organic epica 04-2011 · iii.1 evaluation of primary eye and skin irritation potential 14 skin...

PPPHHHYYYTTTOOOBBBIIIOOOAAACCCTTTIIIVVVEEESSS OOOrrrgggaaannniiiccc EEEPPPIIICCCAAA®®®

GREENTECH S.A. Biopôle Clermont-Limagne - 63360 Saint-Beauzire - France

Tel : 33 4 73 33 99 00 - Fax : 33 4 73 33 91 32 E-mail : [email protected] – web site : www.greentech.fr

ORGANIC EPICA®

ANTI-AGING VEGETAL COMPLEX




2

CONTENT

INTRODUCTION......................................................................................... 3

ORGANIC EPICA® : NATURE and COMPOSITION..................... 4 EXTRACTION PROCESS............................................................................ 6 PLANTS DESCRIPTION. ............................................................................ 7 Black Currant Pinus Tree EFFICIENCY TESTS...................................................................................... 8 I Internal screening tests............................................................................... 8 Anti-Elastase test… 8 Anti-Oxydizing test 9 D.P.P.H. test 10 II IN VITRO Objectivation tests................................................................... 10 Anti-Elastase test 11 Anti-Collagenase test 11 Anti-Hyaluronidase test 13 III IN VIVO tests............................................................................................. 14 III.1 Evaluation of primary eye and skin irritation potential 14 Skin irritation 14 Eye irritation 15 Test on rabbit fibroblasts 15 Het Cam test 16 III.2 Study of anti-wrinkle effect................................................................ 17 Analysis of the shadow 17 3D analysis 17 Subjective assesment of the efficacy of the product

by the subjects 19 Cosmetic qualities of the product 19 Assesment of tolerance 20 FORMULATION EXAMPLE......................................................................... RECOMMENDED AMOUNT OF USE........................................................ 21 TECHNICAL DATA SHEET......................................................................... 22 BIBLIOGRAPHY............................................................................................. 24




3

INTRODUCTION

The quest for everlasting youthfulness is one of the oldest desires of humanity. But, if a young appearance is desirable, it is necessary to prevent, delay and repair signs of ageing. The first signs of ageing appear on the skin. The skin is a mechanical organ composed of three superposed strata and it plays the role of a barrier against toxins, micro-organisms and external aggressions. Cutaneous ageing can be observed by a loss of skin sparkle and flexibility. The skin becomes dehydrated and expression wrinkles become more marked especially in the eye and mouth outline areas. Sometimes anarchic skin pigmentation can be developped. Skin ageing is a complex phenomenon related to conjonctive tissue transformation and loss of cell renewal capabilities. During the course of ageing, tissues loose their extensibility and also their capability to regulate water exchanges. Cutaneous replication becomes less efficient and it can even generate alteration during its renewal. All these phenomena found are visible through the appearance of wrinkles. A recent study shows that the genetic part of ageing represents only 25% in comparison with external factors (health, lifesyle, diet, etc.) which represent 75%. It is therefore essential to protect the skin against external aggressions and to stimulate the organism’s defence systems. To fight against these damages, skincare anti-ageing products can prevent the ageing process with : - Cutaneous hydration preservation - Surface hydrophilic film reconstruction - Free radical scavenging

- Solar radiations protection - timulating substances for cellular multiplication or cutaneous micro- circulation




4

ORGANIC EPICA®: NATURE & COMPOSITION Natural anti-oxydizing molecules are present in plants. Generally these types of molecules are phenolic or polyphenolic compounds and can be Tocopherols, Flavonoïds, Tannins, polyfonctionnal organic acids, etc.. Recent studies have suggested that compounds with anti-oxydizing and radical scavenging activities can inhibit carcinogenesis and delay ageing. Anti-ageing Organic EPICA® complex efficiency, is developed through its different compounds and especially tannins. Two tannin groups are usually identified in the major plants. Firstly, hydrolysable tanins are derived from gallic acid and elagic acid. Secondly, condensed tannins are derived from catechins and contained the well known Pro-Cyanidolic Oligomers (O.P.C.) active class. Biological properties of O.P.C. are related to their complexation capability with free radicals. Free radicals are produced during physiological processes and cell metabolic reactions : mitochondrial respiration, phagocytose, detoxication, etc.. But polluting agents, such as tobacco polycyclic hydrocarbons, solar radiations, inflammations generate excess free radicals. Organic EPICA® complex behaves as a strong free radical scavenger and inhibits superoxyd ion formation. Free radical is a molecule which has an isolated electron on its external orbit. Superoxyd radical is the product of univalent reduction of an oxygen molecule. It is produced by physiological auto-oxidation of molecules during enzymatic reactions in respiration chain or during the aggressions of external factors such as radiations or toxic chemical products. It has been proved that Organic EPICA® complex inhibits collagen and hyaluronidase proteolysis (in vitro test COL 98.458 & HYAL 98.458 VITRO BIO). In a cutaneous extra cellular matricial medium, such macromolecules made from peptidic or glucidic chains, have a reticulated spatial structure because of enzymatic glycosylations. There is a delicate balance between synthesis and enzyme production. During ageing, degradation phenomena are faster than synthesis, and elastin, collagen and proteoglycan fibers have a tendency to disappear.




5

Flavonoïds contained in Organic EPICA® complex are capable of giving an electron to other molecules, organic acids contained in Organic EPICA®

complex potentialize this biological activity specifically on membranar permeation regulation. Organic EPICA® complex is obtained by natural joined extraction of Pine bark and Black Currant leaves. These two plants are an interesting source of anti-oxydizing molecules and assure an efficient protection against free radicals and their destructive activities. A long sun exposure affects the cutaneous structure. The UV radiation energy interacts with the skin lipids and during a peroxydation, lipids produce free radicals which attack the epidermal proteic structures. Organic EPICA®

complex neutralizes these free radicals and protects epidermal elastin and collagen fibers. Therefore Organic EPICA® complex reduces sun exposure damages. Organic EPICA® complex has been developped to be presented as a concentrated form of biological interesting molecules. As it been patented, Organic EPICA® complex is a strong anti-ageing natural product, but can be used as an anti-inflammatory, anti-histaminic, circulating agent and seems to have a beneficial action against psoriasis (data not shown). It is the mixture of different types of molecules, with unequal sizes, with different life times and biological targets which gives to Organic EPICA®

complex its multifunctional capability and which makes it a natural active unique of its kind. Organic EPICA® complex has been developped to fight efficiently against time damages, it is a real “natural skin vitamin”. Organic EPICA® complex gives to the epiderm the strengh of reconstruction, the capability to restore extensibility and hydration. Your skin is healthier, smoother, almost younger ! ...




6

EXTRACTION PROCESS

Plant Mixture Solvent Mixture Pinus Sylvestris Bark Ethylic Alcohol96.3° Black Currant Leaves De-ionised Water EXTRACTION Room Temperature, Mecanical agitation Dark Exposure WRINGING-OUT FILTRATION O R G A N I C E P I C A ® Hydro-alcoholic semi clarified extract Store at 4°C




7

PLANTS DESCRIPTION

I–Blackcurrant(Ribes Nigrum)

From Saxifragaceae family, black currant has a musky and flowery fragrance at the end of spring before becoming globulous black berries much appreciated inpastry-making. It is a shrub whose leaves and pimples are used in traditional medecine. Growing wild in the east of France, it is often harvested in other countries for its fruits : little black berries.

II – Pinus tree (Pinus Sylvestris) From the Pinaceae family, this pine is often named Auvergne Pine or Scotland Pine. Its aromatic pine flakey red-brown bark contrasts with its greeny-blue needles. A great part of the Organic EPICA®

complex activity is given by the pine bark. Flavonoïds and organic acids contained in the bark give to Organic EPICA® complex its anti-oxidizing activity.

Its leaves contain essential oils (containing cymen) and Flavonoïds as dimeric and trimeric prodelphinidols, which give the anti-ageing activity to Organic EPICA® complex.

Located in the northern hemispher, pine is used in traditional medecine as expectorant. It contains an oleoresin (Therebenthin), and this tree is also used in perfume industries for its essential oil (containing Pinen).




8

EFFICIENCY TESTS

I Internal screening tests I.1 - Anti-Elastase test Test method : Organic EPICA® complex anti Elastase activity is based on N-succinyl-Ala-Ala-Ala-p-Nitroanilin (SANA) / Elastase reaction. Elastase is reponsible for elastin degradation which induces the decrease of skin elasticity and flexibility. This test uses a colorless synthetic substrate which is degradeable by elastase. This colorless synthetic substrate produces a yellow colored reaction product after enzyme action. The addition of an anti-elastase product to the reaction medium, induces a decrease of SANA degradation and therefore a variation of optical density (OD) of 390 nm. Results : Organic EPICA® complex inhibits more than 75% of elastase enzymatic activity * C.I. 50 anti-elastase Efficient matter 0.017 - 0.025 % Efficient dose 14.5 - 21.7 % 1.50 % < X** < 2.20 % A 2% formulation assures a 50 % inhibition of elastase enzymatic activity

* : Test realised with a 0.5% dry product matter ** : Amount of mater assuring a 50% inhibition of elastase enzymatic activity




9

I.1 - Anti-Oxydizing test : Test method : Organic EPICA® complex anti oxydizing activity is based on Xanthin/Xanthin Oxydase reaction. Xanthin under Xanthin Oxydase action generates a superoxyd anion and uric acid. The reaction is controlled by cytochrom C (Browny-orange in oxydated form, pink in reduced form) which is reduced by superoxyd anion attack. Addition of a radical scavenger product in the reaction medium induces a decrease of the reduced cytochrom C quantity and a variation of the OD 550 nm. This test can quantify the capability of Organic EPICA® complex to inhibit the oxydation reactions and can be completed by a test with D.P.P.H. Results : Organic EPICA® complex inhibits more than 75% of Xanthin Oxydase enzymatic activity * C.I. 50 Xanthin Oxydase Efficient matter 0.0017 - 0.0025 % Efficient dose 14.5 - 21.7 mg/ml 1.50 % < X** < 2.20 % A 2% formulation assures a 50 % inhibition of elastase enzymatic activity

* : Test realised with a 0.025% dry product matter ** : Amount of product assuring a 50% inhibition of Xanthin Oxydase enzymatic activity




10

I.2 - D.P.P.H. test :

Test method : Organic EPICA® complex anti oxydizing or radical scavenging activity can be shown with the capability of Organic EPICA® to scavange radical of the DiPhenyl Picryl Hydrazyl (D.P.P.H.). Radical scavengering changes the color of the original solution The scavengering effect of Organic EPICA® complex on D.P.P.H. is given by the decreasing percentage of the original solution OD 520 nm. This study completes the Xanthin/Xanthin Oxydase test and confirms the anti-ageing activity of the Organic EPICA® complex . Results : Organic EPICA® complex inhibits more than 90% of Free Radical production * Free Radical Inhibition production amount : 90 % * : Test realised with a 0.5 % dry product matter ** : Amount of product assuring a 50% inhibition of Xanthin Oxydase enzymatic activity

II –In vitro tests

Tests carried out at BIO-HC (COL 98.458/CAEF 98.458/HYAL 98.458). Three tests were carried out on cell cultures in order to determine the precise cosmetic properties of Organic EPICA®. ♦ anti-elastase test ♦ anti-collagenase test ♦ anti-hyaluronidase test




11

II-1 Anti-elastase test

This enables the direct inhibiting action of Organic EPICA® on elastase activity of fibroblasts in human derma to be demonstrated. This assessment allows comparison of the fibroblast elastase activity after 72H treatment of the cells with different concentrations of Organic EPICA®.

The results for each concentration are expressed as the variation in optical density per minute calculated from the regression lines at 405 nm. One elastase unit is defined as the quantity of enzyme liberated by 1 µmole 4-nitroanilide/min. The elastase activity is then expressed as mU/mg protein.

5 concentrations were tested : 1 mg/ml 5 mg/ml 10 mg/ml 25 mg/ml 50 mg/ml

Ethanol controls were carried out simultaneously at the same concentrations. Results

Organic EPICA® is capable of inhibiting, in dose dependant fashion, the catalytic activity of elastase, by direct effect at doses of 25 mg/ml or 2.5% (27% inhibition) and 50 mg/ml or 5.0% (50% inhibition).

II-2 Anti collagenase test

This is the demonstration of the inhibiting capacity of Organic EPICA® on the collagenase activity in human derma. This assessment allows comparison of the fibroblast collagenase activity after 72H treatment of the cells with different concentrations of Organic EPICA®.




12

The mean of optical densities obtained at 630 nm (collagenase dosage) for each concentration were calculated. The collagenase concentrations (ng/ml) were then corrected according to the cell protein levels (µg/ml). Collagenase activity is expressed in ng of collagenase freed per µg cellular proteins.

5 concentrations were tested : 0.1 mg/ml 0.5 mg/ml 1 mg/ml 5 mg/ml 10 mg/ml

Ethanol controls were carried out simultaneously at the same concentrations. Results

Organic EPICA® anti-collagenase activity is dose dependant up to 5 mg/ml or 0.5%. This concentration is capable of reducing cell collagenase activity by 51%.

N° Concentration (mg/ml) Percentage inhibition 1 0.1 6.7% 2 0.5 29.2% 3 1 41.2% 4 5 51.3% 5 10 44.4%

percentage inhibition II-3 Anti hyaluronidase test

EPICA anti-collagenase activity

6.7

29.2

41.2

51.3

44.4

0 10 20 30 40 50 60

1

2

3

4

5

conc

entr

atio

n

pourcentage d'inhibition




13

II-3 Anti hyaluronidase test

This demonstrates the inhibiting capacity of Organic EPICA® relative to the hyaluronidase activity of MDCK epithilial cells.

This assessment allows comparison of the MDCK epithilial cell hyaluronidase activity after 72H treatment of the cells with different concentrations of Organic EPICA®.

The activity is assessed by the rate at which groups of N-acetyl glucosamine derived from degradation of hyaluronic acid are freed. Measurement of the N-acetyl glucosamine (GNAc) levels present in the various concentrations is made using a colorimetric reaction with 4- dimethyl aminobenzaldehyde at 585nm.

One unit of hyaluronidase is defined as being the quantity of enzyme freed by 1µmole N-acetylglucosamine per min, the catalytic concentration expressed in units of hyaluronidase per mg cellular proteins (U/mg prot.) was calculated for each concentration.

5 concentrations were tested: 0.1 mg/ml 0.5 mg/ml

1.1 mg/ml 5.0 mg/ml

10.0 mg/ml Ethanol controls were carried out as comparison at the same concentrations.

Results This study showed that Organic EPICA® is capable of reducing cell hyaluronidase activity. The maximum effect is reached with a concentration of 5 mg/ml or 0.5%. No direct inhibiting action on the isolated enzyme probably means that the Organic EPICA® acts on synthesis of the enzyme rather than by inhibiting hyaluronidase activity.




14

III – In vivo tests III.1 Evaluation of primary eye and skin irritation potential This study was carried out by the BIO-HC laboratory under the

reference IOC 98.691. Skin irritation

Study of the primary skin irritation potential was carried out on the premises of the Dermatology-Allergology Department of Hopital Pellegrin under the experimental control of BIO-HC.

The study was made using a gel containing 10% Organic EPICA® substance, applied without any prior preparation to the back of 10 subjects suffering from a pathology presumed to be of allergic origin, under occlusive dressing for 48 H.

Examination and macroscopic assessment of reactions

A reading was taken between 30 minutes and 1 H after removal of the dressings. When there was any doubt a second reading was taken 24 or 48 H later.

Attention was paid to six parameters which could be observed together or separately, with a weighting using an arbitrary scale with 5 levels (0 to 4)

Presence of erythema Presence of oedema

Presence of blister Skin dryness Skin roughness Reflectivity of the skin

Tolerance of the product was assessed by the dermatologist according to the results observed, their intensity and reproducibility from one subject to another.




15

Results

4 subjects out of 10 reacted to the product, and for 2 of them the allergic reaction recorded was probably due to traces of essential oil in the Organic EPICA® complex. With respect to the skin, the reactions recorded were not connected, à priori, with a strong irritant potential.

Eye irritation

Eye irritant potential was assessed using two alternative and complementary method : cytotoxicity study on fibroblasts.

study of chicken egg chorio-allantoid membrane (HET-CAM). These tests are proposed as an alternative to the DRAIZE test on rabbits for cosmetic and hygiene products. The study used a formulation containing 10% Organic EPICA® complex applied without any prior preparation to the chorio-allantoid membrane, and softened in a water bath prior to the fibroblast cytotoxicity study.

- Test on rabbit fibroblasts Rabbit cornea fibroblasts (SIRC line) were cultivated in complete culture medium with added fœtal calf serum (10%) and antibiotics. The cell suspension was then distributed among the wells of a microplaque (96 wells). The product was then added to the cell suspension and left in contact for 30 minutes, 1 hour and 4 hours. Revelation was by colorimetry (reaction to MTT). Using the optical densities (OD) measured for each contact time, a cytotoxicity percentage was calculated using the following formula : % (t) = [(OD control – OD sample) / OD control] x 100 Based on this percentage, an index of mean cytotoxicity (IMC) was calculated using the formula : IMC = (% 30 minutes + 0.5% 1 hour + 0.125 % 4 hours) / 3

An irritation index equivalent (IO eq) was then calculated which enables the




16

product to be classed according to the DRAIZE test scale presented in the Official Journal of the French Republic dated 10th July 1992. IO eq. = 6.5 The IO eq. index obtained enabled our product to be classed as "slightly irritant".

- HET-CAM test

This test was carried out using 2x3 White Leghorn chicken eggs with embryos weighing between 50 and 60 g., placed in an incubator for 10 days with strict temperature and humidity control. This examination is carried out with a magnifying lamp and consists of assessing the appearance at 30 seconds, 2 and 5 minutes, of the following phenomena at membrane level : Injection Haemorrhage Coagulation . The sum of the scores obtained for the three phenomena gives an individual irritation index. The mean of the individual indexes gives an irritation index (IP-MCA). IP-MCA = 0.3

IP-MCA Class 0 to 0.9 1 to 4.9 5 to 8.9 9 to 21

Non irritant Slightly irritant Moderately irritant Very irritant

♦ Result of evaluation of the irritant potential

IP-MCA = 0.3 IO eq = 6.5

Slightly irritant product




17

III.2 Study of anti-wrinkle effect

This study was carried out by the BIO-HC laboratory under the reference EFR 98-691/692. The main objective of the study was to demonstrate the anti-wrinkle efficacy of Organic EPICA® in a «base» formula, for crows' feet. The study used comparison with a placebo in 12 female volunteers. The imprint of each crows' foot was taken at T0, before any treatment, then at T28 after 28 days of application. Two methods of analysis were chosen: analysis of the shadow and 3D analysis of the wrinkles. The products were applied without any prior preparation to the inside of the elbow for one week in order to check they were innocuous. Analysis of the shadow Analysis of the imprints revealed an improvement at the site treated compared with the placebo site by 8 subjects out of 11 selected for results analysis, with a drop in the surface area of the shadow reaching up to 55% for one of the subjects.

3D analysis A 5 mm2 zone for each pair of imprints (before and after) was selected for each of the two sites (treated site and placebo) and analysed. Three parameters were selected :

- complexity of the wrinkle or visual impact. This corresponds to the extra developed surface area relative to the horizontal surface area ; it is expressed as a percentage.

-Depth of the wrinkle. This corresponds to the mean of the depths at the bottom of the fold ; it is expressed in mm.

-Volume of the wrinkle. This is expressed in mm3.

Analysis of these three parameters revealed an overall improvement at the




18

treated site compared with the placebo site for 7 subjects out of 10. The cross-sections shown below demonstrate very clearly the efficacy of our Organic EPICA® complex.

Subjective assessment of the efficacy of the product by the subjects.

This used analogous scales. The subjects assessed the severity of the large and small wrinkles before application of the products and after the four weeks' of application, at the treated site and the control site.

Before application of Organic EPICA®

After 30 days of Organic EPICA® applications




19

Six subjects out of twelve found a difference in favour of the product studied compared with the placebo with respect to reduction of large wrinkles, and four out of twelve with respect to reduction of small wrinkles. Cosmetic qualities of the products The subjects assessed the cosmetic qualities of the product tested according to nine parameters :

-colour, -perfume, -texture, -ease of application, -penetration of the product, -skin softness, -product overall, -use of the product, -purchase of the product.

The study revealed that seven subjects out of twelve would have liked to continue using the product and that six of them would buy it. Remark: It must be remembered that the formulation used for these tests is basic, in order to check the efficacy of the Organic EPICA® complex and not that of a set of excipients which would improve the cosmetic quality of the finished product (texture, penetration, etc).




20

Assessment of tolerance

No signs of intolerance were noted after the four weeks of application under normal conditions of use.

-Evaluation of tolerance by the subjects : good for eleven subjects fleeting tingling sensation for one subject.

-Evaluation of tolerance by the investigator :

good for all the subjects.




21

Formulation example

APIFIL 12.00 PEG-8 Beeswax Huile de jojoba 5.00 Jojoba Oil Huile d’Amande Douce 3.00 Sweet almond oil PHENONIP 0.50

Phenoxyethanol and methylparaben and butylparaben and ethylparaben and propylparaben

ANTIOX 0.35

Propylen glycol BHA and propylgallate and citric acid

CARBOPOL ULTREZ 0.20

Carbomer

NaOH (10%) 0.40 Sodium hydroxide Organic EPICA® 5.00

Pine and Black currant hydroalcoholic extract Parfum Q.S.P 100.00 Perfum

Eau désionisée Q.S.P. 100.00 De-ionized water

Recommended amount of use 1 to 10 % in a cosmetic formulation according to the biological power desired. This recommended amount of use takes into account the bioavailability of Organic EPICA® complex through the skin barrier.




22

TECHNICAL DATA SHEET

Composition Pine bark and Black Currant leaves hydroethanolic extracts

CTFA name EPICA®

INCI name Water, Alcohol, Pine (pinus sylvestris) extract, Black Currant ribes nigrum) extract.

Solvent used Ethanolic alcohol - Water (60/40)

Organoleptic characters Limpid liquid, browny-orange, characteristic fragrance

20°C density 1.00 ± 0.05

pH 5.00 ± 0.5

Solubilities Soluble in water and diluted alcohols Insoluble in organic solvents and oils

Identification “A” Colorimetric test

Add 1 ml of Hcl (25%) and 150 mg of Magnesium particles to 5 ml of Organic EPICA® complex. A yellow coloration appears indicating the presence of Flavonoids.




23

Identification “B” Chromatographic test

Put 2 ml of Organic EPICA® complex in a 100°C incubator, until total dessication. Dissolve dry residu by 1 ml of 96 ethanolic alcohol. Spot 10 µl on a CCM plate and develope with a migration solvent : Ethyl acetate/ Formic acid/ Acetic acid/ Water (100/10/10/27). Chromatographic migration minimum : 10 cm. Developer : Diphenylboryloxyethylamine and polyoxyethylenglycol 4000.

Read in Visible and UV Rf Visible UV 0.78 Yellow Yellow 0.68 Orange Orange 0.58 Pink Pink 0.55 - Blue 0.43 Orange Orange 0.38 - Blue 0.31 Pink Pink 0.27 - Green 0.18 - Pink

Dry matter yield > 1.00 %

Alcoholic titer 60.00 ° ± 5.0

Polyphenolic compounds 7.00 % ± 1.00

Microbiological count < 100 germ / ml < 10 yeast / ml Absence of enterobacteria




24

Bibliography

R. PARIS et H. MOYSE Matières médicales Masson, Paris, Tome II, 1981. L. BREMNESS Les plantes aromatiques et médicinales Bordas nature, Paris, 1996. J. BRUNETON Pharmacognosie Lavoisier, Paris, 1993. L. BEZANGER-BEAUQUESNE L. – M. PINKAS – M. TORCK Les plantes dans la thérapeutique moderne Maloine, Paris, 1975. A .MELISSOPOULOS et C. LEVACHER La peau – Structure et physiologie Tec et Doc Lavoisier, Paris, 1998. M. PRUNERIAS Précis de cosmétologie dermatologique Masson, Paris, 1981. J. ASSERIN et P. HUMBERT Propriétés viscoélastiques de la peau. Cosmétologie, 16, Octobre/Décembre 1997. AM. ORRECHIONI Produits «anti-âge» : actifs et formes galéniques Cosmétologie, 18, Avril/Juin 1998.

organic epica 04-2011 · iii.1 evaluation of primary eye and skin irritation potential 14 skin...

Documents