FR3069161B1 - ROTARY POLYGONUM MULTIFLORUM EXTRACTS AND USES THEREOF - Google Patents

Abstract

The present invention relates to a root extract of Polygonum multiflorum concentrated in THSG comprising no or very little emodin, a process for preparing such an extract and a cosmetic composition and a pharmaceutical or nutraceutical composition, said compositions comprising active agent at least one root extract of Polygonum multiflorum according to the invention.

Description

ROTARY POLYGONUM MULTIFLORUM EXTRACTS AND USES THEREOF

FIELD OF THE INVENTION

The present invention is in the field of plant extracts and relates to a root extract of Polygonum multiflorum concentrated in THSG with no or very little emodin, a process for preparing such an extract and a cosmetic composition and a pharmaceutical composition or nutraceutical, said compositions comprising as active agent at least one root extract of Polygonum multiflorum according to the invention.

PRIOR ART

Polygonum multiflorum Thunb., A herbaceous plant of the Polygonaceae family, is one of the most popular plants of traditional Chinese medicine. Its dried root called Polygoni Multiflori Padix (RPM, Heshouwu in Chinese) is used in many preparations. In raw form it is used for many purposes including to help digestion (purgative).

Classically, the roots of Polygonum multiflorum are steamed in black soy juice and dried. In this form, Radix Polygoni Multiflori Praeparata (RPMP, Zhiheshouwu in Chinese) is used in particular to detoxify and activate circulation in the liver and kidneys. Recently Polygoni multiflori Radix has been studied for its pharmacological potential. The extract has immunological, anti-cancer, anti-inflammatory, neuroprotective and anti-hyperlipidemic effects

In the cosmetic field, extracts of RPM or RPMP having a protective effect against oxidative stress, are used to prevent the progression of hair whitening or in anti-aging cosmetic compositions. For example, Application KR20150143375 discloses a cosmetic composition containing a Polygonum multiflorum adventitious root extract, exhibiting antioxidant activity, inhibition of collagenase expression, inhibition of elastase activities, and an anti-wrinkle effect. Application KR20160123433 describes a cosmetic composition containing a dried root extract of Polygonum multiflorum has antioxidant and anti-wrinkle properties but also skin whitening (tyrorinase inhibitory activity), anti-inflammatory and anti-atopic functions.

Many compounds have been identified within the Polygonum multiflorum species. These are mostly anthraquinones, stilbenes, flavonoids, among which are 2,3,5,4'-tetrahydroxystilbene-2-0-β-D-glucoside (THSG), emodin and physcion ( Traditional uses, botany, phytochemistry, pharmacology and toxicology of Polygonum multiflorum Thunb .: Lin L, Ni B, Lin H, Zhang M, Li X, Yin X, Qu C, Ni JJ Ethnopharmacol 2015Jan 15; 159: 158 -83).

THSG is supposed to be responsible for the activity of Polygoni Multiflori Radix. It has been identified as having anti-inflammatory and antioxidant activities, prophylactic and therapeutic properties against Alzheimer's disease (Lin et al., 2015). It also acts in the prevention and treatment of hair whitening (W02016 / 101259).

Although Polygonum multiflorum extracts are usually used to treat various liver diseases, a growing number of cases of hepatotoxicity potentially induced by their use has been reported. Studies have shown that quinones and especially emodin can lead to hepatotoxicity. Recently, a toxicological study based on an in silico approach to the analysis of the molecular mechanisms of hepatotoxicity induced by Polygonum multiflorum has shown that emodin is clearly associated with this hepatotoxicity ("Insights into the molecular mechanisms of Polygonum multiflorum"). Thunb-induced liver injury: a Computational Systems Toxicology Approach "Wang YY, Li J, Wu ZR, Zhang B, HB Yang, Wang Q, Cai YC, Liu GX, WH Li, Tang Y. Acta Pharmacol Sin. 27, 719-732.)

According to the EU Inventory of Cosmetic Ingredients, anthraquinones, like emodin, are considered part of the undesirable compounds of plants.

The chemical constituents of Polygonum multiflorum change during treatment of the extract. Thus, the hepatotoxicity of Polygoni Multiflori Radix (RPM) is stronger than that of Radix Polygoni Multiflori Praeparata (RPMP). However, the content of emodin increases with the treatment of Polygonum multiflorum extract (Toxicity of raw and processed roots of Polygonum multiflorum; Wu X, Chen X, Huang Q, Fang D, Li G, Zhang G., Fitoterapia, 2012 Apr ; 83 (3): 469-75). Since emodin is present in the Polygonum multiflorum root, it is therefore necessary to develop a method that allows the selective extraction of THSG or at least one that makes it possible to obtain an extract free of emodin, or comprising emodin in very small quantities.

Techniques for the selective extraction of polyhydroxystilbene, including resveratrol and THSG, have been developed but remain complicated and costly. Aloe vera is a plant whose gel is marketed especially for cosmetic purposes. The anthraquinones contained in the gel must be removed. To this end the international application W02005 / 021018 describes a process using oxidases whose enzymatic activity allows the degradation of aloin and emodin present in the gel of A / oe vera.

International application WO2008 / 092221 describes a system which allows the selective extraction of trans-resveratrol and emodin using toluene as a solvent. Although this process has many advantages it is still long and requires several steps, including root drying and it does not allow to obtain a liquid extract directly usable by the cosmetic industry.

For THSG, as for other polyhydroxystilbenes, the biological activity is mainly related to its antioxidant powers which are related to its polyphenol groups. These groups are very unstable and the THSG is therefore likely to be oxidized and hydrolysed. It is therefore necessary to make extracts in which the molecule will have increased stability through the use of a solvent and an adequate pH.

The inventors have now developed a method for preparing a root extract of Polygonum multiflorum concentrated in THSG with little or no emodin, stable and directly usable by the cosmetic industry. Such a method allows the realization of successive extractions without destroying or altering the survival of the plants. The inventors have also shown that a plant extract according to the invention not only has cosmetic benefits, such as preventing or slowing skin aging, stimulating the renewal of the epidermis or having a soothing effect on the skin or an effect calming of the skin, but also can be used as a medicine to stimulate the antimicrobial defense of the skin, reduce or treat inflammation of the skin and / or promote skin healing, particularly in the case of the closure of the skin. 'a wound.

DESCRIPTION OF THE FIGURES

Figure 1. Evolution of the THSG content in Polygonum multiflorum root extracts at 3 different pH (pH 2.8 represented by the black histograms, pH 4 represented by the gray histograms and pH 6.8 represented by the white histograms) after 2, 4 and 6 months of preservation of the extract at 40 ° C and in the dark.

Figure 2. Chromatogram (UV at 319 nm) of the root extract of Polygonum multiflorum obtained according to Example 1.

Figure 3: Chromatograms (UV at 288 nm) of the root extract of Polygonum multiflorum obtained according to Example 1 (high) and the emodin standard at 10 mg / L (low).

Figure 4. Measurements of the viability of human keratinocytes NHEKs after 24h treatment with five different concentrations (3%, 1%, 0.33%, 0.11% and 0.037%) of an extract of Polygonum multiflorum prepared according to Example 1.4 (shaded dark gray), or its solvent alone at the same concentration (in light gray). The percentages of viability were calculated with respect to the untreated negative control (CTL NT) fixed at 100% and the positive cytotoxicity control (SDS at 0.008%). The histograms represent the average of 3 independent replicates and the error bar is the standard deviation around the mean.

DETAILED DESCRIPTION OF THE INVENTION

The present invention firstly relates to a root extract of Polygonum multiflorum characterized in that it comprises at least 0.8% by weight of THSG, preferably at least 1.5%, more preferably at least 2%, expressed by relative to the total weight of the dry extract, and - it comprises less than 0.01% by weight of emodin, preferably less than 0.001%, more preferably less than 0.0005%, expressed relative to the total weight of the dry extract.

More particularly, the subject of the present invention is a root extract of Polygonum multiflorum characterized in that: it comprises at least 0.8%, at least 1%, at least 1.2%, at least 1.5%, at least 1.7%, at least 2%, at least 2.5%, at least 3%, by weight of THSG, expressed in relation to the total weight of the dry extract, and • that it comprises less than 0 , 01%, preferably less than 0.005%, preferably less than 0.001%, more preferably less than 0.0005% and even more preferably less than 0.0001% by weight of emodin expressed relative to the total weight of the dry extract.

By "root extract of Polygonum multiflorum" is meant a product obtained by the extraction of roots of plants of the species Polygonum multiflorum.

By "THSG concentrate" is meant a root extract comprising a quantity of THSG that must not be less than 0.8%, expressed relative to the total weight of the dry extract.

By "2,3,5,4'-tetrahydroxystilbene-2-O-p-C-glucoside" or "THSG" is meant a compound corresponding to the following general formula (I):

(I)

By "dry extract" is meant the extract obtained by the implementation of the process according to the invention followed by a step of desiccating the extract, the desiccation

being implemented according to any method well known to those skilled in the art, in particular to place the extract in a hot and dry atmosphere.

By "no emodin" is meant a quantity of emodin in the extract below the detection limit and not "very little emodin" is meant an amount of emodin in the extract less than the limit of quantification by supercritical phase chromatography or HPLC / MS.

According to a particular aspect, in a root extract according to the invention, the amount of emodin in the extract is lower than the limit of quantification by supercritical phase chromatography or by high performance liquid chromatography coupled to mass spectrometry. (HPLC / MS).

To obtain a root extract, when the roots are deemed sufficiently developed, they are brought into contact with a solvent by immersion or maceration, and then said solvent is recovered and treated to extract the molecules of interest which were released by the roots of said plants. This type of process is adapted from the process developed by Plant Advanced Technologies (PAT) under the name "PAT Plantes à Traire®" and described in international application WO 01/33942.

According to a particular aspect, the subject of the invention is a root extract of Polygonum multiflorum, more particularly chosen from:

An extract obtained by root exudation in a solvent,

An extract obtained by root maceration in a solvent, and a mixture of at least one extract obtained by root exudation in a first solvent and at least one extract obtained by root maceration in a second solvent;

In a second aspect, the subject of the invention is a method for preparing a Polygonum multiflorum root extract according to the invention, comprising: a) a step of cultivating Polygonum multiflorum under off-soil conditions, and particularly in aeroponics b) optionally a step of stimulating the roots of the plants, c) a step of solid / liquid extraction of the roots optionally stimulated in step b), and d) the recovery of the extract obtained during the step c).

In the context of the invention, the term "cultivation of plants under soil conditions" means any method of cultivation in which the roots of the plant are not in the soil. More specifically, above-ground cultivation is a crop in which the roots of plants rest in a reconstituted environment, detached from the soil. This culture medium is irrigated regularly by nutrient solutions appropriate to the crop plant.

There are different soilless cultivation techniques such as non-substrate systems that require an oxygen enriched nutrient solution, and substrate systems. Among the systems without substrate, we can mention the aquaculture for which the nutritive solution is non-circulating and is contained in a culture tray, Nutrient Film Technique (NFT) for which the nutrient solution is enriched in dissolved oxygen during its displacement by exchange with the air, and the aeropony for which the roots of the plants are not in contact with a solid medium, nor even with a liquid medium: they are fed by a nutritive fog obtained by misting (via a fogger ) of the nutrient solution in a closed medium. Substrate systems include the infra-structure in which the nutrient solution enters the substrate at the bottom and the percolation into which the nutrient solution is distributed by discontinuous irrigation to the upper surface of the system and then drowns downwards. substrate. The substrate, mineral or organic, is neutral and inert like sand, clay or rockwool for example. This substrate can also be of industrial origin.

The term "cultivation of plants in aeroponics" means an aboveground culture mode for which the roots of the plants are not in contact with a solid medium or even with a liquid medium. According to a particular embodiment, the plants are fed with a nutritive mist obtained by misting, via a mist, the nutrient solution in a closed medium.

Typically, in a method according to the invention, plants can be arranged on trays with the aerial part of the plant above the tray and the root part below, the trays are arranged on tables forming a retention zone for collect the excess of a diffused liquid towards the plants, and one transfers the trays on the tables to different stations.

In step a), the plants cultivated in aeroponics are fed by root spraying with a nutritive solution of essential mineral salts (N - nitrogen, Phosphorus - P, Potassium - K), in order to obtain maximum root development and a maximum concentration of molecules of interest, without altering the survival of the plant. The skilled person, with the help of his general knowledge, knows how to adapt the proportions and concentrations of different mineral salts to optimize the root development and concentration of the molecules of interest. The mineral salt concentrations of the nutrient solutions are in this case included in a range of electroconductivity preferably ranging between 0.8 to 1.6 mS / cm, preferably between 1.0 to 1.2 mS / cm, so to promote a higher content of THSG in the extracts.

In step c) of a process according to the invention, the term "solid / liquid extraction" is understood to mean any solvent extraction technique which consists in extracting a chemical species present in a solid and being soluble in said solvent. These extraction techniques include maceration, exudation, infusion, decoction, extraction with Soxhlet and Kumagawa extractors, microwave assisted extraction, ultrasound-assisted extraction. , enzymatic extraction, supercritical fluid extraction (CO2 + DPG). The extraction leads to the recovery of the metabolites contained in one or the other part of the plants, and in particular the roots, by means of a leaching liquid, or solvent, brought into contact with the cut roots and / or attached to the plant, in a particular solvent and for a suitable duration.

According to a particular aspect, in a process according to the invention, step c) of solid / liquid extraction of the roots potentially stimulated during step b), is carried out by means of a process chosen from: exudation and a maceration.

According to a more particular aspect, in a process according to the invention, step c) of solid / liquid extraction of the roots potentially stimulated in step b), is carried out by means of a method chosen from: exudation root and root maceration. This step consists in particular of a "soaking" step, during which the roots of the plants are placed in a dipping tank containing a liquid for a predetermined duration. Typically, the plants are arranged on a tray with the aerial part of the plant above the tray and the root part below, the tray is raised after soaking, then the liquid contained in the soaking tray is collected. In a particular aspect, the soaking step is followed by a cutting step in which the root portion of the plants is partially cut to harvest the cut roots. It is possible to extract the substances from the cut roots, in addition to extracting the substances during soaking.

Preferably, in a process according to the invention, the root exudation is carried out on roots still attached to the plant. In another preferred aspect, the root maceration is carried out on freshly cut roots, that is to say, cut for less than 24 hours, and preferably as soon as possible after cutting, ideally just after cutting, said roots being optionally dried after their section and before maceration. The drying of the roots can be achieved by the implementation of any suitable drying process, known to those skilled in the art, and in particular by placing the roots at a temperature of between 40 ° C. and 60 ° C. for 4 hours, preferably in a dry environment. The drying of the roots can in particular be carried out in a ventilated oven.

More particularly, step c) of a process according to the invention comprises bringing the roots into contact with a solvent chosen from: alcohols, glycerine, glycols and eutectic solvents. Said alcohol is preferably chosen from ethanol and methanol, used pure or in the form of an aqueous solution of alcohol, the latter comprising from 10% to 99.9% of alcohol, more particularly between 40% and 90%, and even more particularly between 50% and 85%. Said glycol is a diol in which the two hydroxyl groups are borne by different carbons, and is chosen from: dipropylene glycol, propane 1,3 diol, propane 1,2 diol and butylene glycol, and is used pure or in the form of an aqueous solution of glycol, the latter comprising from 10% to 99.9% of glycol, more particularly between 40% and 90%, and even more particularly between 50% and 85%. Glycerin is used pure or in the form of an aqueous solution of glycerine, the latter comprising from 10% to 99.9% glycerine, more particularly between 40% and 90%, and even more particularly between 50% and 85%. %. Said eutectic solvent is composed of two or more components, which may be solid or liquid and which, in a particular composition, exhibit a sharp decrease in their melting temperature, thereby rendering the mixture liquid. Natural eutectic solvents are mainly composed of amino acids, organic acids, sugars and choline derivatives. Water may be part of the solvent, but in this case is strongly retained in the liquid and can not be evaporated. The particular combinations of the components constituting said eutectic solvent are chosen from, and in a non-exhaustive manner, choline-glucose chloride (ratio 1: 1), choline chloride-citric acid (ratio 1: 1), chloride of choline - citric acid (2: 1 ratio), choline - sucrose chloride (4: 1 ratio), choline - sucrose chloride (1: 1 ratio), choline chloride - tartaric acid (2: 1 ratio) , choline - xylose chloride (ratio 2: 1), choline - xylose chloride (ratio 3: 1), citric acid - sucrose (ratio 1: 1), citric acid - glucose (ratio 1: 1), glucose - tartaric acid (1: 1 ratio), choline - urea chloride (1: 2 ratio), choline chloride - xylitol - water (2: 1: 3) and lactic acid - glucose - water (5: 1: 3).

By "butylene glycol" is meant butane 1,2 diol, butane 1,3 diol, butane 2,3 diol and butane 1,4 diol.

These solvents, and more particularly glycerin, dipropylene glycol, 1,3-propane diol and 1,2-propane diol make it possible to promote selective extraction; that is, the extraction of a high THSG content, without extracting or extracting at a level very low or lower than the quantization threshold of emodin. In the case of a mixture of root extracts, said first and second solvents may be identical or different.

According to one particular aspect of the invention, the solvent is characterized by an acidic pH, in particular when it is used during the solid / liquid extraction step. According to a particular aspect, said alcohol or said glycol used during a solid / liquid extraction step is characterized by a pH of between 1.5 and 5, preferably between 2 and 4.5, more preferably between 3 and 4. 5. According to another particular aspect, a plant extract according to the invention is characterized by a pH of between 2.5 and 5 and preferably between 3.5 and 4.5. The use of a solvent at acidic pH, that is to say greater than or equal to 1.5 and less than or equal to 5, promotes the solubilization of the compounds of interest in said solvent, limits the chemical or enzymatic degradation of the THSG during the extraction and, in combination with the choice of the solvent, to promote a selective extraction, that is to say the extraction of a high THSG content, without extracting emodin or extracting it at a content very low or lower than the quantization threshold. The acids used for adjusting the pH of the solvent or extract are preferably lactic acid, phosphoric acid, citric acid or hydrochloric acid.

More particularly, said glycol is selected from the following group: dipropylene glycol, propane 1,3 diol, propane 1,2 diol and glycerine.

Propane 1,3 diol, or trimethylene glycol, can be prepared by chemical synthesis according to techniques known to those skilled in the art and described in the literature, it is also commercially available. Said propane 1,3 diol may also be produced by the implementation of a fermentation process under suitable conditions of a living organism, in particular a genetically modified strain of Escherichia coli. The propane 1,3 diol thus obtained is designated by the term "propane 1,3 diol biosourced", such a product is produced and then purified as described in particular in the international application WO 2004/101479 and marketed under the trademark Zemea®. Said propane 1,3 diol may also have an organic certification, for example of COSMOS type.

Even more particularly, in a process according to the invention, step c) of solid / liquid extraction comprises bringing the roots into contact with propane 1,2 diol or with propane 1,3 diol, and in particular propane 1,3 diol biosourced and / or Bio certification.

According to a particular aspect, step c) of a method according to the invention comprises bringing the roots into contact with a solvent for a period of between 15 minutes and 30 minutes for root exudation and for a period of time between 1 hour and 108 hours, especially between 24 hours and 96 hours, especially between 48 hours and 96 hours for root maceration. For root maceration, the ratio of the amount of fresh roots to the amount of solvent varies between 0.2 kg roots / L solvent to 1 kg roots / L solvent.

According to another particular aspect, a method according to the invention comprises a step of sectioning the roots and then drying the severed roots, prior to the step of macerating said roots, the maceration being carried out by placing the roots in contact with a solvent . For root maceration, the ratio of the amount of dry roots to the amount of solvent varies between 0.1 kg roots / L solvent to 0.04 kg roots / L solvent.

The method according to the invention may therefore comprise a first root exudation step carried out by immersion in a solvent of the roots still attached to the plant for a period of between 15 minutes to 30 minutes, followed by a stage of root maceration of the roots. cut from the plant and then dried.

The durations of root exudation and / or root maceration are chosen so as to favor the extraction of a large quantity of compounds of interest while not affecting the survival of the plant and without leading to the exhaustion of the plants. resources. Thus, the plants, after the root exudation step and possibly after the root cutting step, are put back into culture for 1 to 8 weeks in aeroponia according to steps a) and optionally submitted for 1 day to 8 weeks at a time. stimulation stage b) in order to restart their root development and favor the production by the roots of secondary metabolites.

According to a preferred embodiment, the subject of the invention is also a method for preparing a Polygonum multiflorum root extract according to the invention comprising: a) a step of cultivating polygonum multiflorum under off-soil conditions, and particularly in aeroponie, b) a step of stimulating the roots of the plants, c) a step of solid / liquid extraction of the roots stimulated in step b), and d) the recovery of the extract obtained in step c ).

According to another particular aspect, in a method according to the invention, step b) of stimulating the roots of the plants comprises: an elicitation step in which the roots are placed in the presence of a solution comprising at least one agent selected from: a salt, a surfactant, a solvent, an elicitor of fungal or bacterial origin, a derivative of jasmonic acid, in particular methyl jasmonate, salicylic acid, an ethylene generator, a chitin, chitosans and / or their mixture, and / or • a step of placing the roots in contact with a solution deficient in nitrogen, that is to say a solution comprising a proportion of nitrogen less than the proportion of nitrogen usually considered as optimal for the development of the plant, preferably less than 15% nitrogen, and advantageously not including nitrogen, said steps being sequential or simultaneous.

The roots can also be stimulated by placing the plants in contact with a nutrient solution deficient in nitrogen. Placing the plants with a nutrient solution deficient in nitrogen causes a "nitrogen stress" responsible for the stimulation. According to a particular aspect, a solution deficient in nitrogen according to the present invention is a solution comprising less than 15% of nitrogen, preferably less than 10% of nitrogen, advantageously less than 8%, more advantageously less than 6%, less from 5%, less than 4%, less than 3%, less than 2%, less than 1% nitrogen and even more preferably 0% nitrogen. Stage b) of stimulation makes it possible to significantly increase the yields of root biomass.

According to a particular aspect, in a method according to the invention the step of stimulating Polygonum multiforum is carried out in nutrient media of different composition N / P / K. The 0/15/40 medium offers adequate root productivity. Medium 15/10/30 allows limited root growth with very short roots and little branching. Nitrogen rich media, for example 20/20/20 and 28/14/14 did not allow root production.

Advantageously, step b) of stimulating the roots is carried out by spraying or macerating the plant with a solution of elicitors chosen from jasmonic acid derivatives such as, for example, methyl jasmonate, salicylic acid, ethephon and chitosans or by feeding the plant with a nutrient solution N / P / K deficient in nitrogen vaporized on the roots.

According to a particular embodiment of the invention, step b) is carried out by spraying or macerating the plant with a solution chosen from: • a solution of methyl jasmonate at a concentration of between 0.01 and 200 μΜ , advantageously between 0.1 and 100 μM, a solution of salicylic acid at a concentration of between 1 and 500 μM, advantageously between 10 and 50 μM, a chitosan solution at a concentration of 0.002 to 1 g / L, preferably 0.05 to 0.1 g / L, and • a nutrient solution N / P / K comprising less than 6% nitrogen, said solution being preferably sprayed on the roots.

In addition, the step b) of stimulating the roots with a solution of elicitors is advantageously carried out for a period of between 1 day and 21 days, preferably between 1 and 7 days.

In a particular aspect, step b) of stimulating the roots by feeding the plant with a nutrient solution N / P / K deficient nitrogen vaporized on the roots is advantageously carried out for a period of between 7 days and 8 weeks preferably 3 weeks.

In step b), the mineral salt concentrations of the nutrient solutions are in a range of electroconductivity advantageously ranging between 0.8 to 1.6 mS / cm, preferably between 1.0 to 1.2. mS, to promote a higher THSG content of the extracts.

According to a particular aspect of a method according to the invention, step b) is carried out after step a). According to another particular aspect of a process according to the invention, step b) and step a) are carried out simultaneously, the stimulation solution is then incorporated into the nutrient solution or administered by any other mode of administration. administration known to those skilled in the art.

According to an improvement, the "soaking" step is preceded by a washing step in which the liquid diffused towards the plants is clear water. This limits the intake of the elements contained in the nutrient solution or in the possible stimulation solution during the soaking step.

Advantageously, the subject of the invention is a process comprising the following steps: a) a step of cultivation of polygonum multiflorum under off-soil conditions, and particularly in aeroponics, b) a step of stimulating the roots of the plants, c) a step solid / liquid extraction of the roots stimulated in step b), comprising: a first phase of placing the roots still bound to the plant with a solvent, in particular for a period of between 15 minutes to 30 minutes followed by a second phase comprising the section of said roots and maceration of the severed roots in a solvent, said solvent, which is identical or different in the first and second phases, is chosen from alcohols, glycerin, glycols and eutectic solvents, and being used pure or in the form of an aqueous solution of alcohol, glycerine, glycol or eutectic solvent, and d) recovering the extracts obtained during the step c).

According to a particular aspect, in a process according to the invention the pH of the solvent is between 1.5 and 5, preferably between 2 and 5, preferably between 2 and 4.5, preferably between 3 and 4.5, more preferably between 3.5 and 4.5.

A method according to the invention advantageously comprises an additional step of readjusting the solvent content of the extract, to obtain an advantageous content of at least 50%, and / or an adjustment of the pH of the extract, to obtain a pH advantageously between 3 and 5, preferably approximately 4, for the preservation of the extract.

A method according to the invention advantageously comprises at least one additional step of purification and / or treatment of the plant extract, such a step is in particular chosen from: at least one filtration, in particular nanofiltration and / or sterilizing filtration and / or clarifying filtration, liquid / solid extraction, purification, concentration and bleaching of the root extract, such methods of purification and / or treatment are well known to those skilled in the art. This step makes it possible to obtain the final root extract of Polygonum multiflorum concentrated in THSG and containing no or very little emodin.

A chromatogram (FIGS. 2 and 3) of the analysis of a Polygonum multiflorum root extract obtained according to the invention shows that the THSG is predominantly present and that the emodin content is below the limit of quantification.

The present invention also relates to an extract that can be obtained by a process according to the invention.

The third subject of the present invention is a cosmetic composition comprising, as active agent, at least one root extract of Polygonum multiflorum according to the invention or an extract obtained by a process according to the invention, and at least one excipient.

The modes of administration, the dosages and the optimal dosage forms of a cosmetic composition according to the invention can be determined according to the criteria generally taken into account in the establishment of a cosmetic treatment adapted to a subject such as, for example, the type of skin.

The cosmetic composition according to the invention is advantageously intended for topical application. It may especially be in the form of a cream, a milk, a lotion, a gel, a serum, a spray, a mousse, a solution, an ointment, an emulsion, a patch or a mask. A cosmetic composition according to the invention comprises at least one cosmetically acceptable excipient chosen according to the type of administration desired. A cosmetic composition according to the present invention may further comprise at least one adjuvant known to those skilled in the art, chosen from thickeners, preservatives, perfumes, dyes, chemical or mineral filters, moisturizing agents, thermal waters. etc.

A cosmetically acceptable excipient may be chosen from polymers, silicone compounds, surfactants, rheology agents, humectants, penetration agents, oily components, waxes, emulsifiers, film-forming agents, perfumes, electrolytes, pH adjusters, antioxidants, preservatives, dyes, nacres, pigments and mixtures thereof.

A cosmetic composition according to the invention may further comprise at least one other cosmetically active agent, such as another anti-aging agent, a moisturizing agent, an agent having a calming, soothing or relaxing activity, a skin microcirculation stimulating agent. a sebum-regulating agent for the care of oily skin, a cleaning or purifying agent, an anti-radical agent, an anti-inflammatory agent, a chemical or mineral sunscreen, etc.

Advantageously, a cosmetic composition according to the invention comprises at least one root extract of Polygonum multiflorum according to the invention, in a quantity of between 0.01 and 10%, in particular between 0.05 and 5%, more particularly between 0, 1 and 2%, by weight relative to the total weight of the composition.

The fourth subject of the present invention is an extract according to the invention, an extract obtained by a process according to the invention, or a cosmetic composition according to the invention, for preventing or slowing skin aging, and / or for stimulating the renewal of skin aging. the skin and / or have a soothing effect of the skin or a calming effect of the skin following the sensation of irritation.

A cosmetic composition according to the invention may in particular be intended to prevent or slow skin aging. A cosmetic composition according to the invention may also be intended to stimulate the renewal of the epidermis. Also, a cosmetic composition according to the invention may especially be intended to have a soothing effect on the skin or a soothing effect of the skin following the sensation of irritation that may develop after shaving, friction, or contact with the skin. allergens

The fifth subject of the present invention is a pharmaceutical composition comprising, as active agent, at least one extract according to the invention or an extract obtained by a process according to the invention, and at least one pharmaceutically acceptable excipient.

The present invention also has for sixth object a nutraceutical composition comprising, as active agent, at least one extract according to the invention or an extract obtained by a process according to the invention, and at least one nutraceutically acceptable excipient.

In the present invention, the term "pharmaceutically or nutraceutically acceptable" means any ingredient which is useful in the preparation of a pharmaceutical or nutraceutical composition, which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable to a pharmaceutical or nutraceutical composition. veterinary use or in humans.

The term "pharmaceutically or nutraceutically acceptable salts" of a compound means salts which are pharmaceutically or nutraceutically acceptable, as defined above, and which possess the desired pharmacological activity of the parent compound. Such salts include: (1) hydrates and solvates, (2) pharmaceutically or nutraceutically acceptable acid addition salts formed with pharmaceutically or nutraceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with pharmaceutically or nutraceutically acceptable organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid , methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, acid p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and the like, or (3) pharmaceutically or nutraceutically acceptable base addition salts formed when an acidic proton present in the parent compound is replaced by a metal ion, for example a metal ion alkali, an alkaline earth metal ion or an aluminum ion; is coordinated with a pharmaceutically or nutraceutically acceptable organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

The modes of administration, the dosages and the optimal dosage forms of a pharmaceutical composition according to the invention can be determined according to the criteria generally taken into account in the establishment of a pharmaceutical treatment adapted to a subject such as, for example, age or body weight of the patient, severity of his general condition, tolerance to treatment, side effects noted, skin type. Depending on the type of administration desired, the pharmaceutical composition according to the invention may further comprise at least one pharmaceutically acceptable excipient. The pharmaceutical composition according to the present invention may further comprise at least one adjuvant pharmaceutically known to a person skilled in the art, chosen from thickeners, preservatives, perfumes, dyes, chemical or mineral filters, moisturizing agents, water thermal baths, etc.

Advantageously, said pharmaceutical composition comprises at least one extract according to the invention in an amount of between 0.01 and 10%, in particular between 0.05 and 5%, more particularly between 0.1 and 2%, by weight relative to to the total weight of the composition.

A pharmaceutical composition is particularly suitable for oral, nasal, transdermal, parenteral, topical, rectal and mucosal administration. It may be in dry form, such as for example: soft capsule, capsule, tablet, lyophilisate, powder, granule, or patch, or in liquid form, such as: solution, suspension, spray, cream or gel. The pharmaceutically acceptable excipient is known to those skilled in the art and is chosen according to the method of administration of the pharmaceutical composition. By way of example, the pharmaceutically acceptable excipient may be chosen from the group consisting of diluents, binders, disintegrants, dyes, lubricants, solubilizing agents, absorption promoters, film-forming agents and gelling agents. , and their mixtures.

The pharmaceutical composition according to the invention may further comprise at least one compound chosen from the group consisting of emollients, moisturizing active agents, activators of keratin synthesis, kératorégulateurs, keratolytiques, agents restructuring the cutaneous barrier ( skin lipid synthesis activators, PPAR agonists or Peroxysome Proliferator Activated Receptor), activators of keratinocyte differentiation (retinoids, calcidone®, calcium), antibiotics, anti-bacterial agents, antifungal compounds, anti-cancer agents -serials, sebum-regulators, immunomodulators, such as tacrolimus, pimecrolimus, oxazolines, preservatives, anti-irritants, soothing agents, filters and sunscreens, anti-oxidants, growth, healing agents or eutrophic molecules, drugs and anti-inflammatory agents, drugs and anti-Alzheimer's agents.

The subject of the present invention is an extract according to the invention or a pharmaceutical composition according to the invention for its use as a medicament.

More particularly, the subject of the present invention is also an extract according to the invention, an extract obtained by a process according to the invention, or a pharmaceutical composition according to the invention or a nutraceutical composition according to the invention7 for its use as a a medicament for stimulating the antimicrobial defense of the skin, and / or for alleviating or treating inflammation of the skin and / or for promoting skin healing, particularly in the case of wound closure.

More particularly, the subject of the present invention is also an extract according to the invention, an extract obtained by a process according to the invention, or a pharmaceutical composition according to the invention, for its use as a medicament for preventing or slowing down aging. skin, and / or to stimulate the renewal of the epidermis, and / or to have a soothing effect of the skin or a soothing effect of the skin following the sensation of irritation. In this case, said extract will advantageously be associated with a pharmaceutically acceptable excipient and suitable for cutaneous application, and said pharmaceutical composition will advantageously contain a pharmaceutically acceptable excipient suitable for cutaneous application.

The subject of the present invention is also a use of an extract according to the invention, an extract obtained by a process according to the invention for the preparation of a cosmetic composition intended to prevent or delay the appearance of the effects of aging of the skin. skin, and / or to stimulate the renewal of the epidermis and / or to have a soothing effect or a soothing effect of the skin following the sensation of irritation.

The present invention also relates to a cosmetic skin care method aimed at preventing or delaying the appearance of the effects of aging of the skin, and / or stimulating the renewal of the epidermis, and / or having a soothing effect of the skin or a calming effect of the skin following the sensation of irritation, said method being characterized in that it comprises the application, on at least a part of the skin of the body or the face, of a composition cosmetic according to the invention.

Advantageously, in the method according to the invention, the cosmetic composition is applied to a subject in need thereof, in particular in anticipation of or following a single or repeated exposure of the skin to oxidative stress. Indeed, the latter can generate an excess of free radicals that can accelerate the signs of skin aging.

In addition, the invention relates to a method for preventing or slowing skin aging, for reducing inflammation of the skin, for having a soothing effect on the skin or a soothing effect of the skin following the sensation of irritation, and for promoting skin healing, particularly in the case of wound closure, in a subject in need thereof comprising administering to a subject a therapeutically effective amount of a nutraceutical composition according to the invention or a pharmaceutical composition according to the invention.

Examples 1 to 3 which follow and Figures 1 to 4 are intended to illustrate the present invention, without limiting the scope thereof.

EXAMPLES EXAMPLE 1 Preparation and Characterization of Reactive Extracts of Polygonum Multi-Color According to the Invention

The root extracts of Polygonum multiflorum are prepared from plants whose seeds were purchased from a Chinese supplier (Zhong Wei Organic Seeds) in 2012 and 2013. Since 2013, Polygonum multiflorum plants are conserved and grown in a greenhouse by the plaintiff. 1.1 Effect of several culture media as stimulation media on root growth The study was carried out in 12 experimental units of 32 plants each reproducing the culture system in aeroponics. The plants are initially grown for 8 weeks with 15/10/30 culture medium (N / P / K) on 16m2 pilots.

For this experiment, 4 stimulation media were tested, including the initial culture medium. These are ternary fertilizers usually used in horticulture and whose composition differs according to their ratio N / P / K (nitrogen / phosphorus / potassium). The 4 methods are as follows: 0/15/40; 15/10/30; 20/20/20 and 28/14/14. Each category is repeated 3 times according to a random distribution. The plants were grown with different media for 4 weeks.

It is observed that the aerial parts grow well regardless of the medium used. Of the 4 modalities tested, the 0/15/40 medium offers adequate root productivity. Medium 15/10/30 allows limited root growth with very short roots and little branching. The other media rich in nitrogen, that is, the 20/20/20 and 28/14/14 media did not allow the production of roots. 1.2 Study of several solvents for the production of Polygonum multiflorum root extract

Plants of Polygonum multiflorum are grown in aeroponia with a defined nutrient solution, composition N / P / K corresponding to 15/10/30 and electroconductivity of between 1.0 and 1.2 mS / cm for 8 weeks, then with a defined nutrient solution, composition N / P / K corresponding to 0/15/40 and electroconductivity of 1.0 and 1.2 mS / cm for 3 weeks.

The roots are cut and then 500 g of fresh roots are macerated for 48 hours at room temperature in 1 L of different aqueous solvent solution at 70/30 (solventl / water - v / v) at an acidic pH of 1.6 and at an unadjusted pH, i.e. between 5 and 7.5. After maceration, the roots are removed from the solvent.

The four solvents tested are glycerine, dipropylene glycol, propane 1.3 diol biosourced (Zemea ®) and propane 1,2 diol.

The content of emodin in the root extracts is analyzed by HPLC-MS (PDA detector, 254 nm). The quantification of emodin is done using a commercial standard of emodin at 1 mg / L (Supplier = Shagai tauto Biotech, ref = E0053) analyzed in HPLC-MS (PDA detector, 254 nm). Since it was impossible to quantify this compound in the extracts, since emodin is below the quantification threshold of this method, the signal-to-noise ratio (S / N) is measured in order to roughly evaluate its content in the extracts. When the ratio is greater than 3, the amount of emodin is at the limit of detection. When the ratio is greater than 10, the amount of emodin is at the limit of quantification. Table 1 summarizes the information obtained.

Table 1: Signal to noise ratio in the different extracts obtained with 4 different solvents at two pH.

The emodin content in the extracts made with the 4 solvents, namely dipropylene glycol, propane 1,2 diol, Zemea® and glycerol at the two pHs tested is below the limit of detection of emodin. These solvents thus allow selective extraction of THSG without or with very little emodin. 1.3 Stability study of a root extract of Polygonum multiflorum at different acidic pH

The stability test consists of placing a sample of Polygonum multiflorum root extract in the dark at 40 ° C for 6 months. Regular samples are taken to follow the evolution of the THSG content of the extracts by liquid chromatography (HPLC-XR-Prominence, Shimadzu). A standard of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside (THSG) at ΙΟΟμΜ is injected into each analysis sequence in order to normalize the data.

Plants of Polygonum multiflorum are grown in aeroponia with a defined nutrient solution, composition N / P / K corresponding to 15/10/30 and electroconductivity of between 1.0 and 1.2 mS / cm for 8 weeks, then with a defined nutrient solution, composition N / P / K corresponding to 0/15/40 and electroconductivity of 1.0 and 1.2 mS / cm for 3 weeks. The roots are cut, then an extract

rooting is obtained by maceration for 48 hours of 500 g of fresh roots in 1 L of 70% Propylene glycol whose pH (before maceration) was adjusted to the value of 2.8 using citric acid to 3 M.

The pH of the root extract after extraction was then readjusted with 3M citric acid or 5M sodium hydroxide at three different pHs: pH 2.8; pH 4.0 and pH 6.8.

The phytochemical profiles of the extract samples were monitored by HPLC over time for six months. A sample was taken each month. Figure 1 shows the evolution of the THSG content according to the 3 pH at 2, 4 and 6 months.

A clear decrease in the THSG concentration of the extract kept at the initially neutral pH (pH 6.8) is observed. From the fourth month, only traces of the latter remain in the extract. This pH can not be used for the preservation of the extract.

Within the sample, which was initially stored at pH 2.8, it is observed that the THSG content gradually decreases. From the fourth month a significant change in sample concentration is observed, with a 32% loss in the amount of THSG. This pH does not seem ideal either for the preservation of the extract. The sample readjusted at pH 4.0 before storage seems more stable. The THSG content decreases by 8% after 6 months. This pH appears to be the best candidate for the conservation of the Polygonum multiflorum root extract. 1.4 Extract prepared from a glycol solvent

A root extract of Polygonum multiflorum concentrated in THSG and comprising an amount of emodin lower than the limit of quantification by high performance liquid chromatography coupled to mass spectrometry (HPLC / MS), is obtained according to the following method: Culture of Polygonum multiflorum in aeroponia with a defined nutrient solution, composition N / P / K corresponding to 15/10/30 and electroconductivity between 1.0 and 1.2 mS / cm, • Stimulation of the plant during a duration of 2 to 4 weeks by nitrogen stress by the use of a nutrient solution N / P / K comprising: less than 6% nitrogen, 15% phosphorus and 40% potassium, and electroconductivity of 1.0 to 1.2 mS / cm, • Extraction of the molecules of interest by immersion of the roots still attached to the plant for a period of 15 to 30 minutes in a propane mixture 1.2 diol / water, according to a ratio included between 85/15 and 70/30 at room temperature ante and at a pH between 3.5 and 4.5. • Extraction of the molecules of interest by maceration of the cut roots of the plant, during a period of 48 to 96 hours in a propane mixture 1,2 diol / water in a ratio between 85/15 and 70/30, at room temperature and at a pH between 3.5 and 4.5, • Mixture of the extracts obtained in the two preceding extraction steps, • The extract is concentrated by nanofiltration and clarified by clarifying filtration, and • Readjustment of the propane content 1 , 2 diol of the extract (at least 50%) as well as the pH (approximately 4) of the extract for preservation. The root extract of Polygonum multiflorum thus obtained has the following characteristics:

• Content of dry extract: 10.8 g / L • Content of THSG: 270 mg / L (ie 2.5% of the dry extract) • Emodin content: below the limit of quantification • Solvent: 60% • pH: 4.3

The quantification of THSG (2,3,5,4'-tetrahydroxystilbene-2-OpD-glucoside) is carried out by means of a standard range prepared from commercial standards provided by Sigma-Aldrich, reference: SML0834 (Saint-Quentin Fallavier, France) solubilized in the solvent DMSO / water miliQ pH 3.0 (70/30 vol, pH adjusted with 2N HCl).

The quantification of emodin (6-methyl-1,3,8-trihydroxyanthraquinone) is carried out using a standard range prepared from commercial standards provided by Shanghai Tuto Biotech Co LTD, reference: E-0053 (CHINA) solubilized in the solvent DMSO / water miliQ pH 3.0 (70/30 vol, pH adjusted with 2N HCl).

FIG. 2 and FIG. 3 show chromatograms of the Polygonum multiflorum root extract obtained according to Example 1. The apparatus used to analyze the extract is a Shimadzu Nexera X2 HPLC (the LC-30AD pumps, which are the sample SIL-30AC, CTO-20A furnace, SPD-M20A diode array detectors, Kyoto, Japan) operating in reversed phase with Kinetex biphenyl column (00F-4622-AN, Phenomenex, Torrance, CA, USA) 150 mm x 2.1 mm, 2.6 μm. The mobile phase consists of a solvent A (Mili-Q ultrapure water, Merck Millipore + 0.1% formic acid, Carlo Erba, Val-de-Reuil, France) and a solvent B (Acetonitrile, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) whose gradient was programmed as follows:% B: 5-31.4% (0-6 min), 31.4-90% (6-6.1 min), 90 % (6.1-7.9 min), 90-5% (7.9-8.0 min), 5% (8-10 min). The analysis rate is 0.5 mL / min with an oven temperature of 40 ° C. At the output of the column, a diode array detector records the UV spectra between 220 and 370 nm. The device is coupled to a mass spectrometer (Shimadzu LCMS-2020) operating with electrospray ionization (4.5 kV) in positive and negative mode in a range of m / z between 100 and 1000. The software LabSolutions (version 5.60 SP2) is used to operate the system. The extract to be analyzed is filtered on a 0.2 μm filter before injection.

In Figure 2, the chromatogram shows a majority peak for a retention time around 4.40 min corresponds to the THSH. In FIG. 3, no peak is detected for a retention time at around 8.9 (± 5%) min corresponding to the emodin standard, confirming that the amount of emodin in the extract is below the limit. of quantification.

EXAMPLE 2 CHARACTERIZATION OF A RACINARY EXTRACT OF POLYGONUM MULTIFLORUM WITH RESPECT TO A BENEFIT FOR THE SKIN - IDENTIFICATION OF TARGETS BY ANALYZING THE MODIFICATION OF GENE EXPRESSION BY QRT-PCR ON TAQMAN CARDS The study consists in measuring the effects of the Polygonum multiflorum root extract by qRT-PCR on microfluidic TaqMan maps, on the one hand, on the expression of 94 genes involved in dermal biology, connective tissue remodeling and aging ("Dermal" map) Benefits »defined by the company StratiCELL) and, on the other hand, the expression of 94 genes involved in the key functions of the epidermis, such as the barrier function directly related to hydration, the antioxidant response, or pigmentation by melanocytes ("Epidermal Benefits" card defined by StratiCELL).

The protocol consisted in adding Polygonum multiflorum root extract in the monolayer human fibroblasts NHDFs (Normal Human Dermal Fibroblasts) and reconstituted melanized human epidermis (RHE / MEL / 001), and after h, to analyze the different populations of RNA to identify genes differentially expressed by qRT-PCR.

A preliminary cytotoxicity study defined the working concentration of the Polygonum multiflorum root extract for the gene expression study.

TGF-βΙ (Transforming Growth Factor-beta-1) and vitamin D3 (1α, 25-dihydroxyvitamin D3), whose effects are documented in the literature, were used as reference molecules to validate the test systems. and the method of analysis. 2.1. Materials and methods

Extract

The Polygonum multiflorum plants are cultivated in aeroponics according to Example 1. The fresh roots are cut and macerated at room temperature for 48 hours in a 70/30 ethanol / water-v / v solution at 500 g. of roots for one liter of solvent and at an unadjusted pH. Then the extract is filtered. The solvent is removed using a rotary evaporator and the powder dried in a desiccator. An analysis by liquid chromatography coupled to mass spectrography (HPLC / MS), carried out according to the protocol described in Example 1.4, made it possible to verify that the emodin content is below the limit of quantification and that the THSG is present mainly in the extract.

Cellular culture

The first part of the study was performed on human dermal fibroblasts NHDFs (ATCC, CR.L-2522, origin: foreskin) grown in monolayer in DMEM medium (Invitrogen, 31885-049) containing antibiotics (Penicillin / Streptomycin , Invitrogen, 15140-122) but not containing serum. These cells were maintained in a humid atmosphere at 37 ° C containing 5% CO 2.

The second part of the study was performed on reconstituted epidermis (StratiCELL®, RHE / MEL / 001) with or without primary human NHEMs melanocytes (Normal Human Epidermal Melanocytes) from a dark phototype donor (phototype IV to V) (Invitrogen, C2025C, Lot No. 439684). The tissues were cultured at the air-liquid interface for 14 days in a suitable culture medium, and a humid atmosphere at 37 ° C containing 5% CO2. Determination of the range of study concentrations of Polygonum multiflorum root extract by a preliminary cytotoxicity study

In order to determine the optimal analysis concentration for the Polygonum multiflorum racianire extract, a preliminary experiment was performed on NHDFs fibroblasts, human melanocytes NHEMs and reconstituted epidermis.

NHDFs fibroblasts performed 30.1 and 30.7 population doublings and NHEM melanocytes were seeded in 24-well plates 24 h prior to application of the actives.

Reconstituted epidermis (RHE / 001, lot CB0314 / 2) was transferred to a 12-well plate before being treated with the extract. The extract was diluted in the culture medium and then added, without prior filtration, into the culture medium of the human fibroblasts NHDFs containing no serum, in the medium of the primary human melanocytes NHEMs, or in the culture medium of the differentiated epidermis.

This study evaluated the viability of MTS cells (3- (4,5-dimethythiazol-2-yl) -5- (3-carboxy-methoxyphenyl) -2- (4-sulfophenyl) -2H- tetrazolium) (Promega, G3581), 24 hours after the addition of Polygonum multiflorum root extract at 5 concentrations and 3 repeats (n = 3) for NHDFs fibroblasts and NHEM melanocytes, and at 2 concentrations and 3 repetitions (n = 3) for reconstituted epidermis. The 2 concentrations tested for the reconstituted epidermis were chosen on the basis of the cytotoxicity results obtained for the NHEM melanocytes.

SDS (sodium dodecyl sulfate) is toxic to cells and has been used as a positive control to validate the experiment.

At the end of this experiment, a non-cytotoxic concentration was defined in order to measure the expression modifications of the target genes.

The following concentrations, in pg of dry extract / ml, were tested:

NHDFs fibroblasts and melanocytes NHEMs: 0.16 μg / ml, 0.8 μg / ml, 4 μg / ml, 20 μg / ml, 100 μg / ml;

Reconstituted Epiderms: 20 μg / ml and 100 μg / ml.

Analysis of gene expression modifications The extract was added, at a selected concentration, into the NHDFs human fibroblast culture medium (having carried out 30.5 and 30.8 population doublings) in the absence of serum or in the medium. melanized differentiated epidermis cultures (RHE / MEL / 001, lots CB0314 / 3 and CB0314 / 4), without filtration prior to contacting the cells / epidermis.

Reference molecules were studied in parallel, namely, TGF-βΙ for NHDF fibroblasts and vitamin D3 (VD3) for reconstituted epidermis.

Extraction of Total RNA Extraction of total RNAs was performed using the RNeasy Mini Kit (Qiagen, 74106). 24 h after the addition of the extract, the cells were rinsed with PBS and lysed in the ad hoc lysis buffer, while the epidermis were directly immersed in this buffer (culture triples were made for each condition ). The extraction and purification of the RNAs were performed according to the instructions of the supplier. Total RNAs were then stored at -80 ° C.

RNA Qualification by Spectrophotometry and Capillary Electrophoresis

The concentration of the total RNAs was determined by spectrophotometric measurement. The quality and integrity of the RNAs were then verified by capillary electrophoresis (Agilent Bioanalyzer 2100 Platform - Agilent RNA 6000Nano Kit, 5067-1511).

RNA quantification by spectrophotometric measurement: an aliquot of each RNA was diluted in RNase-free water and its concentration was determined using an Ultrospec 1100 Pro spectrophotometer (Amersham).

RNA Integrity by Agilent Bioanalyzer Capillary Electrophoresis: Integrity of total RNA was assessed by visualization of electrophoresis peaks corresponding to ribosomal RNAs. For total RNAs of higher eukaryotes, ribosomal band size should be 1.9 kb for 18S-RNA and 4.7 kb for 28S-RNA. The intensity of the band corresponding to the 28S-RNA must be greater than the intensity of the band corresponding to the 18S-RNA. Small diffuse bands representing lower molecular weight RNAs (tRNA and 5S ribosomal RNA) may be present. When the RNA is degraded, banding of the ribosomal RNA and a background of the higher molecular weight RNAs are observed.

Synthesis of complementary DNA or cDNA

Inverse transcripts (RT) were performed using the "High Capacity RNA-to-cDNA Kit" kit (Applied Biosystems, 4387406). For cDNA synthesis, a mix was prepared according to the supplier's instructions, with 2 μg of total RNA, the ad hoc buffer provided in the kit and the reverse transcriptase enzyme. This reaction was performed at 37 ° C for 1 hour, then 5 minutes at 95 ° C and finally the cDNA samples were set on ice and stored at -20 ° C.

Validation of real-time qPCR test systems using TaqMan-type fluorescent probes

The real-time qPCR method was used to quantify the expression of different specific targets in the NHDF fibroblast populations treated with TGF-βΙ (20 ng / ml) as well as melanized epidermides treated with vitamin D3. (100nM) and ethanol solvent (0.1% EtOH), used to solubilize the VD3.

The target sequences of the genes of interest were amplified by PCR. using the "TaqMan Gene Expression Assays" (Applied Biosystems). These kits include a TaqMan probe and 2 specific primers, which have been premixed at a concentration of 18 μΜ for each primer and 5 μΜ for the probe. This mixture is concentrated 20 times. The TaqMan probes were grafted with a fluorophore (FAM) at the 5 'end of the sequence and with a 3' fluorescence quencher.

PCRs were performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems). The reactions were carried out in a volume of 20 μl. The reaction mixture contains 10 μL of TaqMan Fast Universal Master Mix (Applied Biosystems), 1 μL of TaqMan Gene Expression Assay and 5 μL of RNase-free water.

In each well of a 96-well microplate, 16 μl of the mixture and 4 μl of cDNA (4 ng) were added. For standardization purposes, reaction mixtures with probes and primers corresponding to glyceraldehyde-3-phosphate dehydrogenase (GAPDI-Γ) for fibroblasts and β2-microglobulin {B2M} for melanized epidermis were also prepared with the same cDNA samples. A control without cDNA served as a negative control of amplification. The thermal cycles were programmed with an incubation step at 50 ° C for 2 min, followed by a first denaturation step at 95 ° C for 10 min. The PCR amplification protocol was continued with 40 cycles of 15 sec at 95 ° C followed by a min at 60 ° C.

The levels of expression were quantified according to the method of calculation of the relative expression with respect to a household gene (2 ACt), derived from the calculation of AACt (Pfaffl, A new mathematical model for relative quantification in real-time RT -PCR, Nucleic Acids Res, 29 (9), 2001, 2002-2007, Livak and Schmittgen, Analysis of Relative Gene Expression Using Real-Time Quantitative PCR and the 2 ~ Δα method, Methods, 25 (4), 2001, 402-408) described below:

The relative quantitation of the transcripts was carried out by the use of a calculation method which consists of comparing the average of the control values (Ct) obtained for the TGF-βΙ (20 ng / ml) and VD3 (100 nM) conditions with the respective control conditions (untreated CTL and 0.1% CTL ethanol). These Ct represent the detection threshold at which the amount of DNA is such that the signal differs significantly from the background noise. This average Ct value was normalized to a household gene, namely Glyceraldehyde-3-phosphate dehydrogenase (GAPDI-Γ) for NHDF fibroblasts and p2-microglobulin (B2M) for reconstituted melanized epidermis. the difference in level of expression (RQ) was obtained by using the formula: rq_2 "(ACt condition treated - ACt reference condition) where ACt = Ct (target gene) - Ct (household gene) within the same sample of cDNA.

Preparation of Taqman microfluidic cards, quantitative PCR and Ct analysis

PCR reaction mixtures. Custom-made TaqMan microfluidic cards have been prepared following the detailed instructions in the Applied Biosystems Micro Fluidic Card Getting Started Guide. In summary, 100 ng of cDNA was added to a specific PCR mix (Taqman Universal Master PCR Mix, 4364338, Applied Biosystems) before being injected into the map and dispersed by capillarity. After centrifugation, the card was sealed prior to performing quantitative PCR and analysis with the Applied Biosystems 7900HT system, using the ABI PRISM® 7900 Sequence Detection System software, SDS2.4. The threshold cycles (Ct) were obtained for all the genes represented on the maps and expressed by the melanized reconstructed NHDF fibroblasts and epidermis.

The results were exported from the real-time qPCR device using the SDS RQ Manager software (v1.2.1, Applied Biosystems) and the analysis of the expression modifications was performed using the Data Assist software ( V3.0., Applied Biosystems) designed to perform the relative quantification of gene expression using the Ct comparison method (AACt) (Pfaffl, 2001 and Livak and Shmittgen, 2001) described in the paragraph above and a combination of statistical analyzes. As mentioned above, the AACt calculation method consists in comparing the Ct values obtained for the conditions treated by the extract with the reference condition (DMSO control). These Ct values were themselves normalized with respect to the household gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) for the fibroblasts NHDF and B2M (β-2 microglobulin) for the melanized reconstructed epidermis. The maximum allowable Ct value used as the detection threshold has been set at 36 cycles. 2.2. Results 2.2.1. Determination of the concentration of analysis of the extract by a cytotoxicity study The preliminary study of cytotoxicity on NHDF fibroblasts, on human NHEM melanocytes and on reconstituted epidermis made it possible to define a working concentration (prepared in DMSO ) for the gene expression study. The solvent present in the extract was tested in parallel. SDS at 0.08% (for NHDF) and at 0.05% (for NHEM) was used as a positive cytotoxicity control to validate the experiment. On the basis of these results (data not shown), the following concentrations were chosen for the rest of the study: 100 μg of dry extract / ml (for NHDF fibroblasts) and 100 μg of dry extract / ml ( for reconstituted melanized epidermis). 2.2.2. Qualification of RNAs by capillary electrophoresis

The different RNA populations demonstrate the presence of narrow peaks, corresponding to the 18S and 28S ribosomal RNAs, and a balanced ratio between the two peaks. The absence of intermediate and spreading peaks, characteristic of RNA degradation products, testifies to the integrity of the different populations (data not shown). The quality and the integrity of the extracted RNAs being demonstrated, they have therefore been used for the continuation of the protocol and involved in the synthesis reactions of the complementary DNAs.

2.2.3. Effects of the extract on 94 target genes in NHDF fibroblasts The extract was added to the NHDF fibroblast culture medium (n = 3). Controls treated only with the 1% DMSO solvent (vehicle of the extract) were also analyzed. In parallel, TGF-βΙ (20 ng / ml) was studied as a validation control. After 24 hours of NHDF fibroblast culture, the total tRNA populations were extracted, their integrity was analyzed by capillary electrophoresis, and the gene expression differences were analyzed by qRT-PCR using TaqMan 96 cards. -wells, targeting the key functions of the dermis.

The results are summarized in the form of a table (Table 2) indicating genes representative of a beneficial cosmetic effect, varying significantly, 24 hours after application of the extract on NHDF human dermal fibroblasts.

Table 2: List of genes that vary significantly 24 hours after the application of the Polygonum multiflorum root extract (at 100 μg ES / ml) on human dermis fibroblasts NHDFs. The symbol of genes,

the name of the genes, the relative expression (RQ) with respect to the vehicle DMSO at 1% (RQ> 1: increase) and the value p (p-value) are presented. • Antioxidant defense (5 overexpressed genes) The root extract of Polygonum multiflorum remarkably induces the expression of the superoxide dismutase 2 (SOD2) gene (X 13.8). This regulation is coupled with the overexpression of other genes involved in the oxidative stress response, such as HM0X1 (heme oxygenase 1 (XI, 8)), TXRND1 (thioredoxin reductase 1 (XI, 5)), NQO1 (NAD (H ) dehydrogenase, quinone 1 (XI, 6)) and G6PD (Glucose-6-phosphate dehydrogenase (X1.2)). The superoxide dismutase 2 encoded by the SO2 gene is a mitochondrial protein implicated in the first line defense against reactive oxygen species (ROS). In fact, it converts the superoxide anion into hydroperoxide, which itself is converted into oxygen and water by catalase and peroxyredoxins.

The HM0X1 gene encoding the HO-1 protein or heme oxygenase is often overexpressed in response to stress, and plays a crucial role in protecting and maintaining cellular homeostasis. The HO-1 protein is involved in the degradation of heme (having prooxidant properties) in carbon monoxide, ferrous iron and biliverdin. These last 2 components are the precursors of the antioxidants bilirubin and ferritin. HM0X1 is therefore known for its protective role against oxidative stress.

The thioredoxin system is one of the major antioxidant defense systems. In this system, thioredoxin reductase catalyzes the transfer of electrons from NADPH (Nicotinamide Adenine Dinucleotide Phosphate) to thioredoxin, which itself has a reducing effect on various target proteins. Among the 3 isoforms of thioredoxin reductase, the isoform 1 encoded by the TXR.ND1 gene is the most studied. It is known to be in particular under the transcriptional control of the Nrf-2 transcription factor playing a major role in the antioxidant defense.

NAD (H) dehydrogenase, quinone 1 (NQO1) is another detoxifying enzyme that promotes, by reduction, the formation of hydroquinones from quinones, preventing the production of radical species. The NQO1 gene is overexpressed in response to pro-oxidants, heavy metals, UV or ionizing radiation to protect the cell from its harmful effects.

Glucose-6-phosphate dehydrogenase encoded by the G6PD gene is the first enzyme in the pentose phosphate pathway. It catalyzes the oxidation of glucose-6-phosphate to 6-phosphoglucono-6-lactone. This reaction is coupled with the reduction of a NADP + molecule to NADPH, necessary for the reduction of glutathione GSSG (oxidized form) to glutathione GSH (reduced form), a powerful antioxidant.

The overexpression of 5 genes involved in antioxidant defense demonstrates the ability of the Polygonum multiflorum root extract to reduce the effects of pro-oxidant stress generating excess free radicals, such as UV accelerating signs of aging at the level of the skin, or to fight against various pathologies or proinflammatory skin conditions, also generating reactive species of oxygen.

Structure and homeostasis of the extracellular matrix (ECM) The root extract of Polygonum multiflorum induces the expression of the COL3A1 gene (X1.5) which codes for the alpha-1 subunit of collagen type III, and of the CYR gene .61 (X 1.5) which encodes an ECM CYR 61 protein for "cysteine-rich angiogenic protein 61". The CYR61 protein is expressed at inflammatory sites and cutaneous wounds where it induces the production of new extracellular matrix. Fibrillar collagens are by far the most abundant protein in the skin, constituting more than 90% of its dry weight. Type I collagen accounts for 60 to 80% of collagen in the dermis and hypoderm while collagen type III (COL3A1) accounts for 15 to 25% and type V collagen for 2 to 5%. Collagens of type I, III, and V self-assemble into thicker fibers to form a three-dimensional network throughout the thickness of the dermis. Thus, collagen III (COL3A1) plays a structural role at the level of the ECM and therefore in the resistance and elasticity of the dermis. Induction of the COL3A1 gene by the extract may be of interest for an anti-aging strategy to reduce dermal degradation with age. • Neurotrophins and pain perception

Several genes are also repressed by the extract: this is the case of NTRK2 (X0.6) and NGFR (X0.08). The latter code for neurotrophin receptors, responsible for the sensation of pain. Under-expression of these genes could promote a soothing effect of the skin or a calming effect of the skin due to an inflammatory condition or irritation that develops after shaving, friction, or contact with allergens.

Neurotrophins are part of a family of growth factors controlling the development, maintenance and apoptosis of neuronal cells. Neurotrophins also exert multiple non-neuronal functions: they regulate proliferation and cell differentiation, apoptosis and tissue remodeling, especially in the skin.

Dermal fibroblasts and myofibroblasts synthesize and secrete neurotrophins such as Nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). The effects of neurotrophins are mediated by their receptors also expressed by fibroblasts. There are two classes of receptors: receptors of the tyrosine kinase receptor (Trk) family and the p75 neurotrophin receptor (p75 NTR or NGFR). Each receptor has a specific affinity and specificity with respect to its ligand or ligands. TrkB (encoded by the NTRK2 gene) specifically binds BDNF as well as NT-3 and NT-4 P75 NTR is able to bind all neurotrophins.

Neurotrophins, via their receptors, stimulate the proliferation and migration of fibroblasts as well as the differentiation of fibroblasts into myofibroblasts. They could therefore be involved in the regulation of tissue remodeling during the healing process of skin wounds. NGF is a neurotrophic factor, initially described as a survival factor for neurons in the central nervous system, but also at the level of sensory neurons or nociceptors of the skin. NGF also plays a role in inflammation. In fact, the neutralization of NGF inhibits the sensitization of sensory neurons (nociceptors), with the effect of reducing the perception of pain associated with an inflammatory state. The extract promotes a soothing effect of the skin or a calming effect of the skin due to an inflammatory condition or irritation that develops after shaving, friction, or contact with allergens. • Cutaneous healing The root extract of Polygonum multiflorum induces the expression of the CCL5 gene, coding for the chemokine (C-C motif) ligand 5, also called RANTES. This chemokine has been shown to play an important role in the skin healing process. She is involved in the migration of dermal stem cells (DSC) to the skin lesion site. This process is essential for re-epithelialization, repopulation of fibroblasts in the dermis and angiogenesis. 2.2.4 Effects of the extract on 94 target genes in the melanized reconstructed epidermis (RHE / MEL / 001) • Cutaneous cicatrization The Polygonum multiflorum root extract induces a remarkable amplitude (by a factor of 4.1, p-value = 0.0175) the expression of the metalloproteinase-1 MMP-1 gene. MMPs are metalloproteinases capable of cleaving most of the components of the ECM and modifying, by proteolysis, many molecules important for skin healing. In particular, MMPI plays a major role in the re-epithelialization of keratinocytes. MMPI facilitates ECM assembly, cell elongation and migration, actin cytoskeleton reorganization, and induces ERK kinase activation (extracellular signal-regulated kinase) required for motility and ability to invasion of epithelial cells. In addition, it has been shown that the MMPI protein is overexpressed at the level of the epidermis in response to cutaneous wounds.

The overexpression of the MMP-1 gene at the level of the epidermis by the root extract of Polygonum multiflorum makes it possible to position it as an active agent potentially promoting cutaneous cicatrization, in particular at the level of the closure of the wound. 2.3 Conclusion

In synthesis, the root extract of Polygonum multiflorum is clearly positioned with respect to a cosmetic claim in anti-aging optics related to antioxidant activity and reduction of dermal degradation. The extract, by its role in controlling the production of free radicals, could be used as an active ingredient to ameliorate or treat inflammation of the skin.

In addition, the root extract of Polygonum multiflorum may have a soothing effect on the skin or a soothing effect of the skin due to the sensation of irritation that may develop after shaving, friction, or contact with allergens.

Finally, the root extract of Polygonum multiflorum is optionally positioned as an asset promoting skin healing, particularly in the case of closure of a wound.

Example 3 Effect of Polygonum multiflorum root extracts in propane 1,2 diol on human keratinocytes - "GeneChip Human Gene" microarray study

In order to analyze the possibility that an extract according to the invention has an anti-aging action, a transcriptomic study on a DNA chip was carried out from human keratinocytes (NHEKs) treated for 24 hours by a root extract of Polygonum multiflorum. . Variations in gene expression have been contextualized and interpreted biologically through the proprietary StratiCELL Skin Knowledge database.

A preliminary cytotoxicity study was performed to determine the optimal concentration of the extract used for the study. 3.1. Materials and methods

Extract

A root extract of polygonum multiflorum was prepared according to Example 1.4.

Cell culture The study was carried out on human epidermal keratinocytes NHEKs (Normal Human Epidermal Keratinocytes, Lonza, 0019206, origin: foreskin) grown in monolayer in Epilife medium containing HKGS (Human Keratinocyte Growth Supplement) and gentamycin.

The cells were maintained in a humid atmosphere at 37 ° C containing 5% CO 2. Determination of the range of study concentrations of Polygonum multiflorum root extract by a preliminary cytotoxicity study

NHEK keratinocytes were inoculated in 24-well plates 24 hours before application of the extract in Epilife medium (INVITROGEN, M-EPI-500 A) containing the HKGS supplement (INVITROGEN, S-001-K) and gentamycin (INVITROGEN, 15710-049). The extract was solubilized in the appropriate culture medium and then placed in contact with the NHEK keratinocytes for 24 hours.

This study evaluated the viability of MTS cells (3- (4,5-dimethythiazol-2-yl) -5- (3-carboxy-methoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) ( Promega, G3581), 24 hours after the addition of the extract and its solvent or carrier (propane 1,3 diol 60% pH = 3.5) and this, at 5 concentrations and 3 repetitions (n = 3).

SDS (sodium dodecyl sulfate) is toxic to cells and has been used as a positive control to validate the experiment.

At the end of this experiment, a non-cytotoxic concentration was defined in order to carry out the transcriptomic analysis.

The following concentrations, in percent v / v, were tested for the extract and the solvent: 3%, 1%, 0.33% 0.11%, 0.037%

Treatment of the cells The extract or the solvent were applied to a chosen concentration, for 24 hours, in the culture medium of the NHEK keratinocytes. Culture quadruples were made for each condition (n = 4).

Extraction of total RNA

At the end of the treatments, the total RNA populations were extracted. The extraction was carried out using the RNeasy kit (Qiagen). After 24 hours of treatment, the cells were rinsed with PBS and lysed in ad hoc buffer. The extraction and purification of the RNAs were performed according to the instructions of the supplier. Total RNAs were then stored at -80 ° C for transcriptomic analysis.

RNA Qualification by Spectrophotometry and Capillary Electrophoresis

The concentration of the total RNAs was determined by spectrophotometric measurement. The quality and integrity of the RNAs were then verified by capillary electrophoresis (Agilent Bioanalyzer 2100 Platform - Agilent RNA 6000Nano Kit, 5067-1511).

RNA quantification by spectrophotometric measurement: an aliquot of each RNA was diluted in RNAse-free water and its concentration was determined using an Ultrospec 1100 Pro spectrophotometer (Amersham).

RNA Integrity by Agilent Bioanalyzer Capillary Electrophoresis: Integrity of total RNA was assessed by visualization of peaks corresponding to ribosomal RNAs. For total RNAs of higher eukaryotes, ribosomal band size should be 1.9 kb for 18S-RNA and 4.7 kb for 28S-RNA. The intensity of the band corresponding to the 28S-RNA must be greater than the intensity of the band corresponding to the 18S-RNA. Small diffuse bands representing lower molecular weight RNAs (tRNA and 5S ribosomal RNA) may be present. When RNA is degraded, banding of ribosomal RNA and background noise of the higher molecular weight RNAs are observed.

Hybridization Phase on GeneChip Human Gene 2.0 ST Chips (Affymetrix)

The RNAs were then diluted to 50 ng / μl and 50 μl of each sample (n = 3). The fourth replica serves as a back-up.

The RNA samples (50ng) were amplified using Ribo-SPIA technology, according to 3 steps (Ovation Pico WTA System V2, NuGEN, 3302-12) and purified with the Agencourt RNA Clean up XP Beads Kit ( Agencourt - Beckam Coulter Genomics, A29168). For each sample, 4.5 μg was fragmented and labeled with biotin, using the NuGEN Encore Biotin Module (NuGEN, 4200-12). Hybridization was performed on DNA chips of GeneChip Model Human Gene 2.0 ST (Affymetrix, 902112). The hybridization steps on chip, washing and fixation, were carried out according to the protocol defined by Affymetrix. The hybridization solution was prepared using the Affymetrix Genechip Expression 3 'Amplification Reagent Hybridization Controls kits (Affymetrix, 900454) and Hybridization Module for GeneChip Hybridization, Wash and Stain Kit (Affymetrix, 900720), and blended with complementary DNA (CDNA) amplified in the previous steps. Hybridization was carried out for 18 h in the GeneChip Hybridization Oven 640 (Affymetrix, 800139). The washing and revelation were performed using the GeneChip Fluidics Station 450 fluidic station (Affymetrix, 00-0079) and the intensity measurements were scanned with the GeneChip Scanner 3000 (Affymetrix).

Pretreatment of hybridization data

Raw data processing was performed using R (v3.2.3, Ihaka R and Gentleman T. Journal of Computational And Graphical Statistics 1996, 5 (3), 299-314) and the package 'oligo' (vl.34.2). Benilton S Carvalho And Rafael A Irizarry, Bioinformatics 2010 (Oxford University Press), 26:19, Pp 2363-7, Doi: 10.1093 / Bioinformatics / Btq431) of the BioConductor project (v3.2, Gentleman R, Carey Vj, Bates Dm, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y. Genome Biology 2004, 5, R80). The latest version of the libraries provided by Affymetrix, built on version 19 of the human genome (UCSC Human Genome 19), and the RMA method, described by Irizarri et al. (Irizarry Ra, Hobbs B, Collin F, Beazer-Barclay Yd, Antonellis Kj, Scherf U, Speed Tp., Biostatistics, 2003b, 4 (2), 249-64, Irizarry Ra, SI Ooi, Wu Z, Boeke Jd. Appl Genet Mol Biol 2003a, 2, Epub 2003 Mar 18) were used to guide and perform preprocessing and annotating sequences.

Data analysis, annotation and thematic analysis

The individual statistical analysis of the genes / transcripts was carried out with the methods "Moderated t" and "Moderated F" implemented in the R package

Limma 3.26.8 (Smyth Gk, Statistical Applications in Genetics and Molecular Biology 2004, 3 (3)). Gene annotation and definition of gene clusters for over-representation analysis were made from the StratiCELL Skin Knowledge Database (Salmon M & Berger F. Cosmetic Expression 2014, 30). , 91-94). The over-representation analysis was performed using the hypergeometric test method in order to characterize the dermo-cosmetic themes / groups of interest factors based on the proportion of their detected and differentially expressed targets. The analysis was carried out from the list of genes detected with a p-value of 0.05 and a difference in expression level (rate of change or fold-change or FC) greater than 1.5 (bi-lateral) . 3.2. Results Determination of the analysis concentration of the Polygonum multiflorum root extract by a preliminary cytotoxicity study

A cytotoxicity study was performed on NHEK keratinocytes. The extract and its solvent used for its preparation were applied to the culture media for 24 hours (n = 3). The untreated control was arbitrarily set at 100% viability and the cytotoxicity threshold was conventionally set at 80% viability. The SDS condition is the positive control that validates the experience.

Based on the results of the preliminary cytotoxicity study (Figure 3), the optimal concentrations (in% v / v) selected for the extract and its solvent (propane 1,3 diol) are as follows: 0.33% .

RNA quantification by spectrophotometry

The ratio of absorbance at 260nm and 280nm is used to assess the purity of the RNA. A ratio close to 2 makes it possible to consider the sample as pure and devoid of contamination by proteins. The 260/230 ratio is used as a secondary measure of purity of the sample. The 260/230 value is generally higher than the 260/280 ratio and is expected to be around 2.2. The ratios obtained for all the samples of the present study are therefore satisfactory (data not shown) and were subsequently qualified by capillary electrophoresis.

Qualification of RNAs by capillary electrophoresis

The different RNA populations demonstrate the presence of narrow peaks, corresponding to 18S and 28S ribosomal RNAs, and a balanced ratio between the two peaks. The absence of intermediate and spreading peaks, characteristic of RNA degradation products, testifies to the integrity of the different populations (data not shown).

The quality and the integrity of the extracted RNAs being demonstrated, they were therefore used for the continuation of the protocol and involved in the amplification reactions, purification, biotin labeling and hybridization on microarrays.

Analysis of gene expression modifications induced by the Polygonum multiflorum root extract

At the end of the transcriptomic study on human epidermal keratinocytes NHEKs, the root extract of Polygonum multiflorum could play a potential role in the stimulation of epidermal differentiation and anti-bacterial defense. Hereinafter, the regulations observed in relation to these hypotheses are indicated in parentheses, as follows: gene name - Fold change p-value. • Stimulation of epidermal differentiation The human epidermis is a stratified epithelium constituting a barrier whose layers are defined by the stage of differentiation of the cells that compose them. As they are differentiated, the keratinocytes are loaded into keratin filaments and contain a matrix retaining the water, all surrounded by the horny envelope. Terminal differentiating keratinocytes are isolated by intercorneocyte lipids and are attached to each other by corneodesmosomes.

Basal keratinocytes progress through the four layers of the epidermis: basal, thorny, granular and horny.

The basal layer is formed by a single layer of cubic or prismatic keratinocytes linked together by desmosomes. In this layer the cells divide, one of the two cells remains in the basal layer while the other migrates to the upper layers. Basal keratinocytes are attached by hemi-desmosomes to a basement membrane that separates the epidermis from the dermis and forms the dermal-epidermal junction.

The spinous layer is formed by 5 to 15 layers of polygonal keratinocytes linked together by desmosomes. These cells contain tonofilaments, the precursor filaments of keratin.

The granular layer is formed by 3 layers of keratinocytes which contain keratohyaline grains and lamellar granules.

The stratum corneum is formed from 5 to 15 layers of corneocytes, i.e. differentiated keratinocytes which no longer have nuclei and organelles, but are surrounded by a horny envelope. They are loaded with keratin filaments.

Formation of keratin filaments:

During epidermal differentiation, keratin filaments (called tonofilaments) become more numerous and begin to aggregate in the cells of the spinous layer to form the tonofibrils. At the level of the granular layer, they associate with keratohyaline granules. The keratohyaline granules are composed of loricrin, involucrine (IVL -2.24 p = 0.0002), trichohyaline and profilaggrin which will be transformed into filaggrin by various proteases, including caspase 14 (CASP14 - 4.76 p = 0.00002 ). The crosslinking of filaggrin with keratin accelerates the process of aggregation and alignment of tonofilaments to form the keratinous cables that make up 85% of the volume of corneocytes. These are then associated with desmosomes during the formation of the horny envelope by transglutaminases (TGM1 - 1.98 p = 0.00025). Keratin plays the role of rigid mechanical structure in association with the horny envelope to ensure the maintenance of epidermal integrity.

At the level of the basal layer, the keratinocytes mainly express the keratins 5 (KRT5 - 1.54 p = 0.0039) and 14 whereas the upper layers contain rather the keratins 1 (KRT1 - 2.65 p = 0.000056), 2 and 10 (KRT10 - 7.81, p = 0.0000013).

Formation of the horny envelope:

During the differentiation of keratinocytes stimulated by various transcription factors such as zinc finger protein 750 (ZNF750 - 2.59 p = 0.00004), the plasma membrane is gradually replaced by a rigid shell called horny envelope. The initiation of this change results in an intracellular protein assembly coordinated by transglutaminases (TGM1 - 1.98 p = 0.00025). Periplakin, envoplakin and involucrine (IVL - 2.24 p = 0.0002) form an armature which gradually covers the inner face of the plasma membrane. At the same time, the protein structure is reinforced by other proteins such as loricrin, hornerine, "small proline rich" proteins (SPRR1A - 2.48 p = 0.000011; SPRR1B - 2.61 p = 0.00037; SPRR4 - 1.73 p = 0.0084), the S100 proteins (S100A8 - 2.51 p = 0.000056; S100A9 - 1.57 p = 0.014), and the late-cornified envelope proteins (LCE1C - 1.53 p = 0.031; LELP1 - 1.77 p = 0.049).

Intercellular junctions:

Desmosomes represent a type of junction between keratinocytes. Among the components of this type of structure, the desmocollins {DSC1 - 7.9 p = 0.000002; DSC2 - 2.43 p = 0.0000095; DSC3 - 1.57 p = 0.00046) and desmogleines (DSG1 - 7.62 p = 0.0000042; DSG3 -1.84 p = 0.00011) are transmembrane glycoproteins belonging to the family of cadherins. Their extracellular parts provide intercellular adhesion while their cytoplasmic parts associate with intracellular proteins such as plakoglobin (JUP - 1.73 p = 0.00031), plakophilin (PKP1 - 2.01 p = 0.00014) or the desmoplakin (DSP -1.64 p = 0.00028). Desmoplakine interacts itself with the keratin filaments thus allowing attachment of the cytoskeleton to the adhesion complex. During the cornification, the desmosomes are modified, the desmosomal plate is incorporated into the horny envelope and the corneodesmosine is integrated therein. They are then called corneodesmosomes. • Stimulation of the anti-bacterial defense

To fight against pathogenic aggressions, keratinocytes can produce anti-microbial peptides, responsible for the attraction and activation of immune cells.

Anti-microbial peptides have broad spectrum activity and destroy organisms by disrupting their membrane integrity. Some of them are constitutively expressed but most are inducible. Keratinocytes express peptides of this type in particular calgranulin A (S100A8 - 2.51 p = 0.000056) and calgranulin B (S100A9 - 1.57 p = 0.014) which form an antimicrobial heterodimeric complex called calprotectin. Calprotectin has been shown to exert its antibacterial activity against Escherichia coli, Klebsiella spp., Staphylococcus aureus, Staphylococcus Epidermidis, Capnocytophaga sputigena and Borrelia burgdorferi (Lyme disease). On the other hand, the transcriptomic study made it possible to demonstrate a very important increase in the expression of the gene coding for the chemokine (C-X-C motif) ligand 14 (CXCL14 - 16.7 p = 0.00000013). This chemokine is constantly produced in the epidermis and dermis in healthy conditions. However, in addition to its chemo-attracting effects, it has recognized bactericidal activity against Escherichia coli, Staphylococcus aureus, Finegoldia magna, Streptococcus pyogenes.

Different mechanisms are put in place to modulate the proliferation of bacteria, among them, we distinguish the desquamation process that eliminates unwanted microorganisms installed on corneocytes. The desquamation is based on the equilibrium between the synthesis of structural proteins of corneodesmosomes and their degradation by specific proteases such as kallikreins (KLK5 - 2.57 p = 0.000011, KLK7 - 2.17 p = 0.000029) and L2 cathetinsin (CTSV - 3 , 41 p = 0.00000083). 3.3 Conclusion

By positively modulating the genes encoding the proteins involved in epidermal differentiation, the root extract of Polygonum multiflorum could potentially stimulate epidermal renewal. In addition, it may play a role in the antimicrobial defense of the epidermis by increasing the expression of genes encoding certain antimicrobial peptides or chemokines involved in anti-bacterial defense.

Claims (10)

1. Polygonum multiflorum root extract characterized in that: it comprises at least 0.8% by weight of THSG, preferably at least 1.5%, more preferably at least 2%, expressed relative to the total weight of dry extract, and it comprises less than 0.01% by weight of emodin, preferably less than 0.001%, more preferably less than 0.0005%, expressed relative to the total weight of the dry extract. 2. Polygonum multiflorum root extract according to claim 1, characterized in that the amount of emodin in the extract is below the limit of quantification by supercritical phase chromatography or by high performance liquid chromatography coupled with spectrometry. mass (HPLC / MS). 3. Process for the preparation of a Polygonum multiflorum root extract according to claim 1 or 2, comprising: a) a step of cultivating Polygonum multiflorum under off-ground conditions, and particularly in aeroponics, b) optionally a step of stimulating plant roots, c) a step of solid / liquid extraction of the roots optionally stimulated in step b), and d) the recovery of the extract obtained in step c). 4. The method according to claim 3, wherein step c) comprises a first phase of placing the roots still bound to the plant with a solvent, in particular for a period of between 15 minutes and 30 minutes, followed by a second phase comprising the section of said roots and maceration of the severed roots in a solvent, said solvent, which is identical or different in the first and second phases, being chosen from alcohols, glycerin, glycols and eutectic solvents, and being used pure or in the form of an aqueous solution of alcohol, glycerine, glycol or eutectic solvent. 5. Cosmetic composition comprising, as active agent, at least one extract according to claim 1 or 2, or an extract obtained by a process according to claim 3 or 4, and at least one excipient. 6. Extract according to claim 1 or 2, extract obtained by a process according to claim 3 or 4, or cosmetic composition according to claim 5, for preventing or slowing skin aging and / or for stimulating the renewal of the epidermis, and / or have a soothing effect of the skin or a soothing effect of the skin following the sensation of irritation. A pharmaceutical composition comprising, as active agent, at least one extract according to claim 1 or 2, or an extract obtained by a process according to claim 3 or 4, and at least one pharmaceutically acceptable excipient. 8. Nutraceutical composition comprising, as active agent, at least one extract according to claim 1 or 2, or an extract obtained by a process according to claim 3 or 4, and at least one nutraceutically acceptable excipient. 9. Extract according to claim 1 or 2, extract obtained by a process according to claim 3 or 4, pharmaceutical composition according to claim 7 or nutraceutical composition according to claim 8, for its use as a medicament for stimulating the antimicrobial defense of the skin and / or to attenuate or treat inflammation of the skin and / or to promote skin healing, particularly in the case of closure of a wound. 10. Use of an extract according to claim 1 or 2, or an extract obtained by a process according to claim 3 or 4, for the preparation of a cosmetic composition intended to prevent or delay the appearance of the effects of aging. skin and / or stimulate the renewal of the epidermis, and / or have a soothing effect of the skin or a calming effect of the skin following the sensation of irritation.