EP4661889A1

EP4661889A1 - Cosmetic use of a combination of plant extracts

Info

Publication number: EP4661889A1
Application number: EP24703423.4A
Authority: EP
Inventors: Corinne SOLÉAU; Dominique Chollet
Original assignee: Crodarom SAS
Current assignee: Crodarom SAS
Priority date: 2023-02-06
Filing date: 2024-01-26
Publication date: 2025-12-17
Also published as: FR3145488A1; WO2024165939A1; CN121013726A

Abstract

The present invention provides the use of a combination of plant extracts comprising at least an extract of Scutellaria, an extract of Glycyrrhiza, an extract of Ziziphus and an extract of Paeonia, or a composition comprising said association and a physiologically acceptable medium, for a topical non-therapeutic cosmetic treatment. The extracts can be obtained by different techniques, preferably by digestion and/or by means of microwaves. The cosmetic treatment can be a soothing treatment, a moisturizing treatment, an anti-aging treatment and/or a slimming treatment.

Description

Cosmetic use of a combination of plant extracts.

The present invention relates to the use of a combination of plant extracts or a composition comprising said combination for a topical cosmetic treatment.
« Cosmetic treatment » means a treatment which treats healthy skin and/or its appendages, said treatment being intended to improve or beautify their appearance and condition. Such treatment has no therapeutic purpose.
The present invention concerns in particular to the cosmetic industries, which manufacture and/or use products intended for the treatment of the skin, including the scalp, mucous membranes and appendages (such as hair, eyelashes, eyebrows, nails) of mammals, animals or humans, for improving their appearance and/or their general condition.
These industries are in increasing demand for new products, in particular in demand for new active ingredients which are derived from plants because they offer a combination of efficacy, limitation of risks of irritation and allergy, reduction of side effects, and biodegradability, with the possibilities of labelling / certification and matching with a logic of sustainable development and/or fair trade.
The skin is the body's first protective barrier against the environment. It is made up of two compartments which interact, one superficial, the epidermis, and the other deeper, the dermis.
The epidermis is divided into a plurality of layers:
- the deeper layer is the basal layer, comprising keratinocytes which migrate from the deepest layer to the most superficial layer, by differentiating themselves into corneocytes.
- the most superficial layer is the stratum corneum or cornified layer consisting of a plurality of keratinocyte layers at the terminal stage of their differentiation and called corneocytes. This highly water-insoluble corneal envelope is composed of anucleate corneal cells, without cytoplasmic organelles and completely keratinised.
The stratum corneum is a semi permeable protective layer that prevents water loss and keeps the skin hydrated. It is composed of the corneocytes, lipids, lamellar bodies, and natural moisturizing factors (NMF - substances capable of binding water in the stratum corneum) which maintain the level of skin moisture at an optimum level.
The trans-epidermal water loss (TEWL) characterises the evaporation of water contained in the skin: the more damaged the skin barrier is, the more easily water evaporates. Dermis provides the epidermis with a solid support. It is also its nourishing element. It consists mainly of fibroblasts and an extracellular matrix composed mostly of collagen and elastin molecules.
The cohesion between the epidermis and dermis is provided by the dermo-epidermal junction (DEJ).
The balance of the skin barrier and mucous membranes is dependent on complex biological mechanisms involving numerous growth factors, hormones, enzymes and mediators within the epidermis and dermis. This balance can be modified, for example, when the skin is attacked. The skin barrier reacts by modifying its composition and can lead to skin reactions of discomfort resulting in sensory phenomena such as tingling, tightness and itching.
Discomfort skin reactions of the face or body, including the scalp, can be induced by contact with chemical products such as cleansers, permanents, hair colouring or result from mechanical actions such as shaving, exfoliation, peeling, hair removal, or originate from the action of temperature, climate, UV, atmospheric pollution, or from xenobiotics such as undesirable microorganisms and allergens, or from internal attacks such as psychological, hormonal stress or sweating.
These aggressions cause the permeabilization of the skin barrier. It becomes deficient in lipids (ceramides) which will modify the organisation of the lipid cement and lead to an alteration of the barrier function thus increasing trans-epidermal water loss (TEWL) and a modification of the natural hydration factors. This results in skin dehydration and also in skin discomfort, which is an unpleasant sensory phenomenon.
All these skin disorders described above appear on the most exposed areas of the body: face, hands and scalp. An amplification these disorders appears with age.
These discomfort feelings appear more particularly in people with dry skin.
Therefore, there is a significant need to prevent a decrease of the skin barrier function and/or to strengthen it, in particular to prevent and/or reduce sensations of skin discomfort, tingling, tightness, heating and itching, or improve the hydration state of the skin.
The aim of the present invention is to provide a cosmetic treatment to beautify and improve the general skin condition, including the scalp and its appendages, and more particularly a treatment to reinforce the skin barrier to impart a more comfortable skin.
« Comfortable skin » means a « normal » skin, which does not present unpleasant sensory effects such as oily skin that shines or dry skin that tingles, feels tight and/or itches.
« More comfortable skin » means a skin that tends to approach a normal skin by reducing these unpleasant sensory effects.
To this aim, the present invention provides the use of a plants extract combination comprising a least a Scutellaria extract, a Glycyrrhiza extract, a Ziziphus extract and a Paeonia extract for a topical non-therapeutic cosmetic treatment.
Advantageously, combined cosmetic effects are obtained by the combination of the four plant extracts, or even synergistic cosmetic effects. Test results are given below in the description.
The four plants are of the genus of Scutellaria, Glycyrrhiza, Ziziphus and Paeonia.
Concerning the Scutellaria genus, the Applicant was particularly interested in the Scutellaria baicalensis species.
Baikal Skullcap (Scutellaria baicalensis) is a plant of the Lamiaceae family, native to eastern Asia. The root is used in Chinese medicine to combat fever and lung and liver conditions.
Concerning the Glycyrrhiza genus, the Applicant was particularly interested in the Glycyrrhiza glabra species.
Liquorice (Glycyrrhiza glabra) also known as Gan Cao is a plant of the Fabaceae family. The root is used in Chinese medicine as a remedy for gastrointestinal problems, coughs, bronchitis and arthritis.
Concerning the Ziziphus genus, the Applicant was particularly interested in the Ziziphus jujuba species.
Jujube (Ziziphus jujuba Mil or Ziziphus vulgaris Lam) is also known under the common name jujube or Chinese date. It is a plant of the Rhamnaceae family. In China, ripe and dried fruits are consumed as tonics with the ability to eliminate diseases and prolong life.
Concerning the Paeonia genus, the Applicant was particularly interested in the Paeonia lactiflora species.
The Chinese peony (Paeonia lactiflora) also called White peony (Paeonia albiflora) is a species of plant in the Paeoniaceae family, native to central and eastern Asia (from eastern Tibet through northern China to east of Siberia). Since ancient times, the Chinese peony has been considered as magical plant and is used to treat skin rashes, cyclical fevers and promote wound healing.
The combination of these four plants is also known in traditional Chinese medicine. It corresponds to the PHY906 prescription called “Huang Qin Tang” which is used to treat intestinal disorders such as diarrhoea, nausea and vomiting. Prescriptions are made by plants decoction then ingested by patients. Nothing in traditional Chinese medicine suggests topical application of the prescriptions.
In the description, the terms « plant combination » and « plant mixture » are use synonymously.
According to preferred features, the plant extracts can be made from the whole plant or from one or more part(s) of the plant such as the leaf, the stem, the flower, the fruit, the seed, the root, the tuber and/or the pericarp.
According to the invention, the extract of Scutellaria, Glycyrrhiza and Paeonia are roots extract and the extract of Ziziphus is a fruit extract. Preferably, the fruit extract is made without the stone.
According to other preferred features, the mixture of said plants is obtained by extraction of each of the plants. Each extract can be obtained by the usual solid/liquid extraction techniques including, for example, maceration, simple decoction, infusion, leaching, extraction under reflux, extraction by subcritical or supercritical fluid, extraction by means of ultrasound or microwaves or any other physical and/or chemical method, such as percolation, digestion, cryoextraction and enzymatic digestion. If the technique of extraction used for several plants is identical, the plants can be extracted together.
« Maceration » means a process consisting of soaking a plant or part of a plant in an extraction solvent at room temperature.
« Infusion » means a process consisting of bringing the extraction solvent to the boil before pouring it onto the plant or part of the plant.
« Decoction » means a process consisting of blending the plant or the part of plant with the extraction solvent, then heating the mixture at the boiling point.
« Digestion » means a process consisting of blending the plant or the part of plant with the extraction solvent, then heating the mixture below boiling point.
Preferably according to the invention, at least one of extract is obtained by digestion or by extraction by means of microwaves.
Preferably, according to the invention, the extract is obtained by digestion in at least one aqueous, alcoholic, glycolic or lipidic solvent, or mixture thereof, such as a C1, C2, C3 or C4 alcohol, or a polyol selected from pentanediol, sorbitol, butylene glycol, pentylene glycol, propylene glycol, hexanol, caprylic/capric triglyceride (GTCC), a vegetable oil, or a mixture thereof. Preferably, the extraction solvent is a mixture of glycolic and aqueous solvent, and more preferably, a mixture of glycerin and water. The digestion duration can vary between 30 minutes and 3 hours, preferably between 1 hour and 2 hours.
According to other preferred features, the extraction by means of microwaves is performed in at least one solvent selected from: water, glycerin, propylene glycol, butylene glycol, polyethylene glycol of any molecular weight, monoethyl ether of diethylene glycol, hexylene glycol, but also ethanol, propanol, butanol and other polyols (pentanediol, hexanols, sorbitol and other liquid sugars) and mixtures thereof. More preferably, the extraction solvent is a mixture of at least one glycolic solvent and at least one aqueous solvent, and more preferably, a mixture of glycerin and water. The extraction duration can vary between 10 and 300 seconds, preferably between 30 and 100 seconds. The power of microwave generators can vary between 100 and 2000 Watts, the microwave processing frequency being limited between 2000 and 3000 MHz.
On the one hand, digestion extraction is adapted for difficult-to-extract materials such as roots requiring high extraction temperatures for a long time period.
On the other hand, microwave extraction is adapted for the softest materials such as fruits and sensitive molecules like vitamins. Furthermore, microwave extraction limits side reactions such as polymerization or the Maillard reaction, and the extraction time is also faster than extraction by digestion. Microwave extraction provides a less coloured, more stable extract with a low ecological impact because it consumes less energy than digestion.
Preferably according to the invention, Scutellaria extract and Glyc yrrhiza extract are obtain by digestion. Preferably according to the invention, Ziziphus extract and Paeonia extract are obtain by microwave extraction to optimize all these parameters. Then, all extracts obtained are mixed together.
According to another aspect of the invention, the ratios of plants extract are between 1: 1: 1: 1 (Scutellaria: Glycyrrhiza: Paeonia: Ziziphus) and 3: 1: 3: 1.
Unexpectedly, the combination of the four plant extracts according to the invention is particularly effective for a cosmetic treatment, in particular a treatment selected from:
- a treatment for soothing skin discomfort sensations, in particular to reduce pro-inflammatory mediators (PGE₂ and IL-6), having the effect of reducing skin redness,
- a moisturizing skin treatment, in particular to reduce insensible water loss via the reinforcement of the skin barrier and to promote hyaluronic acid synthesis,
- a slimming treatment, in particular for an anti-lipogenesis effect, and
- an anti-aging skin treatment, in particular to strengthen the dermo-epidermal junction, reduce glycation and matrix metallo-proteinases (MMPs), increase cell viability, and for an antioxidant and astringent effect.
In vivo , in vitro and self-assessment tests results are given below in the description showing these specific cosmetic activities, beneficial for the skin including the scalp.
With age, the epidermis thickness is reduced, keratinocytes lose their renewal capacity, hyaluronic acid synthesis collapses, and the quality and cohesion of the dermo-epidermal junction decline. Consequently, the skin tissue is thinner and more fragile than young skin and weakens the skin which becomes less firm, less elastic, fragile and less hydrated. The skin can also lose its radiance, show an increase in the pore diameter, or fine lines and wrinkles can appear. A product with an anti-aging activity allows to compensate for these signs of the age, and thus allows to have a skin that is visibly more aesthetic.
According to the invention, the anti-ageing treatment is adapted for firming the skin, making it more supple, more radiant, reducing the pores size, preventing and/or treating wrinkles and fine lines to make them less visible.
The combination of plant extracts can be used as it is or in a composition, diluted in a physiologically acceptable medium. The nature of the medium is defined as a function of the properties of the combination of plant extracts, and also as a function of the destination of the formed composition: a simple ingredient or a more sophisticated galenic form of a final composition for the consumer.
"Physiologically acceptable medium" means according to the present invention, without limitation, an aqueous or hydro-alcoholic solution, a water-in-oil emulsion, an oil-in-water emulsion, a micro-emulsion, an aqueous gel, an anhydrous gel, a serum, a dispersion of vesicles, or a powder.
"Physiologically acceptable" means that the compositions are suitable for topical or transdermal use, in contact with mucous membranes, appendages (nails, hair and body hair), scalp and skin of mammals, particularly human, compositions which may be ingested, or injected into the skin, without risk of toxicity, incompatibility, instability, allergic response, and others. This "physiologically acceptable medium" forms what is commonly called the excipient of the composition.
According to the invention, the physiologically acceptable medium can be an aqueous, hydroglycolic or hydroalcoholic solution, or a water-in-oil emulsion, an oil-in-water emulsion, or a microemulsion. Preferably, it is hydroglycolic. Preferably, the physiologically acceptable medium is a mixture of glycerin and water.
Therefore, the present invention covers a non-therapeutic topical cosmetic treatment adapted for beautifying or improving the appearance and general condition of skin and/or its appendages by reducing pro-inflammatory mediators, trans-epidermal water loss, glycation, matrix-metallo-proteinases (MMPs), oxidation and/or lipogenesis, by increasing hyaluronic acid synthesis and astringency, by strengthening the dermo-epidermal junction, comprising the topical application to the skin of a subject in need thereof of an effective amount of the combination of plant extracts according to the invention or a composition comprising it in a physiologically acceptable medium.
The effective amount of the combination of extracts according to the invention, that is to say its dosage, depends on the destination of the composition. It depends on various factors, such as the age, the condition of the patient, the severity of the disorder and the administration mode. An effective amount means a non-toxic amount enough to achieve the desired effect.
In a cosmetic composition according to the invention containing the combination of extracts according to the invention, to be present in an effective amount, it is generally present in an amount ranging from 0.000001% and 15% based on the total weight of the composition, preferably ranging from 0.00001% and 10%, depending on the destination of the composition and the more or less pronounced desired effect. More preferably, the effective amount is between 0.0001% and 5% based on the total weight of the composition.
All percentages and ratios used herein are by weight of the total composition and all measurements are made at 25°C unless it is otherwise specified.
For indication, for a cosmetic face treatment, the European standard dosage of a cream is 2.72 mg/cm²/day/person and for a cosmetic body treatment the European standard dosage of a lotion is 0.5 mg/cm²/day/person.
According to other features, the cosmetic treatment method according to the invention can be combined with one or more other treatment methods targeting the skin such as luminotherapy, heat or aromatherapy treatments.
According to the invention, devices with several compartments or kits may be proposed to apply the treatment described above which may include for example and non-restrictively, a first compartment containing the combination of plant extracts of the invention, and in a second compartment an additional active ingredient, the compositions contained in the said first and second compartments in this case being considered to be a combination composition for simultaneous, separate or stepwise use in time, particularly in one of the treatment methods recited above.
According to other advantageous features, the combination of extracts according to the invention may be associated with one or more other active ingredients at effective concentrations that can act synergistically or additionally for reinforcing and achieving the desired effects described for the invention, such as the following agents: filtering radiations, in particular UVA, UVB, IR or generated by blue light, hydrating, moisturizing, humectant, calming, muscle relaxant, slimming, restructuring, firming, replumping, lifting, smoothing, acting on blood microcirculation, inflammation, free radicals, anti-aging, anti-fine lines and wrinkles, lightening, acting on complexion, anti-glycation, anti-carbonylation, pro- pigmenting, acting on stratum corneum, on dermal -epidermal junction, on firmness, elasticity and tone of skin, on hair growth or anti-regrowth (including eyelashes and eyebrows), on eye contours (dark circles and under eye bags), peptides, vitamins, etc. These active ingredients can be obtained from plant materials, such as plant extracts or products of plant cell culture or fermentation production methods.
The Personal Care Products Council ("International cosmetic ingredient dictionary & handbook" published by the "Cosmetic, Toiletry, and Fragrance Association, Inc.", Washington, D.C.) describes a non-limited wide variety of cosmetic and pharmaceutical ingredients conventionally used in the skin care industry that can be used as additional ingredients in the compositions for the present invention, as long as they are physically and chemically compatible with the other ingredients of the composition and especially with the active ingredients of the present invention. Also, the nature of these additional ingredients should not unacceptably alter the benefits of the active ingredient of the invention. These additional ingredients can be synthetic or natural such as plants extracts or issued from a bio-fermentation process.
Further skin care actives that are particularly useful combined with the composition can be found in the commercial literature of Croda, Crodarom, Alban Muller international and Sederma, and on the website www.croda.fr.
More specifically, the combination of extracts according to the invention can be combined with at least one compound chosen among vitamin compounds, group B C, E, F, D and A, especially compounds as niacinamide or tocopherol, retinoids compounds as retinol, hyaluronic acid, hexamidine, α-lipoic acid, resveratrol or DHEA, peptides, aluminium salts, phosphate salts, magnesium oxide, zirconium hydrochloride salts, ceramides, which are classic active ingredients used in topical cosmetic or dermo-pharmaceutical compositions.
Furthermore, the present invention provides the use of said combination of plant extracts according to the invention, as described above, for the manufacture of a composition for a cosmetic treatment, as described above.
The composition for the use according to the invention can be provided in any galenic form (examples are given below) defined according to the composition destination and application site.
A composition according to the invention can be applied to the face, body, neckline, scalp, hair, eyelashes, body hair, in any form or vehicle known to those skilled in the art, in particular in the form of a solution, dispersion, emulsion, paste or powder, individually or as a premix or vehicle individually or as a premix in a bound form, incorporated or adsorbed in vectors such as macro-, micro-, or nanocapsules, macro-, micro- or , nanospheres, liposomes, oleosomes or chylomicrons, macro-, micro-, or nanoparticles or macro-, micro or nanosponges, micro- or nanoemulsions, or adsorbed on organic polymer powders, talcs, bentonites, spores or exines, and other inorganic or organic supports.
In cosmetics, applications can be proposed in particular in the ranges of skin care for the face, body, hair and body hair and ranges of make-up treatments, in particular eyelashes and eyebrows.
For example, the galenic form of the composition can be a lotion, a cream, a butter, a milk, a solid form, a foam, a gel, a deodorant, an antiperspirant, a shampoo, a conditioner, a hair mask, a face mask, a shower gel, etc.
The galenical formulations can enter in different product ranges for personal care and/or beauty products including skin care, cleaning, makeup, cleansing, sunscreen, artificial tanning, pre-shave, shaving or aftershave, moisturizer, humectant, emollient, conditioning, exfoliating, astringent, depilatories or antiperspirant, deodorant, etc.
The composition may be incorporated onto a non-woven or woven material, with natural or synthetic fibres, wool, or any material intended to come into contact with skin and that can be used in clothing, including tights and socks, shorty, day or night underwear, tissues, handkerchiefs or fabric to exert its cosmetic effect via the contact skin/textile and enable continuous topical delivery (cosmetic-textiles).According to the invention, it is thus also provided a woven or non-woven fabric comprising at least one a combination of extracts according to the invention as describe above, for use in a non-therapeutical cosmetic treatment.

DETAILED DESCRIPTION

The present invention will be better understood in the light of the following description of embodiments and studies described below.

1. Examples of obtaining a combination of plant extracts according to the invention and a composition comprising it

1.1. Example of preparation of a combination of plant extracts according to the invention

After harvesting, the roots of liquorice, skullcap and peony are dried and cut into pieces of 1 to 5 millimetres and the pitted fruits of Chinese dates are dried before cutting.

On the one hand, liquorice roots and skullcap roots are extracted together for 2 hours by digestion in a water/glycerin mixture (in a ratio between 100:0 and 20:80) at a temperature around 80°C. After cooling, the extract obtained is centrifuged and the supernatant is recovered.

On the other hand, Chinese date fruits and peony roots are extracted together at 2000 Watts by means of microwaving in a water/glycerin mixture (in a ratio between 100:0 and 20:80) at a temperature around 80°C.

After cooling, the extract obtained is centrifuged. The supernatant is collected and mixed with the first supernatant obtained by digestion described above. The whole is filtered. Thus, the said combination of plant extracts according to the invention is obtained. The mixture can be filtered if necessary.

The ratio of each plant is approximately 1:1:1:1. The total percentage by weight of dry plants is between 2 and 5%.

1.2. Example of the preparation of a cosmetic composition containing the combination of plant extracts according to the invention, said composition forming an active ingredient.

The combination of the four plant extracts obtained in point 1.1. above, is mixed with a water/glycerin mixture (in a ratio between 100:0 and 20:80), as well as preservative additives.

This composition constitutes the active ingredient according to the invention.

pH can be adjusted as needed, preferably between 2 and 8, more preferably between 4 and 5.5. This ingredient can be filtered again to ensure sterilisation.

2. Example of galenic formulation

A cosmetic formulation is described below, using an active ingredient according to the invention as described above at point 1.2. Additional active ingredients, acting in support and/or in addition to the activity of the combination of extracts according to the invention may be added in the appropriate phase according to their hydrophobic, hydrophilic or amphiphilic nature. These ingredients can be of any category according to their function(s), the place of application (scalp, face, body, neck, bust, hands, etc.), the desired end effect and the targeted consumer, for example specific anti-ageing, anti-wrinkle, moisturizing, anti-dark circles, firming, anti-glycation, slimming, volumizing, soothing, myo-relaxing, anti-redness, sensitive skin, anti-stretch marks, detoxifying, mattifying etc.

The ingredient according to the invention is recommended in a composition at a level of 1 to 5%, preferably 3%. These levels can vary without departing from the scope of the present invention, depending on the more or less pronounced effects sought.

Example: Cream form

Raw materials	%	INCI name
Phase A
Water	qs.	Aqua / water
Carbomer	0.2	Carbomer
Phase B
Arlacel™ 170	1.0	Glyceryl Stearate (and) PEG-100 Stearate
Crodacol™ CS90-PA-(RB)	1.5	Cetearyl alcohol (and) Dicetyl Phosphate (and) Ceteth-10 Phosphate
Crodamol™ GTCC	3.0	Caprylic/Capric Triglyceride
Phase C
Crodamol™ AB-LQ-(RB)	1.5	C12-15 Alkyl benzoate
Pemulen™ TR2	0.2	C10-30 Alkyl Acrylate Crosspolymer
Phase D
Glycerol	2.0	Glycerine
Caprylyl glycol	0.5	Caprylyl glycol
Phase E
Phenoxyethanol	0.8	Phenoxyethanol
Phase F
Potassium sorbate	0.2	Potassium sorbate
Phase G
Water	4.0	Aqua / Water
NaOH 35%	0.4	Sodium hydroxide
Phase D
Ingredient according to the invention	3.0

Protocol:

Swell the carbomer in water (part A), then heat. Weigh and heat B. Weigh C. Weigh and heat D, homogenize and let cool. Add E to D, then add D+E to A with stirring, mix. Add B to A+D+E, then add C. Homogenize. Add F to A+B+C+D+E. Add G to obtain a pH between 5 and 6.5. Add H to A+B+C+D+E+F+G and homogenize.

3. Evaluation of the different activities of the combination of plant extracts according to the invention by in vitro tests

The in vitro tests below were carried out on equivalents of the active ingredient according to the invention titrated at 0.69% by weight of dry extract (hereinafter called “active ingredient according to the invention”) at 1 %, 3%, 3.2% and 10% of this active ingredient. These doses correspond to those at which the active ingredient according to the invention will be recommended for a cosmetic treatment according to the invention.

3.1. Skin soothing 3.1.1. Pro-inflammatory mediators

Inflammation mediators such as PGE₂ and IL-6 are very present in micro-inflammation phenomena. Reducing the presence of these mediators has the effect of reducing feelings of discomfort in sensitive skin and redness.

Furthermore, PGE2 decreases the expression of collagen I and increases the expression of MMP1, and IL-6 modulates the migration and activity of MMPs in dermal fibroblasts.

Protocol

Normal Human Dermal Keratinocytes are cultured at confluence. At this stage, they are contacted with the ingredient according to the invention for 24 hours. After this contact period, the cells are irradiated with UVB, and the cells are then contacted with the active ingredient of the invention for another 24-hour period. The quantities of PGE₂ and IL-6 synthesized are measured in the culture supernatants by ELISA assay. The number of cells is assessed to normalize the data. An analysis of variance and a Student t-test for non-paired series are performed to evaluate the significance of the results.

Results

Variation of IL-6 and PGE2 production by keratinocytes irradiated with UVB. Effect of the equivalent of 3% of the active ingredient according to the invention compared to the control irradiated with UVB:

	IL-6 (pg/10⁶ cell)	PGE₂ (pg/10⁶ cell)
Control	Reference	Reference
Variation (%); significance	- 56%; p<0 . 01	- 95%; p<0 . 0 1

Thus, advantageously, the active ingredient according to the invention strongly and significantly reduces both pro-inflammatory messengers, protecting the skin against major aggressions, reducing skin discomfort sensations and redness.

3.2. Skin hydration 3.2.1. Hyaluronic acid synthesis

Hyaluronic acid is a major constituent of the epidermis contributing to the barrier function as well as to the maintaining of a satisfactory hydration of the skin. It is capable of absorbing 1,000 times its own weight in water. It is in the form of an aqueous and nourishing gel which fills the spaces between the keratinocytes. It prevents from dryness of the skin, which is known to alter the texture of the skin, giving it a coarse and rough touch.

Protocol

Human keratinocytes are cultured at sub-confluence and then contacted or not (for the control cases) with the active ingredient according to the invention. After this contact, the synthesis of hyaluronic acid is assayed using ELISA methods. The cell concentration is estimated using the Hoechst method.

Results

Variation of hyaluronic acid production by keratinocytes. Effect of the equivalent of 3% of the active ingredient according to the invention at 0.26% compared to the control (n=5):

	Variation (%); significance
Control	Reference
3% of the ingredient according to the invention	+ 64%; p<0 . 01

These results show that the ingredient according to the invention significantly increases hyaluronic acid synthesis by keratinocytes, thus participating to the barrier function of the skin and its hydration.

3.3. Anti-ageing 3.3.1. Cell viability

Cell viability refers to the number of healthy cells relative to unhealthy cells in a population of cells. With age, the epidermis cells are fewer in number and renew more slowly than in young skin, leading to a reduction in the epidermis and the stratum corneum thickness.

Protocol

Keratinocytes are contacted with the active ingredient according to the invention for 24 hours and 72 hours. Cell viability is assessed by the neutral red absorption test. Optical density is measured at 540 nm.

Results

Variation of cell viability by keratinocytes. Effect of the equivalent of 3.2% and 10% of the active ingredient according to the invention compared to the control (n=6):

	Control	3.2% of the ingredient according to the invention	10% of the ingredient according to the invention
Optical density	0.676 ± 0.014	0.712 ± 0.019	0.744 ± 0.023
Variation (%); significance	Reference	+ 5.4%; p<0 . 01	+ 10.1%; p<0 . 01

These results show that the ingredient according to the invention significantly increases the cell viability. Therefore, the ingredient according to the invention can fight against skin aging linked to a reduction in the number of healthy cells.

3.3.2. Strengthening the dermo-epidermal junction (DEJ)

DEJ assures the cohesion between the dermis and the epidermis. During aging, a decrease of synthesis of its components (especially collagen VII and laminin) is observed. The aging of the DEJ has significant repercussions on the resilience of the skin and the loss of its dynamism.

Protocol

Human keratinocytes (HK) are cultured at sub-confluence and are brought into contact or not (for the control cases) with the ingredient according to the invention. After this contact, the syntheses of collagen VII and laminins are evaluated using ELISA type kits. The number of cells is estimated using the Hoechst method and used to standardize the results.

Results

Variation of collagen VII (n=5) and laminins (n=5) production by keratinocytes. Effect of the equivalent of 3% the active ingredient according to the invention compared to the control:

	Laminins (µg / mL / 10⁶ cell.)	Collagen VII (µg / mL / 10⁶ cell.)
Control	Reference	Reference
Variation (%); significance	+118%; p<0 . 01	+ 62 %; p<0 . 01

These results show that the active ingredient according to the invention significantly increases the production of collagen VII (+62%; p<0 . 01) and laminins (+118%; p<0 . 01) by keratinocytes. Therefore, it has a direct action of DEJ reinforcement by stimulating these syntheses.

The ingredient according to the invention can improve the skin aging linked to a DEJ disorganization, by counteracting the loss of flexibility and elasticity that it causes.

3.3.3. Non-enzymatic glycation of BSA

Glycation of proteins with reducing sugars in the skin is also responsible for skin aging.

Indeed, the formation of many glycated proteins whose functional, enzymatic, structural properties are altered, has consequences on the correct functioning of the cell or the organism.

Mechanical and elastic properties of the dermal extracellular matrix are impaired, the dermis becoming less flexible, more rigid, but also more flaccid and less reactive, also resulting in a dull complexion.

Protocol

The study of the non-enzymatic glycation is done between a model protein, the serum albumin, which serves as a target, and an edible reducing sugar from fruits. The protein is gradually glycated (bound to sugar) in an irreversible manner, in the presence or not of the active ingredient according to the invention. This change is followed by fluorescence.

Results

Variation of glycation (n=4). Effect of the equivalent of 3% of the active ingredient according to the compared to the control:

	Non-enzymatic glycation (Average variation in fluorescence unit)	Variation (%); significance
Control	77340 ± 819	Reference
3 % of the active ingredient according to the invention	9989 ± 320	- 87 %; p<0.01

The results show the strong anti-glycant potential of the active ingredient according to the invention that can fight against skin aging.

3.3.4. MMP1

Matrix-metallo-proteinases (MMPs) are enzymes responsible of the cellular waste elimination (keratinocytes and fibroblasts). Under the influence of external aggressors, such as UV rays, wind, cold or pollution, these MMPs become more active and attack the dermal extracellular matrix (ECM). In the ECM, there are collagen and elastin fibres which are responsible for the tone of the blood micro-capillaries. If the ECM is weakened, the micro-capillaries lose their tone, dilate, and become visible on the surface and create an unsightly red effect.

Protocol

Keratinocytes are contacted with the active ingredient according to the invention for 24 hours, and then are radiated with UVB to trigger an experimental increase in MMP1 synthesis. The cells received again the active ingredient according to the invention for 48 hours and the supernatants are assayed using ELISA tests. The number of cells is estimated using the Hoechst method and used to standardize the results.

Results

Variation of MMP1 synthesis in UVB-irradiated keratinocytes. Effect of the equivalent of 1% of the active ingredient according to the invention compared to the control (n=3):

	MMP1 (ng/mg cellular protein)	Variation (%); significance
Irradiated control	61.79 ± 0.74	Reference
1% of the extract according to the invention	27.32 ± 1.56	- 85%; p<0 . 01

These results show that the active ingredient according to the invention significantly decreases the MMP1 synthesis induced by an UVB stress. This inhibition of MMP1 allows strengthening of the ECM.

3.3.5. Antioxidant capacity

Oxidative stress plays a central role in the cutaneous response to various stresses. Free radicals (H₂O₂, OH•, O₂-, O₂, ¹O₂ …) lead to protein, lipid, and DNA damages, causing premature aging of the skin.

Protocol

Lipid peroxidation is evaluated by using a test to monitor the oxidation appearance (measurement of the quantity of conjugated dienes formed) of lipid membranes in the form of liposomes after oxidizing UVA radiation. Reduction by an antioxidant is sought. In this test, the active ingredient according to the invention is introduced after irradiation.

Results

Variation of the lipid peroxidation production (n=2). Effect of the equivalent of 3% of the active ingredient according to the invention compared to the control:

	Lipid peroxidation
Control	Reference
Variation (%); significance	- 67 %; p<0.01

The results show that the ingredient according to the invention significantly reduces lipid peroxidation with respect to induced UVA pro-oxidant stress. Therefore, the active ingredient according to the invention has a strong antioxidant capacity to effectively fight against premature aging of the skin.

3.3.6. Astringency

With age, the pore size in the skin increases and pores become visible. A product with astringency activity therefore can compensate for this increase and make the skin visibly more aesthetic.

Principle

The in tubo assay for determining the astringency capacity of a product is based upon tannin-polymer interaction resulting in the formation of insoluble polymer tannin complexes which then precipitate. The difference in optical density (OD) before and after precipitation corresponds to the astringency capacity of the product.

Protocol

The active ingredient according to the invention on the one hand, and red wine on the other, are incubated with and without methylcellulose and the optical density at 280nm is measured by spectrophotometer. The precipitated part of the extract corresponds to the astringent tannin fraction.

Results

Astringency of the active ingredient according to the invention (n=4) compared to red wine (n=12):

	Means tannins precipitated with methylcellulose (MCP) (equivalent epicatechin ppm)	% Variation
Tannic red wine	1334 ± 222	Reference
Extract according to the invention	1357 ± 107	+1.7 %; nsd*

* n s d : non-significant data.

Tannic red wine is considered as an astringent positive control. Its astringency is of 1334ppm. The active ingredient according to the invention shows an astringent capacity equivalent to that of tannic red wine.

3.4. Anti-lipogenesis effect

Glycerol-3-Phosphate Dehydrogenase (G3PDH) is a fat storage enzyme whose expression increases strongly during the differentiation of pre-adipocyte fibroblasts into adipocytes. Its inhibition will reduce therefore the quantity of adipocytes and therefore the adipose tissue.

Protocol

Mature human adipocytes are formed from an immature state using a hormonal cocktail. A series receives the active ingredient according to the invention during this phase. The increase in adipose reserves is compared to control cases visually and by measuring the activity of the fat storage enzyme (G3PDH). A viability test is carried out in parallel.

Results

Variation of G3PDH activity in adipocytes in the way of differentiating (n=3). Effect of the equivalent of 3% of the active ingredient according to the invention compared to control:

	Variation (%); significance
Control	Reference
0.66% of the active ingredient according to the invention	- 65%; p<0.01

These results show that the active ingredient according to the invention slows down the differentiation of the adipocytes significantly.

4. Evaluation of the different activities of the combination of plant extracts according to the invention by in vivo tests

Tested product

The cream described in point 2. above vs. a placebo cream, i.e. same cream as of point 2. without the active ingredient according to the invention.

Principle and protocol of in vivo tests

Surfactants are the main constituents of detergents. They cause significant damage to the lipid and protein structures of the stratum corneum because they modulate surface tension. An example of a surfactant is the Sodium Lauryl Sulfate (SLS) which is an anionic surfactant commonly used in detergent products such as laundry detergent, shampoo, and bubble bath.

SLS alters the barrier property and the permeability of the skin, increases the blood flow, and induces the feeling of dryness and roughness. Additionally, SLS increases the production of inflammatory mediators such as PGE2 and IL-6.

The test was conducted in an Asian city known to be polluted. Air quality can be assessed using the Air Quality Index (AQI) which is based on the levels of five pollutants which are ozone, particulate matter, carbon monoxide, sulfur dioxide and nitrogen dioxide.

When the AQI is between 101 and 150, the air quality is slightly polluted and the most sensitive people (people with lung or heart disease, children, and older adults) may experience health effects.

In the city where the tests were conducted, the average AQI was 124 during the study period.

Protocol

The aim of this study is to evaluate the soothing effect of the cream according to the invention on the skin barrier and on skin redness.

Specific inclusion criteria

The study was carried out on a first panel of 24 healthy adult subjects, 6 men and 18 women with an average age of 49 years (between 29 and 60 years).The following criteria also had to be respected for the recruited panel: being an Asian man or woman, being between 18 and 60 years old, having sensitive and dry skin on the forearms and responding positively to the SLS patch, i.e. the trans-epidermal water loss should increase after removal of the SLS patch.

Type of study, duration, applications

First, a patch containing 1% SLS was applied to both forearms of each volunteer. D-1 corresponds to the day when the patch with 1% SLS is applied.

After 24 hours, the patch is removed, and the skin is rinsed. Applying this patch with 1% SLS alters the skin barrier. D0 corresponds to the day when the patch with 1% SLS is removed. Then, for 8 days, from D0 to D7, the volunteers applied to the forearms, twice a day, the cream containing the ingredient according to the invention and the placebo cream contralaterally. The first application is made one hour after removing the patch.

Statistics

For the quantification of the redness, statistical studies were performed using the Student t test for parametric values.

For the questionnaire values, statistical studies were performed using the Khi² tests. The level of 0.05 is selected as the minimum acceptance point for statistical significance.

4.1. Skin soothing

The spectrophotometer converts the colours perceived by humans into a digital code. The a* parameter allows the study of the spectrum from green to red. A decrease of the a* parameter over time indicates a reduction in skin redness.

Protocol

Skin colorimetric measurements were carried out on the treated areas on the forearms with a MINOLTATM CM700-d Spectrophotometer before application of the SLS patch, one hour after removal it and after 4 days. Each measurement is an average of three acquisitions on the same skin area. The mean value of the measurements was calculated for each subject.

Results

The values measured on D-1 are subtracted from all the results to obtain results relating to the induction of redness while eliminating the initial conditions, i.e., the skin natural red tint.

Variation of the redness (a* parameter) (N=24). Effect of the cream according to the invention.

a* (arbitrary units)	Cream according to the invention		Placebo cream
	D0	D+4	D0	D+4
Mean ± Standard deviation	3.39 ± 0.19	2.86 ± 0.24	3.71 ± 0,23	3.42 ± 0.37
Variation (%); significance vs. T0	- 16%; p<0 . 05		- 8%; n sd
Significance vs. Placebo	- 8%; n sd

The skin redness increase observed after removal of the skin barrier by the SLS patch was reduced by 16% (p<0.05 vs. T0) after 4 days use of the cream according to the invention. On the same time, application of the placebo cream only resulted in a non-significant and more moderate increase of 8% (n sd vs. T0).

Therefore, the results show a significant reduction of skin redness with the cream according to the invention.

4.2. Skin hydration

When the skin barrier is damaged, for example by the SLS patch, the trans-epidermal water loss (TEWL) increases considerably, reflecting damage to the skin barrier. Applications of soothing care products can rebuild the skin barrier integrity until it returns to its initial state by reducing the TEWL.

Protocol

The effect of the cream according to the invention on the skin barrier is compared to the values obtained one hour after removal of the SLS patch (D0) and after 8 days (D+7).

Skin barrier measurements are carried out using the Tewameter^TM 300® from Courage & Khazaka.

Results

Variation of TEWL differences observed after SLS patch removal (N=23). Effect of the cream according to the invention compared to the placebo cream.

TWEL (g/h/m²)	Cream according to the invention			Placebo cream
TWEL (g/h/m²)	D-1	D0	D+7	D-1	D0	D+7
Mean ± Standard deviation	7.94 ± 0.28	16.90 ± 0.61	9.58 ± 0.45	8.12 ± 0.28	16.61 ± 0.46	10.26 ± 0.37
Variation (%); significance vs. T0	/	- 82%; p<0.01		/	- 75%; p<0.01
Significance vs. Placebo	/	- 7%; p<0.05		/

The TEWL increase observed after removal of the skin barrier by the SLS patch is reduced by 82% (p<0.01 vs. T0) after 8 days of use of the cream according to the invention. The latter promotes better resistance of the skin barrier to stress, mimicking the use of conventional detergents on the skin.

At the same time, the application of the placebo cream leads to a more moderate strengthening of 75% (p<0.01 vs. T0).

4.3. Self-assessment test of the effectiveness of the cream according to the invention

Protocol

After 8 days of the cream according to the invention application, 7 questions on the effectiveness of the cream according to the invention were asked to the volunteers. The answer could be “totally agree”, “agree”, “disagree”, “strongly disagree” or “not applicable”.

Results

Results are expressed as a percentage of volunteers who responded “totally agree” or “agree”.

	Cream according to the invention		Placebo cream		Cream according to the invention vs. placebo cream
	Totally agree	Agree	Totally agree	Agree	Totally agree	Agree
My skin is less tight	42%	54%	29%	67%	+13%; n s d	- 13%; n s d
My skin is less stinging	50%	46%	29%	63%	+21%; n s d	-17%; n s d
My skin is less burning	54%	46%	33%	58%	+21%; n s d	-13%; n s d
My skin is less tingling	46%	46%	21%	67%	+25%; p<0 . 07	-21%; n s d
My skin is less itchy	67%	29%	29%	63%	+38%; p<0 . 01	-33%; p<0 . 05
My skin feels more comfortable	79%	17%	33%	63%	+46%; p<0 . 01	-46%; p<0 . 01
The treatment improves visually the quality of my skin	75%	17%	21%	75%	+54%; p<0 . 01	-58%; p<0 . 01

All answers to the asked questions show an improvement of the skin condition. Generally, the volunteers present a higher satisfaction rate of the cream according to the invention (mostly “totally agree”) compared to the placebo cream (mostly “agree”). There are statistically more volunteers who “totally agree” that the cream according to the invention is more effective than the placebo on tingling (+25%; p<0.07), itching (+38%; p<0.01), skin comfort (+46%; p<0.01) and visually improve skin (+54%; p<0.01).

Claims

Use of a combination of plant extracts comprising at least an extract of Scutellaria, an extract of Glycyrrhiza, an extract of Ziziphus and an extract of Paeonia, or of a composition comprising said association and a physiologically acceptable medium, for a topical non-therapeutic cosmetic treatment.
Use according to claim 1, wherein the Scutellaria extract is an extract of Scutellaria baicalensis and/or the Glycyrrhiza extract is an extract of Glycyrrhiza glabra and/or the Ziziphus extract is an extract of Ziziphus jujuba and/or Paeonia extract is an extract of Paeonia lactiflora.
Use according to claim 1 or 2, wherein the extracts of Scutellaria, Glycyrrhiza and Paeonia are root extracts.
Use according to claim 1 or 2, wherein the extract Ziziphus is a fruit extract.
Use according to anyone of the preceding claims, wherein at least one of the plant extracts is obtained by digestion.
Use according to anyone of the preceding claims, wherein at least one of the plant extract is obtained by means of microwaves.
Use according to claim 5, wherein the Scutellaria and Glycyrrhiza extracts are obtained by digestion.
Use according to claim 5, wherein the Ziziphus and Paeonia extracts are obtained by means of microwaves.
Use according to any one of the preceding claims for a cosmetic treatment selected from:
A soothing treatment; and/or

A moisturizing treatment; and/or

An anti-ageing treatment; and/or

A slimming treatment.
Use according to claim 9, wherein the anti-aging treatment is adapted to firm the skin, make it more flexible, more radiant, to reduce pore size, to prevent and/or treat wrinkles and fine lines to make them less visible.