Tens’UpTM

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INSTANT BEAUTY

Our modern lifestyle demands a perfect image during long days over

which fatigue is accumulated and this is reflected on our skin.

The cosmetic response to immediately eliminate these signs of fatigue

and restore skin to all its glory can be found in the products with the so-

called lifting effect. The "lifting" is an immediate tightening effect that

visibly improves the appearance of skin as it reduces wrinkles, providing

comfort and softness.

The active ingredients with a lifting action exert an immediate mechanical

tension on the skin, resulting in a smoothing of the skin surface and showing a softer skin appearance.

TENS'UPTM

LIFTING EFFECT FOR IMMEDIATE BEAUTY

TENS’UPTM

is an innovative active ingredient with a marked lifting or tightening effect, with both an immediate

action and a medium-term action, as shown in in-vivo efficacy studies. It can be incorporated into facial

products, makeup bases and specific treatments for the eye, neck and décolleté area to obtain this immediate

tightening effect that visibly improves skin appearance.

TENS’UPTM combines its lifting action with anti-aging activity, as it increases

collagen synthesis, so there is a dual action of immediate improvement in skin

appearance and anti-aging treatment in the longer term.

Lifting effect

The immediate lifting or tightening action on skin surface is a cosmetic property

exerted by macromolecules of different origins: animal origin (collagen, chitosan,

keratin, bovine serum, hyaluronic acid), vegetable origin that are generally

biopolymers (proteins of wheat, rice, soybeans, polysaccharides), and synthetic

origin derivatives.

The mechanism of action of these molecules is to exert enough mechanical

tension on the skin as to smooth the skin surface and instantly provide a soft and

radiant appearance. These macromolecules form a superficial film that smoothes

skin, erases wrinkles and expression lines, and provides a more radiant and brighter appearance. The

extension of this tightening effect depends solely on the film that is formed.

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Provital S.A. has developed an innovative dual action vegetable origin cosmetic ingredient with a lifting effect

that also facilitates the penetration of its biomolecules, increasing collagen synthesis and prolonging the

tightening effect on skin.

TENS’UPTM

is a novel galactomannan tridimensional hydrocolloid matrix, which sequentially releases

oligosaccharides obtained from the chicory root (Cichorium intybus L.). This tridimensional mesh is adsorbed

on the skin surface to form a continuous film that enables this immediate lifting effect and a prolonged effect

for the release and later absorption of oligosaccharides.

BOTANY AND CHEMISTRY

CHICORY

Chicory, succory, coffeeweed and, in some countries, radicchio are the common

names used to refer to the “Cichorium intybus L.” species. It is a biennial or

perennial plant belonging to the Asteraceae family that can grow up to one meter

high. The flowers appear in the summer, they are sky blue-violet in color and they

form star-shaped capitula.

Chicory is native to Europe and temperate regions of Asia. It was used by

Egyptians as a medicinal plant. Dioscorides mentioned it in his treatise on

medicinal plants, and Galen referred to it specifically for liver disorders. The

Greeks and Romans ate it in salads with mallow flowers.

It was considered as a magical plant in the Middle Ages, capable of giving invulnerability to those who

consumed it. Chicory leaves are an important component for salads in Europe, especially in France, Belgium

and Holland, and in the United States. The root was ground and used as a coffee substitute.

In traditional medicine, the leaves have been used for the treatment of liver, kidney and

urinary diseases. The juice from its leaves has also been applied topically to treat warts

and skin irritations. Flower decoctions have been applied in eye treatments. Preparations

made with its roots are used in cases of loss of appetite and dyspepsia, and to remove

kidney stones.

The chicory root contains high amounts of inulin (>40%), saccharose (14%), cellulose (5%), proteins (6%),

chlorogenic and isochlorogenic acid, sesquiterpene lactones: lactucin and lactupicrine and mineral elements

(4%). Other minor components are flavonoids, tannins, coumarins, gums, pectins, and volatile components.

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Chicory oligosaccharides

Chicory is rich in fructans such as inulin (figure 1) and inulonose, in fructooligosaccharides such as 1-kestose

(figure 2) and nystose (figure 3), and sugars such as fructose, sucrose and glucose (van Den Ende et al.,

1996).

Inulin consists of fructose molecules linked by -(21) fructosyl-fructose bonds. The fructose chains have the

particularity of ending in a glucose unit linked by a -(1,2) bond (D-glucopyranosyl residue), but also the

terminal monomer of the chain can correspond to a residue of -D-fructopyranosyl. These carbohydrates have

a predominantly linear polymeric structure.

The process of obtaining the oligosaccharides from chicory (OCs) is performed by a physical separation by

column chromatography, where previously there has been a hot water extraction, filtration and enzymatic

depolymerization.

Figure 1. Chemical structure of inulin. GFn with a terminal glucose molecule and Fn with a terminal fructose

molecule (Madriga et al. 2007).

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Oligosaccharides in TENS’UPTM

have a polymerization degree (PD) < 20 in more than 85%.

Figure 2. 1-Kestose Figure 3. Nystose

TARA

“Caesalpinia spinosa (Mol) O. Kuntze” is commonly known as tara.

The species is native to Peru, but it is widely distributed in Latin

America. It grows wild in the forests and valleys of the Andes, in

heights of approximately between 1000 and 3100 meters. Tara has

a high potential for reforestation, it grows well in dry environments

and it develops in soils ranging from sandy to rocky.

Its fruits are flattened pods containing 4 to 7 seeds. Inside the shell

of the seeds two parts can be differentiated: the germ (embryo) and

the endosperm (albumen). This endosperm is mainly composed of

mannose and galactose polysaccharides known as

galactomannans.

The galactomannans are high molecular weight polysaccharides

that have a natural moisturizing function in the seed. They prevent

the desiccation of the embryo during its development thanks to its high capacity to capture and retain water.

Structurally, they are linear chains of mannose mutually linked by glycosidic bonds with lateral branches of a

galactose residue, with a mannose:galactose ratio of 3:1 and a fairly regular distribution of galactose along the

chain, which gives it good solubility in water.

These galactomannans are the ones that make up the matrix that will contain the

active ingredient. This matrix is obtained using an innovative technology, exclusive

to Provital, by multicapillary injection.

The galactomannans tridimensional matrix contains a minimum of 35% of fructooligosaccharides from the root

of Cichorium intybus L.

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TENS’UP™ TECHNOLOGY AND STRUCTURE

In the preparation stage, the fraction of polysaccharides or galactomannans that will form the matrix is

subjected to a humectation phase with controlled temperature increase until complete solubility is achieved.

Figure 4. Multicapillary injection.

A hydrocolloid pre-gel is formed which is needed to accurately incorporate the oligosaccharides from chicory.

The galactomannan pre-gel is circulated through a tube at constant temperature and at a certain point a

multicapillary injection of the concentrate of oligosaccharides (COS) is applied, followed by an immediate

cooling of the system. The injection enables a critical concentration of solutes, which combined with the

drastic drop in temperature causes the reaction of the galactomannan, forming a tridimensional matrix that

captures the active ingredients. The injection of COS is performed continuously and at high pressure, ensuring

a homogeneous incorporation of small molecules to the galactomannan matrix.

With this innovative technology, the unique structure in TENS’UPTM

is obtained, which determines its

mechanism of action and the extended release of Cichorium intybus oligosaccharides.

TENS’UP PROPERTIES

The vast majority of these "flash" effect active ingredients of vegetable origin are proteins or semisynthetic

derivatives of proteins or combinations with chemical polymers. This group is characterized by a marked

tightening effect, but it has the disadvantages associated to the use of proteins (characteristic color, odor).

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Another group consists of combinations of protein hydrolysates with natural gums (acacia, xanthan, carob).

The purpose of these active ingredients is always the same: to obtain a powerful immediate tightening effect

and to improve the sensory characteristics on the skin.

Currently polysaccharides and oligosaccharides are replacing proteins in flash-type

formulations. The objective remains to improve the sensory properties after product

application, maintaining an optimum tightening effect.

TENS’UPTM represents a breakthrough in this type of active ingredients due to its

dual effect, and it combines its superficial tightening action and anti-aging

treatment with pleasant sensory properties.

In order to assess the lifting effect of TENS’UPTM

, two studies have been carried out:

- an in vivo study, aimed at observing the changes on the skin and obtaining the results of a sensory

panel.

- an ex vivo study, to demonstrate the anti-aging benefits by increasing collagen synthesis.

IN VIVO EFFICACY

The objective of the assay was to study the in vivo efficacy of TENS’UPTM

as a cosmetic active ingredient with

lifting effect. In order to carry out this assessment, images have been taken (Fotofinder Dermoscope ver. 2.0)

to visualize the changes on the skin. A subjective evaluation has also been performed.

Methodology

A panel of 15 volunteers (40 - 65 years) has performed a single application of an aqueous solution at 10% of

TENS’UPTM

and its effect has been assessed at 5 minutes and at 2 hours after its application.

An image of the periocular area is taken before product application and 5 minutes and 2 hours after its

application.

Results

Fotofinder Dermoscope Ver. 2.0

Figures 5, 6 and 7 show the images obtained with Fotofinder Dermoscope Ver. 2.0 of the area treated with a

solution at 10% of Tens’Up™, at time zero (T0) before and after (T5’, T2h) its application.

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T0’

Figure 5. Images obtained at T0 before treatment with Tens’Up.

T5’

Figure 6. Images obtained at T5’ after treatment with Tens’Up.

T2h

Figure 7. Images obtained at T2h after treatment with Tens’Up.

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The application of TENS’UPTM

visually improves skin appearance and a visible attenuation of wrinkles occurs

around the eyes.

TENS’UPTM has a marked lifting effect on the skin.

Subjective assessment

To carry out the subjective assessment, volunteers completed a questionnaire both five minutes (T5’) and two

hours (T2h) after the application of TENS’UPTM

.

Results obtained using TENS’UP™ at 5 minutes (T5’) and two hours after its application (T2h) are shown in

figures 8 and 9.

The subjective assessment of TENS’UPTM

provides excellent feedback regarding its lifting effect. More than

90% of volunteers confirm this immediate tightening effect, both at 5 minutes and at 2 hours after application.

Anti-wrinkle action is also highly appreciated by the volunteers.

Very satisfactory results are also given to the softness and comfort provided by the active ingredient, as well

as to the overall effect on the skin.

Figure 8. Questionnaire results obtained at T5’ after treatment with Tens’Up.

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Figure 9. Questionnaire results obtained at T2h after treatment with Tens’Up.

EX VIVO EFFICACY

The ability of TENS’UPTM

to increase collagen levels in the dermis and act as an anti-aging treatment, in

addition to the tightening effect, is evaluated.

The quantification of collagen content has been performed using the modified Miller's stain on skin biopsies.

Materials and methods

The products to be tested have been two cosmetic formulations containing 5 and 10% TENS’UPTM

respectively, Placebo, Control and Positive control (Retinol 0.01%) and they have been applied topically on

skin samples, every day for 6 days.

Table 1 shows the components of the cosmetic formulations and placebo used in this study. The positive

control is Retinol at 0.01% dissolved in dimethyl sulfoxide (DMSO).

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INGREDIENT Tens’Up 5% Tens’Up 10% Placebo

Ethylhexyl Stearate,

Phenoxyethanol, Sorbitan

Laurate, Polyglyceryl-4 Laurate,

Dilauryl Citrate

4.2 4.2 4.2

Cetyl Ricinoleate 0.80 0.80 0.80

Xanthan Gum 0.72 0.72 0.72

Water (Aqua) QS 100 QS 100 QS 100

Tens’Up 5.0 10.0 --

Aqua (Water), Citric Acid 0.05 0.05 0.05

Table 1. Cosmetic formulations tested in the in vivo study.

After six days of treatment, the skin samples are stained using the modified Miller's stain (Miller’s elastina

stain kit, Clin-Tech Ltd), which presents collagen fibers with a red coloration. Quantification is performed by

studying the intensity and distribution of staining, using an appropriate image analysis software.

Results

Figure 10 shows the comparative results of collagen assessment of the different treatments, after six days in

culture.

The application of TENS’UPTM

causes an increase in collagen levels.

If results are compared with Control, there is a 15% and 25% increase for TENS’UPTM

at 5% and 10%,

respectively.

If results are compared with Placebo, there is an 8% and 17% increase for TENS’UPTM

at 5% and 10%,

respectively.

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Figure 10. Assessment of collagen content after 6 days of treatment.

Values in red show the percentage compared with Control.

It is worth noting the good results that have been obtained, especially if we consider that they are compared

with Retinol, a well-known anti-wrinkle agent; the increase in collagen synthesis is higher in the case of 10%

TENS’UPTM

.

It is observed that the increase in collagen levels is dose-dependent.

Figure 11 shows some of the images obtained for the quantification of collagen depending on the different

treatments. A higher density of staining means a greater amount of collagen. An improvement in the synthesis

of this protein after the application of TENS’UPTM

can be clearly seen.

17%

6%

15% 25%

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Control (DMSO) Retinol 0.01%

Placebo

Tens’Up 5% Tens’Up 10%

Figure 11. Illustrative images of the different treatments with the modified Miller's stain.

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CONCLUSION

TENS’UPTM

is a new approach to active ingredients with an

immediate lifting effect, as its superficial tightening action is

combined to the anti-aging activity through the proven increase

of collagen synthesis, which has a wrinkle-replenishing effect

comparable to that of retinol.

Its dual action enables the formulation of "flash" products with

the anti-aging added value.

In addition, the sustained-release mechanism extends the lifting effect, so there is both an immediate

action and a long-term action.

Due to its sensory properties, it is also particularly suitable for products related to makeup, as it

facilitates makeup application and allows for longer-lasting makeup.

The positive assessment of the lifting effect by 90% of volunteers is also important, in a type of

product in which the perception of the immediate effect of the applied cosmetic is essential.

COSMETIC APPLICATIONS

- “Flash” action products.

- Product lines for the neck and décolleté areas.

- Eye contour

- Intensive masks

- Fluid makeup products.

- Makeup bases.

RECOMMENDED DOSAGE

The recommended dosage of TENS’UP™ is 5-10%.

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BIBLIOGRAPHY

Alonso J. Tratado de Fitofármacos y Nutracéuticos. Rosario: Corpus. 2004.

Benaiges A., Guillén P. Botanical extracts. In: Analysis of cosmetic products. Salvador A and Chisvert A (eds.)

Elsevier, Amsterdam (2007) p. 345-363.

Bland E.J., Keshavarz T, Bucke C. The influence of small oligosaccharides on the immune system.

Carbohydrate Research. 2004, 339:1673-1678.

Epstein E.H., Munderloh N.H. Human skin collagen. The Journal of Biology Chemistry. 1978, 253(5): 1336-

1337.

Jouandeaud M., Bordes S., Soulie C., Closs B. The influence of oligosaccharides on skin aging: an alternative

to retinoids. Cosmetics & Toiletries. 2004, 119(6):67-75.

Madrigal L., Sangronis E. La inulina y derivados como ingredientes claves en alimentos funcionales. Archivos

Latinoamericanos de Nutrición. 2007, 57(4):387-396.

Mansell P.W.A. Polysaccharides in skin care. Cosmetics & Toiletries. 1994, 109(9):67-72.

Matsui M.S., Muizzuddin N., Arad S., Marenu K. Sulfated polysaccharides from red microalgae have

antiinflammatory properties in vitro and in vivo. Applied Biochemistry and Biotechnology. 2003, 104(1):13-22.

Disponible en: http://www.springerlink.com/content/63rx4482p4p5583u/

Péterszegi G., Isnard N., Robert A.M., Robert L. Studies on skin aging: Preparation and properties of fucose-

rich oligo-and polysaccharides. Effect on fibroblast proliferation and survival. Biomed Pharmacother. 2003,

57(5-6):187-194. Disponible en: http://www.ncbi.nlm.nih.gov/pubmed/12888253

Petrovic J., Stanojkovic A., Comic L.J., Curcic S. Antibacterial activity of Cichorium intybus. Fitoterapia. 2004,

75(7-8):737-739.

Van Beek T.A., Maas P., King B.M., Leclercq E., Vora A.G.J., de Groot A. Bitter sesquiterpene lactones from

chicory roots. J Agric Food Chem. 1990, 38:1035-1038. Disponible en:

http://pubs.acs.org/doi/abs/10.1021/jf00094a026.

Van Den Ende W., Mintiens A., Speleers H., Onuoha A.A., van Laere A. The metabolism of fructans in roots

of Cichorium intybus during growth, storage and forcing. New Phytol. 1996, 132:555-563.