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www.provitalgroup.com
Linefill™
All-in-one
wrinkle filler
and lip
plumper
ANTI-WRINKLES
V02‐07/14 72150‐1
FOR A HARMONIOUS AND REJUVENATED FACE…
As we age, our skin undergoes a number of
changes. One of the most important is the loss
or redistribution of facial volume and
subcutaneous fat, which modifies the face’s
balance and which is one of the most visible
signs of aging.
This loss means that, as we age, our folds and
wrinkles become more accentuated, especially
those that form around the mouth and eyes, as
well as a loss of lip volume (Wong et al., 2010;
Buckingham, 2013; Ilankovan, 2013).
Dermal fillers are currently among the most popular methods used to fill wrinkles and expression lines and
replace facial volume. The American Society of Plastic Surgeons has stated that, in 2012, these fillers were
the second most popular non‐surgical anti‐aging treatment, after Botox, with more than 2 million
applications in the USA. In addition, lip augmentation is also a common treatment for retaining a young,
attractive appearance and to increase self‐esteem (Park, 2006).
However, these invasive procedures can often give rise to unexpected results and they can cause a loss of
harmony between the injected features and the rest of the face, giving it an unnatural appearance.
The restoration of a natural volume distribution of is one of the main objectives of facial rejuvenation,
and is one that can now be obtained through the use of cosmetics. Linefill™ stimulates the body’s natural
mechanisms, allowing it to promote a more natural and attractive redistribution of facial volume in the
area around the mouth and lips.
V02‐07/14 72150‐2
ADIPOSE TISSUE AND ITS FORMATION
THE COMPONENTS OF ADIPOSE TISSUE
Adipose tissue or body fat is a specialized type of connective tissue. It is formed when adipocytes join
together along reticular fibers (collagen), forming small clumps that make up fatty lobes.
Body fat is predominantly composed of white adipose tissue, which is distributed throughout the body,
mainly in the subcutaneous layer. White adipose tissue has traditionally been considered to be a passive
store of energy, however, it has recently been established that it plays an active role in hormone regulation
and in homeostasis, as well as determining the shape of the body's surface.
One of this tissue's main characteristics is that it has a highly heterogeneous cell population, however, the
majority of cells are mature adipocytes (Rodríguez, 2002):
Preadipocytes are cells that are predestined to differentiate only into adipocytes. They still retain a
phenotype similar to that of a fibroblast, but they have already developed the enzymatic
machinery of an adipocyte, although they are not able to synthesize or accumulate lipids.
Adipocytes are the most common cells in adipose tissue. They have a rounded shape when they
are isolated and become polyhedral when they are grouped into lobes. One of their functions is
the synthesis and accumulation of lipids (in a large triglyceride‐filled vacuole).
THE DIFFERENTIATION PROCESS
During adipogenesis (Figure 1), the cells look increasingly less like fibroblasts as they become more
spherical. Large changes in cellular morphology also take place at this time, both in the cytoskeleton and in
the extracellular matrix (Gregoire et al., 1998).
Figure 1. Process of adipogenesis.
V02‐07/14 72150‐3
This phenomenon is a process comprised of multiple steps involving various transcription factors, which
regulate the activity of more than 2,000 genes and allow the development and differentiation of adipocytes
(Symonds, 2012). Adipogenesis involves two different phases:
1. Determination phase: conversion of stem cells into preadipocytes.
2. Terminal differentiation phase: the preadipocytes develop the characteristics of mature
adipocytes. These, in turn, acquire the cellular machinery necessary for the transport and synthesis
of lipids.
The key to this process is the level of transcription factors, especially C/EBP (CCAAT/enhancer binding
protein) and PPARγ (peroxisome proliferation‐activated receptor γ). The differentiation phase starts with
the expression of C/EBPβ, which increases the expression of PPARγ. PPARγ becomes activated when bound
to a ligand, stimulating the expression of C/EBPα, which in turn also increases the expression of PPARγ,
forming a positive feedback loop (Kirkland, 2002).
This process causes the formation of lipid droplets in the cell cytoplasm, which increase over time and fuse
until one or two large lipid droplets are formed that occupy a large part of the adipocyte.
During our youth, all these phases of
adipogenesis take place without
complications, giving rise to
subcutaneous adipose tissue that is
diffuse and homogenously distributed
(Ilankovan, 2013). However, as we age,
even though the determination phase
remains active and the stem cells continue their subsequent differentiation into preadipocytes in the later
stages, they do not complete their final differentiation into functional adipocytes capable of synthesizing
and accumulating fat (Niemela et al., 2008). This results in a loss of subcutaneous fat, particularly around
the eyes, on the forehead and around the mouth.
Linefill™ activates the adipogenesis to achieve more mature adipocytes capable of synthesizing and
accumulating fat
V02‐07/14 72150‐4
Linefill™ restores lost facial volume,
reducing lines and making lips fuller
Linefill™’s MODE OF ACTION
It is possible to reverse the aging process by
stimulating the first phases of the preadipocyte’s final
differentiation; this decreases the effects of the loss of
subcutaneous fat.
Linefill™ is able to activate PPARy by acting as a ligand
or agonist, which stimulates the conversion of
preadipocytes to adipocytes, resulting in more mature
adipocytes that are able to synthesize and accumulate
fats.
COMPOSITION
Linefill™ is a fraction rich in sesamin, which is obtained from sesame seeds (Sesamum indicum).
BOTANY
Sesamum indicum is an annual shrub belonging to the Pediliaceae
family, commonly known as sesame. Depending of conditions, plants
grow from 0.5 to 2.5 m tall. The leaves are ovate, opposite and deeply
veined. Its flowers are bell shaped and are white with a hint of blue,
red or yellow. The fruit is about 2.5 cm in size and is an oblong capsule
with small seeds (Chakraborthy et al., 2008)
The seeds are flat, small and can be white, grey or black. They are between 2 and 4 mm long and 1 to 2 mm
wide. Sesame is widely cultivated for its seeds, which are rich in oil.
It is thought that sesame originated in Ethiopia and that it was brought to America by slaves, who used the
seeds to season a wide variety of dishes. The plants are currently found in tropical and subtropical
temperate zones, particularly in India, China, South America and Africa.
V02‐07/14 72150‐5
Figure 2. Structure of sesamin.
CHEMISTRY
Sesame seeds are rich in sesamin (Figure 2), which belongs
to a group of compounds called lignans. Lignans are a widely
occurring class of natural compound.
The last investigations show that sesame oil is able to
increase the expression of PPARγ (Periasamy et al., 2014) and that sesamin itself increases the activity of
transcriptional PPARγ in some cell types (Liu et al., 2014).On the other hand, it has been demonstrated that
sesamin has a series of effects at different levels: reduces inflammation and expression of certain cytokines
(Jeng et al 2005), reduces arteriosclerosis, diminishes the resistance to insulin and glycemic index and
reduces the levels of cholesterol, as well as the levels of circulating triglycerides degrading them to fatty
acids, that can be already stored (Hong et al., 2013). All these activities can be explained by the activation of
PPARγ receptor, demonstrating its direct implication in all these processes (Houseknecht et al., 2002; Wen
et al, 2010). All these studies support the described mechanism of sesamin to stimulate adipogenesis.
TRADITIONAL USES
Sesame oil plays an important role in Indian Ayurvedic medicine. It
is rubbed onto the skin during abhyanga, a type of Indian massage
that improves energy flow and helps eliminate impurities from the
body. It is also used as a base for many oils due to its antioxidant
and moisturizing properties (Chakraborthy et al., 2008; Hazra and
Panda, 2013)
The oil is also used as an antibacterial agent in mouthwash preparations and in preparing Iodinol and
Brominol, which are used for both external and internal use.
It is also used for the production of edible oil and margarine. It is appreciated in the countries where it is
consumed for its pleasant and palatable flavor, especially in the Far East (China and Japan).
V02‐07/14 72150‐6
IN VITRO EFFICACY
IN VITRO STUDY PROTOCOL
The ability of Linefill™ to enhance adipocyte differentiation was studied in order to ascertain its effect as a
lipofiller. In order to achieve this, an adipocyte cell model was chosen using the 3T3‐L1 cell line, which is
one of the most widely used for investigating the adipogenesis process. The protocol was designed to
mimic the body’s natural process and takes approximately 10 days to develop (Figure 3):
1. Once the preadipocyte culture was ready, Linefill™ was added at three different
concentrations (43, 143 and 430 ppm), along with the MD1 differentiation medium, and the
preparation was incubated for 48 hours.
2. After this, the medium containing the active ingredient was removed and the MD2
differentiation medium was added (without active ingredient). The preparation was incubated
for a further 5 days, changing the medium every 2‐3 days.
3. At the end of 13 days, samples were taken in order to measure total triglycerides and to assay
using the Oil‐Red O staining method.
By adding Linefill™ during the early phases of differentiation, along with the MD1 culture medium, and by
only incubating for two days, it was ensured that the effect of the active ingredient occurred in the initial
phases of terminal differentiation (the preadipocyte to mature adipocyte step). This is a key stage in the
adipogenesis process, which includes the activation of PPARy by a ligand or agonist in order to complete the
adipogenesis process.
1. QUANTIFICATION OF TOTAL TRIGLYCERIDES
Triglyceride quantification is a biochemical indicator of adipocyte differentiation or adipogenesis.
Preparation of
preadipocytes
Initial phases of
differentiation
Final phases of
differentiation
Mature
adipocytes
Linefill™ Linefill™ removed Samples taken
MD1 MD26 days 2 days 5 days
Figure 3. 13 day In vitro study protocol.
V02‐07/14 72150‐7
In order to quantify triglyceride concentrations in the adipocytes, the cells were lysed and the triglycerides
(TG) hydrolyzed to obtain glycerol (Figure 4), which is measured by optical density.
The results are expressed as µM of glycerol as shown on Graph 1 (Evaluation of total TG) as a percentage of
triglyceride accumulation compared to the control.
It can be seen that Linefill™ is able to increase the amount of triglyceride stored in adipocytes for all
concentrations tested. Triglyceride levels increased by some 30% at a Linefill™ concentration of 430 ppm.
Linefill™ is therefore a potent stimulator of the early stages of adipogenesis (preadipocyte differentiation
to mature adipocytes) and increases lipid synthesis and storage capacity.
STRUCTURE OF A TRYGLYCERIDE
GLYCEROL
Fatty Acid 1
Fatty Acid 2
Fatty Acid 3
HYDROLYSIS
Triglyceride Glycerol Fatty Acids
Figure 4. Structure and hydrolysis of triglycerides.
5.6%
11.7%
30.2%
0
5
10
15
20
25
30
35
Linefill™ (43ppm) Linefill™ (143ppm) Linefill™ (430ppm)
% Variation triglyceride
(Glycerol)
Evaluation of total triglycerides
Graphic 1. Quantification of total triglycerides.
V02‐07/14 72150‐8
2. OIL‐RED O STAINING
Oil‐Red O staining was carried out to allow the variation in triglyceride accumulation obtained by
stimulating adipogenesis to be seen under the microscope (Figure 5):
The number and size of the lipid vesicles was seen to increase, showing that the preadipocytes have
successfully differentiated into fully functional, mature adipocytes that are able to accumulate more
triglycerides.
Absorbance or optical density was then measured at 540 nm (Graph 2). This demonstrated an increase in
absorbance, which represents an increase in triglyceride concentration, with respect to the control for all
concentrations tested. An increase of 48% was recorded at the highest dose (430 ppm).
Figure 5. Photograph of adipocyte cultures; the adipocytes of the linefill™ group show a greater accumulation of triglycerides and
more voluminous vacuoles (larger and more intense red points).
Control culture (D0) Culture with Linefill™ (D13)
V02‐07/14 72150‐9
IN VIVO EFFICACY
IN VIVO STUDY PROTOCOL
A double blind comparative study was conducted in order to evaluate the in vivo efficiency of Linefill™ as a
wrinkle filler and lip volumizer when compared to a placebo. The study was carried out in line with the
following protocol:
41 volunteers (women) with wrinkles in perioral area: nasolabial folds, supralabial and lateral
wrinkles.
2 groups: 20 women applied a formulation with 2% Linefill™, while the control group of 21 women
applied the same formulation without Linefill™ (placebo).
Age: 45 ‐ 65 years of age.
Application area: perioral area, including the nasogenian folds and lips.
Twice a day for 28 days.
Measurements were made on Day 0 (D0), before the application, and again on Day 28 (D28).
10.2% 12.7%
48.2%
0
10
20
30
40
50
60
Linefill™ (43ppm) Linefill™ (143ppm) Linefill™ (430ppm)
% Absorbence variation
Oil Red O
Graph 2. Quantification of total triglycerides.
Linefill™ can clearly increase the quantity of adipose tissue, since it increases both
the size and the number of adipocytes
(Larger lipid vacuoles and more intense red points)
V02‐07/14 72150‐10
1. EVALUATION OF WRINKLE REDUCTION AND ROUGHNESS IN THE PERIORAL AREA
The reduction in wrinkling and roughness was determined using the capture and comparison of 3D images
by fringe projection (PRIMOS) both on D0 and on D28. The efficacy of Linefill™ was evaluated in three areas
with different types of wrinkles, in order to demonstrate that it is effective against all types of wrinkles:
nasolabial fold, supralabial area (barcode wrinkles) and the lateral wrinkles that appear at the ends of the
lips (marionette lines).
Nasolabial fold
Linefill™ decreased the volume of the nasolabial fold by 9.2% compared to the start of the study (D0). This
result was 7.8 percentage points (pp) better than that obtained by the placebo (Graph 3).
‐9.2%
‐1.4%
‐10
‐8
‐6
‐4
‐2
0
Linefill™ Placebo
Nasolabial fold volume (%)
7.8
Graph 3. Decrease in volume of nasolabial fold after 28 days of application.
D0 D28
Figure 6. Visible decrease in nasolabial fold volumen obtained by Linefill™.
V02‐07/14 72150‐11
Supralabial wrinkles
1. Number of supralabial wrinkles
Linefill™ reduced the number of supralabial wrinkles by 45.6% compared with D0 and with a significant
difference of 19 pp compared with the placebo (Graph 4).
2. Volume of supralabial wrinkles
The volume of the wrinkles in this area decreased by 12.1% in the group treated with Linefill™, while in the
placebo group this value increased by 10.7%. There was therefore a statistically significant variation
between the two groups, with a difference of 22.8 pp (Graph 5).
‐45.6%
‐26.6%
‐50
‐40
‐30
‐20
‐10
0
Linefill™ Placebo
Number of supralabial wrinkles (%)
19.0
Graph 4. Decrease in the number of supralabial wrinkles after 28 days of application.
‐12.1%
10.7%
‐15
‐10
‐5
0
5
10
15
Linefill™ Placebo
Volume of supralabial wrinkles (%)
22.8
Graph 5. Decrease in volume of supralabial wrinkles obtained by Linefill™ after 28 days of application.
Linefill™
V02‐07/14 72150‐12
3. Cutaneous microrelief
Shallow regular grooves form in young skin, but as the skin ages, this relief will change and deeper grooves
form without actually forming a wrinkle, but giving the skin a more aged and rough appearance.
The roughness of the skin can be measured to provide an estimate of cutaneous microrelief: Ra (average
roughness) and Rz (an average of the 5 most pronounced peaks and valleys). An improvement for both
parameters was seen in the group treated with Linefill™, compared to initial values (Graph 6):
Ra: was decreased by Linefill™ by 6.7%, while in the placebo group it increased by 3.5%, giving a
difference of 10.1 pp between the active ingredient and the placebo.
Rz: treatment with Linefill™ produced a decrease of 5.4%, while with the placebo there was an
increase of 3.6%. There was therefore a difference of 8.6% compared with the placebo.
Figure 7. Visible decrease in supralabial wrinkles obtained by Linefill™.
D0 D28
‐6.7%‐5.4%
3.5% 3.3%
‐8
‐6
‐4
‐2
0
2
4
Ra Rz
Cutaneous microrelief (%)
Linefill™
Placebo10.1 8.6
Graph 6. Decreased roughness (Ra and Rz) of supralabial wrinkles obtained by Linefill™ after 28 days of application.
V02‐07/14 72150‐13
Lateral wrinkles
In the case of lateral wrinkles, or marionette lines, the number and volume of the wrinkles was measured.
A comparison of the decrease in number and volume of these wrinkles in the treated group when
compared to the placebo group showed that the efficacy of Linefill™ was statistically significant.
1. Number of lateral wrinkles
The number of lateral wrinkles for the group treated with Linefill™ decreased by 58.8% when compared
with D0, with a significant difference of 19.2 pp compared to the placebo (Graph 7).
2. Volume of lateral wrinkles
Linefill™ produced an 18.5% decrease in lateral wrinkle volume, with a significant percentage difference
of 17.9 pp compared to the placebo (Graph 8).
‐18.5%
‐0.6%
‐20
‐15
‐10
‐5
0
Linefill™ Placebo
Volume of lateral wrinkles (%)
17.9
Graph 8. Decrease in volume of lateral wrinkles after 28 days of application.
‐58.8%
‐39.6%
‐80
‐60
‐40
‐20
0
Linefill™ Placebo
Number of lateral wrinkles (%)
19.2
Graph 7. Decrease in the number of lateral wrinkles after 28 days of application.
V02‐07/14 72150‐14
2. EVALUATION OF THE ANTIAGING EFFECT THROUGH IMAGE ANALYSIS
Standardized photographs were taken using the VISIA‐CA system under normal, polarized and ultraviolet
lighting in order to provide an overall confirmation of the antiaging effect, supported by analysis of texture
and redness parameters. With age, the skin’s texture and appearance becomes increasingly affected by the
appearance of imperfections, blemishes, wrinkles and expression lines that give a rougher and irregular
texture and a more aged look to the skin.
Thanks to its overall antiaging effect, Linefill™ reduced blemishes by 6.3%, giving a significant difference of
11 pp compared to the placebo.
Linefill™ visibly decreases all types of wrinkles and refines roughness of the skin, thus
blurring the imperfections caused by the passage of time
Figure 8. Visible decrease in lateral wrinkles obtained by Linefill™.
D0 D28
D0 D28
Figure 9. Visible decrease in redness (darkest spots) obtained by Linefill™.
V02‐07/14 72150‐15
The skin’s texture provides us with general information regarding its roughness, which increases with age.
Linefill™ reduced the number of imperfections caused by the skin’s roughness by 5.2%, and this
improvement was statistically significant compared to the placebo, with a difference of 5.5 pp (Graph 9).
3. INSTRUMENTAL EVALUATION OF THE INCREASE IN LIP VOLUME
Instrumental evaluation
Fringe projection (PRIMOS) was used to evaluate the increase in lip volume.
As well as reducing wrinkles and redness, Linefill™ also increased lip volume by 23.1%, in a way that was
clearly superior and statistically significant when compared to the placebo, with a difference of 24.8 pp
(Graph 10).
23.1%
‐5
0
5
10
15
20
25
Linefill™ Placebo
Lip Volume (%)
24.8
Graph 10. Increase in lip volume obtained by Linefill™ after 28 days of application.
‐1.7%
‐5.2%‐6
‐5
‐4
‐3
‐2
‐1
0
1
Linefill™ Placebo
Texture (%)
5.5
Linefill™ softens roughness and soothes redness of the skin, for an overall younger
appearance.
Graph 9. Decrease in the number of imperfections obtained by Linefill™ after 28 days of application.
0.3%
V02‐07/14 72150‐16
4. EVALUATION OF HYDRATION
The skin’s hydration is a parameter that has a certain influence on the condition of wrinkles and on the
skin’s appearance. If the skin is kept hydrated, this will optimize an active ingredient’s anti‐wrinkle effects.
A Corneometer® was therefore used to analyze the amount of water in the skin’s upper layers.
Linefill™ hydrates the skin, showing a 22.5% increase, with a difference of 11.2 percentage points with
respect to the placebo (Graph 11).
Linefill™ hydrates the skin, improving both the status of wrinkles and the overall appearance
of the skin
22,5%
11.3%
0
5
10
15
20
25
Linefill™ Placebo
Hydration (%)
11.2
Graph 11. Increase in cutaneous hydration after 28 days of application.
Linefill™ increased lip volume, thus achieving more voluminous and sensual lips
Figure 10. Visible increase in lip volume obtained by Linefill™.
V02‐07/14 72150‐17
5. SUBJECTIVE QUESTIONNAIRE
At the end of the study, the volunteers filled in a questionnaire to evaluate the efficacy of Linefill™ or of the
placebo. A total of 95% considered that the product containing Linefill™ was good or very good and 85%
wanted to buy it; in contrast only 52.4% thought that the placebo product was good or very good and only
47.5% intended to buy the product.
In addition, the following graph (Graph 12) shows the percentage of volunteers that were satisfied with the
other subjectively evaluated parameters:
Linefill™ is an active lipo filler that decreases wrinkles, increases lip volume and improves the
overall condition of the skin
Graph 12. Results of subjective questionnaire on the effectiveness of Linefill™ after 28 days of application.
0
20
40
60
80
100
General lipimprovement
Lip volumeincrease
More hydrated lips
sSofter skin
More hydrated skin
Supralabialwrinkles reduction
Lateral wrinklesreduction
Nasolabial foldreduction
Uniform skin tone
Beautiful skin
Very good product
Purchase intention
Subjective questionnaire
Linefill™
Placebo
V02‐07/14 72150‐18
CONCLUSIONS
Linefill™ is a vegetable based active ingredient that is able to
increase the accumulation of lipids in the adipose tissue, providing
a greater volume to the areas that have lost their shape over the
years.
Thanks to its action, Linefill™ is able to perceptibly refill wrinkles
and cutaneous folds, as well as having a potent volumizing effect on the lips, giving a rejuvenated, attractive
and relaxed face.
COSMETIC APPLICATIONS
Anti‐aging lines.
Lip and eye contour creams.
Lip fillers.
Targeted body volumizers (breasts, buttocks, cheeks etc.).
Anti‐aging hand treatments.
Firming treatments.
RECOMMENDED DOSE
The recommended dose is between 1 and 3%.
BIBLIOGRAPHY
Buckingham ED. Poly‐L–Lactic Acid Facial Rejuvenation: An Alternative to Autologous Fat? Facial Plast Surg
Clin North Am. 2013, 21(2):271‐84.
Chakraborthy GS, Sharma G, Kaushik, KN. Sesamum Indicum:A Review. Journal of Herbal Medicine and
Toxicology. 2008, 2(2):15‐19.
V02‐07/14 72150‐19
Gregoire, FM, Smas CM, Sul HS. Understanding Adipocyte Differentiation. Physiological Reviews. 1998,
78(3):783‐809.
Hazra J, Panda AK. Concept of Beauty and Ayurveda Medicine. J Clin Exp Dermatol Res. 2013, 4:3.
Ilankovan V. Anatomy of ageing face. Br J Oral Maxillofac Surg. 2013, 1‐8.
Kirkland JL, Tchkonia T, Pirtskhalava T, Han J, Karagiannides I. Adipogenesis and aging: does aging make fat
go MAD? Exp Gerontol. 2002, 37(6):757‐67.
Niemelä S, Miettinen S, Sarkanen JR, Ashammakhi N. Adipose Tissue and Adipocyte Differentiation:
Molecular and Cellular Aspects and Tissue Engineering Applications Topics in Tissue Engineering. 2008, 4:1‐
26.
Park, JI. Asian Facial Cosmetic Surgery. Philadelphia: Saunders Elsevier, 2006. 448 p. ISBN‐13: 978‐1‐4160‐
0290‐1; ISBN‐10: 1‐4160‐0290‐1.
Periasamy S, Hsu DZ, Chang PC, Liu MY. Sesame oil attenuates nutritional fibrosing steatohepatitis by
modulating matrix metalloproteinases‐2, 9 and PPAR‐γ. J Nutr Biochem. 2014, 25(3):337‐44.
Rodríguez, ML. Diferenciación Adipocitaria y Factores Reguladores de la Biogénesis Mitocondrial. Efectos de
los Fármacos Antiretrovirales. Barcelona: Universidad de Barcelona. 2002, 93 p.
Symonds ME (Ed). Adipose Tissue Biology. London: Springer, 2012, VI. 414 p. ISBN: 978‐1‐4614‐0964‐9;
eISBN 978‐1‐4614‐0965‐6
Wong WW, Davis DG, Camp MC, Gupta SC. Contribution of lip proportions to facial aesthetics in different
ethnicities: A three‐dimensional analysis. Journal of Plastic, Reconstructive & Aesthetic Surgery. 2010, 63:
2032‐2039.