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Resurrection
in cosmetics
Paper Mulberry and its Preparations as Tyrosinase Inhibitors and
Skin Lightening
Agents
Resurrection in cosmetics
Dr. Raphael Plüss, Dr. Marion Fröschle, Dr. Katrin Bojarski and André Peter, Induchem AG
Introduction
The protection and restructuring of the skin is a matter of
major concern in cosmetics. Mother Nature is an excellent
guide in the search for new concepts. On the one hand, our
skin is subjected to stress by abiotic factors such as heat,
coldness, dryness and other influences. On the other hand, the
living world shows us surprising possibilities for protecting our
skin from the harsh aspects of our environment.
Water is one of the most important and limiting factors for
plants, animals and humans. The human body consists of 6065 % water and loses daily up to several litres through the skin.
The regulation of water content is therefore very significant.
Plants especially have developed fascinating physiological and
structural strategies to minimize water loss and survive periods
of dryness. The so called “resurrection plants” are examples of
such survival artists. These plants have the ability to dry out
completely and to survive a dry period of several months in a
state of anabiosis without harm. Anabiosis is a state of reduced
metabolism. After such plants have picked up moisture again,
the green colour of their leaves is restored and the plant can
resume its normal growth within one to two days (Figure 1).
Such plants are mostly algae, lichen and ferns(1). In the
case of the flowering plants, there are only a few
“resurrection plants” to be found. They settle in different
arid sites. Some grow in dry cavities or on the top of rocks
where the plants grow in the hot sun and may use rain water
for a short period of time. Others grow in swamps that dry
Figure 1: The resurrection fern Selaginella lepidophylla in the
dry state (left) and two days after watering.
out for several months per year and others prefer the semi
shade protected from the hot sun.
During the drying out period, these plants undergo
intensive morphological and physiological changes in order to
be able to transfer the existing cell components in an ordered
form into the anabiosis reduced phase of activity. In this phase
specific protective substances are accumulated(2). Many of these
protective substances are special, low molecular weight sugars
such as trehalose and raffinose which are not present in most
other plants or occur only at low concentrations.
Figure 2 : Chemical structure of the trisaccharide raffinose.
Raffinose consists of one of each of the sugar molecules
galactose, glucose and fructose
Most of these low molecular protective substances possess a
so-called cosmotropic property, i.e. they have a structure
enhancing property for other molecules. This prevents crystal
formation when the plant is drying out and protects the main
structures of the cell such as the mitochondria, chloroplasts or
cell membranes. At a finer structural level, such as that
presented by proteins, cosmotropic substances protect proteins
from denaturation(3). For proteins, it is important in order to
maintain the tertiary structure that they are continuously
surrounded by a film of water even when this only consists of
one or two layers. Tests have shown that cosmotropic
substances form around a protein in such a way that when the
protein dries out it is still surrounded by a sheet of water(4).
In contrast, denaturing substances have a destabilizing
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Resurrection in cosmetics
effect on proteins as they can penetrate through the water
sheet of a protein, attach themselves directly to it and
change its spatial structure. Such substances are called
chaotropic substances.
1.1 Concept of the active complex S-61*
The aim of this active complex development was to
incorporate the fantastic abilities of resurrection plants –
protection and restructuring of cells – into a cosmetic active
complex. This was achieved due to intensive research and
development based on current scientific knowledge about
resurrection plants and about cosmotropic substances.
In choosing resurrection plants we allowed ourselves, in
addition to scientific knowledge, to be guided by the
teachings of Paracelsus, an outstanding physician of the
sixteenth century. In his teachings, by way of analogy, the
effect of shape, smell, behavior, etc. of a plant on the human
body are examined. The best plant chosen was Haberlea
rhodopensis (Figure 3). This thrives optimally in a shaded
environment with steady, warm temperatures. It has broad,
soft leaves which grow in the shape of a rosette. Its fine,
delicately colored flowers remind us of older people who
avoid gleaming sunshine and place a lot of value on
appearance. The protection, and if possible, the
restructuring of weakening parts of the body is of general
relevance, but is especially so for older people.
Further components are especially selected extracts from
Beta vulgaris (sugar beet) and Saccharomyces cerervisiae (baker’s
yeast). Such extract fractions containing a high proportion of
cosmotropic substances were specifically chosen so that they
would mutually complement each other.
Figure 3 : A blooming Haberlea rhodopensis
*The active complex S-61 is available as Unisurrection S-61
from Induchem AG, Switzerland
104 COSMETICS AND TOILETRIES MANUFACTURE WORLDWIDE
2. Materials and methods
2.1 Tests with the enzyme isocitrate dehydrogenase (ICDH)
As the basis for the experimental set-up, a commercial
enzyme test kit for D-isocitric acid was chosen(5). The test is
based on the principle that D-isocitric acid is converted into
2-oxoglutarate and CO2 by ICDH. This involves the
reduction of NADP⊕ to NADPH. The test kit contains in
separate solutions buffer, enzyme, substrate (isocitric acid)
and the coenzyme NADP⊕. The progress of the reaction was
monitored by the absorption at 340 nm and from this the
enzyme activity was calculated.
In order to demonstrate the protective effect of the active
complex S-61, the following procedure was used. A test
solution consisting of enzyme, substrate and active complex
S-61 was mixed with urea and left standing for 2.5 minutes at
24°C. Then the enzyme reaction was started by adding
NADP⊕. A reaction control solution was used without the
addition of the active complex.
In order to demonstrate the restructuring of the enzyme
ICDH, the following system was chosen. A test solution
consisting of enzyme and substrate was mixed with urea and
left standing for 5 minutes at 24°C. During this time the
enzyme is more or less severely denatured depending on the
concentration of urea. Then the mixture was separated into
three separate solutions. In the case of two of these solutions
the active complex S-61 was added with an end
concentration of 1 and 5% respectively. The equivalent
amount of water was added to the third solution. Then the
solutions were left for 45 minutes so that the enzyme could
restructure itself. After this time the enzyme reaction was
started by adding NADP⊕ solution and the reaction
monitored by observing the change in absorption at 340 nm.
2.2 Tests with cell cultures
The spontaneously immortalized keratinocyte cell line
HaCaT was used for the test. The cells were cultivated in a
DMEM medium with 10% serum, up to a cell density of
200,000 cells/ml. The cells were then distributed on 96 well
plates with 20,000 cells per well. After an incubation period
of 12 hours, during which the cells adhered to the well, the
active complex S-61 was added, whereby end
concentrations of 5 µg/ml (5 ppm) and 50 µg/ml (50 ppm)
respectively were used. The cells were then cultivated for a
further 72 hours. After the 72 hours cultivation, the
following tests were carried out: DNA synthesis rate by
means of the BrdU test(6), mitochondrial activity by means
Resurrection in cosmetics
of the MTT test(7) and vitality by means of the LDH test(8).
Per measured point n=12 well were used for calculation.
The tests were carried out by Hochschule Wädenswil, 8820
Wädenswil, Switzerland.
2.3 Efficacy tests in vivo
The skin moisture was measured using the Corneometer
CM825/MPA 8 and the skin surface structure using the
instrument SELS 2000/Visioscan VC98. Three various
cosmetic formulations were tested, namely 1) placebo, 2)
placebo plus 3% S-61 and 3) placebo plus 5% S-61. Five
healthy volunteers applied the corresponding test cream
twice daily to a specific area on the forearm. The test
parameters were measured before treatment, as well as one
and two weeks respectively after the treatment. The tests
were carried out by Laboratory Iderma AG, 4141
Münchenstein, Switzerland.
3. Results
The effectiveness of the active complex S-61 is proven at
various levels which differ in their degree of complexity:
• At a sub-cellular level with the enzyme isocitrate
dehydrogenase
• At a cellular level with keratinocyte cultures
• At skin level using volunteers
3.1 Test on sub-cellular level shows increased protection
and restruction
In order to demonstrate the protective effect of the active
complex S-61 the enzyme ICDH was mixed with the active
complex S-61 and urea. Urea belongs to the chaotropic
substances and inactivates the enzyme ICDH as can see in
Figure 4. The control curve without added active complex
S-61 shows that the urea concentration of 0.5 mol/l damages
enzyme activity by more than 50% and at a urea
concentration of 1 mol/l there is practically no enzyme
activity left. The curve labeled “1% S-61” shows that the
active substance complex S-61 does an outstanding job of
protecting the enzyme. Even at a highly denaturing urea
concentration of 2 mol/l, there is still a considerable level of
enzyme activity.
In order to demonstrate the restructuring effect of the
active complex S-61 the enzyme ICDH was mixed in a
solution with urea and left standing for 5 minutes at 24°C.
Then the mixture was divided into three separate solutions.
Figure 4 : Protection of the enzyme ICDH from the denaturing
effect of urea using 1% active complex S-61
In the case of two of these solutions the active complex
S-61 was added with an end concentration of 1 and 5%
respectively. The equivalent amount of water was added to
the third solution. Then the solutions were left for 45
minutes so that the enzyme could restructure itself. After
this time the enzyme reaction was started by adding
NADP⊕ solution.
The results are gathered together in Figure 5. It is seen
that the active complex S-61 possesses the rare ability to
restructure a denatured enzyme. At high urea
concentrations, the end concentration of a 5% active
complex S-61 is clearly more effective than a 1% solution.
Figure 5: Restructuring of the enzyme ICDH by the active
complex S-61 at various urea concentrations
3.2 Test on cellular level shows increased proliferation
behavior, mitochondrial activity and vitality
In this chapter the revitalization of the human keratinocyte
cell line HaCaT is reported, by showing the influence of the
active complex S-61 on proliferation behaviour,
mitochondrial activity and vitality of the cell line.
The proliferation behavior of a cell culture shows how
intensive the cells divide and grow. The DNA synthesis rate is
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Resurrection in cosmetics
Figure 6 : Increase of HaCaT cell activity by the active complex
S-61 measured as the DNA synthesis rate
a good measurement parameter for this since the amount of
DNA can be strongly correlated with the number of cells. The
evaluation of the DNA synthesis rate is shown in Figure 6.
The active complex S-61 increases the proliferation activity of
the cells within the dosage values of 5 to 50 µg/ml as compared
to the untreated sample (i.e. the experimental control). This
points to a strong stimulation of growth.
The mitochondrial activity of a cell culture demonstrates
how intensively the metabolism in the cell is activated. The
MTT test is such that activity of the enzyme succinate
dehydrogenase can be directly determined. This is a key
enzyme of the citric acid cycle found in the mitochondria.
Figure 7 shows the evaluation of the MTT tests. Significant
increases in the mitochondrial activity by the active complex
S-61, within a dosage range of 5 to 50 µg/ml, compared to the
experimental control are observed.
The vitality of a cell culture can be recorded by means of
the activity of the enzyme lactate dehydrogenase (LDH) within
the cell culture medium. LDH is a sensitive indicator for any
cell damage, as cell damage or even the death of a cell leads to
Figure 7 : Influence of Unisurrection S-61 on the mitochondrial
activity (MTT-Test) of HaCaT cells
106 COSMETICS AND TOILETRIES MANUFACTURE WORLDWIDE
Figure 8: Increase in the vitality of HaCaT cells by the active
complex S-61 represented by means of the LDH activity in the
culture medium
a destruction of cell integrity and hence to a discharge of LDH
present in the cell fluid into the culture medium. Therefore a
low LDH concentration in the culture medium implies the
presence of a healthy cell culture with intact cells.
The evaluation of the LDH test is shown in Figure 8. The
results show that there is a significant decrease in LDH activity
in the cell cultures treated with the active complex S-61
compared with the untreated experimental control. This is due
to the substance having a highly effective protective effect on
the cells. Essentially, the results with the keratinocytes, i.e.
the increase in the rate of DNA synthesis and mitochondrial
activity with a simultaneous decrease of the LDH activity in
the culture medium, can be interpreted in terms of S-61
protecting and vitalizing the cells.
3.3 Efficacy test in vivo shows visible anti-wrinkle effect
The results of skin moisture measurements are detailed in
Figure 9. Test areas which were treated with a cream
Figure 9 : Influence of the active complex S-61 on skin moisture
Resurrection in cosmetics
Figure 10 : Influence of the active complex S-61 on intensity
of wrinkles
containing the active complex S-61 showed a clear and
intensive increase in skin moisture. This favorable result was
reached already after one week and subsequently remained.
Test areas treated with the placebo cream or with no
treatment remained at the initial level or even had decreased
skin moisture levels.
The observation of the skin surface structure showed that
the intensity of wrinkles was reduced after two weeks of
treatment (Figure 10). Four out of five volunteers noticed at
the end of the test period that their skin clearly looked better.
These results can be explained by a microscopically
detectable swelling of the corneocytes on the skin surface.
These corneocytes can restructure their water content,
hence causing wrinkles to be filled out.
4. Conclusion
Resurrection plants possess the fantastic ability to dry out and
then reach their normal state again and continue growing a
short time after being remoisturized. For this to be possible
there has to be an ingenious protection system in place in the
plant during the drying-out phase as well as an extraordinary
ability to restructure the plant’s cell structure during the
remoisturization period. The potential of resurrection plants
could be incorporated in the active complex S-61. As the test
results showed, a short treatment of 1 – 2 weeks with a cream
containing the active complex S-61 improves the skin’s
appearance, with the skin looking clearly revitalized. The skin
is seen to have restructured itself, wrinkling is significantly
reduced and the suppleness of the skin can be felt as being
improved. The skin’s ability to resist wear and tear from its
external environment is seen to improve.
References
(1) Scott P. Resurrection Plants and the Secrets of Eternal
Leaf, Annals Bot. 85, 159-166 (2000).
(2) Farrant J.M. et al. The effect of drying rate on the survival
of three desiccation-tolerant angiosperm species, Annals Bot.
84, 371-379 (1999).
(3) Galinski E. et. al. The kosmotropic (structure-forming)
effect of compensatory solutes. Comp. Biochem. Physiol.
117 (3), 357-365 (1997).
(4) Timasheff S.N. The control of Protein stability and
association by weak interactions with water: How do solvents
affect these processes? Annu. Rev. Biophys. Biomol. Struct.
22, 67-97 (1993).
(5) Anonym. ENZYTECTM D-Isocitric Acid, ID-No. 1 002
807, of Scil Diagnostics GmbH Martinsried, Germany.
(6) Porstmann T. et. al. Quantitation of 5-bromo-2-deoxyuridine
incorporation in to DNA: an enzyme immunoassay for the
assessment of the lymphoid cell proliverative response. J.
Immunol. Methods 82 (1), 169-179 (1985).
(7) Mosmann T. Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity
assays. J. Immunol. Methods 65 (1-2) 55-63 (1983).
(8) Deters A. et. al. High molecular compounds
(polysaccharides and proanthocyanidins) from Hamamelis
virginiana bark: influence on human skin keratinocyte
proliferation and differentiation and influence on irritated
skin. Phytochemistry 58, 949-958 (2001).
Abbreviations
ATP
BrdU
FAD
FADH2
ICDH
LDH
MTT
NADP⊕
Adenosine-5’-triphosphate
5-Bromo-2-Deoxyuridine
Flavin-Adenine-Dinucleotide, oxidised form
Reduced FAD
Isocitrate dehydrogenase
Lactate dehydrogenase
Dimethyl thiazolyl diphenyl tetrazolium bromide
Nicotinamide-Adenine-Dinucleotide phosphate,
oxidized form
NADPH reduced NADP⊕
Authors
Dr. Raphael Plüss, Dr. Marion Fröschle, Dr. Katrin Bojarski
and André Peter
Induchem AG
Industriestrasse 26
CH-8604 Volketswil Switzerland
COSMETICS AND TOILETRIES MANUFACTURE WORLDWIDE 107