<i>Myrciaria dubia </i>fruit extracts rich in organic acids, cosmetic compositions comprising same and cosmetic uses thereof

Abstract

A pulp and skin extract from dried, seed-free Myrciaria dubia fruit. The Myrciaria dubia fruit has a maturity of at least 70%, a soluble solids/acidity ratio between 2.5 and 3, and it is rich in organic acids. The extract is obtained by solid-liquid extraction in a hydro-polyalcohol solvent selected from a hydro-glycerol and/or hydro-gylcolic solvent under agitation or with ultrasound. The extract is an active agent in cosmetic compositions, which reduce the signs of skin fatigue.

Claims

1. A process for obtaining an extract, the method comprising: providing pulp and skin of the seed-free dried fruit of Myrciaria dubia, having a ripeness of at least 70% and a soluble solids/acidity ratio of 2.5 to 3; dispersing and extracting the pulp and skin of the seed-free dried fruit in a hydro-polyalcohol solvent; separating a liquid phase loaded with compounds of interest from the solid phase of the pulp and skin of the seed-free dried fruit and hydro-polyalcohol dispersion; and microfiltering the liquid phase to remove solid plant particles.

2. The process according to claim 1, wherein the hydro-polyalcohol solvent is a mixture comprising from 10% to 90% by weight of glycerol and/or 1,3-propanediol in demineralised water in relation to the total weight of solvent plus water.

3. The process according to claim 2, wherein extracting is a solid-liquid extraction with a hydro-glycolic solvent composed of 60% 1,3-propanediol and 40% demineralised water.

4. The process according to claim 1, wherein extracting is carried out at a temperature between 4° C. and 100° C. for a time between 10 minutes and 4 hours stirring or with ultrasound.

5. The method according to claim 1, wherein separating comprises decanting and/or centrifuging.

6. The method according to claim 1, wherein separating comprises clarifying filtering.

7. The method according to claim 1, wherein, after microfiltering the liquid phase, the liquid phase is purified by ultrafiltration and/or nanofiltration.

8. The method according to claim 1, comprising drying the extract by spraying, freeze-drying, or zeodration.

Description

(1) The invention and the advantages deriving therefrom will be better understood by reading the following description and the non-limiting embodiments, which are described in relation to the annexed figures, in which:

(2) FIG. 1 shows the ATP measurement on dermal fibroblast cultures;

(3) FIG. 2 shows the measurement of creatine kinase activity in dermal fibroblast cultures;

(4) FIG. 3 shows the qPCR measurement of COL1A1 type collagen on reconstructed dermis;

(5) FIG. 4 shows the qPCR measurement of 11b-HSD1 and 11b-HSD2 in normal human keratinocyte (KHN) cultures;

(6) FIG. 5 shows the haematoxylin/eosin (HE) staining on reconstructed epidermis;

(7) FIG. 6 shows the immunostaining intensity of PFKFB3 on reconstructed epidermis; and

(8) FIG. 7 shows the measurement of creatine kinase activity on reconstructed epidermis.

EXAMPLE 1: PREPARATION OF A PLANT EXTRACT FROM PULP AND SKIN POWDER OF THE SEEDED MYRCIARIA DUBIA FRUIT

(9) The pulp and skin of the freshly seeded Myrciaria dubia fruit, 70% ripe, are placed for drying in a hot air tunnel system heated to 60° C. under humidity control. The resulting powder (200 g) is placed in a glass reactor, equipped with a stirring means and heating by means of a double jacket in the presence of a heat-transfer fluid.

(10) The solvent used (3800 g) for maceration is a mixture of 60% 1,3-propanediol and 40% demineralised water.

(11) The Myrciaria dubia pulp and skin powder is dispersed in the solvent for 2 hours at 30° C., then the mixture is separated on a decanter, centrifuged to remove the pellet, and the supernatant is recovered and filtered. After filtration, the supernatant is a 3220 g extract, in solvent medium 60% 1,3-propanediol and 40% water.

(12) The extract thus obtained is called “E1 Myrciaria extract” in the following examples. This extract E1 contains 2.0% dry matter including itself (results expressed in relation to the dry matter (DM)): 35.3%/DM of organic acids, including malic acid, citric acid and total ascorbic acid (ascorbic acid and dehydroascorbic acid); 10.1%/DM of sugars, including glucose, fructose, sucrose and maltose; 1.8%/DM of minerals, including potassium, magnesium, calcium, sodium, manganese, zinc and iron; traces of peptides/proteins and traces of CA1 compounds; 1.5%/MS of polyphenols in ellagic and gallic form.

(13) The dry extract described above is obtained by a gravimetric method based on the mass before and after evaporation of the solvent present in the liquid extract.

(14) Total ascorbic acid (ascorbic acid+dehydroascorbic acid) was determined using an HPLC/external calibration method.

(15) Citric and malic acids were determined by an HPLC/DEDL method by external calibration.

(16) The total sugars were determined using an HPLC/DEDL method by external calibration.

(17) The polyphenols were determined by HPLC/DAD by external calibration.

(18) The minerals were assayed according to a method adapted from the NF EN ISO 11885 standard (the NF EN ISO 11885 method is an analytical method by ICP/OES).

(19) List of Abbreviations: HPLC: High-Performance Liquid Chromatography DAD: Diode Array Detector DEDL: Evaporative Light-Scattering Detector ICP-OES: Inductively Coupled Plasma Optical Emission Spectrometry

EXAMPLE 2: EFFECT OF E1 MYRCIARIA EXTRACT ON THE SYNTHESIS OF ATP ON DERMAL FIBROBLAST CULTURES IN A TIRED CONDITION

(20) The aim of this study is to determine the level of ATP synthesis on dermal fibroblast cultures “tired” by replications and a nutrient-poor medium. These cultures are treated with the E1 extract at 0.1% (volume/volume), i.e. diluted 1/1000th in the culture medium.

(21) Protocol: Dermal fibroblast cultures are “tired” by a number of replications (cells used between passes 15 and 20) and are fed with a nutrient-poor medium (without glucose) for 3 days. These cultures are treated with 0.1% E1 Myrciaria extract, from the second day of stress and for the remaining two days of stress, i.e. 48 hours of treatment. On the fourth day, an application of 0.1% E1 Myrciaria extract is carried out for 1 minute, and then the ATP is determined by luminescence (Abcam—ref: ab113849).

(22) Results: After 48 hours of treatment with E1 Myrciaria extract, the ATP level is increased by +104% compared to untreated “tired” fibroblasts. After 1 min of treatment with E1 Myrciaria extract, the ATP level is increased by +82% compared to untreated “tired” fibroblasts. The results are shown in FIG. 1.

(23) Conclusion: Treatment of “tired” fibroblasts with 0.1% E1 Myrciaria extract leads to an increase in ATP synthesis.

EXAMPLE 3: EFFECT OF E1 MYRCIARIA EXTRACT ON CREATINE KINASE ACTIVITY ON DERMAL FIBROBLAST CULTURES IN A TIRED CONDITION

(24) The aim of this study is to determine the level of creatine kinase activity in dermal fibroblast cultures. The function of this enzyme is to catalyse the conversion of creatine to phosphocreatine, thus providing a rapidly usable energy reserve.

(25) This activity measurement is performed on cultures of “tired” dermal fibroblasts treated with 0.1% E1 Myrciaria extract (volume/volume), i.e. diluted 1/1000th in the culture medium.

(26) Protocol: Dermal fibroblast cultures are “tired” by a few replications (cells used between passes 15 and 20) and are fed with a nutrient-poor medium (without glucose) for 3 days. These cultures are treated with 0.1% E1 Myrciaria extract, or with vitamin C at 4 μg/mL as a positive control, from the second day of stress and for the remaining two days of stress. Two days after the start of treatment, a measurement of creatine kinase activity is performed using a kit (Abcam—ref: ab155901).

(27) For this purpose, the fibroblasts are detached with the CK buffer contained in the kit, recovered and centrifuged at 4000 g for 5 min at 4° C. The activity is then measured for each sample according to the kit protocol. Kinetics are obtained by a colorimetric reading with the Synergy 2 spectrometer (Biotek) coupled to Gen5 software.

(28) Results: In the condition where cells are “tired”, creatine kinase activity decreases by −51%. With the treatment with 0.1% E1 Myrciaria extract, this enzymatic activity increases by +62% compared to untreated “tired” cells and by only +44% with the vitamin C treatment. The results are shown in FIG. 2.

(29) Conclusion: The treatment of cells with 0.1% E1 Myrciaria extract prevents the decrease of creatine kinase activity on “tired” cells and is more important than the effect observed with vitamin C.

EXAMPLE 4: EFFECT OF E1 MYRCIARIA EXTRACT ON QPCR MEASUREMENT OF COL1A1 TYPE COLLAGEN ON RECONSTRUCTED DERMIS IN A “TIRED” CONDITION

(30) The aim of this study is to determine the level of collagen I messenger RNA (mRNA) on equivalents of dermis reconstructed from fibroblasts tired by replications and a nutrient-poor environment. These cultures are treated with 0.1% E1 Myrciaria extract (volume/volume) i.e. diluted 1/1000th in the culture medium.

(31) Protocol: Dermal fibroblast cultures are “tired” by a number of replications (cells used between passes 15 and 20) and are fed with a nutrient-poor medium (without glucose) for two days. These cultures are also treated during these two days with 0.1% E1 Myrciaria extract (volume/volume), i.e. diluted 1/1000th. A fibroblast culture (pass between 4 and 10) maintained in DMEM 1 g/L medium in glucose supplemented with 10% (v/v) foetal calf serum, 2 mM L-glutamine (Lonza) and 100 μg/mL Primocine (Invivogen), is used as a control.

(32) From these fibroblast and collagen cultures, dermis equivalents are reconstituted. Once the reconstitution is done, the 0.1% E1 Myrciaria extract is added once at D0, D1 and D2. At D5 the dermis are frozen at −80° C.

(33) The dermis are then thawed in order to extract the total RNAs.

(34) To do this, the dermis are crushed using Gentle MACS (Mylteni) and the total RNAs extracted using the mirVana* miRNA Isolation Kit (AM1561, Ambion). The RNAs are then reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (4374966, Life technologies). Finally, the real-time PCR reaction is performed on a StepOnePlus thermal cycler (Applied Biosystems) with the TaqMan* Gene Expression Master Mix (4369514, Life technologies) and the TaqMan* Gene Expression Assays (Life technologies). The Ct (Threshold Cycle) comparison method is used for relative quantification of the targets, and the StepOne* software (Applied Biosystems) for data processing.

(35) Results: In the situation where the fibroblasts are “tired”, the level of collagen I messenger RNA (mRNA) is decreased compared to non-tired fibroblasts by −58%. When these fibroblasts were cultured with the 0.1% E1 Myrciaria extract, the level of collagen I messenger RNA (mRNA) increases by +773%. The results are presented in FIG. 3.

(36) Conclusion: The treatment of “tired” fibroblasts with 0.1% E1 Myrciaria extract, before and after reconstitution of equivalent dermis leads to an increase in messenger RNA (mRNA) of collagen I.

EXAMPLE 5: EFFECT OF E1 MYRCIARIA EXTRACT ON THE QPCR MEASUREMENT OF 11B-HSD1 AND 11B-HSD2 ON NORMAL HUMAN KERATINOCYTE (KHN) CULTURES

(37) The aim of this study is to determine the messenger RNA (mRNA) level of 11b-HSD1 and 11b-HSD2 on keratinocytes. 11b-HSD enzymes catalyse the interconversion of active cortisol to inactive cortisone. Cortisol levels have been associated with anxiety and psychological stress, which may contribute to signs of skin fatigue. These cultures are treated with 0.1% E1 Myrciaria extract (volume/volume), i.e. diluted 1/1000th in the culture medium.

(38) Protocol: Keratinocytes are cultured and treated with 0.1% E1 Myrciaria extract once daily for 24 hours and 48 hours. The messenger RNA (mRNA) levels of 11b-HSD1 and 11b-HSD2 are measured by real-time PCR.

(39) To do this, total RNAs are extracted using the mirVana* miRNA Isolation Kit (AM1561, Ambion) and then reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (4374966, Life technologies). Finally, the real-time PCR reaction is performed on a StepOnePlus thermal cycler (Applied Biosystems) with the TaqMan* Gene Expression Master Mix (4369514, Life technologies) and the TaqMan* Gene Expression Assays (Life technologies). The Ct (Threshold Cycle) comparison method is used for relative quantification of the targets, and the StepOne* software (Applied Biosystems) for data processing.

(40) Results: In KHN treated with E1 Myrciaria extract, the level of 11b-HSD1 decreased by −67% and −84% respectively after 24 and 48 hours of treatment. The level of 11b-HSD2 increased respectively by +15% and +23% after 24 and 48 hours of treatment. The results are shown in FIG. 4.

(41) Conclusion: Treatment of keratinocytes with 0.1% E1 Myrciaria extract leads to a decrease in the level of 11b-HSD1 messenger RNA (mRNA) and an increase in the level of 11b-HSD2 messenger RNA (mRNA).

EXAMPLE 6: EFFECT OF E1 MYRCIARIA EXTRACT ON THE MORPHOLOGY OF RECONSTRUCTED EPIDERMIS IN A TIRED CONDITION

(42) The aim of this study is to create a model of reconstructed “tired” human skin. To do this, the skin is reconstructed using adult donor keratinocytes and a respiratory chain inhibitor (rotenone) to create a lack of energy.

(43) The use of a respiratory chain inhibitor such as rotenone creates mitochondrial dysfunction, including ATP depletion. Therefore, lack of cellular energy was equated in our model to a state of cellular fatigue (Han G et al, The mitochondrial complex I inhibitor rotenone induces endoplasmic reticulum stress and activation of GSK-3β in cultured rat retinal cells, Invest Ophthalmol Vis Sci. 31; 55(9):5616-28, 2014). These cultures are treated during the reconstruction with 0.1% E1 Myrciaria extract (volume/volume), i.e. diluted 1/1000th or 1% (volume/volume), i.e. diluted 1/100th in the culture medium.

(44) Protocol: On day 12 of epidermal reconstruction, rotenone is added to the culture medium. A control condition is maintained without adding rotenone but changing the culture medium anyway. After 3 h incubation with the inhibitor, the medium is changed to a new medium containing 0.1% E1 Myrciaria extract and the epidermis is incubated again for 48 h. The placebo condition contains PBS (phosphate buffer saline) in the culture medium. The morphology of the skin is then observed by a Haematoxylin-Eosin (HE) staining.

(45) For this, the reconstructed epidermises are fixed with formaldehyde and then embedded in paraffin. Cuts of 4 μm are then made.

(46) For staining, the cuts are dewaxed by xylene and ethanol baths and then rinsed. Staining is done by a haematoxylin and then eosin bath.

(47) The cuts are then examined under a microscope (Eclipse E600, Nikon) and photos are taken with the Q-capture acquisition software (QImaging*).

(48) Results: In the condition where reconstructed epidermises are treated with rotenone, we observe a thinner epidermis than the control and a more damaged basal layer. When these are treated with 0.1% E1 Myrciaria extract, the epidermis seems to show less damage in its structure. The results are shown in FIG. 5.

(49) Conclusion: Treatment of reconstructed epidermis with 0.1% E1 Myrciaria extract seems to partially compensate for the lack of energy induced by the inhibition of the respiratory chain by rotenone.

EXAMPLE 7: EFFECT OF E1 MYRCIARIA EXTRACT ON THE EXPRESSION OF PFKFB3 ON A RECONSTRUCTED EPIDERMIS IN A TIRED CONDITION

(50) The aim of this study is to determine the level of expression of PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) in a model of “tired” reconstructed human skin.

(51) PFKFB3 is involved in the glycolysis process. It is an inducible enzyme that is expressed by the active epithelial cells of the epidermis. It enables the activation of the enzyme PFK-1 (phosphofructokinase-1), which limits this glycolysis process.

(52) To obtain “tired” skin, this is reconstructed using adult donor keratinocytes and a respiratory chain inhibitor (rotenone) to create a lack of energy (see example 6). These cultures are treated during reconstruction with 0.1% E1 Myrciaria extract (volume/volume), i.e. diluted 1/1000th in the culture medium.

(53) Protocol: On day 12 of epidermal reconstruction, rotenone is added to the culture medium. A control condition is maintained without adding rotenone but changing the culture medium anyway. After 3 h incubation with the inhibitor, the medium is changed to a new medium containing 0.1% E1 Myrciaria extract and the epidermises are incubated again for 48 h. The placebo condition contains PBS (phosphate buffer saline) in the culture medium. Detection of PFKFB3 expression is performed by an indirect immunofluorescence technique.

(54) For this, the reconstructed epidermises are fixed with formaldehyde and then embedded in paraffin. Cuts of 4 μm are then made.

(55) For staining, the cuts are dewaxed by xylene and ethanol baths and then rinsed. To unmask specific antigen binding sites, the cuts are heated in a citrate buffer bath in a microwave oven and then treated with a trypsin solution. After saturation with 5% BSA for 30 minutes to block non-specific sites, the cuts are then incubated with a PFKFB3 antibody solution for 1.5 hours and then with a solution of secondary anti-rabbit antibody coupled to a fluorochrome (Alexa Fluor® 488, Invitrogen) for 1 hour. The cuts are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). A quantification of the fluorescence, using Volocity® image analysis software (PerkinElmer, Inc.), is performed from the obtained photographs.

(56) Results: In the condition where reconstructed epidermises are treated with rotenone, we observe a decrease in PFKFB3 expression of −20% compared to unstressed epidermises. When these are treated with 0.1% or 1% E1 Myrciaria extract, its expression increases by +19% and +46% respectively. The results are shown in FIG. 6.

(57) Conclusion: Reconstructed epidermises treated with rotenone show a decrease in PFKFB3, reflecting reduced glycolysis. Treatment of these “tired” reconstructed epidermis with 0.1% E1 Myrciaria extract seems to partially compensate for this lack of energy observed by stimulating PFKFB3 synthesis.

EXAMPLE 8: EFFECT OF E1 MYRCIARIA EXTRACT ON THE ACTIVITY OF CREATINE KINASE ON A RECONSTRUCTED EPIDERMIS IN A TIRED CONDITION

(58) The aim of this study is to determine the level of creatine kinase activity in a model of reconstructed human skin. The function of this enzyme is to catalyse the conversion of creatine to phosphocreatine, thus providing a rapidly usable energy reservoir.

(59) This measure of activity is done on a model of “tired” reconstructed human skin obtained using adult donor keratinocytes and a respiratory chain inhibitor (rotenone) to create a lack of energy. These cultures are treated during reconstruction with 0.1% Myrciaria extract (volume/volume), i.e. diluted 1/1000th in the culture medium.

(60) Protocol: On day 12 of epidermal reconstruction, rotenone is added to the culture medium. A control condition is maintained without adding rotenone but changing the culture medium anyway. After 3 h incubation with the inhibitor, the medium is changed to a new medium containing 0.1% E1 Myrciaria extract and the epidermises are incubated again for 48 h. The placebo condition contains PBS (phosphate buffer saline) in the culture medium. A measurement of creatine kinase activity is performed using a kit (Abcam—ref: ab155901).

(61) To do this, the reconstructed epidermises are crushed using a glass pestle with the CK buffer contained in the kit, recovered, and centrifuged at 4,000 g for 5 min at 4° C. The activity is then measured for each sample according to the kit protocol. Kinetics are obtained by a colorimetric reading with the Synergy 2 spectrometer (Biotek) coupled with the to Gen5 software.

(62) Results: In the condition where reconstructed epidermises are “tired”, creatine kinase activity decreases by −40% compared to non-“tired” epidermises. With treatment with 0.1% E1 Myrciaria extract, this enzymatic activity increases by +29% compared to the “tired” model. The results are shown in FIG. 7.

(63) Conclusion: The treatment of reconstructed epidermises with 0.1% E1 Myrciaria extract prevents the decrease in creatine kinase activity observed on a model of “tired” reconstructed skin.

EXAMPLE 9: VELVET CREAM FORMULATION

(64) TABLE-US-00001 Ingredient/Trade name INCI name % Phase A Purified water Water 70.48 Phase B Natrosol ™ Plus 330 CS Cetyl Hydroxyethylcellulose 0.50 Solagum AX* Acacia Senegal Gum & Xanthan Gum 1.00 Phase C Simulgreen 18-2* Hydroxystearyl Alcohol & Hydroxystearyl Glucoside 3.00 Phase D Ceraphyl ™ 791 ester Isocetyl Stearoyl Stearate 5.00 Macadamia Nut Oil CP RBD Macadamia Temifolia Seed Oil 5.00 Virgin Prunus Oil Prunus Domestica Seed Extract 2.00 Orchid ™ Complex OS ester Caprylic/Capric Triglyceride (and) Cymbidium 5.00 Grandiflorum Flower Extract Phase E Optiphen ™ BSB-N preservative Benzyl Alcohol (and) Glycerin (and) Benzoic Acid 1.00 (and) Sorbic Acid Zemea* Propanediol 5.00 Phase F Extract E1 1,3-Propanediol (and) Water/Aqua (and) Myrciaria 1.00 Dubia Fruit Extract Phase G Purified water Water 1.00 Sodium Hydroxide 0.02

(65) Method of Preparation: 1. In the main beaker, add the water and begin to homogenise by heating to 70-75° C. 2. Sprinkle in Natrosol Plus 330 CS and mix well for 30 minutes. 3. Sprinkle in Solagum AX and mix well for 20 minutes. 4. Add the phase ingredients to a second beaker and heat to 70-75° C. 5. Add phase C to phase A while homogenising. 6. At 70-75° C., add phase D to the main beaker and mix well. The emulsion should be homogeneous. 7. Start cooling down. Add phase E ingredients one by one at 50° C., mixing well after each addition. 8. At 25° C., add phase F and homogenise. 9. Premix phase G until a clear and uniform mixture is obtained. Add to the main beaker and mix well. 10. Stop at 25° C. 11. Appearance: White emulsion; opaque. pH: 4.4-4.8; viscosity (D0): 12000-20000 cps (Brookfield RVT/Spindle B/5 RPM/1 minute/25° C.) 12. The preservation of the formula was validated by a double test of effectiveness after 28 days. However, the preservatives were not optimised at their lowest level of effectiveness.

EXAMPLE 10: VITAFRUIT CREAM GEL FORMULATION

(66) TABLE-US-00002 Ingredient/Trade name INCI name % Phase A Purified water Water/Aqua Qs. 100 Tetrasodium EDTA Tetrasodium EDTA 0.05 Lubrajel ™ Oil Free hydrogel* Glycerin (and) Glyceryl Acrylate/Acrylic Acid 7.00 Copolymer (and) PVM/MA Copolymer Optiphen ™ HD preservative 1,2-Hexanediol 3.00 booster Butylene Glycol Butylene Glycol 7.00 Glycerol Glycerin 5.00 Phase B FlexiThix ™ polymer PVP 2.00 Phase C Montanov ™ 68* Cetearyl Alcohol (and) Cetearyl Glucoside 0.20 Optiphen 200 condom Phenoxyethanol (and) Caprylyl Glycol 0.90 Ceraphyl ™ SLK ester Isodecyl Neopentanoate 2.50 Lexfeel ™ N5* Diheptyl Succinate (and) Capryloyl Glycerin/Sebacic 1.00 Copolymer Ganex ™/Ganex ™ Sensory VP/Acrylates/Lauryl Methacrylate Copolymer 0.25 polymer Phase D Surfin ™ 96* Alcohol 10.00 Phase E RapiThix ™ A-60 polymer Sodium Polyacrylate (and) Hydrogenated Polydecene 0.50 (and) Trideceth-6 Phase F Extract E1 1,3-Propanediol (and) Water/Aqua (and) Myrciaria 1.00 Dubia Fruit Extract Phase A Smart 5* Isododecane (and) Hydrogenated 5.00 Tetradecenyl/Methylpentadecene Unicert Yellow 08005-J (Sol. 1%) CI 19140 (yellow 5) 0.35 Unicert Red 07004-J (sol. 1%) CI 14700 (red 4) 0.25 Timiron ™ Silk Gold * CI 77891 (Titanium Dioxide) (and) Mica (and) Tin 1.00 Oxide PF Caipirinha Fresh G11724962 Fragrance/Parfum (and) Benzyl Salicylate (and) Citral 0.30 (and) Limonene (and) Linalool (and) Butylphenyl methylpropional (Filial)

(67) Method of Preparation: 1. In the main beaker add the ingredients of phase A, one by one, heating to 70° C. while mixing until completely homogenised. 2. Sprinkle in the polymer FlexiThix™ and homogenise until a smooth mixture is obtained. 3. In a separate beaker, heat phase C (except Antaron™/Ganex™ Sensory polymer) to 70° C. until homogenised. Then immerse the Antaron/Ganex Sensory polymer in phase C under stirring. 4. At 70° C., add phase C (Antaron/Ganex Sensory polymer are in wet powder form) to the main beaker and homogenise. 5. Cool. 6. At 25° C., add phase D slowly and mix until completely homogenised. 7. Add phase E and mix until completely homogenised (viscosity increases). 8. At room temperature, pre-mix phase F with stirring, add the contents of the main beaker and mix well. 9. Stop when mixture is uniform in colour. 10. Appearance: orange opalescent cream gel; pH: 6.0-6.5; viscosity (D0): 25000-40000 cps (Brookfield RVT/Spindle B/5 RPM/1 minute/25° C.). 11. The preservation of the formula was validated by a 3-month accelerated ageing test and an efficacy test after 28 days. However, the preservatives were not optimised at their lowest level of effectiveness.

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