Sunlight and man. Tokyo : University of Tokyo Press ; 1974.

(4) Smith C, O’Hare K, Allen J. Selective cytotoxicity of hydroquinone for melanocyte-deri-ved cells is mediated by tyrosinase activity but independent of melanin content. Pigment Cell Res 1988 ; 1 : 386-389.

(5) Chakraborty AK, Funasaka Y, Komoto M, Ichihashi M. Effect of arbutin on melanogenic proteins in human melano-cytes. Pigment Cell Res 1998 ; 11 : 206-212.

(6) Nakajima M, Shinoda I, Fukuwatari Y, Hayasawa H. Arbutin increases the pigmentation of cultured human mela-nocytes through mechanisms other than the induction of tyrosinase activity. Pigment Cell Res 1998 ; 11 : 12-17.

(7) Lerner A, Fitzpatrick T, Calkins E, Summerson W. Mammalian tyrosinase. Preparation and properties. J Biol Chem 1949 ; 178 : 185-195.

(8) Kahn V. Effect of kojic acid on the oxidation of DL-DOPA, norepi-nephrine, and dopamine by mushroom tyrosinase. Pigment Cell Res 1995 ; 8 : 234-240.

(9) Nakagawa M, Kawai K. Contact allergy to kojic acid in skin care products. Contact Dermatitis 1995 ; 32 : 9-13.

(10) Kameyama K, Sakai C, Kondoh S, Yonemoto K, Nishiyama S, Tagawa M, Murata T, Uhnuma T, Quigley J, Dorsky A, Bucks D, Blanock K. Inhibitory effect of magnesium L-ascorbyl-2-phosphate (VC-PMG) on melanogenesis in vitro and in vivo. J Am Acad Dermatol 1996 ; 34 : 29-33.

(11) Tirosh O, Katzhendler Y, Barenholz Y, Ginsburg I, Kohen R. Antioxidant properties of amidothionophosphates : novel antioxidant molecules. Free Rad Biol Med 1996 ; 20 : 421-432.

(12) Peterkofsky B, Prather W. Cytotoxicity of ascorbate and other reducing agents towards cultured fibroblasts as a result of hydrogen peroxyde forma-tion. J Cell Physiol 1977 ; 90 : 61-70.

(13) Maeda K, Fukuda M. In vitro effectiveness of several whitening cosmetic compo-nents in human melanocytes. J Soc Cosmet Chem 1991 ; 42 : 361-368.

(14) Deshpande V, Wakharkar P, Rama Rao A. Wood phenolics of Morus species: Part V - Isolation of a new flavone, mulberranol and a novel phenol, alboctalol from Morus alba. Ind J Chem 1976 ; 14 : 647-650.

(15) Konno C, Oshima Y, Hikino H. Morunisol, isoprenoid flavone from Morus root barks. Planta Medica 1977 ; 32 : 118-124.

(16) Nomura T, Fukai T, Yamada S, Katayanagi M. Studies on the constituents of the cultivated mulberry tree. I. Three new prenylflavones from the root bark of Morrus alba. Chem Pharm Bull 1978 ; 26 : 1394-1402.

(17) Nomura T, Fukai T, Katayanagi M. Studies on the constituents of the cultivated mulberry tree. III. Isolation of four new flavones, kuwanon A, B, C and oxydi-hydromorusin from the root bark of Morrus alba. Chem Pharm Bull 1978 ; 26 : 1453-1458.

(18) Nomura T, Fukai T. On the structures of mulberrin, mulberrochromene, cyclo-mulberrin anc cyclomulberrochromene. Heterocycles 1979 ; 12 : 1289-1295.

(19) Niwa Y, Sakane T, Miyashi Y, Kanoh T, Somiya K. Decrease in generation of oxygen radicals by neutrophils from patients with infections monoclueosis – Rôle of suppres-sor T lymphocytes. Blood 1984 ; 64 : 994-999.

Ames B, Mc Cann J, Yamasaki E. Methods of detecting carcinogens and mutagens with the salmonella/mammalian microsome mutagenicity test.. in

“B. J. Kilbey et al. (Eds.) Handbook of mutagenicity Test Procedures”. Elsevier, Amsterdam, 1-17 (1977)

Bruneton J. Pharmacognosie, phytochimie, plantes médecinales. Paris : Tech & Doc ; 1993.

Cesarini J. Structure et fonctions du système pigmentaire. In : Prunieras M, Agache P. Précis de cosmétologie dermato-logique. Paris ; New York ; Barcelone : Masson ; 1981.

Mishima Y, Hatta S, Ohyama Y, Inazu M. Induction of melanogenesis suppression: cellular pharmaco-logy and mode of differential action. Pigm Cell Res 1998 ; 1 : 367-374.

Morison W. What is the function of melanin ? Arch Dermatol 1985 ; 121 : 1160-1163.

Prota G. Melanins and melanogenesis. London : Academic Press ; 1992.

Sansei Seiyaku K. Whitening cosmetic compositions containing kojic acid which inhibit the action of tyrosinase and supress the formation of melanin. Brevet 76JP-076778 (760628), Numéro de publication J53003538 A 780113

Shibuya T, Murota T, Sakamoto K, Iwahara S, Ikeno M. Mutagenicity and dominant lethal test of kojic acid. J Toxibol Sci 1982 ; 7 : 255-262.

Rival D, Perrier E. Use of sulfites and metabisulfites for manufacturing cosmetic or pharmaceutical compositions, notably in dermatology, with melanogenesis-inhibiting effect or with depigmenting activity. (France: FR 2 764 189), (Europe: EP 887 072), (USA: US 5 989 596), (India, Japan, Korea: Pending).

About Engelhard

At Engelhard, we believe it's absolutely essential to deliver technologies that provide lasting business value and significantly affect people's lives for the better.

Personal care materials are just one example of how we apply our expertise in surface and materials science. From the development of catalytic converters to keep automotive pollutants from the air to special effect pigments that add glimmer to lipstick, we provide enhanced aesthetics, improved product performance and increased manufacturing efficiency. We match our unique technologies to market needs, thereby delivering significant value to our customers.

Which is why, more and more, companies turn to Engelhard to change the nature of their products. They get results that change their markets and our world.

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process or its selection or use is provided WITH NO WARRANTY OF ANY KIND. All sales are subject to Engelhard's Terms and Conditions of Sale, which are available upon request. ALL IMPLIED WARRANTIES ARE DISCLAIMED. Remedies for

any breach and Engelhard's liability, including that for patent infringement, are limited as provided in Engelhard's Terms and Conditions of Sale. Engelhard is not liable for consequential, incidental, or special damages. Nothing should be

construed as a recommendation or inducement to infringe any patent. No assumption should be made that all safety or environmental protection measures are indicated, or that other measures may not be required.

© 2005 Engelhard Corporation

For more informationplease contact:

Europe and South America83, rue de Villiers92200 Neuilly-sur-Seine,FranceTel: +33 (0) 1 47 45 35 00Fax: +33 (0) 1 47 45 11 [email protected]

AsiaUT Building, 7th floor1-5-13 Hirakawa-cho, Chiyoda-kuTokyo 102-0093, JapanTel: +81 (0) 3-5226-0440Fax: +81 (0) [email protected]

North America545 Fifth Avenue, Suite 1108New York, NY 10017Tel: +1 212-450-8280Fax: +1 [email protected]

50 Health Sciences DriveStony Brook, NY 11790-3350Tel: +1 631-689-0200Fax: +1 [email protected]

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