The olive: A natural supplier of active ingredients for skin lightening and age spot reduction

Abstract

Plant extracts became an important element as agents for skin lightening, since substances like hydroquinone, arbuti, and kojic acid that were used for a long time came into negative discussions about their skin toxicology and compatibility. The used plants extracts are, for example, polyphenol-rich licorice extract or corresponding extract combinations like: mallow (Malva sylvestris), peppermint (Mentha piperita), primrose (Primula veris), lady's mantle (Alchemilla vulgaris), veronica (Veronica officinalis), lemon balm (Melissa officinalis), and common yarrow (Achillea millefolium). Most extracts are tyrosinase inhibiting [1]. A further plant with skin-light-ening properties is the well-known and much-valued olive. The olive tree, Olea europaea, is native to the Mediterranean basin and parts of Asia Minor. Native olive oil has often been used in cosmetic formulations, but other interesting ingredients from the fruit and the leaves were for a long time not investigated for the use in cosmetic products. The fruit and the leaves contain high amounts of polyphenols. The major polyphenol is oleuropein. With increasing maturity of the olive, hydroxytyrosol will be released, which is even stronger in anti-oxidative power than the oleuropein. The used olive active ingredient, containing higher amounts of polyphenols and hydroxytyrosol, could be expected to provide skin-whitening and age spot-reduction effects. A direct measurement, melanin reduction, and an indirect measurement, glutathione (GSH) increase, were conducted in vitro on primary human melanocytes. A high GSH level in melanocytes directs the production of melanin into soluble and lighter brown pheomelanin instead of dark and insoluble eumelanin. An in vivo test was designed as a double blind, placebocontrolled study on the hands of 12 Caucasian female volunteers, aged between 46 and 72. The study duration was three months, with readings before the first application, after four, eight, and twelve weeks. A significant melanin reduction of up to 50% and a significant increase of the GSH level up to 55% could be measured in vitro. The confirmation in vivo showed not only a significant reduction of age spot color but also a color reduction of the whole skin area on the hands.

Full text

JAHRBUCH
NATURKOSMETIK
2011
naturkosmetikverlag
e. dambacher
Maria Lüder

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The Olive – A Natu-
ral Raw Material Sup-
plier for Cosmetics
Active Ingredient Carrier for Skin Lightening and the Reduction of Pig-
ment Spots
The appearance of our skin is like our business card. It is considered to
be particularly beautiful when it has an even tone. Many people like to
cover up their pigment spots, freckles, broken blood vessels or red patch-
es with make up or camoufl age products. This even skin tone is asso-
ciated with being young or young at heart. Skin lighteners are useful in
this area, along with make up and self-tanning products 1).
People with paler skin tend to want to look more tanned, while people
with naturally darker pigmentation often prefer lighter skin. This is par-
ticularly true in Asia, where whitening products are booming. Whiten-
ing products make the skin look light even when no make up or camou-
fl age products are being worn.
Plant extracts have become important ingredients in skin lightening
products, as whitening agents such as hydroquinone, arbutin and ko-
jic acid are fi nding themselves increasingly in the fi ring line because of
their harmful side effects. Plant extracts used include concentrates rich
in polyphenols derived from licorice root or other combinations of ex-
tracts from mallow (malva sylvestris), peppermint (mentha piperita),
cowslips (primula veris), lady’s mantle (alchemilla vulgaris), speedwell (ve-
ronica offi cinalis), lemon balm (melissa offi cinalis) and yarrow (achillea
millefolium). Most of these extracts act as tyrosinase blockers1. These
skin-lightening plants include the well-known olive, a plant which is high-
ly-valued by many consumers.
The olive tree, olea europaea, is native to the Mediterranean region and
a few parts of Asia. Virgin olive oil is used widely in cosmetics formula-
tions, but its fruit and leaves also contain interesting substances which
have not yet been studied for cosmetic purposes. The fruit and leaves
contain large amounts of polyphenols. The main polyphenol is oleuro-
pein, the substance responsible for the slightly bitter taste of virgin olive
oil. As the olive ripens, hydroxytyrosol is released from the oleuropein.
This has an even stronger antioxidant effect than oleuropein.
1 H. Lautenschläger, Whitening – Heller Teint erwünscht, Kosmetik International 2004
(4), 45- 47.
Maria Lüder
Abstract

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The water-soluble olive extracts which we are considering in this ar ticle
contain large amounts of polyphenols and hydroxytyrosol and it could
be surmised that this composition of antioxidants has properties which
promote skin-whitening and reduce pigment spots. Direct measure-
ment of melanine reduction and indirect measurement of glutathione
increases were carried out in vitro on primary human melanocytes. A
high glutathione content in the melanocytes means that melanin pro-
duction tends more towards the production of soluble, light brown ph-
eomelanins. An in vivo test was carried out as a double blind study us-
ing placebos on 12 light-skinned women aged 46-72 with liver spots on
their hands. The study lasted three months, with examinations carried
out before the start of the trial and at months four, eight and twelve of
the study.
A signifi cant reduction in melanin of up to 50 % and a signifi cant rise in
glutathione content of up to 55 % was observed in vitro. This was con-
fi rmed in vivo, with the pigment spots on the hands becoming much pal-
er and a reduction in the surrounding unspotted skin.
Skin whitening products ar e available on the market to make skin appear
lighter. They are also used for clinical treatments for pigment disorders
such as melasma or post-infl ammatory hyperpigmentation. Skin-light-
ening agents can be active on the skin at various stages of melanin pro-
duction. Many of the substances are known as tyrosinase inhibitors, the
key enzyme involved in melanogenesis. Other agents prevent enzyme
maturation or the transport of the pigment particle from the melano-
cytes to the keratinocytes.
Melanocytes are located in the basal layer which separates the epidermis
and the dermis. A melanocyte is surrounded by approximately 36 ke-
ratinocytes. Together they make up what is known as the melanin unit.
The melanin which is produced is stored in melanocytes in the melano-
somes (small organelles fi lled with melanin) and transported to the sur-
rounding keratinocytes via dendrites.
The substances found in olives can affect melanogenesis in two ways.
They act as tyrosine inhibitors and also steer the melanin formation to-
wards the soluble, light pheomelanins.
The olive has been valued for its healing properties since ancient times.
Hippocrates used olive leaves to help heal wounds and Dioscorides used
them to treat abrasions, infl ammations, abscesses, fungal infections and
slow-healing wounds. Nowadays the results of numerous trials and ad-
vances in cell biology have shown us that the olive’s high polyphenol
content and attendant strong antioxidant properties is responsible for
this healing effect.
Keywords: Skin lightening,
reduction of liver spots, olea
europaea, hydroxytyrosol,
oleuropein, pheomelanin,
eumelanin, glutathione
Introduction

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Depending on the ripeness of the olives, these secondary plant com-
pounds2 are found in cold-pressed olive oil (approx. 0.1 %). They have a
similar effect to the much-lauded omega-3 fatty acids. We should now
take a closer look at the oleuropein in olives. As olives ripen, hydroxy ty-
rosol is produced from the oleuripein, which has an even stronger anti-
oxidant effect (Fig. 1). Just like resveratrol in grapes, the amphiphile hy-
droxytyrosol is a very strong antioxidant. As hydroxytyrosol is soluble
in oil and water, it can act as a free-radical scavenger in both cell plasma
and cell membranes. It supports the proper functioning of the cells and
also the exchange of information and matter with the surrounding cells
and the extra-cellular matrix.
Scientifi c studies have shown that hydroxytyrosol protects human
melanocytes in vitro against protein damage induced by long-wave UV
radiation. It reduces the distribution of infl ammatory mediators such as
Cox-2 in macrophages3.
Glutathione is a peptide consisting of three amino acids: glutamic acid,
cysteine and glycin. It can be found in high concentrations in almost all
cells and is one of the most important natural antioxidants in the hu-
man body. Glutathione protects cellular macromolecules such as pro-
teins and membrane lipids against free radicals (ROS reactive oxygen
species). Higher concentrations of glutathione not only protect against
ROS but also stimulate the formation of lighter and more easily soluble
pheomelanins as opposed to the darker, insoluble eumelanin4. Tyrosi-
nase, the enzyme which determines the development of melanogenesis,
catalyzes the hydroxylation of L-tyrosinase to dopa and the oxidation of
dopa to dopaquinone. If cysteine or glutathione are present, they react
with dopaquinone to cysteinyldopa and the benzothiazine derivative of
the phaomelanin5. The olive extract used was developed with a high
concentration of active agents and tested both in vitro and in vivo for
its effect on pigment spots and to demonstrate skin lightening.
The in vitro effect of the olive component was tested twice. The mela-
2 Informationsgemeinschaft Olivenöl (München): Wissenschaftliche Erkenntnisse über
Olivenöl in der Ernährung. Hintergrundinformation Nr. 1 des Instituts für Arteriosk-
lerosef Forschung an der Westfälischen Wilhelms -Universität, Münster (2001).
Maiuri M.C. et al., Hydroxytyrosol, a phenolic compound from virgin olive oil, pre-
vents macrophage activation. Naunyn Schmiedebergs Archives of Pharmacology;
371(6):457-65; (2005).
3 D’Angelo S. et al., Hydroxytyrosol, a natural antioxidant from olive oil, prevents pro-
tein damage induced by long-wave ultraviolet radiation in melanoma cells, Free Radi-
cal Biology and Medicine 1;38(7):908-19, (2005).
4 Slominski A., Tobin D.J., Shibahara S., Wortsman J. Melanin Pigmentation in Mam-
malian Skin and Its Hormonal Regulation, Physiol Rev 84: 1155–1228, (2004).
5 Ebanks J.P., Wickett R.R., Boissy R.E., Mechanisms Regulating Skin Pigmentation: The
Rise and Fall of Complexion Coloration, Int. J. Mol. Sci., 10, 4066-4087 (2009).
Fig. 1: Structure of oleuropein

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nin reduction and glutathione increase resulting from a treatment with
this substance on primary human melanocytes was measured. The glu-
tatione increase was measured in order to prove that melanin produc-
tion tends towards soluble pheomelanin.
The olive extract (Cayoma® Olive, Qenax AG, Zug, Switzerland) con-
tains 8 % hydroxytyrosol and 12 % olive polyphenols and was used for
the in vitro studies. All tests were carried out by independent testing
centres. The in vitro tests were carried out by CELLnTEC Advanced
Cell Systems (Bern, Switzerland) and the in vivo test by Skin Test Insti-
tute (Neuchâtel, Switzerland).
In the in vivo study a handcream with 0.2 % olive extract was the only
agent used for skin lightening.
Primary human melanocytes (Lifeline Technologies) were exposed to ac-
tive agents in concentrations between 0 – 0.0078 % for 48 hours.
The cells were incubated with a medium containing 0.1 % L-dopa in or-
der to trigger melanogenesis. OD (optical density) 405nm readings were
taken after 0 and 140 minutes.
The melanin content was then calculated from these. The results are
shown as a percentage of the negative control (untreated cells). Cytotox-
icity tests using the same or higher concentrations of active substances
which were carried out before this study showed no harmful effects.
The test is based on the conversion of a luciferin derivative to luciferin,
which presently occurs from glutathione catalyzed by glutatione S-trans-
ferase (GST). The signal generated in the coupled reaction with fi refl y
luciferase is proportional to the amount of glutathione in the sample6.
Primary human melanocytes were treated with olive extract for 48 hours
and their glutathione content analysed before and after the test. Cyto-
toxicity tests using the same concentration of active substances which
were carried out before this study showed no harmful effects.
The objective of the study was to demonstrate the properties of the
olive extract in reducing pigment spots and lightening skin in a double-
blind, placebo-controlled trial. The study lasted 3 months with measure-
ments taken before the test started and at 4, 8 and 12 weeks. Of the 12
test subjects aged between 46 and 72, 4 had normal skin (33 %), 3 had
dry skin (25 %) and 5 had very dry skin (42 %).
The subjects had to apply the handcream containing 0.2 % olive extract
three times per day in 0.5 gram amounts. One hand was treated with
6 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA,
GSH-Glo™ Glutathione Assay, Technical Bulletin, (2008).
Material and measurement
methods
1) Melanin reduction
2) Glutathione increase
3) In vivo study on pigment spot
reduction in test subjects

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the cream and the other hand with a placebo cream. The test subjects
were asked to avoid direct sunlight during the period of the trial.
The measurements were carried out using the Skin Pigmentation Ana-
lyzer SPA 99 from Courage & Khazaka. The measuring head has a diam-
eter of 2 mm and pigment spots of 3 mm and more are large enough
to avoid false positive results.
In the SPA 99, the measuring head emits three specifi c wavelengths onto
the skin surface in a controlled environment. A receiver module meas-
ures the light that is refl ected by the skin and the microprocessor in the
device calculates the amount of light absorbed. The result is displayed
digitally as a fi gure between 0-99 (Fig. 2).
Four suffi ciently large pigment spots were selected and measured at
each of the set times. The surrounding unspotted skin was measured in
order to ascertain the overall effect of the cream in direct comparison
with the spots. In addition, high-resolution photos of the hands were
taken before the test began and every time the spots were measured.
The melanin index of the selected skin zones was also measured. All
measurements were taken under controlled conditions (T 22.5±1.5°C;
RH 50±10 %).
The melanin index is derived from the refl ectance spectrum and can
be used as a primary measurement of skin colour7. The melanin index
is a relative, unitless, continuous variable which allows skin colour to be
quantifi ed objectively8.
1) Melanin reduction
With a coverage of 10,000 cells per well, the highest melanin reduction
was observed in the highest concentration (0.00078 %) of olive extract
tested. 48 hours after treatment began an average reduction of 50-65 %
was identifi ed compared to the control. Fig. 3).
A similar picture was observed when 5,000 cells per well were used in-
stead of 10,000 cells per well. The highest concentration of extract in-
duced the greatest amount of melanin reduction, with fi gures of 55-70 %
compared to the control.
7 Dawson JB, Barker DJ, Ellis DJ, Grassam E, Cotterill JA , Fisher GW, Feather JW, A
theoretical and experimental study of light absorption and scattering by in vivo skin,
Phys Med Biol;25(4)695-709, (1980).
Verkruysse W., Svaasand L.O., Franco W., Nelson J.S., Remittance at a single wave-
length of 390 nm to quantify epidermal melanin concentration, J Biomed Opt. 14(1),
(2009).
8 Takiwaki H., Measurement of skin color: practical application and theoretical consid-
erations, The Journal of Medical Investigation Vol.44, (1998).
Fig. 2: Principe SPA 99
spring
photo detector
16 light emitting
diodes arranged
circularly
skin diffusive refl ections
Results

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The tests show that olive ex-
tract in concentrations between
0.002 % – 0.008 % can signifi cantly
reduce melanin content in primary
human melanocytes.
2) Glutathione increase
Glutathione content grew signifi cantly in cells which were treated with
the olive extract. The effect varies according to dosage, with the steep-
est increase (+55 %) occurring when using the highest concentration of
the active ingredient (0.0078 %).
The tests show that the olive ex-
tract signifi cantly increases glutath-
ione content in primary human
melanocytes after treatment last-
ing 48 hours when applied in con-
centrations between 0.002 % and
0.008 % (Fig 4).
3) In vivo study on pigment spot reduction in test subjects
After 8 to 12 weeks, the active treatment (cream with 0.2 % olive ex-
tract) showed that it had a signifi cant effect on the reduction of pigment
spots compared to the placebo (Fig 5-1)
The active treatment showed a signifi cant effect after 8 weeks and an
extremely signifi cant effect after 12 weeks in lightening the surrounding
unspotted skin compared to the placebo (Fig 5-2).
The degree of skin lightening achieved by the active treatment was
stronger in unspotted zones than on pigment spots; the statistical signif-
icance was approx. 10 % higher in the unspotted zones after 12 weeks
of treatment.
Fig. 4:
Increase of cells
Fig. 3:
Cell Reduction

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The photos on Day 0 show irregular pigmentation of the skin treated
with the placebo and the active treatment on the backs of the hands.
On the hand being treated, a dark patch (red circle) on Day 0 became
signifi cantly lighter by Day 84. The overall skin tone became more even
and much lighter during treatment with the handcream which contained
olive extracts.
The changes in the degree of brownness of the placebo-treated skin
between Day 0 and Day 84 can be explained by the seasonal effect of
reduced sun exposure (the trial began in autumn). However, the pig-
ment spots marked (red arrow) did not fade during the 3 month peri-
od (Fig. 6).
Skin lightening is an important part of the cosmetics industry in Asian
countries. Natural beauty is associated with a light skin tone. Skin light-
ening and pigment reduction is also becoming more signifi cant in Europe
and the USA, partly because of demographic changes in the population
on both continents. People are getting older, but they do not feel old
because they still lead active lives, even when they are advanced in year s.
Looking younger means having fewer wrinkles but also having an
even skin tone. This is why cosmetic products with active substances
which fi ght these signs of aging are now very popular.
Fig 5-1:
Melanin index of pigment spots.
Active treatment: Before the treat-
ment, D0
Fig. 5-2
Melanin index of surrounding unspot-
ted skin zones. Active treatment: Af-
ter the treatment D84
Fig 6-1:
Melanin index of pigment spots
Placebo: Before the treatment, D0
Fig. 6-2
Melanin index of surrounding unspot-
ted skin zones
Placebo: After the treatment D84
Conclusions

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In the past, ingredients such as hydroquinone, arbutin and kojic acid
dominated skin lightening products in the cosmetics industry. Hydroqui-
none and ingredients containing hydroquinone are still used in cosmet-
ics in certain parts of the world. But European regulations have placed
a total ban on the use of hydroquinone. In the USA, hydroquinone has
been classifi ed as a medicine by the FDA, and its use in cosmetic prod-
ucts has also been banned.
Hydroquinone occurs naturally in plants and animals in a range of dif-
ferent forms. It has been found in non-volatile extracts of coffee beans
and as arbutin (a glucoside of hydroquinone) in the leaves of blueber-
ries, cranberries and bearberries11).
The natural toxicity of hydroquinone has led cosmetic scientists to shift
their focus to safer natural and nature-identical molecules with similar
functions but without the signifi cant side-effects. Hydroquinone is known
to have serious side-effects when used for an extended period9.
The use of kojic acid and arbutin is still widespread because these ingredi-
ents always prove to be effective skin lighteners. Mulberry and licorice ex-
tracts are currently the most popular natural ingredients in skin lighteners.
Lemon extract is also a powerful skin lightener which is used in cosmetic
formulations, but it can only be used in low concentrations because it of-
ten leads to skin irritations. Various active substances have been extract-
ed from species of sophora (types of pagoda tree) which act as powerful
inhibitors to the tyrosinase and pigment production. Sophora extract can
be found in Synerlight from LiBiol. Here it is combined with kiwi fruit (ac-
tinidia chinensis), which contain fl avonoids (e.g. quercetin) which may act
as tyrosinase inhibitors. Niacinamide can be found in Lucederm and also
plays a part in inhibiting tyrosinase in melanosome transfer to the kerati-
nocytes. The Revitol skin lightener contains Lumiskin with certain patent-
ed ingredients, e.g. diacetylboldin which inhibits the production of tyro-
sinase. Bayer’s mandresy extract contains two components, luteolin and
verbascoside, which not only inhibit the production of tyrosinase and pig-
ment but also infl uence the link between keratinocytes and melanocytes
by reducint the production of dendrites. Some of the products contain
a mixture of several extracts with well-known tyrosinase inhibitors such
as mulberries, licorice, sophoria and peonia, but also with other extracts
which may have antioxidant and anti-irritant effects. Some companies still
offer single, synthetic substances, such as Lipotec, which sells dimetylmeth-
oxy chromanyl palmitate under the trade name Chromabright. This sub-
stance has been shown to have a lightening effect on the skin of 20 Asian
9 Gillner M., Moore G.S., Cederberg H., Gustafsson K.,Hydroquinone, Published un-
der the joint sponsorship of the United Nations Environment Programme, the In-
ternational Labour Organisation, and the World Health Organization, (1994).

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test subjects after 30 and 60 days.
So developments in the area of natural and nature-identical substances
for skin lightening have taken off over the last few years. The use of sin-
gle substances which act as tyrosinase inhibitors has in many cases been
expanded to include complex mixtures designed to have effects on such
things as tyrosinase inhibition, transport to keratinocytes, antioxidation
and anti-irritation10 .
It can be seen that most skin lighteners used today are produced with
botanic or other natural extracts. Consumers are familiar with plants
which have long been used in the nutrition sector or as traditional rem-
edies and so they feel positive and confi dent about using them. Olives
and olive oil have been used for hundreds of years. The strong antioxi-
dants found in the fruit and leaves have been shown to have an anti-
aging effect on the skin3+13). Hydroxytyrosol is one of the main anti-
oxidants found in olives and it works in different ways. It increases anti-
oxidant levels in the body, making it more resistant to illness because
of the increased glutathione content of the cells. This increased glutath-
ione level also causes melanin production to contain more of the soluble
pheomelanin. The pheomelanin and overall reduction in melanin levels
causes the skin to become signifi cantly lighter11.
It is now clear that effective skin lightening formulations can be developed
which have no harmful effects on the skin and which are produced using
natural ingredients. At the same time, these natural ingredients can pro-
tect the skin by triggering the body’s own defence mechanisms..
Qenax AG
Sumpfstrasse 26
6301 Zug
Schweiz
Tel. +41 41 544 60 50
Fax +41 41 544 60 55
info@qenax.com
www.qenax.com
Qenax produces active ingredients for use in cosmetics. The company
develops and markets quality cosmetic ingredients. The Cayoma® range
constitutes natural products made using plant extracts. The products con-
tain high levels of active ingredients which have been tested to provide
10 Lueder M., Blank J., Hydroxytyrosol from Olives, an interesting radical scavenger for
cosmetic applications, CSC-Conference Amsterdam (2008).
11 Tomohiro C., Whitening effect of hydroxytyrosol containing Oleaceae plants., Fra-
gr. J, Vol.32; (8); 41-48, (2004).
Maria Lüder
Geschäftsführerin

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evidence for any claims. The individuality and versatility of the products is
down to the natural plant extracts used.
The industry is trying to develop effective cosmetic products for custom-
ers who are becoming increasingly demanding. QENAX® is playing its
part in this by offering its customers effective ingredients.
QENAX® has been continually launching new products over the last few
years and has featured strongly in the cosmetics trade press.
Cayoma®- cosmetic active ingredients from QENAX®
• High levels of active ingredients
• Proven effectiveness
• Advertising claims
• Low application concentrations
Cayoma® Olive
• Counters oxidative stress and pigment spots
Cayoma® Ginkgo
• Stops irritation and actively protects cells (DNA and proteins)
Cayoma® Grapefruit
• Destroys toxins, promotes anti-glycation and stimulates the skin’s own
repair mechanisms.