FR3131215A1 - Fermented extract of mangosteen fruit peel - Google Patents

Abstract

The present invention relates to a fermented extract of mangosteen fruit peel, comprising: - a gluconic acid content greater than 50 mg/g, - an acetic acid content greater than 200 mg/g, and weight to weight dry extract of the fermented extract.

Description

Fermented extract of mangosteen fruit peel FIELD OF THE INVENTION

The present invention relates to the cosmetic field and in particular to a fermented extract of mangosteen fruit peels as well as the fermentation process making it possible to obtain it, a cosmetic composition containing said fermented extract of mangosteen fruit peels, and its use in a cosmetic process for the care and/or makeup of keratin materials.

STATE OF THE ART

We know that aging is induced by endogenous factors (e.g. stress, hormones, etc.) and exogenous factors (e.g. temperature, climate, UV radiation, atmospheric pollution, cigarette smoke, etc.) leading to a slowdown and/or alteration of different cellular functions. In terms of the skin, aging results in various clinical signs, in particular a loss of firmness and elasticity of the skin, a reduction in the thickness of the skin, the appearance of wrinkles or fine lines, appearance of a less homogeneous complexion, and/or without radiance.

There is therefore a constant need to find new agents capable of preventing and/or reducing the signs of skin aging, and/or making it possible to stimulate and/or promote the biomechanical resilience of the skin, as well as the vitality of skin cells.

Unexpectedly, the Applicant has demonstrated the 'anti-aging' effects of a fermented extract of mangosteen fruit peels in vitro on a culture of human cells (keratinocytes or fibroblasts). Indeed, the fermented extract of mangosteen fruit peel advantageously makes it possible to improve the biomechanical properties of the skin by acting on the expression of essential proteins of the extracellular matrix. Furthermore, the fermented extract of mangosteen fruit peel, in combination with a kombucha extract, has a protective effect equivalent to vitamin C against the production of ROS (reactive oxygen species), advantageously making it possible to reduce oxidative stress in skin cells. These effects therefore make it possible to consider the use of this fermented extract of mangosteen fruit peel to promote and/or reinforce the firmness, density and/or tension of the skin, and/or promote and/or reinforce the elasticity, extensibility and biomechanical resilience of the skin and thus prevent and/or reduce the alteration of the flexibility of the skin and/or the formation of wrinkles and fine lines and/or prevent and/or reduce the signs of aging skin, but also to prevent and/or reduce oxidative stress on skin cells and thus promote and/or stimulate the vitality of skin cells, and/or promote and/or stimulate the homogeneity or radiance of the complexion.

According to a first aspect, the invention relates to a fermented extract of mangosteen fruit peels, comprising:

- a gluconic acid content greater than 50 mg/g, and

- an acetic acid content greater than 200 mg/g,

in weight relative to the weight of dry extract of the fermented extract.

Preferably, said fermented extract of mangosteen fruit peels according to the invention is in liquid form and it comprises at least one preservative, preferably selected from butylene glycol, glycerin, propylene glycol, sodium benzoate, potassium sorbate and propanediol.

Another aspect of the invention relates to a process for fermenting mangosteen fruit peels comprising the following steps:

a) the incubation of a consortium of microorganisms in a solution comprising at least water, a carbohydrate and black tea leaves infused at a temperature between 12 to 45°C for 1 to 21 days in order to 'obtain a preparation of symbiotic ferments, said consortium comprising at least one lactic acid bacteria belonging to the genus Lactobacillus or Pediococcus , at least one yeast belonging to the genus Saccharomyces , Schyzosaccharomyces or Torulaspora , and at least one acetic acid bacteria belonging to the genus Acetobacter , Gluconobacter or Komagataeibacter ;

b) grinding mangosteen fruit peels to obtain particles having an average size ranging from 0.5 mm to 1 mm;

c) maceration of the particles obtained in step b) in water in the presence of a carbohydrate;

d) filtration of the maceration mixture to remove plant particles;

e) adding the preparation of symbiotic ferments obtained in step a) to the filtrate of step d) to obtain a fermentation mixture,

f) incubating the fermentation mixture at a temperature between 21 and 35°C for 15 to 25 days;

g) sieving then filtering the fermentation mixture in order to obtain a fermented extract of mangosteen fruit peels, and

h) optionally, the addition of at least one preservative.

Preferably, according to said process, the mangosteen fruit peel particles are added to the maceration medium of step c), at a concentration ranging from 30g to 70g per liter, preferably 40g to 60g per liter and preferably at a concentration of approximately 50 g of particles per liter.

Preferably, according to said process, the carbohydrate is added to the maceration medium of step c), at a concentration ranging from 30g to 70g per liter, preferably 40g to 60g per liter and preferably at a concentration of approximately 50 g of carbohydrate per liter.

The present invention also relates to a fermented extract of mangosteen fruit peels obtained by the process as described above.

According to another aspect, the present invention relates to the combination of a fermented extract of mangosteen fruit peels, as described above, and an extract of kombucha.

The present invention further relates to a cosmetic composition comprising, in a physiologically acceptable medium, a fermented extract of mangosteen fruit peels as described above.

According to a particular embodiment, said cosmetic composition is characterized in that it also comprises a kombucha extract.

Preferably, said cosmetic composition is characterized in that the fermented extract of mangosteen fruit peels is present in a content ranging from 0.0000012% to 0.0825%, preferably from 0.000006% to 0.033%, plus preferably from 0.000012% to 0.0165% by weight of active ingredient (dry matter of fermented extract of mangosteen fruit peel) relative to the total weight of said composition.

Preferably, said cosmetic composition is characterized in that the kombucha extract is present in a content ranging from 0.01% to 10%, in particular from 0.02% to 5%, preferably from 0.05% to 4%. % by weight of raw material (product weight) relative to the total weight of the composition.

Preferably, said cosmetic composition according to the invention is characterized in that it further comprises at least one cosmetic adjuvant chosen from the group consisting of antioxidant agents, emollient agents, moisturizing agents, anti-aging agents, perfumes, and their mixtures.

Preferably, said cosmetic composition according to the invention is characterized in that it is in the form of a milk, a cream, an ointment, a water-in-oil or oil-in-oil emulsion. water or a multiple emulsion, a balm, a stick, a solution, a suspension, gel, a lotion, a serum, or even a powder.

The present invention further relates to a cosmetic process for the care and/or makeup of keratin materials, in particular of the skin and/or lips, comprising the application to said keratin materials of at least one layer of a composition. cosmetic as defined above.

Preferably, said cosmetic process is characterized in that it is intended to promote and/or reinforce the firmness, density and/or tension of the skin, and/or promote and/or reinforce the elasticity, extensibility and the biomechanical resilience of the skin and thus prevent and/or reduce the alteration of the flexibility of the skin and/or the formation of wrinkles and fine lines and/or prevent and/or reduce the signs of skin aging.

The present invention further relates to the cosmetic use of a fermented extract of mangosteen fruit peels as defined above as a cosmetic active ingredient to promote and/or reinforce the firmness, density and/or tension of the skin, and/or promote and/or strengthen the elasticity, extensibility and biomechanical resilience of the skin and thus prevent and/or reduce the alteration of the flexibility of the skin and/or the formation of wrinkles and fine lines and /or prevent and/or reduce the signs of skin aging.

The present invention further relates to the cosmetic use of at least one fermented extract of mangosteen fruit peels as defined above and of at least one kombucha extract, as a combination of cosmetic active ingredients intended to prevent and/or reduce oxidative stress on skin cells and thus promote and/or stimulate the vitality of skin cells, and/or promote and/or stimulate the homogeneity or radiance of the complexion.

DESCRIPTION OF FIGURES

Effect of fermented mangosteen fruit peel extract on collagen I production in normal human fibroblasts (FHN).

Effect of fermented mangosteen fruit peel extract on elastin production in normal human fibroblasts (NHF).

Effect of fermented mangosteen fruit peel extract on MMP2 production in normal human fibroblasts (NHFs).

Effect of fermented mangosteen fruit peel extract on Vinculin production in normal human fibroblasts (FHN).

Effect of fermented mangosteen fruit peel extract on fibronectin production in normal human fibroblasts (NHFs).

Effect of fermented mangosteen fruit peel extract and Kombucha Nepal extract, alone or in combination on ROS production in normal human keratinocytes (KHN).

DETAILED DESCRIPTION OF THE INVENTION Garcinia mangostana or mangosteen

Mangosteen, whose scientific name is Garcinia mangostana , is a tropical tree in the Clusiaceae family. Garcinia mangostana is a hermaphrodite tree, which contains at the end of each branch one or two male or female flowers which transform into fruit after fertilization. The fruit, spherical and smooth in shape, initially presents a greenish-white color which turns purple red once ripe. The ripening of mangosteen fruit generally lasts between two and three months. The fruit peel is 6 to 10 mm thick and surrounds the edible part of the fruit. Considered a superfruit, Garcinia mangostana is a fruit very rich in antioxidants.

In the context of the present invention, the fermented extract of Garcinia mangostana or mangosteen is obtained from the fruits of the Garcinia mangostana or mangosteen plant and preferably obtained from fruit peels or pericarp of the Garcinia mangostana or mangosteen plant . For simplification, we will use in this description the expression “mangosteen fruit peel” but this expression designates the peel of the fruit of Garcinia mangostana .

Fermented extract of mangosteen fruit peel

The peels of mangosteen fruit or pericarp are used in a fermentation process in order to generate a fermented extract of mangosteen fruit peels, which is the subject of the present invention. Reference may also be made in the rest of the description to a "fermented mangosteen extract", but it is understood that, in the context of the present invention, the term "fermented mangosteen extract" refers to a "fermented extract of mangosteen". mangosteen fruit peel.

It is specified that in the present description, the expression “fermented extract” designates the product resulting from the fermentation process, preferably after filtration, as well as any product having undergone additional stages, such as a formulation stage (for example by adding a preservative).

According to a particular embodiment, the fermented extract of mangosteen fruit peels, also designated "fermented mangosteen extract", according to the invention is in the form of an aqueous solution of fermented extract comprising the fermented dry extract , water and a preservative. The fermented extract in the form of an aqueous solution is also called 'raw material' in the description, as opposed to 'dry matter' or fermented dry extract.

According to a particular mode, the aqueous solution of fermented extract comprises the fermented extract in an active content (dry matter) ranging from 0.5 to 4% by weight, in particular 1 to 3% by weight, preferably 1.7 to 2.7% by weight, and in particular 1.2 to 1.9% by weight of active material relative to the total weight of raw material (aqueous solution of fermented extract), water and a preservative, preferably propanediol. The water content will generally range from 60 to 80% by weight, in particular from 65 to 75%, in particular from 68 to 69% by weight relative to the total weight of raw material (aqueous solution of fermented extract) and the preservative content. (for example propanediol) will generally range from 20 to 40% by weight, in particular from 25 to 35% by weight, in particular 30% by weight relative to the total weight of raw material (aqueous solution of fermented extract).

Thus, the invention also relates to a fermented extract of mangosteen fruit peels in the form of a solution comprising 1 to 2% by weight of active material of fermented extract of mangosteen fruit peels, 65 to 75 % by weight of water and 25 to 30% by weight of propanediol.

Preferably, the aqueous solution of fermented extract, otherwise called 'raw material' whose characterization is illustrated below, comprises 1.2 to 1.9% in active material (dry matter) of fermented extract, 28.1 at 28.8% water and 30% propanediol relative to the total weight of raw material (aqueous solution of fermented extract).

According to a particular and preferred mode, the fermented extract of mangosteen fruit peels according to the invention is characterized by a gluconic acid content greater than 50 mg/g, and an acetic acid content greater than 200 mg/g, in weight relative to the weight of dry extract of the fermented extract.

According to a particular mode, the fermented extract of mangosteen fruit peels comprises a gluconic acid content greater than 5%, preferably ranging from 5 to 15%, and an acetic acid content greater than 20%, preferably ranging from 20 to 50%, by weight relative to the weight of dry extract (dry matter) of the fermented extract.

Preferably, the fermented extract of mangosteen fruit peels comprises a gluconic acid content ranging from 5 to 15%, more preferably ranging from 6 to 10% by weight relative to the weight of dry extract of the fermented extract. .

Preferably, the fermented extract of mangosteen fruit peels comprises an acetic acid content ranging from 20 to 50%, more preferably ranging from 25 to 35% by weight relative to the weight of dry extract of the fermented extract.

The fermented extract of mangosteen fruit peels may include other organic acids, such as malic acid, pyruvic acid, quinic acid, succinic acid, and citric acid. Also, the fermented mangosteen extract may include:

- a malic acid content ranging from 0.1 to 0.5%, preferably from 0.15 to 0.7%, more preferably from 0.17 to 0.5%, by weight relative to the weight of dry extract fermented extract,

- a pyruvic acid content ranging from 0.01 to 1%, preferably from 0.02 to 0.5%, more preferably from 0.04 to 0.1%, by weight relative to the weight of dry extract of the 'fermented extract,

- a quinic acid content ranging from 0.1 to 1.5%, preferably from 0.2 to 1%, more preferably from 0.4 to 0.7%, by weight relative to the weight of dry extract of the 'fermented extract,

- a succinic acid content greater than 0.1% by weight, preferably ranging from 0.1 to 1.5%, preferably from 0.2 to 1% more preferably from 0.4 to 0.7%, in weight relative to the weight of dry extract of the fermented extract, and/or

- a citric acid content ranging from 0.05 to 1%, preferably from 0.08 to 0.7%, more preferably from 0.1 to 0.5%, by weight relative to the weight of dry extract of the fermented extract.

Preferably, the fermented mangosteen fruit peel extract does not include glucuronic acid or tartaric acid.

The fermented extract of mangosteen fruit peels may also include one or more preservatives. By “preservative” we mean a compound which aims to preserve the composition of the extract or the cosmetic composition containing it from any physicochemical and/or microbial alteration. Preferably, at least one preservative is chosen from butylene glycol, glycerin, propylene glycol, sodium benzoate, potassium sorbate, and propanediol.

According to a preferred embodiment, the fermented extract of mangosteen fruit peels according to the invention is in liquid form and it comprises at least one preservative, preferably selected from butylene glycol, glycerin, propylene glycol, sodium benzoate, potassium sorbate, and propanediol. More preferably the preservative is propanediol.

The fermented extract may in particular comprise one or more preservative(s) at a final concentration of between 10 and 40%, preferably between 20 and 38%, preferably between 25 and 35%, more preferably 30% by weight relative to the total weight, preferably at least one of the preservatives described above is chosen from butylene glycol, glycerin and propanediol.

Fermentation process

According to one aspect of the invention, the fermented extract of mangosteen fruit peels is obtained by a fermentation process comprising the steps of:

a) the incubation of a consortium of microorganisms in a solution comprising at least water, a carbohydrate and black tea leaves infused at a temperature between 12 to 45°C for 1 to 21 days in order to 'obtain a preparation of symbiotic ferments, said consortium comprising at least one lactic acid bacteria belonging to the genus Lactobacillus or Pediococcus , at least one yeast belonging to the genus Saccharomyces , Schyzosaccharomyces or Torulaspora , and at least one acetic acid bacteria belonging to the genus Acetobacter , Gluconobacter or Komagataeibacter ;

b) grinding mangosteen fruit peels to obtain particles having an average size ranging from 0.5 mm to 1 mm;

c) maceration of the particles obtained in step b) in water in the presence of a carbohydrate;

d) filtration of the maceration mixture to remove plant particles;

e) adding the preparation of symbiotic ferments obtained in step a) to the filtrate of step d) to obtain a fermentation mixture,

f) incubating the fermentation mixture at a temperature between 21 and 35°C for 15 to 25 days;

g) sieving then filtering the fermentation mixture in order to obtain a fermented extract of mangosteen fruit peels, and

h) optionally, the addition of at least one preservative.

According to another aspect, the present invention relates to a process for fermenting mangosteen fruit peels comprising the steps of:

a) the incubation of a consortium of microorganisms in a solution comprising at least water, a carbohydrate and black tea leaves infused at a temperature between 12 to 45°C for 1 to 21 days in order to 'obtain a preparation of symbiotic ferments, said consortium comprising at least one lactic acid bacteria belonging to the genus Lactobacillus or Pediococcus , at least one yeast belonging to the genus Saccharomyces , Schyzosaccharomyces or Torulaspora , and at least one acetic acid bacteria belonging to the genus Acetobacter , Gluconobacter or Komagataeibacter ;

b) grinding mangosteen fruit peels to obtain particles having an average size ranging from 0.5 mm to 1 mm;

c) maceration of the particles obtained in step b) in water in the presence of a carbohydrate;

d) filtration of the maceration mixture to remove plant particles;

e) adding the preparation of symbiotic ferments obtained in step a) to the filtrate of step d) to obtain a fermentation mixture,

f) incubating the fermentation mixture at a temperature between 21 and 35°C for 15 to 25 days;

g) sieving then filtering the fermentation mixture in order to obtain a fermented extract of mangosteen fruit peels, and

h) optionally, the addition of at least one preservative.

Microorganism Consortium

By “fermentation process” we mean a process making it possible to obtain a fermented extract of mangosteen fruit peels by culturing a consortium of microorganisms as defined here in a medium containing a carbohydrate and allowing their growth. Fermentation can take place under batch conditions, continuous culture, or continuous-discontinuous conditions ( Fed batch ). Fermentation can be carried out aerobically, microaerobically and/or anaerobically.

By “consortium of microorganisms” we mean an assembly of at least three species of microorganisms that can coexist in a stable and reproducible manner.

Advantageously, the microorganisms make it possible to combine complementary activities. As a non-limiting example, the use or assimilation of a carbon source by a first species makes it possible to produce metabolites which can then be metabolized by a second species.

The consortium of microorganisms according to the present invention comprises:

i) at least one microorganism belonging to the lactic acid bacteria group,

ii) at least one microorganism belonging to the yeast group, and

iii) at least one microorganism belonging to the group of acetic bacteria.

Preferably, at least one microorganism belonging to the group of lactic acid bacteria is a microorganism of the order Lactobacillales of the family Lactobacillaceae, in particular those belonging to the genera Lactobacillus and Pediococcus , such as Lactobacillus plantarum and/or Lactobacillus acidophilus , more preferably L. plantarum .

Preferably, at least one microorganism belonging to the group of yeasts is a microorganism of the order of Saccharomycetales of the family Saccharomycetaceae or of the order of Schyzosaccharomycetales of the family of Schyzosaccharomycetaceae, in particular those belonging to the genera Saccharomyces , Schyzosaccharomyces or Torulaspora .

Preferably, at least one microorganism belonging to the yeast group is Saccharomyces cerevisiae, Torulaspora delbrueckii, Saccharomyces boulardii and/or Schyzosaccharomyces pombe .

Preferably, at least one microorganism belonging to the group of acetic bacteria is a microorganism of the order Rhodospirillales of the family Acetobacteraceae, in particular those belonging to the genera Acetobacter , Gluconobacter and Komagataeibacter (also called Gluconacetobacter ). Acetic bacteria can originate from unpasteurized cider or wine vinegar with an acidity level of 4% to 5%, preferably from a commercial cider vinegar, labeled “Organic farming” according to the definition of the European Union. Preferably, the addition of acetic bacteria to the consortium is done by direct addition of an aliquot of vinegar to the composition comprising the other species of microorganisms. In vinegars, alongside the dominant acetic bacteria, there is a complex natural indigenous flora, some of which will be able to coexist with the added micro-organisms. Another part will collapse during the establishment of the consortium, in the same way that the symbiotic development of all the microorganisms forming the consortium prevents the development of undesirable species, including pathogenic species.

According to a preferential mode, the consortium includes:

- at least one lactic acid bacteria belonging to the genus Lactobacillus or Pediococcus, more preferably chosen from Lactobacillus plantarum and Lactobacillus acidophilus,

- at least one yeast belonging to the genus Saccharomyces , Schyzosaccharomyces or Torulaspora more preferably chosen from Saccharomyces cerevisiae, Saccharomyces boulardii, Schyzosaccharomyces pombe and Torulaspora delbrueckii , and

- at least one acetic bacteria from a cider vinegar or a wine vinegar, preferably belonging to the genus Acetobacter , Gluconobacter or Komagataeibacter.

Step a): preparation of a culture of microorganisms or also called preparation of symbiotic ferments

According to a first step, a), of the method, a culture of microorganisms (in other words, a consortium comprising the microorganisms as described above) is prepared. This step advantageously makes it possible to activate and multiply the microorganisms while establishing a stable and balanced consortium. During this step, the different microorganisms that will be present in the consortium are introduced into a solution comprising a carbohydrate (otherwise called a “nutrient medium”) and possibly also an aqueous extract of tea leaves in order to generate a microbial suspension. . The suspension is then incubated for 1 to 21 days to prepare the culture of the microorganisms (in other words, the consortium).

The solution includes at least one carbohydrate, generally one or more mono- or disaccharides. It may be a pure sugar, such as sucrose, glucose or fructose, or a processed or co-produced product, such as molasses. Also, according to a preferred mode, the solution comprises a carbohydrate chosen from sucrose, glucose or fructose, molasses, or a mixture of one or more of these. Preferably, the carbohydrate is sucrose or a mixture of glucose and fructose. Preferably, at least one carbohydrate is present at a concentration between 20 g/l and 100 g/l, preferably between 50 to 95 g/l of solution. Preferably, the nutrient medium comprises sucrose, or a mixture of glucose and fructose, at a concentration of 50 g/l.

According to a particular embodiment, the solution may also comprise an aqueous extract of tea leaves ( Camellia sinensis ). Preferably, the aqueous extract is obtained from an infusion of tea leaves, preferably black tea leaves. Preferably, the aqueous extract of tea leaves may be an infusion of black tea prepared with 2 g/l to 50 g/l of tea leaves, more preferably with 5 g/l to 10 g/l of tea leaves. .

The tea is prepared by adding dry leaves infused in water at a temperature of 70°C to 90°C for 10 to 60 min. Preferably, we use 5 g/l to 10 g/l of tea leaves infused for 15 minutes at 80°C. The infusion can be used as is with the leaves, or more conveniently the aqueous phase is filtered.

Microorganisms can be introduced into the nutrient medium in dry and/or wet form; the inoculation rate can in particular be between 10 ³ CFU/g and 10 ⁵ CFU/g of solution. The microbial suspension can then be incubated ranging from 12°C to 45°C, preferably from 17°C to 38°C, preferably from 25°C to 35°C, more preferably at 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C even more preferably at 26°C. According to a particularly advantageous mode, the incubation takes place in an open reactor, without needing to ensure aseptic conditions.

The suspension can be incubated for 1 to 21, preferably for 1 to 15 days, preferably for 1 to 10 days, more preferably for 3 to 4 days, even more preferably for 3 days or 4 days. At the end of the incubation time, the total flora in the suspension may be between 10 ⁴ and 10 ⁷ CFU/g.

The consortium thus obtained is capable of reacting with a natural substrate, such as mangosteen fruit peel, in an aqueous medium to make accessible, and possibly transform, a wide diversity of the compounds it contains.

Step b): preparation from the peel of the fruits of m angosteen

According to step b), the peel of mangosteen fruits are coarsely ground to obtain particles. The bark particles advantageously have an average size ranging from 0.05 mm to 5 mm, preferably ranging from 0.1 mm to 3 mm, preferably ranging from 0.2 mm to 2 mm, even more preferably ranging from 0 .5mm to 1mm. Preferably, the peels are raw. Preferably, the peels are separated from the pulp of the mangosteen fruit. Preferably the barks are dried before being crushed.

Step b) can be carried out either before, at the same time or after step a).

Step c): maceration of mangosteen fruit peel particles

According to step c), the particles obtained in step b) are macerated in water in the presence of at least one carbohydrate.

By “maceration” we mean that the particles obtained in step b) remain in a liquid (in this case a mixture of water and carbohydrates) to extract active and/or nutritional principles and/or to obtain a modification of said particles, such as for example their softening or their hydration.

The maceration solution comprises at least one carbohydrate, generally one or more mono- or disaccharides. It may be a pure sugar, such as sucrose, glucose or fructose, or a processed or co-produced product, such as molasses. Also, according to a preferred mode, the solution comprises a carbohydrate chosen from sucrose, glucose or fructose, molasses, or a mixture of one or more of these. Preferably, the carbohydrate is sucrose or a mixture of glucose and fructose. Preferably, at least the carbohydrate is added to the maceration medium of step c), at a concentration ranging from 30g to 70g per liter, preferably 40g to 60g per liter and preferably at a concentration of approximately 50 g per liter. liter.

The mangosteen fruit peel particles are introduced into the maceration medium as described above, at a concentration ranging from 30g to 70g per liter, preferably 40g to 60g per liter and preferably at a concentration of approximately 50 g per liter.

The maceration solution can be incubated for 1 to 21, preferably 1 to 15 days, preferably 1 to 10 days, at a temperature ranging from 12°C to 45°C, preferably from 17°C to 38°C. , preferably from 20°C to 30°C, more preferably at 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C , 29°C, or 30°C.

Step d) filtration of the maceration mixture

The maceration mixture is then filtered during step d). By “filtration” we mean here a step allowing the solid and liquid fractions to be separated. The solid fraction may in particular include the remaining mangosteen fruit peel particles. Advantageously, filtration makes it possible to remove all the plant particles present in the mixture, in order to obtain a mangosteen fruit peel macerate free of solid residues, also called filtrate. This filtration step can be carried out by using one or more sieves and/or filters. Preferably, step d) is a sieving step.

Step e): preparation of a fermentation mixture

According to step e) of the process, the preparation of symbiotic ferments comprising the culture of the microorganisms obtained according to step a) as well as a carbohydrate, is added to the filtrate of step d) in order to obtain a mixture of fermentation.

Preferably, the preparation of symbiotic ferments obtained in step a) is added to the filtrate of step d) at a concentration of between 5% and 15% by weight relative to the weight of fermentation medium, preferably between 7%. and 13%, more preferably 10%.

Preferably, the consortium of microorganisms is present in the fermentation medium at a concentration ranging from 10 ³ CFU/g and 10 ⁶ CFU/g of fermentation medium.

Step f): incubation

According to step f) of the process, the fermentation mixture obtained in step e) is then incubated in order to ensure the fermentation of the filtered macerate of mangosteen fruit peels.

Incubation takes place at a temperature between 12°C and 45°C, preferably between 25°C and 35°C, more preferably at 25°C, 26°C, 27°C, 28°C, 29°C. C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C, even more preferably at 26°C. The incubation is carried out for a period of 10 to 30 days, preferably for 15 to 25 days, more preferably for 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days, even more preferably 20 days.

In one particular aspect, the incubation takes place in an open reactor, without the need to ensure aseptic conditions. According to a particular aspect, the incubation takes place at a humidity level of between 20% to 60%, preferably between 30% to 50%, more preferably 40%. According to a particular aspect, the fermentation is carried out under static conditions, i.e., without agitation.

Step g): filtration

The fermentation mixture is then filtered during step g). By “filtration” we mean here a step allowing the solid and liquid fractions to be separated. The solid fraction may in particular comprise microorganisms. Advantageously, filtration makes it possible to remove all of the microorganisms present in the mixture, in order to obtain a fermented extract of sterile mangosteen fruit peel. Filtration can be carried out by using one or more sieves and/or filters. Also, according to one embodiment, step g) corresponds to one or more filtration steps, preferably comprising at least one sieving step and/or at least one sterilizing filtration step. Preferably, step g) comprises a sieving step and a sterilizing filtration step. Preferably, the sterilizing filtration is carried out on a filter having an average pore size of between 0.1 µm to 0.2 µm, preferably 0.2 µm. Advantageously, sieving makes it possible to remove a large part of the microorganisms, in particular the living cells clumped together. Advantageously, sterilizing filtration makes it possible to remove all non-sporulating living microorganisms quickly and inexpensively. Preferably, at least one of the filters is a cellulose filter.

Stage h ): concentration and/or formulation (optional)

The process described above makes it possible to obtain a fermented extract of clarified mangosteen fruit peels. However, in some cases it may be advantageous to carry out one or more additional steps, for example concentration or formulation. The process according to the invention can thus further comprise at least one additional step subsequent to step g) of concentration or formulation.

In a particular embodiment, the process according to the invention further comprises, after filtration step g), one or more of the following steps aimed in particular at adapting the composition to a particular formulation:

- a concentration stage; and or

- a formulation stage.

Each optional step identified above may be present as a single additional process step, or in combination with one or more other optional steps. Preferably, the fermentation process comprises a step h) of adding at least one preservative to the fermented extract of mangosteen fruit peels. Preferably, said preservative is chosen from butylene glycol, glycerin, propylene glycol, sodium benzoate, potassium sorbate and propanediol, more preferably propanediol. In some cases, two or more preservatives may be added to the fermented mangosteen fruit peel extract. Preferably, the preservative is added to the fermented extract of mangosteen fruit peels from step h) at a final concentration of between 20 and 40%, preferably between 25 and 35%, more preferably 30%, by weight relative to the total weight of the extract in the form of a solution. When the preservative is added to the fermented extract of mangosteen fruit peels from step g) at a final concentration of between 20 and 40%, said preservative is selected from butylene glycol, propanediol and propylene glycol.

The fermented extract of mangosteen fruit peel is preferably stored at approximately 4°C (4 ± 2°C) and/or in the dark after step g) of filtration or after any other step when an additional step is present after step g).

When the process includes the addition of at least one preservative, the fermented extract of mangosteen fruit peels is preferably stored at room temperature.

The fermented extract of mangosteen fruit peels is referenced under the international nomenclature, INCI: “WATER, PROPANEDIOL, GARCINIA MANGOSTANA FRUIT PEEL EXTRACT, LACTOBACILLUS FERMENT LYSATE, YEAST FERMENT EXTRACT”. This product contains in particular 1.2 to 1.9% by weight of dry extract of fermented extract of mangosteen fruit peels in a water/propanediol mixture.

Combination of an extract of mangosteen fruit peel according to the invention and an extract of Kombucha

Kombucha

Kombucha is a tea that has undergone a microbiological transformation. This transformation is a fermentation by a symbiont of yeast of the genus Saccharomyces which nests within a matrix of polysaccharides produced by Xylinum bacteria. The exact composition of this symbiosis (in particular the proportions of each species) varies with geographical and climatic conditions and depends on local wild subspecies of yeast and bacteria; nevertheless we can cite, among others, without this list being exhaustive: Saccharomyces ludwigii , Saccharomyces apicalutus , Bacterium xylinoides, Bacterium gluconicum, Schizosaccharomyces pombe, Acetobacter ketogenum , Torula species, Pichia fermentans and other yeasts. In the literature on kombucha, this symbiont of yeast and bacteria is also called “mushroom”, “long-lived champion” and numerous synonyms.

This fermentation is completely original since the alcohol produced by the yeast from the sugar is transformed by the bacteria into different acids, glucuronic, lactic, usnic and especially acetic which gives its tangy taste to the kombucha with a final pH of between 2 , 5 and 4. On the other hand, the Xylinum bacteria, described as “acetic bacteria”, also uses the sugar present in tea and transforms the sucrose into cellulose microfibrils, thus constituting the support membrane in which the yeast nests and develops. .

The products of yeast metabolism are excreted in kombucha and consist of many vitamins such as B1, B2, B3 and B12, essential cofactors for bacterial growth.

Kombucha, also called “long-lived mushroom tea”, is a popular therapeutic remedy long known by different names [FRANK G., 1999]. It is a refreshing drink with a yellow-amber color and a sweet-sour taste of cider. Its effect has been known and appreciated for generations by many people, especially in the countries of Eastern Asia. The tea used for making kombucha can be of any kind and of any origin, including Camellia sinensis sinensis or assamica varieties. All varieties of green tea, semi-fermented tea, black tea, smoked black tea, yellow tea, dark tea, white tea, plant or fruit herbal tea, infusion, can serve as a basis for making kombucha. We preferentially use all varieties of green tea, semi-fermented tea, black tea, smoked black tea, yellow tea, dark tea, white tea (all Camellia sinsensis ) and more particularly black tea.

The term "kombucha" in the present invention includes synonyms such as combucha, cajnyj kvas (Russian), Combuchagetrânk (German), Kargasoktee (German), komboecha-drank (Dutch), Kombuchakwal (German), tea-beer and tea- cider (English), etc...

In the same way, the symbiont, "long-lived mushroom" is also called, in particular: cajnyj grib (Russian), Japanese or Chinese mushroom, miracle mushroom, comboucha, combucha (Japanese), fungojapon, fungus japonicus (pharmaceutical designation), funko cinese (Italian), ganoderma japonicum, japanischer Teepilz (German), kombucha, ling zhi (Chinese), mandschurischer Schwamm, russische Blume, tea fungus kombucha, Wolgaqualle (German), Zauberpilz, etc…

Surprisingly, the Applicant was able to show by in vitro tests that the combination of the fermented extract of mangosteen fruit peel and a kombucha extract made it possible to reduce oxidative stress in normal human keratinocytes ( see example 5). This result is all the more unexpected as each of these extracts, taken individually, does not achieve this type of effect.

Thus, one aspect of the invention concerns the combination of a fermented extract of mangosteen fruit peels according to the invention (as described above) and a kombucha extract, preferably a kombucha extract from Nepal. According to a particular embodiment, the kombucha extract used in combination in the present invention is the product marketed by SEDERMA under the commercial reference KOMBUCHKA™. The process for obtaining this extract is described in particular in French patent FR 2 843 023. Advantageously, this same process for obtaining can be used to obtain other kombucha extracts, in particular for obtaining a kombucha extract from Nepal.

Thus, according to another particular and preferred embodiment, the kombucha extract used in combination in the present invention is a kombucha extract from Nepal obtained according to the process described in French patent FR 2 843 023 or the process described below. After.

According to one embodiment, said kombucha extract, preferably said kombucha extract from Nepal, is prepared in the following manner: in a glass container with a wide opening, leave sweet tea to ferment, containing at least 5 g of tea ( black for example) per liter and at least 70 g of sugar per liter, with the symbiote (the “long-lived kombucha mushroom”), of which a piece of approximately 25 to 100 g/l is added to the sweet tea. Place the fermentation container in a warm place for several (5 to 12) days. After filtration, the kombucha is ready, and the “mushroom” is recovered for the following fermentation. It is possible in the manufacture of kombucha to add 10% of kombucha obtained during a previous production to the mixture of sweet tea and mushroom, in order to initiate fermentation more easily.

Cosmetic composition

Surprisingly, the inventors have demonstrated that the fermented extract of mangosteen fruit peel has beneficial effects on the skin, making it possible in particular to improve the biomechanical properties of the skin by acting on the expression of essential proteins. extracellular matrix such as type I collagen, elastin, fibronectin, metalloprotease 2 (MMP2), or even vinculin. Thus, the fermented extract of mangosteen fruit peel helps fight against the signs of age-related skin aging, in particular the loss of firmness and/or elasticity.

According to the invention, the fermented extract of mangosteen fruit peels is used in an effective amount to achieve the desired effect. It can be used as is or advantageously incorporated into a cosmetic composition suitable for topical application to keratin materials.

In particular, the fermented extract of mangosteen fruit peel is used as is or is present in a cosmetic composition.

Also, another object of the invention relates to a cosmetic composition comprising, in a physiologically acceptable medium, a fermented extract of mangosteen fruit peels according to the invention.

By “cosmetic composition” is meant any composition for cosmetic purposes, that is to say aesthetic, which can be brought into contact with the superficial parts of the human body and more particularly with keratin materials, in particular human skin and/or lips. .

By “physiologically acceptable medium” is meant any excipient suitable for topical use, in contact with keratin materials, without risk of toxicity, incompatibility, instability and/or allergic response.

By “keratin materials” according to the invention is meant the skin and/or its integuments, and more particularly the human skin and/or lips. In particular, it will concern the skin of the face and/or neck and/or body, and the lips, preferably the skin of the face and/or neck.

The keratin materials according to the invention are in particular healthy keratin materials (“healthy” subjects), that is to say not presenting disorders or disorders which would relate to a pathological state (“unhealthy” subjects, affected of a pathology). We will speak indifferently of healthy skin and/or lips or of skin and/or lips in the rest of the description.

The fermented extract of mangosteen fruit peels is present in the composition of the invention in a content ranging from 0.0001% to 5%, preferably from 0.0005% to 2%, more preferably from 0.001% to 5%. 1% by weight of raw material (fermented extract solution obtained according to the process described above) relative to the total weight of the composition. Thus the fermented extract of mangosteen fruit peels is present in the composition of the invention in a content ranging from 0.0000012% to 0.095%, preferably from 0.000006% to 0.033%, more preferably from 0. 000012% to 0.0165% by weight of active ingredient (fermented extract of mangosteen fruit peels) relative to the total weight of the composition.

According to certain embodiments, the cosmetic composition of the invention may also comprise, in addition to the fermented extract of mangosteen fruit peels, at least one kombucha extract, in particular the product marketed by SEDERMA under the trade reference KOMBUCHKA™ or according to a preferred embodiment, a kombucha extract from Nepal obtained according to the process described in French patent FR 2 843 023 or the process described previously in the description.

The kombucha extract is present in the compositions of the invention in an effective quantity to obtain the desired effect, in particular to allow, when combined with the fermented extract of mangosteen fruit peels, to prevent and/or reduce oxidative stress on skin cells and thus promote and/or stimulate the vitality of skin cells, and/or promote and/or stimulate the homogeneity or radiance of the complexion. In particular, said kombucha extract is present in a content ranging from 0.01% to 10%, in particular from 0.02% to 5%, preferably from 0.05% to 4% by weight of raw material (weight of product) relative to the total weight of the composition.

The physiologically acceptable medium generally represents 1 to 99% by weight, relative to the total weight of said composition. The physiologically acceptable medium of the composition according to the invention comprises water and optionally the preservative as defined above.

In a particular embodiment, said cosmetic composition comprising a fermented extract of mangosteen fruit peels according to the invention, and optionally the kombucha extract as described above, is a care and/or makeup composition. keratin materials, in particular of the skin and/or lips and in particular of the skin of the face and/or neck.

The cosmetic composition of the invention generally comprises, in addition to the fermented extract of mangosteen fruit peels and the physiologically acceptable medium, one or more acceptable cosmetic excipients among those known to those skilled in the art with a view to obtaining a composition for topical application for example in the form of a milk, a cream, an ointment, a water-in-oil or oil-in-water emulsion or a multiple emulsion, a balm , a stick, a solution, a suspension, gel, a lotion, a serum, or even a .

The cosmetic composition of the invention can also be in the form of a patch or a mask, in particular a mask in the form of a thick cream, or even in the form of a cellulose mask soaked in fermented extract of mangosteen fruit peel or a composition containing it.

In a preferred embodiment, said composition is in the form of a cream, an emulsion, a solution, a suspension, a gel, a milk, a lotion, or 'a serum. When the composition is in the form of an emulsion, the emulsion may be an oil-in-water emulsion, or water-in-oil or multiple emulsion.

The cosmetic composition of the invention can be presented in any galenic form suitable for topical application on keratin materials in particular of the skin and/or lips and in particular of the skin of the face and/or neck comprising the fermented extract mangosteen fruit peels and at least one cosmetic ingredient chosen from antioxidants, perfumes, vitamins, thickening agents, emollient agents, moisturizing agents, anti-aging agents, lifting agents, tightening agents, plumping agents, soothing agents, anti-pollution agents, lightening or depigmenting agents, fillers, pearls and their mixtures.

Also, according to a preferred object of the invention, the cosmetic composition may further comprise at least one cosmetic adjuvant chosen from the group consisting of antioxidant agents, emollient agents, moisturizing agents, anti-aging agents, perfumes and their mixtures.

Depending on the nature of the composition, one or more cosmetically acceptable excipients will be selected from emulsifiers, polymers, surfactants, rheology agents, electrolytes, pH adjusters, anti-oxidant agents, preservatives, dyes. , and their mixtures.

As a particular example, the cosmetic composition may comprise hydrophilic gelling agents, antioxidants, preservatives and mixtures thereof.

Typically, the additional ingredients used advantageously in the composition of the invention can be present in a content ranging from 0.0001% to 10%, preferably from 0.001% to 5% by weight (of raw material) relative to the total weight. of the composition.

Cosmetic process

The present invention also relates to a cosmetic process for the care and/or makeup of keratin materials, in particular of the skin and/or lips and in particular of the skin of the face and/or neck, comprising topical application on said keratin materials, at least one layer of the fermented extract of mangosteen fruit peels or of a cosmetic composition containing it as described according to the invention.

The cosmetic process of the present invention is particularly intended to promote and/or reinforce the firmness, density and/or tension of the skin, and/or promote and/or reinforce the elasticity, extensibility and biomechanical resilience of the skin. the skin and thus prevent and/or reduce the alteration of the flexibility of the skin and/or the formation of wrinkles and fine lines and/or prevent and/or reduce the signs of skin aging.

By “promoting and/or reinforcing the firmness, density and/or tension of the skin, and/or promoting and/or reinforcing the elasticity, extensibility and biomechanical resilience of the skin” we mean that the extract fermented mangosteen fruit peel is intended to promote the expression of proteins essential to the architecture and function of the extracellular matrix.

By “preventing and/or reducing the signs of skin aging” we mean in particular that the fermented extract of mangosteen fruit peel is intended to reduce and/or delay the loss of firmness, the loss of elasticity, the loss of density, dryness of the skin, loss of flexibility, and/or the appearance of wrinkles and/or fine lines.

According to a particular mode, said fermented extract of mangosteen fruit peels according to the invention or said cosmetic composition containing it is applied to aged skin or skin showing signs of aging.

The present invention also relates to a cosmetic process for the care and/or makeup of keratin materials, in particular of the skin and/or lips and in particular of the skin of the face and/or neck, comprising topical application on said keratin materials, at least one layer of the combination of the fermented extract of mangosteen fruit peels according to the invention and a kombucha extract, as described above, or of a composition cosmetic containing these two extracts.

According to this embodiment, said cosmetic process is intended in particular to prevent and/or reduce oxidative stress on skin cells and thus promote and/or stimulate the vitality of skin cells, and/or promote and/or stimulate the homogeneity or the radiance of the complexion.

By “promoting and/or stimulating the vitality of skin cells”, we mean in particular that the fermented extract of mangosteen fruit peel is intended to promote the general condition of the skin and its radiance.

By “preventing and/or reducing the alteration of the homogeneity or radiance of the complexion” we mean in particular that the fermented extract of mangosteen fruit peels is intended to reduce and/or delay the appearance of imperfections. or microreliefs of the skin, dark circles, a blurred, dull and/or uneven complexion.

According to a particular mode, said fermented extract of mangosteen fruit peels according to the invention or said cosmetic composition containing it is applied to aged skin or showing signs of aging, preferably aged skin or showing signs of aging.

According to a particular mode, said fermented extract of mangosteen fruit peels according to the invention, alone or associated with said kombucha extract as described above, or said cosmetic composition containing it is applied to aged skin or skin showing signs of aging and/or skin exposed to stress, and/or tired, and/or presenting a dull and uneven complexion.

By “skin exposed to stress”, we mean that the skin is subjected to stress of endogenous or exogenous origin, in particular oxidative stress likely to induce unsightly non-pathological skin reactions – e.g. the skin is visibly tired and/or dull and lacks radiance.

The skin and lips to which the compositions of the invention are applied are healthy, that is to say not presenting disorders or disorders which would result from a pathological state (“unhealthy” subjects, suffering from a pathology).

Advantageously, the compositions comprising fermented extract of mangosteen fruit peels used in the context of the cosmetic process of the invention are those described above.

Use s fermented extract mangosteen fruit peel

Another object of the invention relates to the cosmetic use of the fermented extract of mangosteen fruit peels according to the invention for topical application to keratin materials, in particular the skin and/or lips and in particular of the skin of the face and/or neck, as a cosmetic active ingredient intended to promote and/or reinforce the firmness, density and/or tension of the skin, and/or promote and/or reinforce elasticity, extensibility and biomechanical resilience of the skin and thus prevent and/or reduce the alteration of the flexibility of the skin and/or the formation of wrinkles and fine lines and/or prevent and/or reduce the signs of skin aging.

Another object of the invention relates to the cosmetic use of at least one fermented extract of mangosteen peel fruits according to the invention and of at least one kombucha extract, as described above, for a topical application to keratin materials, in particular the skin and/or lips and in particular the skin of the face and/or neck, as a combination of cosmetic active ingredients intended to prevent and/or reduce oxidative stress on skin cells and thus promote and/or stimulate the vitality of skin cells, and/or promote and/or stimulate the homogeneity or radiance of the complexion.

EXAMPLES

The invention is illustrated by the following non-limiting examples. The % are expressed in weight of raw material relative to the total weight of the composition, unless otherwise indicated.

Example 1 : Obtaining of A extract fermented fruit peel mangosteen according to the invention

1.1. Material and methods Microorganism Consortium

Microorganisms included in the consortium include:

- at least one lactic acid bacteria belonging to the genus Lactobacillus or Pediococcus ,

- at least one yeast belonging to the genus Saccharomyces , Schyzosaccharomyces or Torulaspora , and

- at least one acetic acid bacteria belonging to the genus Acetobacter , Gluconobacter or Komagataeibacter.

Preparing mangosteen fruit peels

Mangosteen fruits are dried beforehand then the peels are collected and crushed to obtain particles with a size between 0.5mm and 1mm.

Preparation of nutrient medium

A tea is prepared from 5 g/l to 10 g/l of tea leaves infused for 10 to 60 min at 70°C to 90°C. Sucrose is then dissolved in the aqueous phase at a concentration of between 20 g/l and 100 g/l, generally 50 g/l. Sucrose can in particular be replaced by a mixture of glucose and fructose.

Fermentation process

A fermentation of mangosteen fruit peels was carried out according to a process comprising the following steps:

a) the incubation of a consortium of microorganisms in a solution comprising at least water, a carbohydrate and infused black tea leaves (in other words the “nutritive medium”) at 26°C for 3 to 4 days in order to obtain a culture also called preparation of symbiotic ferments;

b) grinding previously dried mangosteen fruit peels to obtain particles, as described above;

c) maceration of the particles obtained in step b) in water in the presence of a carbohydrate:

- mangosteen fruit peel particles are added at a concentration of 50 grams per liter of water;

- the carbohydrate is added at a concentration of 50 grams per liter of water;

d) filtration of the maceration mixture to remove plant particles;

e) the addition, at a concentration of 10% (w/w), of the preparation of symbiotic ferments obtained in step a) in the filtrate of step d) to obtain a fermentation mixture,

f) incubating the fermentation mixture at a temperature of 26°C for 20 days;

g) filtration of the fermentation mixture by coarse sieving followed by filtration on a cellulose filter having an average pore size of 0.2 µm in order to obtain a fermented extract of mangosteen fruit peel, and

h) the addition of propanediol (solvent and preservative) to the fermented extract of mangosteen fruit peels at a final concentration of 30% by weight relative to the total weight of the extract in liquid form.

1.2. Results

A fermented extract of mangosteen fruit peels is obtained in the form of a translucent aqueous solution having a concentration of 1.7 to 2.7% by weight of active material (dry matter) of mangosteen fruit peels by relative to the total weight of the extract.

The effect of this extract will be illustrated in the following examples.

Example 2 : Obtaining of a mangosteen extract watery (non- fermented ) – comparative extract

20 g of coarsely ground mangosteen fruit peels were added to a capped Erlenmeyer flask containing 200 mL of MilliQ H2O. The Erlenmeyer flask was sonicated for 20 minutes. The mixture was then vacuum filtered with a Whatman 47 mm GF/F filter. The filtrate was partially dried using the GENEVA EZ-2 PLUS centrifugal evaporator, then drying was completed using the ZEODRY+PLUS ZD+L, under vacuum at 5 mbar following a drying gradient. A brown-red dry extract is thus obtained. A dry extract yield ranging from 12% to 13.5% is obtained relative to the weight of raw material.

Example 3 : VS phytochemical characterization of s extracts fermented mangosteen ( extract according to the invention obtained in example 1 ) and unfermented ( comparative extract obtained in example 2)

Objective of the study : The objective is to analyze the effect of fermentation on aqueous extracts of mangosteen fruit peels, by carrying out a phytochemical study by HPTLC (high performance thin layer chromatography) and analyzes qualitative and quantitative by HPLC-DAD-MS of polyphenols and organic acids.

3.1. Material and methods Liquid chromatography (HPLC)

The identification of polyphenols and the quantification of organic acids were carried out by liquid chromatography, on an Agilent InfinityLab LC/MSD chain coupled to a DAD detector and mass spectrometry (Agilent). HPLC allows the separation of the compounds present in the extracts, then the UV chromatograms are obtained by UV detection with DAD at several wavelengths: 210, 254, 280, 320, 360, and 520 nm. The separated molecules are then ionized at the source by an electrospray and analyzed by a simple quadrupole. MS detection is carried out by an electromultiplier. An analysis in TIC (Total ion chromatogram) mode is carried out for masses between 100 and 1000 Da in positive and negative mode. Data acquisitions and processing were carried out with Control Panel software.

High performance thin layer chromatography coupled with mass spectrometry (HPTLC-MS)

Analysis by high-performance thin layer chromatography coupled with mass spectrometry is a relatively rapid and effective technique for characterizing the molecules of an extract. The desorption of the spots was carried out with the solvent MeOH/H ₂ O (1/1) (v/v) and 0.1% formic acid using the Agilent InfinityLab chain pump, then the analysis was carried out. conducted by mass spectrometry via the CAMAG TLC-MS interface.

3.2. Results

The fermentation process generates a fermented extract of mangosteen fruit peels (according to the invention) having a very different composition from the extracts of unfermented mangosteen fruit peels (comparative extract of Example 2).

In particular, the extracts contain a variety of polyphenols and sugars with a different content between the non-fermented extracts and the fermented extracts but, for each of these two extracts, few or no amino acids and non-polar molecules. Fermentation reduces the quantity of certain polyphenols but we observe that the fermented extract is richer in organic acids.

Table 1 : Organic acids present in the fermented extract of mangosteen fruit peels compared to an aqueous extract of mangosteen fruit peels (non-fermented) (in mg relative to g of dry extract)

Malic acid Pyruvic acid Quinic acid Succinic acid Citric acid Acetic acid Gluconic acid mg/g mg/g mg/g mg/g mg/g mg/g mg/g Fermented extract of mangosteen fruit peel
(invention) 1,942 0.357 5,153 4,423 2,222 295,425 75,744 Unfermented extract of mangosteen fruit peel
(comparative) 4,358 0 15,324 0 9,595 0.698 37,268

The fermentation of mangosteen fruit peels makes it possible to increase the content:

• gluconic acid, succinic acid and pyruvic acid which have beneficial effects on the texture of the skin by increasing the production of glycoproteins, collagen and elastic fibers; And
• acetic acid, known for its antibacterial properties.

Example 4 : Effect of the' extract mangosteen fermented on the expression of cellular matrix markers

Objective of the study : The effect of the fermented mangosteen extract according to the invention was evaluated on the expression of proteins present in the extracellular matrix (ECM) and/or ensuring the anchoring of fibroblasts in the ECM. These proteins therefore have biological functions linked to the firmness, density, tension of the skin, as well as its elasticity, extensibility and biomechanical resilience.

4.1. Material and methods

Cell culture and treatment

The cells used for this study are Normal Human Fibroblasts (NHF) from an abdominoplasty from a 37-year-old female donor.

The cells were thawed in DMEM 1g/L glucose medium supplemented with 10% Fetal Bovine Serum (FCS) at passage 5, at 1.10 ⁶ cells in a T150 flask.

The FHNs were trypsinized at pre-confluence and seeded in 96-well plates (µ-plate 96 well, ibiTreat from Ibidi) at a density of 10,000 cells per well in complete DMEM medium (1g/L glucose +10% of SVF), then placed in the incubator at 37°C, 5% CO2.

After 3 days of incubation, the cells were treated with fermented mangosteen extract (as obtained by the process described in Example 1) at concentrations 0.5% and 1% (by weight of extract). , or with aqueous mangosteen extract (non-fermented) at a concentration of 0.01% (by weight of extract) in complete DMEM medium (1 g/L glucose + 10% FCS). The treatment was carried out for each condition (8 replicates), for 5 days with a renewal of the culture medium including the fermented mangosteen extract (retreatment) on the 3rd day of culture following the initial treatment. For the untreated control condition, the medium was replaced with fresh culture medium.

After a total of 5 days of treatment, the cells were rinsed twice with PBS (PBS Tablets, Invitrogen GIBCO). The cells were then fixed with formalin (Formalin Solution 10%, Neutral Buffered, Sigma) for 10 minutes then rinsed 3 times with PBS.

Immunostaining

The membranes of the fixed cells were permeabilized, or not (depending on the primary antibody used), with a solution of PBS/Triton 0.1% (Triton x-100, Sigma) for 10 minutes at room temperature then rinsed with PBS.

The permeabilized cells were incubated in a 1% PBS/BSA solution (Bovine Serum Albumin, Sigma) for at least 30 minutes at room temperature in order to saturate the non-specific sites.

The primary antibodies targeting the proteins of interest (see Table 2) were diluted in a 1% PBS/BSA solution. The cells were incubated for 1 night at 4°C in the presence of the primary antibody solution.

At the end of the incubation time, the cells were rinsed 3X with PBS and covered with a 1% PBS/BSA solution containing the labeled secondary antibodies targeting the specific primary antibody (see Table 3) and DAPI ( 4',6'-diamidino-2-phenylindole, dihydrochloride, Molecular Probes Life Technologies; diluted 1/500). DAPI is a DNA intercalating agent used to reveal cell nuclei.

The cells were then incubated for one hour in the dark and at room temperature with the secondary antibody solution and DAPI. Finally, the cells were rinsed 3X with PBS and each well of the plates was filled with 200 µL of PBS.

Primary Antibodies Target Supplier Reference Species Dilution Permeabilization Collagen 1 Novotec 20111 Rabbit 1/300 - Elastin Novotec 25011 Rabbit 1/200 - Fibronectin Abcam Ab2413 Rabbit 1/500 - MMP2 Abcam Ab92536 Rabbit 1/300 + Vinculin Neomarkers MS-1209-P1 Mouse 1/1000 +

Secondary Antibodies Antibody name Supplier Reference Species Dilution DAPI Alexa Fluor 568 Invitrogen A11011 Goat anti-Rabbit 1/400 + Alexa Fluor 568 Invitrogen A11004 Goat anti-Mouse 1/400 +

Acquisition and analysis of images via the High Content screening platform

The plates were scanned with the ArrayScan XTi from Thermo Cellomics, with the following acquisition parameters:

• DAPI detection with the XF53_386_23 filter
• Detection of the Alexa Fluor 568 antibody with the XF53_572_15 filter
• A resolution of 1104 x 1104
• The 10x dry objective
• 25 images per well (i.e. 25 x 8 replicates = 200 images per condition).

The images were analyzed with the “ Spot detector ” image analysis application in Studio software (Thermo). This software makes it possible to detect the marking (colored in red) of the targeted protein, corresponding to its expression in cells. The area of the measurement area was delimited and defined as the entire image area. Cell number was determined by counting DAPI-labeled nuclei. The results are expressed by the intensity of the labeling detected per number of cells to obtain quantification.

Statistical analyzes

Statistical analyzes were carried out using Microsoft Excel software. These analyzes consist of a calculation of the mean, the standard deviation and the confidence interval (α = 0.05) of the ratio of total marking intensity per number of cells, averaged in the well analyzed. The significance of the results was calculated using a two-tailed Student's t test. Results are expressed as mean ± standard error (SEM) and the difference is considered significant at p < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001).

4.2. Results

The results are shown in Figures 1 to 5.

They show the protein expression of Collagen I, Elastin, Metalloprotease 2, Vinculin or Fibronectin (expressed as total intensity per cell) in each of the conditions evaluated, that is to say:

• Unprocessed control (also called UNPROCESSED CONTROL in Figures 1 to 5).
• Condition treated with fermented mangosteen extract at 0.5% dilution (as obtained in example 1, also called FERMENTED MANGOUSTAN SR in figures 1 to 5),
• Condition treated with fermented mangosteen extract at 1% dilution (as obtained in example 1, also called MANGOUSTAN SR FERMENTED in figures 1 to 5),
• Condition treated with non-fermented mangosteen extract at 0.01% dilution (as obtained in example 2, also called EXT. DRY AQUEOUS MANGOSTANO in figures 1, 2, 4 and 5)

Note that a concentration of fermented mangosteen extract at a 1% dilution corresponds to the same quantity of raw material (mangosteen fruit peel) as a concentration of non-fermented mangosteen extract at a 0.01 dilution. %.

Effect from the extract of m fermented angosteen on the expression of Collagen I

Type I collagen is the main collagen present in the skin. Collagen I promotes firmness and density of the skin on the one hand, and on the other hand gives it elasticity and extensibility which contribute to the biomechanical resilience of the skin. Together, the effects of collagen help reduce wrinkles.

The data show that fermented mangosteen extract induces, compared to the untreated control, a significant increase (+28% at the 0.5% dose and +24% at the 1% dose) in the production of collagen I in FHN ( ). The increase induced by the fermented mangosteen extract is therefore significantly greater than that observed with the extract of non-fermented mangosteen fruit peel (+8%; ).

Effect from the extract of m fermented angosteen on the expression of Elastin

Elastin is a protein from the structural fibrous protein family. Secreted by fibroblasts, it is the major component of the skin's elastic fibers. Elastin therefore promotes the elasticity and bounce of the skin.

The data show that fermented mangosteen extract induces, compared to the untreated control, a significant increase (+88% at the 0.5% dose and +145% at the 1% dose) in elastin production in FHN ( ). No increase is observed with the extract of unfermented mangosteen fruit peel ( ).

Effect mangosteen extract fermented on the expression of Metalloprotease 2 (MMP2)

Metalloprotease 2 is a secreted or membrane proteolytic enzyme that acts on various proteins of the extracellular matrix and connective tissue such as collagen, gelatin, and elastin.

The data show that fermented mangosteen extract induces, compared to the untreated control, a significant increase (+15% at the 0.5% dose and +52% at the 1% dose) in the production of MMP2 in FHNs. ( ).

Effect from the extract of m fermented angosteen on the expression of Vinculin

Vinculin is a cytoskeletal protein associated with cell-cell and cell-matrix junctions. Vinculin has a high affinity for actin filaments/microfilaments and is involved in anchoring F-actin to the membrane. It therefore plays a key role in maintaining the shape of fibroblasts by creating a tension effect. Thus, vinculin promotes tension, elasticity and firmness of the skin.

The data show that fermented mangosteen extract induces, compared to the untreated control, a significant increase (+36% at the 0.5% dose and +44% at the 1% dose) in vinculin production in FHNs. ( ). No increase is observed with the extract of unfermented mangosteen fruit peel ( ).

Effect from the extract of m fermented angosteen on the expression of Fibronectin

Fibronectin plays a key role in adhesion between the extracellular matrix and fibroblasts. It has a strong affinity for collagens I and III. It contributes to the integrity and biomechanical properties of the skin, as well as its firmness.

The data show that fermented mangosteen extract induces, compared to the untreated control, a significant increase (+39% at the 0.5% dose and +40% at the 1% dose) in the production of fibronectin in FHNs. ( ). No increase is observed with the extract of unfermented mangosteen fruit peel ( ).

4.3. Conclusions

The results obtained with fermented mangosteen extract are therefore significantly superior to those obtained with non-fermented mangosteen fruit peel extract. These results show that the fermented mangosteen extract according to the invention stimulates the expression of proteins of the ECM or associated with the ECM, such as collagen 1, elastin, MMP2, vinculin, and fibronectin. Thus, these results demonstrate that the fermented mangosteen extract according to the invention can be used to promote and/or reinforce the firmness, density and/or tension of the skin, and/or promote and/or reinforce elasticity. , extensibility and biomechanical resilience of the skin and thus prevent and/or reduce the formation of fine lines and wrinkles.

Example 5 : Antioxidant effect from the extract of m fermented angosteen and from an extract from kombucha , tested alone, or in combination

Objective of the study : This study aims to evaluate the effect of the fermented mangosteen extract according to the invention and an extract of Kombucha Nepal (prepared according to the process described previously in the description) each alone or both in association, on the production of ROS in response to oxidative stress induced by TBHP ( tert -Butyl hydroperoxide ) . This study aims to evaluate whether the fermented mangosteen extract according to the invention has protective antioxidant properties.

5.1. Material and methods

Cell culture and treatment

This study was carried out on normal human keratinocytes (NHK) from an abdominoplasty of a 47-year-old Caucasian woman.

After thawing, the keratinocytes were seeded in passage 4 in 150 cm² flasks (T150) in Epilife medium supplemented with HKGS (human keratinocyte growth supplement) for amplification.

After trypsinization at pre-confluence, the KHNs were seeded at passage 5 in black 96-well plates with a transparent bottom (TC-treated Corning Costar), at a rate of 15,000 cells per well.

After 24 hours of culture, the cells were pretreated or not (control condition) for 24 hours with the active ingredients (tested alone) Kombucha Nepal at 3% (obtained according to the process described previously), FERMENTED MANGOUSTAN at 1% (obtained by the process described in Example 1), or in combination Kombucha Nepal at 3% + FERMENTED MANGOUSTAN at 1% in Epilife medium supplemented with HKGS. Vitamins E and C were included as positive controls in the experimental design, at respective concentrations of 25µM and 100µg/mL.

Determination of reactive oxygen species (ROS) using the H2DCF-DA probe

The Dichlorodihydrofluorescein Diacetate (H ₂ DCF-DA) probe is a permeable and stable probe used for the detection of ROS ( Reactive Oxygen Species ). When used in a cellular application, the acetate groups are cleaved by membrane esterases. The non-fluorescent product Dichlorodihydrofluorescein (H ₂ DCF) is thus obtained. The distribution of this molecule is cytoplasmic and is non-specific to a single type of ROS. When the probe encounters ROS or nitric oxide (NO), it is oxidized into a fluorescent product, Dichlorodifluorescein (DCF), which can be measured with a spectro-fluorometer.

This test highlights the antioxidant power of an active ingredient through its ability to reduce the quantity of ROS produced within the cell, under stressed conditions compared to a corresponding control.

Preparation and incorporation of the H ₂ DCF-DA probe

The H ₂ DCF-DA probe (molecular weight = 487.29 g/mol) should be handled protected from light. After 24 hours of pretreatment with the active ingredients in Epilife medium supplemented with HKGS, the latter was removed and the microplates were rinsed with PBS heated to 37°C. Then 200 µL of the H ₂ DCF-DA probe (Invitrogen), prepared at 25 µM in PBS + Ca ²⁺ + Mg (Gibco) from a 25 mM stock solution in DMSO, was deposited in each of the well. The microplates were placed at 37°C 5% CO ₂ for 30 minutes.

ROS induction

After incubation of the probe, the cells were rinsed with hot PBS before receiving TBHP ( tert -Butyl hydroperoxid ) , which is a chemical compound with strong oxidizing power and generator of ROS. The cells were placed in the presence or absence of TBHP at 200 μM for 30 mins at 37°C 5% CO ₂ , in Epilife medium without HKGS. At the end of the incubation, the microplates were rinsed twice with PBS, then the fluorescence reading was carried out with the FLUOstar BMG Labtech (reading at the bottom, gain = 1500, λex = 485 nm, λem = 520 nm ).

BCA dosage

After reading, the PBS was evacuated and the microplates were frozen at -80°C while awaiting the protein assay carried out 24 hours later, by BCA method. The reagents for the assay were prepared immediately: reagent A (Bicinchoninic Acid from Sigma) and reagent B (Copper Sulfate from Sigma) were mixed at the ratio 1 to 50. The colorimetric assay is based on the reduction of the copper (B) by proteins, making it reactive with the Bicinchoninic acid present (A). The previously obtained ROS fluorescence quantification results (RFU) were then normalized with the quantity of proteins measured in each well (in µg).

Statistical analyzes

Statistical analysis of the results was carried out using Microsoft Excel software. This analysis consists of a calculation of the mean, standard deviation and confidence interval (α = 0.05) of the measured fluorescence (RFU) normalized by the quantity of protein per well (µg). The significance of the differences observed between the control samples and the treated samples is measured by the Student test, which is said to be significant if its value is less than 0.05 (* p<0.05; ** p<0.01; ***p<0.001).

5.2. Results

The results are shown in .

They show the production of ROS in KHN (expressed in units of relative fluorescence) in each of the conditions evaluated, that is to say:

• Non-stressed control,
• Control stressed with TBHP (200µM),
• Control stressed with TBHP (200µM), treated with vitamin E (positive control),
• Control stressed with TBHP (200µM), treated with vitamin C (positive control),
• Condition stressed with TBHP (200µM), treated with fermented mangosteen extract at 1% dilution (as obtained in example 1),
• Condition stressed with TBHP (200µM), treated with kombuchaNepal extract (prepared according to the process described previously) at 3% dilution,
• Condition stressed with TBHP (200µM), treated with the Fermented Mangosteen association (as obtained in Example 1) at 1% dilution + Nepal kombucha (prepared according to the process described previously) at 3% dilution.

The results confirm that TBHP, which is a chemical compound with strong oxidizing power, induces a very strong increase in ROS production in KHN stressed by TBHP with an increase of + 1583%, compared to the non-stressed control.

The results also confirm that vitamin E and vitamin C have antioxidant properties described in the literature (used as positive controls). We observe for vitamin E tested at 25µM a decrease in ROS production -65% and for vitamin C tested at 100µg/mL a decrease in ROS production of -37%, compared to the control stressed with TBHP.

Concerning the extracts used, we observed that the fermented mangosteen extract (tested at 1%) and Kombucha Nepal (tested at 3%), tested alone, did not present an inhibitory effect on the production of ROS.

However, and surprisingly, we note that the combination of these two extracts (fermented mangosteen extract at 1% and Kombucha Nepal at 3%) allows a strong and significant reduction in the production of ROS (-36% compared to the stressed witness).

5.3. Conclusions

These results therefore show that the combination of a fermented mangosteen extract and a kombucha extract has a protective effect equivalent to vitamin C (positive control) against the production of ROS in normal human keratinocytes stressed by TBHP.

Thus, these results demonstrate that the combination of fermented mangosteen and kombucha extracts has strong antioxidant activity.

Example 6: Examples of cosmetic compositions

The fermented extract of mangosteen fruit peels as prepared in Example 1 is formulated in the following illustrative and non-limiting compositions, prepared according to conventional cosmetic formulation methods.

6.1. Composition in the form of a lotion

Ingredients % in weight Glycerin 6.0000 Butylene Glycol 5.0000 Cetyl Alcohol 1.0000 Hydrogenated Polyisobutene 9,000 Fermented mangosteen extract* 0.2000 Perfume, Neutralizers, Preservatives qs Purified water qsp 100%

* prepared according to example 1

Applied to the skin, particularly on the face, this composition provides an anti-aging action with beneficial effects on the firmness and/or elasticity of the skin.

6.2. Composition in the form of a serum

Ingredients (INCI name) % in weight Hydrogenated Polyisobutene 8.0000 Caprylic/Capric Triglyceride 4.0000 Glyceryl Stearate 1.5000 Peg-100 Stearate 2.5000 Cera Alba 1.5000 Cetearyl Isononanoate 1.5000 Cetyl Alcohol 1.0000 Stearyl Alcohol 1.6000 Dimethicone 2.0000 Phenoxyethanol 1.7000 Fermented mangosteen extract* 0.5000 Caprylyl Glycol 0.3000 Xanthan Gum 0.1800 Acrylates/C10-30 Alkyl Acrylate Crosspolymer 0.1500 Sodium Hydroxide 0.0300 Tocopherol 0.0004 Perfume, Neutralizer, Preservative qs Purified water qsp 100%

*prepared according to example 1

The serum is applied to the entire face and particularly to areas showing signs of aging. The serum comprising the fermented extract of mangosteen fruit peels according to the invention promotes the density and elasticity of the skin, for firmer and younger-looking skin.

6.3. Composition in the form of a rich cream

Ingredients (INCI name) % in weight Glycerin 7.5000 Acrylates/C10-30 Alkyl Acrylate Crosspolymer 0.3000 Xanthan gum 0.2000 Cetyl Alcohol/Glyceryl Stearate/PEG-75/Ceteth/Steareth-2 5.2500 C10-C18 Triglycerides 5.0000 Hydrogenated Coco-Glycerides 2.5000 Cetyl Alcohol 1.0000 Hydrogenated Polyisobutene 8.2000 Citric acid 0.0200 Trisodium Citrate 0.1400 Fermented mangosteen extract* 0.1000 Kombucha Nepal 0.0800 Tocopheryl Acetate 0.2000 Perfume, Neutralizer, Preservative Qs Purified water qsp 100%

* prepared according to example 1

Applied to the skin, particularly on the face, alone or following the application of a serum, this composition provides an antioxidant action, promoting the homogeneity and radiance of the complexion.

Claims (17)

Fermented extract of mangosteen fruit peels, comprising:
- a gluconic acid content greater than 50 mg/g, and
- an acetic acid content greater than 200 mg/g,
in weight relative to the weight of dry extract of the fermented extract. Fermented extract of mangosteen fruit peels according to claim 1, characterized in that it is in liquid form and that it comprises at least one preservative, preferably selected from butylene glycol, glycerin, propylene glycol, sodium benzoate, potassium sorbate and propanediol. Process for fermenting mangosteen fruit peels comprising the following steps:
a) the incubation of a consortium of microorganisms in a solution comprising at least water, a carbohydrate and black tea leaves infused at a temperature between 12 to 45°C for 1 to 21 days in order to 'obtain a preparation of symbiotic ferments, said consortium comprising at least one lactic acid bacteria belonging to the genus Lactobacillus or Pediococcus , at least one yeast belonging to the genus Saccharomyces , Schyzosaccharomyces or Torulaspora , and at least one acetic acid bacteria belonging to the genus Acetobacter , Gluconobacter or Komagataeibacter ;
b) grinding mangosteen fruit peels to obtain particles having an average size ranging from 0.5 mm to 1 mm;
c) maceration of the particles obtained in step b) in water in the presence of a carbohydrate;
d) filtration of the maceration mixture to remove plant particles;
e) adding the preparation of symbiotic ferments obtained in step a) to the filtrate of step d) to obtain a fermentation mixture,
f) incubating the fermentation mixture at a temperature between 21 and 35°C for 15 to 25 days;
g) sieving then filtering the fermentation mixture in order to obtain a fermented extract of mangosteen fruit peels, and
h) optionally, the addition of at least one preservative. Process according to claim 3, in which the mangosteen fruit peel particles are added to the maceration medium of step c), at a concentration ranging from 30g to 70g per liter, preferably 40g to 60g per liter and preferably at a concentration of approximately 50 g of particles per liter. Process according to claim 3, in which the carbohydrate is added to the maceration medium of step c), at a concentration ranging from 30g to 70g per liter, preferably 40g to 60g per liter and preferably at a concentration of approximately 50 g of carbohydrate per liter. Fermented extract of mangosteen fruit peels obtained by the process according to any one of claims 3 to 5. Combination of a fermented extract of mangosteen fruit peel according to claim 1, 2 or 6 and a kombucha extract. Cosmetic composition comprising, in a physiologically acceptable medium, a fermented extract of mangosteen fruit peels according to claim 1, 2 or 6. Cosmetic composition according to claim 8, characterized in that it further comprises a kombucha extract. Cosmetic composition according to claim 8 or 9, characterized in that the fermented extract of mangosteen fruit peels is present in a content ranging from 0.0000012% to 0.0825%, preferably from 0.000006% to 0.033% , more preferably from 0.000012% to 0.0165% by weight of active ingredient (dry matter of fermented extract of mangosteen fruit peels) relative to the total weight of said composition. Cosmetic composition according to any one of claims 9 or 10, characterized in that the kombucha extract is present in a content ranging from 0.01% to 10%, in particular from 0.02% to 5%, preferably from 0.05% to 4% by weight of raw material (product weight) relative to the total weight of the composition. Cosmetic composition according to any one of claims 8 to 11, characterized in that it further comprises at least one cosmetic adjuvant chosen from the group consisting of antioxidant agents, emollient agents, moisturizing agents, anti-aging agents , perfumes, and their mixtures. Cosmetic composition according to any one of Claims 8 to 12, characterized in that it is in the form of a milk, a cream, an ointment, a water-in-oil or oil-emulsion. in water or a multiple emulsion, a balm, a stick, a solution, a suspension, gel, a lotion, a serum, or even a powder. Cosmetic process for the care and/or makeup of keratin materials, in particular of the skin and/or lips, comprising the application to said keratin materials of at least one layer of a cosmetic composition as defined in one any of claims 8 to 13. Cosmetic process according to claim 14, characterized in that it is intended to promote and/or reinforce the firmness, density and/or tension of the skin, and/or promote and/or reinforce elasticity, extensibility and the biomechanical resilience of the skin and thus prevent and/or reduce the alteration of the flexibility of the skin and/or the formation of wrinkles and fine lines and/or prevent and/or reduce the signs of skin aging. Cosmetic use of a fermented extract of mangosteen fruit peels as defined according to one of claims 1, 2 or 6 as a cosmetic active ingredient to promote and/or reinforce the firmness, density and/or tension of the skin , and/or promote and/or strengthen the elasticity, extensibility and biomechanical resilience of the skin and thus prevent and/or reduce the alteration of the flexibility of the skin and/or the formation of wrinkles and fine lines and/ or prevent and/or reduce the signs of skin aging. Cosmetic use of at least one fermented extract of mangosteen fruit peels as defined according to one of claims 1, 2 or 6 and of at least one kombucha extract, as a combination of cosmetic active ingredients intended to prevent and /or reduce oxidative stress on skin cells and thus promote and/or stimulate the vitality of skin cells, and/or promote and/or stimulate the homogeneity or radiance of the complexion.