SENSITIVE

SKIN

TM
Agascalm

www.provitalgroup.com
Mind-skin connection
We live in a very demanding society that keeps us on the move and where multitasking is essential. Work, home,
children, sports, leisure, a lot of activities that we try to combine in our daily lives leading to physical and
psychological fatigue. Our brain is overwhelmed and this can cause stress, a situation that affects our mind and the
other organs in our body, including our skin. Skin and mind are connected and, the same way we can experience pain or
pleasure through our skin, the mind can transmit to skin stressful situations that deteriorate it.

Skin separates our body from the outside, acting as a protective barrier against external agents, but it is also a living
organ. It is interconnected with the other organs and it can also be altered due to internal factors in our body, such as
our psychological well-being (Slominski 2007).

Stress can become a chronic symptom affecting our body and therefore our skin. As if they were a mirror, skin
symptoms can reveal internal imbalances with a potential psychological link (Chen 2014).

In Provital Group we have created a product that treats skin hypersensitivity, acting on the biochemical mechanisms
that cause inflammation and the appearance of redness in particular due to disorders caused by our mind, such as
stress.

TM
Agascalm neutralizes the negative effects caused by psychological stress on the skin, such as inflammation
and redness.

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Neurogenic inflammation
Neurogenic inflammation is produced by neurons that locally release inflammation mediator substances such as
Substance P, Calcitonin Gene-Related Peptide (CGRP), neurokinin A (NKA), and endothelin-3 (ET-3) (Chen 2014)
(Geppetti 2008). Substance P is responsible for initiating inflammation through the translocation and activation of NF-
kB.

This process seems to play an important role in the pathogenesis of many diseases, including psoriasis (Schön 2005),
eczema, rosacea, dystonia and multiple chemical sensitivity (Bascom 1997).

During psychological stress, the neuroendocrine system and peripheral sensory nerves are activated leading to the
release of mediators, which are capable of activating mast cells, inflammatory cells associated with the sensory nerves
located in the skin (Slominski 2000). At the same time, mast cells release mediators that can excite and stimulate C-
fibers, increasing psychological stress and therefore promoting the neurogenic inflammation in a loop that, if prolonged
in time, can negatively affect the skin.

TM
Agascalm increases skin's resilience against psychological stress, protecting it from damage caused by this
type of stress.

Psychological stress mechanism on the skin

The pro-inflammatory reaction that is triggered by psychological stress is managed by the nuclear factor kappa B (NF-
κB). It is a DNA transcription factor found in the cytoplasm of most cells that is capable of regulating the expression of
more than 150 genes, including genes involved in immunity and inflammation, in anti-apoptotic processes, cell
proliferation and also those responsible for self-limiting its own activity (Blas 2004; Aberg 2007; Moynagh 2005).

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In cells found in normal conditions, NF-κB is retained in the cytoplasm by the protein IkB. This protein does not allow it
to reach into the nucleus, preventing its action of transcribing part of the DNA.

When stress occurs, the protein IkB degrades, releasing NF-κB and allowing it to enter the nucleus to transcribe the
DNA genes that trigger inflammation (Blas 2004; Calixto 2003; Moynagh 2005; Aktan 2003). This reaction is not a
negative one, unless it continues for a long time causing a chronic inflammation that affects skin and deteriorates it.

Agascalm™, due to its acacetin and tilianin content, is able to counteract the effects of psychological stress on the
skin, reducing the degradation of protein IkB that retains NF-kB in the cytoplasm, therefore inhibiting its movement
towards the nucleus (Ha 2012). By decreasing the amount of NF-kB that moves towards the nucleus, DNA transcription
will be reduced for the appearance of cytokines, chemokines and other inflammation-related substances, which
damage the skin when expressed continuously over time.

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Botany and chemistry
Agascalm™ is a natural active ingredient derived from the
plant Agastache mexicana, belonging to the Laminacea family
and native to southern North America, Mexico. It can reach up
to 1.5 meters in height. Its stems are square and the leaves
are spear-shaped, with toothed edges. Its flowers are
arranged in clusters, with tubular form and red or red-purple
in color. Its fruits are coffee-colored. It is present in warm,
semi-warm and temperate climates from sea level to 780
meters. It is associated with tropical forests and thorn forests.

Agastache mexicana is rich in acacetin and tilianin, flavonoids with marked anti-inflammatory activity.

Traditional uses
Agastache mexicana is a medicinal plant widely used
traditionally in Mexico for treating diseases or symptoms
related to nerves, such as “shock and horror”. Traditionally
this plant has been linked to the treatment of psychological
stress caused in individuals by a situation of horror. It is for
this reason that Provital became interested in it in order to
develop an active ingredient that could be used in skin care
cosmetics, taking into account the scientific evidence linking
flavonoid components of this plant with the biochemical
mechanisms of psychological stress and its skin
symptomatization.

TM
Agascalm does not avoid psychological stress but it does block its effects on the skin.

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AgascalmTM: social and environmental value
Respect for the environment is one of the
fundamental values in Provital Group. This
includes the social value in the development of
its products. Through an agreement with the
University of Queretaro (Mexico) for the
cultivation of the plant Agastache mexicana,
TM
from which the Agascalm active ingredient is
obtained, an organic garden involving families
living in the surrounding area has been created
in the Carbonera region. Currently there are
several local women working in the
development project of the organic farming
center and in the planting and cultivation of
Agastache mexicana.

Agascalm™ is a sustainable active ingredient. It is respectful of the environment and the biodiversity and it creates jobs
in the geographical area of cultivation, through the participation in a university social project and in a context of fair
trade.

Efficacy of AgascalmTM
TM
In order to verify the efficacy of Agascalm in the treatment of skin inflammation and redness, in vitro and in vivo
studies of the active ingredient were carried out.

In vitro efficacy
TM
Three experiments on keratinocytes were conducted to demonstrate that Agascalm is able to reduce the effects of
psychological stress on the skin and to determine its mechanism of action at the molecular level:

1. The active ingredient capacity to inhibit the translocation of NF-κB towards the nucleus was assessed. The
TM
purpose of this test is to verify that Agascalm acts through a mechanism linked to the effects of stress on
the skin.

2. The modulation of the expression of NF-κB-dependent genes was studied. The aim was to verify whether
the inhibition of the transcription factor caused by the active ingredient (and demonstrated in the previous
experiment) leads to a decrease in its nuclear activity.

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 3. The inhibition at the protein level was quantified. With this the intention was to demonstrate that the
activity is carried out at both the mRNA and protein levels and that, therefore, it has the final biological
activity we believe it has.

1. Evaluation of the effect on NF-kB translocation towards the nucleus

In order to study NF-kB translocation towards the nucleus, a cellular model of normal human epidermal keratinocytes
(NHEK) was chosen. These keratinocytes were stimulated with TNF-α. When stimulated, NF-kB will move towards the
cell nucleus to transcode the inflammation-triggering compounds in the DNA. To measure the amount of NF-κB in the
cells and therefore to know to what extent its movement to the cell nucleus has been inhibited due to the use of our
active ingredient Agascalm™, they were first labeled with an anti-NFkB antibody that binds to NFkB. Then a second
antibody that binds to the first one and emits fluorescence that can be measured was included. By measuring the
fluorescence we can know the amount of NF-kB in the cell and the amount that has moved from the cytoplasm to the
nucleus.

Figure 1. Quantification of NF-kB by fluorescence.

Agascalm™, tested at 0.03, 0.11 and 0.30%, provides a clear inhibition of translocation of 22, 36 and 70% respectively.
At a concentration of 0.11%, the same inhibition as with the reference substance is achieved.

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 EFFECT ON THE NF-kB TRANSLOCATION
120
(%) Translocation inhibition
100

80

60

40

20

0
Non-stressed Stressed + Stressed Stressed + Stressed + Stressed +
NF-kB Agascalm™ Agascalm™ Agascalm™
inhibitor III 0.03% 0.11% 0.30%

Graph 1. Inhibitory effect of translocation.

Below there is a series of photographs of the translocation study in human keratinocytes. In the non-stressed culture it
can be seen how NF-κB, shown in green, is distributed throughout the cytoplasm of cells, leaving the nucleus empty
(shown in black). By contrast, in the stressed culture it can be seen how the green color prevails inside the nucleus,
while the cytoplasm has less intensity. When Agascalm™ is applied, virtually the entire nucleus can be seen in black.
TM
Therefore, this demonstrates that Agascalm prevents NF-kB from moving towards the nucleus and transcoding the
compounds that trigger inflammation.

Figura 2. Visualization by fluorescence of NF-kB location.

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 2. Assessment of the transcriptional effects of AgascalmTM

In order to study the gene expression modulation of Agascalm™ a model of normal human epidermal keratinocytes
stimulated with Poly(I:C) was used. This is a synthetic substance capable of triggering an immune response and
stimulating cytokine release. It also shares some steps in the cascade of events triggered by psychological stress, such as
NF-κB activation.

TM
Fifteen inflammation-related and NF-κB-activated genes were chosen to verify whether Agascalm influences their
expression: IFNB1, TSLP, IL6, TNF, CCL20, CCL3, CCL5, CCL27, IL8, CSF2, IL23A, IL1A, IL1B, IL1RN and TLR3.

To quantify the expression of the selected genes, the cells (keratinocytes) are pre-incubated for 24 hours with or
without Agascalm™, the reference compound (Bafilomycin) or the solvent (DMSO). Then poly(I:C) is added or not, to
cause cellular stress or not, during 4 and 24 hours.

TM
After analyzing the results at 4 hours, Agascalm (at 0.30%) intensively reduces the expression of 7 of the
inflammation-related genes selected to be studied. After 24 hours, a decrease in all analyzed markers is observed. In
TM
fact, Agascalm almost gets reverse cellular conditions to non-stressed state.

3. Assessment of the effects on cytokine release

The amount of cytokines released by the cells is directly proportional to the degree of inflammation.

The release of 4 cytokines was measured: IL-6, IL-8, IL-1β and TNF-α. These are frequently quantified in studies related
to psychological stress (Hansel et al 2010; Hawkley).

TM
To conduct this analysis, cells are pre-incubated for 24 hours in a medium containing or not Agascalm . Subsequently
Poly(I:C) is added to cause cellular stress or is not added to have the non-stimulus reference.

% INHIBITION
CYTOKINE
0.11 % 0.23 % 0.30 %
Agascalm™ Agascalm™ Agascalm™

IL-1β 79 94 104

IL-6 71 93 99

IL-8 56 86 96

TNF-α 71 98 100

Table 1. Inhibition of cytokines.

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Its action as a strong inhibitor of cytokines in keratinocytes was verified in the three different concentrations of
TM TM TM
Agascalm . At the higher dose, Agascalm inhibits cytokine synthesis by between 96 and 104%. Agascalm gets
completely block the effects of stress in cells.

In vivo efficacy
Many skin diseases or disorders related to inflammation, such as dermatitis, psoriasis or rosacea worsen and/or
outbreaks occur because of psychological stress (Slominski 2001). Psychological stress produces several negative
consequences on the skin. These are the most important ones:

1. Alteration in the skin's barrier function: When substances released by stress, such as inflammatory cytokines
and glucocorticoids, remain active for long periods of time, an increase in keratinocyte proliferation occurs. Epidermal
differentiation is impaired and there is a decrease in the density and size of corneodesmosomes. The skin barrier is
altered (Garg 2001; Altemus 2001), transepidermal water loss increases and, therefore, dryness, wrinkled and rougher
skin and even itchiness appear (Altemus 2001; Choi 2005; Garg 2001)

2. Vasodilation of skin capillaries: NF-κB is capable of releasing iNOS, which produces NO, a known powerful
vasodilator agent. Continued vasodilation causes redness and extravasation. It also prevents capillaries from contracting
the way they should (Vila 2004; Elewa 2013), and it may be a contributing factor to rosacea.

3. Redness, caused by an increase in detrimental vasodilatation of skin capillaries (Calixto 2003).

4. It is believed that IL-8 produced during inflammation is capable of reducing collagen reorganization of
fibroblasts at a dermal level, which will also produce lower skin radiance (Han 2001). Although radiance and overall skin
optical properties are often associated with the presence of chromophores such as melanin and hemoglobin, it has
been observed that the role of collagen fibers is also very important. There is a relationship between light scattering on
contact with the skin and the thickness and alignment of collagen fibers.

In order to conduct this study, a group of 45 female volunteers was chosen. 30 of them suffered psychological stress
and 15 of them were prone to develop rosacea. Their ages ranged from 19 to 62. The volunteers applied a cream
TM
containing Agascalm at 2% on one half of the face twice daily for 28 days, and a placebo on the other half.
Measurements were taken at day 0, before the first application, at day 7 and at day 28.

Before including the volunteers with stress, both a psychological questionnaire and a quantification of cortisol in saliva
were conducted. Subjects with rates higher than 27 nmol/L were selected.

1. Assessment of microvascular tonicity

The microvascular system was monitored directly, using a monochromatic low-energy laser that penetrates the tissue
and illuminates the blood vessels and blood cells in circulation. It was also measured indirectly through thermography.
Doppler flowmetry (LDF) and the concentration of moving red cells (CMBC), which are expressed in arbitrary units (AU),
were measured using the laser. The more these factors are reduced the lower the microvascular dilation will be.

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Using the thermography, measured with infrared radiation, the hotter areas are visualized, ie, those presenting
vasodilation and inflammation.

1.1 Direct measurements (laser)

After 28 days of treatment with Agascalm™ a clear reduction in the two parameters measured regarding vasodilation is
achieved:

- 30% CMBC reduction

- 25% LDF reduction

At the end of the treatment (D28), the active ingredient shows statistically significant differences versus baseline, while
placebo does not. Moreover, at D28 Agascalm™ results are also statistically better than those of the placebo.

Microvascular tonicity
Vasodilation variation (%)

10

-10

-20

-30

-40
CMBC CMBC D28 LDF D7 LDF
D7 D28

Graph 2. Measure of tonicity of the whole panel.

If the panels are considered separately, that is, on the one hand the results obtained from volunteers who had
psychological stress and on the other hand the results obtained from volunteers with rosacea:

Panel with stress Panel prone to develop rosacea

CMBC reduction  29% CMBC reduction  31%

LDF reduction  20% LDF reduction  35%

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 Microvascular wellness
(stress and rosacea panel separately)

10
Variation of vasodilation (%)

-10 Stressed panel

-20 Rosacea prone

panel

-30

-40
CMBC CMBC D28 LDF D7 LDF D28
D7

Graph 3. Measure of tonicity of the two panels separately.

TM
Agascalm shows very positive effects on volunteers with rosacea.

Agascalm™ reduces continued vasodilation by between 25 and 30%. It also enables capillaries, arterioles and venules
to recover their ability to contract and improve their strength and integrity, reducing skin redness.

1.2 Indirect measurements (thermography)

When the microvasculature dilates, the passage of blood increases and the temperature of the skin area rises. The
thermal camera can show the temperature of the different skin areas to be analyzed, in this case the face, and measure
TM
the beneficial effect of Agascalm .

TM
Agascalm reduced skin temperature by 0.4 degrees in subjects with stress and/or skin prone to develop rosacea. In
TM
the following images, taken on day 28, the change in color between the side of the face treated with Agascalm and
the side treated with the placebo can be observed. The more purple and less yellow, the lower the skin temperature
and the lower its stress level.

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 Figure 3. Skin temperature reduction in stressed panel.

Figure 4. Skin temperature reduction in panel with rosacea.

2. Assessment of facial redness

When a microvascular dilation occurs the passage of blood increases, causing skin reddening. Two techniques are used
to measure redness: chromatometry and VISIA image analysis. This technique is capable of quantifying spots and
redness and it is a more suitable technique for measuring skin with rosacea, as it can appear as scattered spots and not
as a single red area.

2.1 Chromatometry

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Using a Chromameter that measures the light reflected on the skin, it was found that Agascalm reduced redness by
9%.

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 Anti-redness efficacy
(stressed panel)
5
Redness variation (%)

0 a*: value that

measures the red
pigmentation
-5

-10

-15
a* D7 a* D28

Graph 4. Evaluation of redness in stressed panel.

2.2 Image analysis

The images are taken with the VISIA-CA system using a cross polarized light flash. This equipment easily visualizes
rosacea, spider veins and redness in general.

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As a result, Agascalm reduced redness by 10%.

Anti-redness efficacy
(panel prone to rosacea)

3
1
Redness variation (%)

-1
-3
-5
-7
-9
-11
-13
-15
redness D7 redness D28

Graph 5. Evaluation of redness in rosacea panel.

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 TM
The capacity of Agascalm to reduce redness can be clearly observed in the following images.

Figure 5. Reduction in skin redness with Rosacea-prone skin.

Figure 6. Rosacea-prone skin. Reduces redness and increases the skin radiance.

3. Assessment of the barrier function of skin

TM
To quantify the effect of Agascalm on the barrier function and moisturization, two techniques were used:

- Tewametry (TEWL) to evaluate the barrier function by measuring transepidermal water loss.

- Corneometry (CORNEO) to evaluate the amount of water in the outermost skin layers.

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 TM
After applying Agascalm for 28 days, a 13% reduction in skin water loss using the TEWL technique and a 6% increase
in moisturization using the CORNEO technique were measured.

Skin barrier and moisturization

10

5
Variation vs D0 (%)

-5

-10

-15

-20
TEWL TEWL CORNEO CORNEO
D7 D28 D7 D28

Graph 6. Measure values associated with hydration.

Skin barrier and Moisturizing

(stress and rosacea panel separately)

10

5
Variation vs D0 (%)

Stress panel
0

-5
Rosacea
-10 prone panel

-15

-20
TEWL D7 TEWL D28 CORNEO D7 CORNEO
D28

Graph 7. Measurement values related to hydration in the two separate panels.

TM
Agascalm prevents the deterioration of the barrier function and maintains the level of skin moisturization

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 4. Improvement in skin radiance and overall skin complexion.

This study was conducted through the observation of the photographs taken of the volunteers at days 0 and 28 of
TM
Agascalm application.

Figure 7. Stressed panel. Less redness and more skin radiance.

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Images show that, 28 days after the application of Agascalm , skin's own radiance increases and its complexion is
improved and unified.

Conclusion
TM
Agascalm is an active ingredient with excellent anti-inflammatory activity that modulates the biochemical
mechanisms that link psychological stress with skin deterioration:

-it reduces redness

-it moisturizes

-it restores luminosity and a more even skin tone.

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Cosmetic applications
TM
Agascalm can be applied to any cosmetic product designed to repair and protect skin, anywhere in the body, against
the negative consequences caused by psychological stress. It would be highly recommended for cosmetic products for
“sensitive skin” used to improve skin prone to suffer atopic dermatitis, psoriasis and rosacea.

Recommended dosage
TM
The recommended Agascalm dosage ranges between 1 and 3%.

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PM (2007). Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of
cutaneous infections in mice. The Journal of Clinical Investigation, 117(11) 3339–3349.

Aktan F (2004). iNOS-mediated nitric oxide production and its regulation. Life Sciences 75 (2004) 639–653.

Altemus M, Rao B, Dhabhar FS, Ding W, and Granstein RD (2001). Stress-Induced Changes in Skin Barrier Function in
Healthy Women. The Journal of Investigative Dermatology 117(2), 309-317

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Bascom, R; Meggs, WJ; Frampton, M; Hudnell, K; Killburn, K; Kobal, G; Medinsky, M; Rea, W (1997). "Neurogenic
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Slominski A, Zbyteka B, Nikolakisd G, Mannae PR, Skobowiata C, Zmijewskif M, Lig W, Janjetovica Z, Postlethwaitec A,
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PROVITAL. S.A.
Pol. Ind. Can Salvatella
Gorgs Lladó, 200
08210 Barberà del Vallès
Barcelona (España)
Tel. (+34) 93 719 23 50

info@provitalgroup.com
www.provitalgroup.com

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